Pathology of pain and its implications for therapeutic interventions

Abstract

Pain is estimated to affect more than 20% of the global population, imposing incalculable health and economic burdens. Effective pain management is crucial for individuals suffering from pain. However, the current methods for pain assessment and treatment fall short of clinical needs. Benefiting from advances in neuroscience and biotechnology, the neuronal circuits and molecular mechanisms critically involved in pain modulation have been elucidated. These research achievements have incited progress in identifying new diagnostic and therapeutic targets. In this review, we first introduce fundamental knowledge about pain, setting the stage for the subsequent contents. The review next delves into the molecular mechanisms underlying pain disorders, including gene mutation, epigenetic modification, posttranslational modification, inflammasome, signaling pathways and microbiota. To better present a comprehensive view of pain research, two prominent issues, sexual dimorphism and pain comorbidities, are discussed in detail based on current findings. The status quo of pain evaluation and manipulation is summarized. A series of improved and innovative pain management strategies, such as gene therapy, monoclonal antibody, brain-computer interface and microbial intervention, are making strides towards clinical application. We highlight existing limitations and future directions for enhancing the quality of preclinical and clinical research. Efforts to decipher the complexities of pain pathology will be instrumental in translating scientific discoveries into clinical practice, thereby improving pain management from bench to bedside.

Introduction

Pain is defined as an unpleasant sensory and emotional experience associated with, or resembling that associated with, actual or potential tissue damage.¹ It is considered the most primitive and widespread human experience. Owing to its subjective nature, the interplay of nociceptive, cognitive, emotional and social components collectively shapes the pain experience.² Acute pain acts as a defense mechanism against noxious stimuli, infection, homeostasis dysfunction and secondary insults.^3,4 Patients suffering from congenital insensitivity lack the ability to avoid damage, potentially leading to a predisposition toward self-mutilation.⁵ In contrast, chronic pain is inherently distressing and often the primary reason for patients to seek medical care. It poses a vast socioeconomic burden globally,⁶ with prevalence rates ranging from 10% to 40% and a relatively low recovery rate of only 5%.^7,8,9 Pain relief has been a requisite and an important index for clinical treatment.

Pain serves as a crucial nexus between primary diseases and secondary outcomes. It can trigger a dynamic and detrimental interplay among biological, social and psychological factors, leading to disability and poor prognosis for patients. Pain-related psychiatric disorders, such as insomnia, depression, anxiety and impaired social interaction, can exacerbate the progression of primary diseases. These pathological deteriorations also negatively impact social relationships and self-esteem as evidenced by notable increases in divorce, substance abuse, and suicide rates.^10,11,12 Chronic pain also undermines the survival benefits of cancer treatment.¹³ Notably, pain is not equal to suffering. The outcomes of pain are affected by various factors unique to an individual. For instance, massage can elicit pleasant sensations despite transient pain, and an individual in a positive emotional state may exhibit greater pain tolerance. These examples underscore that pain extends beyond a mere biological event and is intricately processed by the brain.

Analgesic drugs are the mainstay of acute and chronic pain management. Despite their short-term effectiveness, significant concerns regarding drug dependence, addiction and other side effects have been raised.^14,15 The misuse of analgesics has also garnered international attention. New insights into the mechanisms underlying pain sensitivity and recovery are gradually being reported. The development of new therapeutic modalities, drug delivery systems and nonpharmaceutical adjuvant therapies has potential value in pain management. However, these varied interventions still fall short of fully addressing the needs of an individual’s quality of life.

This review will introduce the basic knowledge concerning pain research and then discuss current advances in understanding the pathology of pain perception and modulation. Two hot topics, sexual dimorphism and pain comorbidity, will also be discussed. Management approaches for pain will be summarized and remarked for fully displaying the status quo of pain research. Finally, we will discuss the existing limitations and propose future directions for enhancing the research and clinical practice of pain.

Historical milestones of investigations into pain therapy

The history of human development is intertwined with the struggles against pain (Fig. 1). Opioid alkaloids, derived from the opium poppy, have been used for analgesia and euphoria for thousands of years. In 1806, Friedrich W. Sertürner pioneered the extraction of pure opioids. This event opened a new chapter in fighting with pain using modern medicine. Another representative drug, acetylsalicylic acid, also called aspirin, was synthesized by Felix Hoffman in 1897. Since then, non-steroid anti-inflammatory drugs (NSAIDs) have gradually become a mainstay in pain management. The discovery of their mechanisms was awarded the Nobel Prize in 1982. With the growing understanding of psychological factors of pain, psychologist Aaron Beck summarized the achievements and proposed cognitive behavioral therapy (CBT) in 1960s. The efficacy of CBT in treating mental disorders, including pain, has been substantiated by numerous cases. This finding underscores the tight link between pain and psychological factors. Advances in computers and algorithms have enabled rapid processing of complex data. In 1965, Melzack and Wall proposed the Gate Control theory. This theory depicted the important functions of spinal dorsal horn in modulating pain signals, offering novel insights into pain pathology and approaches to clinical pain management.¹⁶ A clinical trial explored the analgesic effects of spinal cord stimulation to treat eight painful patients after 2 years. Half of the patients obtained longstanding pain relief within 2 min, which first proved the superiority of spinal cord stimulation.¹⁷ In 1976, the opioid receptor was identified in the primate spinal cord.¹⁸ In the same year, Yaksh and Rudy conducted intrathecal opioid delivery of narcotics in rats. It effectively exerted potent analgesia only at the spinal level. This exploring experiment laid the foundations for the development of spinal cord stimulation therapy.¹⁹ In 1988, artificial intelligence (AI) was first applied in a clinical trial focusing on the pain diagnosis. The results demonstrated that AI outperformed clinicians in differential diagnosis, highlighting its potency in the pain field.²⁰ Six years later, Edelman et al. utilized magnetic resonance imaging (MRI) to detect brain region activities, laying the groundwork for exploring regions involved in pain perception.²¹ At the end of 20th century, David Julius and colleagues identified the ion channel TRPV1, which is responsive to heat and then produce pain signals. This finding paved the way for discovering other temperature sensors. David Julius was honored with the Nobel Prize in 2021 for this breakthrough.

Fig. 1

The brief timeline of historic milestones in the field of pain therapy. Morphine was first extracted in 1806, which opened the chapter in fighting with pain using the fruits of modern medicine. Since then, many intervention methods for pain management were discovered and came into clinical application, such as CBT, spinal cord stimulation, monoclonal antibody therapy and gene therapy. The progress in the research on pain mechanisms and interdisciplinary collaboration boosted advances in pain therapy. In recent years, the wide application of high-throughput biotechnologies has further deepened the understanding in pain pathology and has contributed to the development of individualized pain management. Key milestones of pain therapy are chronologically illustrated in the figure. The achievements awarded by the Nobel prizes are marked with the medals

Entering new century, advanced technologies have been employed in basic research and pain management. The first clinical trial on a monoclonal antibody in neuropathic pain was reported in 2003.²² The effectiveness and safety of gene therapy were proved by a phase I clinical trial in 2011.²³ A year later, the technique for converting pluripotent stem cells into nociceptors was established. This progress has provided a better in-vitro model for pain research.²⁴ The associations between microbiota and pain have been revealed long before. However, it was commonly believed that microbiota activated nociceptors only through inducing inflammatory responses or secreting specific metabolites. A basic study in 2013 showed that gut microbiota could directly stimulate nociceptor neurons and induce pain sensation.²⁵ The revelation shifted previous perceptions in this field and marked a milestone in microbiota and pain research. Over the last decade, research breakthroughs have continued to emerge. The organ-on-a-chip technique was applied to create a spinal microphysiological system for investigating pain and opioid-induced tolerance.²⁶ It represents another significant advancement in experimental pain research tools. The latest milestone is the brain cell atlas, described using multi-omics by the BRAIN Initiative Cell Census Network project, which was reported in the special column of Science journals. This pioneering work parses brain structures at the single-cell level, providing valuable data for elucidating pain mechanisms.

Categories of pain

Pain can be classified as nociceptive, neuropathic or nociplastic pain according to its etiology. One pain event tends to involve multiple categories. For instance, in a serious car accident, acute pain induced by open wounds can cause nociceptive pain. Spinal cord injury caused by a car crash may bring about perennial neuropathic pain. Posttraumatic stress disorder (PTSD) may also be triggered by this life-challenging event, resulting in somatic nociplastic pain. The etiology of cancer pain is more complicated, involving nerve invasion, organ damage, immune dysregulation and other unknown factors. Therefore, the clarification of pain categories is conducive to the development of pain research.

Nociceptive pain

Nociceptive pain refers to pain induced by a physiological protective system that protects against noxious stimuli,²⁷ which is the most frequent type of pain. It is by nature a transient response to actually or potentially harmful factors, triggering evasive action and protective behaviors. Inflammatory pain is one of the most representative subtypes of nociceptive pain. Somatic nociceptive pain is usually perceived in the dermis layer and is often described as lancinating, sharp or burning pain. In contrast, the sensation of visceral nociceptive pain is blurry and diffuse. The pain generated by cutting, burn and corrosion injuries can be classified as nociceptive pain.

Neuropathic pain

Neuropathic pain is defined as pain arising as a direct consequence of a lesion or disease affecting the somatosensory system, including central neurons and peripheral fibers (Aβ, Aδ, and C fibers). According to epidemiological investigations, 7–10% of the general population experiences neuropathic pain, accounting for 20–25% of patients suffering from chronic pain.^28,29 The prevalence of neuropathic pain is dramatically increased in individuals with specific chronic diseases due to its mechanistic particularity. Diabetic polyneuropathy, cancer, herpes zoster, multiple sclerosis and spinal cord injury are important diseases with secondary involvement in neuropathic pain. Patients with neuropathic pain typically experience a series of manifestations, such as burning and electrical-shock sensations. Persistence and poor responses to analgesics create enormous health burdens for patients, usually accompanied by psychiatric disorders, such as depression, anxiety and insomnia.

Nociplastic pain

Some patients with explicit pain phenotypes fail to present with organic lesions and therefore cannot be classified as either of aforementioned types. In 2016, the concept of nociplastic pain was proposed and defined as a mechanistic descriptor for chronic pain states not characterized by clear activation of nociceptors or neuropathy but exhibiting clinical and psychophysical findings suggestive of altered nociceptive function. Its prevalence in the general population ranges from 5% to 15%, and there is a significant female preference.³⁰ Nociplastic pain is divided into five categories: chronic widespread pain, chronic primary musculoskeletal pain, chronic primary visceral pain, chronic primary headache pain and complex regional pain syndrome.³¹ Genetic, psychosocial, and environmental factors jointly contribute to the progression of nociplastic pain.³²

Animal models applied for current research on pain

Experimental animal models are indispensable tools for basic and preclinical investigations into occurrence, diagnosis and treatment of pain. As pain is a multimodal event, an ideal pain model should encompass both biological and psychological factors. A diverse array of model preparation methods has been developed, including physical damages, chemical and biological irritants and psychosocial stressors (Fig. 2). Regrettably, standardized modeling approach that perfectly replicates pain development is still lacking. Most current models fail to accurately represent the mechanisms of specific pain types, potentially compromising the validity of basic research findings. In this section, we summarize the commonly employed methods of pain model generation to provide the swift access to pain research field for readers.

Fig. 2

Current animal models in pain research. Physical damages, chemical irritants, cancer cell implantation and psychosocial stressors constitute the three primary methods for preparing pain models. Furthermore, composite regimens that combine several of the aforementioned methods have been employed as pain is a multifactorial event. CCI chronic constriction injury, CFA complete Freund’s adjuvant, DSS dextran sulfate sodium, IBD inflammatory bowel disease, IBS irritable bowel syndrome, MS maternal separation, NLB neonatal limited bedding, PTSD posttraumatic stress disorder, SNI spared nerve injury, SNL spinal nerve ligation, TNBS 2,4,6-trinitrobenzene sulfonic acid, WAS water avoidance stress

Physical damages

Surgery is a common method for generating nociceptive and neuropathic pain models. Chronic constriction injury (CCI), spared nerve injury (SNI) and spinal nerve ligation (SNL) are classical approaches for inducing neuropathic pain. CCI is produced by placing loosely constrictive ligatures around the common sciatic nerve. SNI entails the incision of tibial and common peroneal nerves, sparing the sural nerve. Therefore, a key advantage of SNI is better observation of impacts of injured and non-injured nerves. Following these procedures, the metapedes of both models typically develop hyperalgesia, and the mechanical withdrawal threshold decreases. Hyperalgesia usually peaks after 7 days of surgery and persists over two months. The spontaneous ongoing pain also becomes detectable after 7 days. The spinal nerve, due to its accessible anatomical position and significant physiological functions, is another idea target. Commonly, the L5 spinal nerve, located near the dorsal root ganglion (DRG) is selected for SNL modeling.³³ Pain perception typically develops within in 1–3 days, sooner than in CCI and SNI models. The mechanical and heat hyperalgesia can sustain 10 and 3 weeks, respectively. The spontaneous pain phenotype develops after one month of SNL.³⁴ The significant advantage of SNL is better investigations into the impacts on DRG. It is noteworthy that neonatal subjects may not experience mechanical allodynia or undergo delayed-onset pain sensitivity following SNI, CCI, and SNL modeling,^35,36 suggesting their unsuitability for early-life neuropathic pain studies. Although these three methods simulate physical nerve injury, it still remains unclear whether they can recapitulate the common diseases of neuropathic pain, such as diabetic neuropathy, neuropathic low back pain and radiculopathy.³⁷ Therefore, it should be cautious to draw conclusions concerning associations between etiological factors and clinical neuropathic pain based on these models.

Given the organ and tissues specificity of innervation, some studies exploring topical pain-associated diseases involve surgical damage to specific nerves to induce hyperalgesia at targeted sites. For example, T9 laminectomy combined with radical contusion damage is used to simulate spinal cord injury.³⁸ Trigeminal nerve root compression in inferior orbital fissure or inferior alveolar nerve is performed to generate animal models of trigeminal neuropathic pain.^39,40 Furthermore, paw incision is an effective approach to imitate the status of postoperative pain or acute pain, which is extensively applied due to the simplicity and reproducibility.⁴¹

However, there are two significant limitations of physical damage models. First, despite precise intervention, inflammatory pain following operations, particularly in the acute phase, is inevitable. Consequently, research conclusions should be interpreted cautiously and comprehensively. Second, most methods are “all or nothing”. They lack the capability to control the extent of damage, rendering them unsuitable for studies investigating the effects of varying degrees of nerve damage, with partially different underlying mechanisms. Electrocautery tends to progress into persistent allodynia,⁴² making it more suitable for the research on pain chronicity. Additionally, electrical stimulation is also employed to trigger pain sensations. Its non-invasive nature is noteworthy. Potential inflammatory responses following invasive operations can be significantly reduced. Furthermore, some studies have verified the antalgic role of electrical stimulation.⁴³ Differentiating its pain-inducing and pain-relieving effects requires further investigation.

Chemical irritants and cancer cell implantation

Complete Freund’s adjuvant (CFA) is a water-in-oil solvent composed of mineral oil, dead Mycobacterium tuberculosis and an antigen salt solution. It is extensively used in preparing topically inflammatory pain or arthritis models by injection into the paw or arthrosis, respectively. Paw injection of CFA can induce pain hypersensitivity and non-evoked ongoing pain after 24 h and it will last for 1–2 weeks. Joint pain occurs after 7 days of intra-articular injection. High-dose CFA is one of few approaches to generate models at the chronic phase of pain. Furthermore, CFA elicits synovitis and bone resorption without cartilage alteration, thus it has been evaluated as a robust model for the research on rheumatic arthritis.⁴⁴

Formalin is a protein coagulant commonly employed for tissue and cell fixation. Subcutaneous injection of formalin diluent into animal hind paws can generate local pain. Formalin-induced evoked pain and spontaneous ongoing pain are characterized by a two-phase response. The first phase (0–5 min) results from the activation of peripheral nociceptors, whereas the second phase (10–40 min) reflects the development of inflammation and central sensitization.⁴⁵ Low-dose formalin directly activates nociceptors, while injection of high-dose formalin can exert additional tissue damage and inflammatory stimuli.⁴⁶ Hence, the evidences indicate a significant time and dose-dependent manner of formalin-induced pain. It is usually employed for investigations into pain mechanisms. Additionally, topical injection of carrageenan is mainly used for preparing transient joint inflammation. The hyperalgesia and spontaneous nociceptive behaviors occur within 3–5 h and lasts for 24 h. Zymosan is a typical agent for acute inflammation research. It can induce thermal and mechanical hyperalgesia after 30 min in a dose-dependent manner. Spontaneous pain can be observed after 24 h of high-dose injection of zymosan.⁴⁷ Capsaicin is commonly used for construction of skin inflammation and inflammatory bowel disease (IBD), as well as examination of analgesic drug efficacy. It promptly triggers evoked pain perception and fades within 1 h. Spontaneous ongoing pain occurs primarily within 5 min. Compared to the sustained and biphasic pain induced by formalin, it exhibits shorter lasting and monophasic duration.⁴⁸ Intriguingly, high-dose or continuous treatment reversely lead to neuronal desensitization and analgesic effects. The modeling regimens should be carefully investigated before generating pain modeling using capsaicin.

Notably, chemical pain inducers play a crucial role in generating models of gastrointestinal disorder-associated pain. Intrarectal administration of dextran sulfate sodium (DSS) and oral treatment with 2,4,6-trinitrobenzene sulfonic acid (TNBS) are classical methods for inducing IBD. The symptoms of visceral hypersensitivity are detectable within several weeks. The pathology induced by DSS shares more features of ulcerative colitis, while the immunological and histopathological mechanisms underlying Crohn’s disease progression are following TNBS treatment.⁴⁹ Researchers should choose proper chemical irritants according to disease types. For the research on irritable bowel syndrome (IBS), intracolonic injection of zymosan or acetic acid is commonly used, whereas with different treatment periods. Zymosan-induced visceral hypersensitivity can be detected only after 3 days.⁶¹ Water avoidance stress (WAS) is another method to achieve movement restraint. Mice are placed on a small platform inadequate for standing by all fours and surrounded by water, which forces the subjects to remain continuously vigilant, resulting in a strong stress response. Repeated WAS can induce typical visceral hypersensitivity. Nevertheless, there have been no studies examining the effects of NLB and WAS on spontaneous ongoing pain.

The social relationship damage is also employed in pain model generation. Maternal separation (MS) is the severance between juvenile individuals and their dependent subjects, which affects nervous system development and increases the risks of adult psychiatric disorders.⁶² Therefore, MS has also been conducted in basic research on nociplastic pain. Current studies using this method have focused on visceral hypersensitivity. Animals undergoing early-life MS will suffer from pain hypersensitivity at the adult phase. Separation time is a critical factor in the effects of MS. Brief separation, more parallel to mother scavenging for food, has a relatively mild impact on juvenile subjects, whereas severe anxiety behaviors are observed following long-term separation. Likewise, the studies focusing on MS and spontaneous ongoing pain is still lacking.

Composite models

Despite the apparent pain-inducing effects of the above single-factor models, their limitations on disease reducibility are obvious due to the etiological complexities of pain in patients. Some studies tried to simultaneously use several methods to corroborate each other.^63,64 Furthermore, the comprehensive modeling strategies based on existing approaches can maximize the simulation capabilities of pain models. For example, the TC-IBS method includes trinitro-benzene-sulfonic acid treatment and subsequent chronic unpredictable mild stress, with properties of both inflammatory induction and psychiatric strike.⁶⁵ Similarly, MS and chemical stimulation have been assembled to prepare a pain hypersensitivity model.⁶⁶ In the research on PTSD-related pain, chemical irritants and restraint stress are used simultaneously. Nervous system homeostasis and neurologic functions are typically disrupted, akin to PTSD symptoms.⁶⁷ Although these models are based on the superposition of different factors, they offer valuable insights for develo** more scientific models for pain research.

Basic circuits of pain

Pain perception is a complex physiological process involving both the central nervous system (CNS) and peripheral nervous system (PNS). Numerous nervous structures, cells and molecules collectively underlie the transduction, transmission, modulation and perception of pain signals (Fig. 3). This section provides an overview of the basic mechanisms of pain perception for readers to better understand the subsequent contents in this review.

Fig. 3

Schematic illustration of pain sensation pathways. The exposure to pain-inducing events changes activity of specific receptors and activates action potential of peripheral nociceptors. The signals are then transmitted from DRG located to the spinal cord via afferent nerves. The nerves are categorized into Aβ, Aδ and C fibers. During neuronal transmission, the presynaptic membrane releases various neurotransmitters into the subsynaptic membrane, inducing potential alterations in the subsequent neuron. The figure shows some representative neurotransmitters in during pain perception. Additionally, neurogliocytes, immune cells and other types of neurons collaboratively modulate pain signals. The DRG, as the relay station, is responsible for ascending transmission to the corresponding sensory cortex, which modulates the ultimate pain sensation. The descending regulatory pathways also play a role in pain modulation. ASIC acid-sensing ion channel, AMPAR α-amino-3-hydroxy-5- methylisoxazole-4-propionate receptor, CGRP calcitonin gene-related peptide, GABA gamma-aminobutyric acid, GPCR G protein-coupled receptor, mGluR metabotropic glutamate receptor, NGF nerve growth factor, NMDAR N-methyl-D-aspartate receptor, P2X3 purinergic receptor 3, TrkA tropomyosin-related kinase A, TRPA1 transient receptor potential ankyrin 1, TRPM8 transient receptor potential melastatin 8, TRPV1 transient receptor potential vanilloid 1, TRPV2 transient receptor potential vanilloid 2, TRPV3 transient receptor potential vanilloid 3, TRPV4 transient receptor potential vanilloid 4, VGCC voltage-gated calcium channel, VGPC voltage-gated potassium channel, VGSC voltage-gated sodium channel

Peripheral transmission of pain signals

Nociceptors, peripheral transducers of pain signals, are located in the skin, mucosa, muscles, surface and interior of tendons, periosteum, vasculature, and internal organs. They are morphologically free or undifferentiated nerve endings, the cell bodies of which reside in the DRG and trigeminal ganglion. According to the received noxious stimuli, they can be divided into thermo-sensitive, mechanical-sensitive and injury signal-sensitive types. Compared to other sensors, the activation thresholds of nociceptors are relatively higher, ensuring that human body perceives normal tactile information without pain. Nociceptors are regarded as the gatekeepers and initiators of pain sensation.

The peripheral terminals of nociceptors have many types of ion channels, which can perceive external stimuli, code signals and generate membrane excitability. Ion channels produce electrical signals through regulating the ion current across membranes. The adjacent voltage-sensitive channels are forced open in a chain reaction. According to the precipitating factors of channel opening, they can be generally divided into two categories, voltage-gated ion channels and ligand-gated ion channels.

Voltage-gated ion channels refer to a kind of transmembrane proteins whose conformation is determined by membrane potentials. They play a crucial role in converting receptor potentials into a series of action potentials. Voltage-gated sodium channel (VGSC) family comprises 9 members, including Nav1.1 to Nav1.9. VGSCs rapidly adopt open conformations following cell membrane depolarization, allowing sodium to flow into cells down a concentration gradient. This process initiates action potentials and produces pain signals at nerve endings. VGSCs have typical differences in species, spatial and temporal distributions, as well as electrophysiological characteristics.^68,69 Nav1.7, in particular, has garnered significant attention. Mutations in the Nav1.7 encoding gene Scn9a are associated with various pain disorders, such as inherited erythromelalgia, paroxysmal extreme pain disorder and small-fiber neuropathy.⁷⁰ Inhibiting Nav1.7 functions effectively mitigates neuropathic pain and stimulates the production of endogenous opioids.⁷¹ The role of Nav1.7 varies with different types of pain. For instance, it contributes to the development of neuropathic pain, whereas bone cancer pain and oxaliplatin-induced pain do not depend on Nav1.7-postive nociceptors.⁷² Other VGSCs, like Nav1.1, Nav1.6, and Nav1.8, also play important roles in pain modulation.^68,73,74

Voltage-gated calcium channels (VGCCs) are distributed in all types of excitable cells. They are composed of four subunits: α1, β1-4, α2δ1-4, and γ1-8. Each VGCC type has a unique subunit composition, with α2δ being a crucial component. α2δ interacts with α1 and β subunits, enhancing peak potentials and rates of channel activation and inactivation. Noxious stimuli can upregulate α2δ expression in both the CNS and PNS, subsequently augmenting pain signals.^75,76 The functions of calcium channels in sensory neurons are finely tuned by various factors, like adiponectin, neuromedin B and non-coding RNAs.^77,78,79

In contrast to VGSCs and VGCCs, voltage-gated potassium channels (VGPCs) primarily facilitate potassium outflow from neurons, inducing membrane hyperpolarization and neuronal excitability attenuation. Noxious stimuli, such as mechanical force, heat and algogens, can downregulate potassium channel expression and inhibit their activity,⁸⁰ leading to ectopic spontaneous discharges in nociceptors.⁸¹

Transient receptor potential (TRP) channels, the most representative ligand-gated ion channels, are extensively distributed in both the CNS and PNS. TRP family members act as molecular sensors of pain and itch, responding to physical and chemical stimuli. Currently, 28 TRP members have been identified, with well-documented biological functions for TRPV1, TRPV2, TRPV3, TRPV4, TRPA1, and TRPM8.⁸² The mechanistic associations of TRPV1 and TRPA1 with pain modulation have been largely investigated. Their activation states and expression levels are positively associated with pain sensation.^83,84 Intriguingly, variouNMDARs natural biotoxins induce pain perception just through targeting TRPV1 and TRPA1,^85,86 demonstrating the ingeniousness of interspecies evolution. The TRP channel antagonists, like V116517 and BCTC, have shown significant potential in pain management.^87,88

N-methyl-D-aspartate receptors (NMDARs), consisting of various GluN subunits, are particularly sensitive to mechanical stimulation. Calcium influx through NMDARs is a critical inducer of electrical signal activation.⁸⁹ NMDARs interact with calcium channel subunit α2δ, tonically activating primary afferent neurons.⁹⁰ The ion-specific permeability is controlled by Mg²⁺, and neuronal depolarization contributes to the activation of NMDARs. Both presynaptic and postsynaptic NMDARs modulate excitatory synaptic transmission and CNS synaptic plasticity, facilitating hyperalgesia.^150,151 In addition to internal networks, the abnormality of DNM connectivity with other brain regions has been extensively discovered, involving the insula, ventral lateral/posterolateral nucleus and postcentral gyrus.^152,153,154 The advances in DMN research provide strong proof for identifying mechanisms underlying emotional changes that affect pain perception. For instance, mind wandering restores the ectopic connectivity between PAG and DMN, redirecting spontaneous attention away from pain.¹⁵⁵ The thalamic-DMN decoupling has been proved as an important mechanism of mindfulness meditation.¹⁵⁶ On the other hand, negative mood promotes pain hypersensitivity through influencing DMN functional connectivity during the progression of chronic pain.¹⁵⁷ Notably, despite close associations between DMN and chronic pain shown by most studies, acute pain can likewise induce alterations in oscillatory activity and functional connectivity of DMN, which underpins attentional processes in the presence of pain.¹⁵⁸

Molecular mechanisms of pain modulation

In addition to the basic circuits and corresponding molecules as introduced above, a series of molecular mechanisms underlie pain perception under intricate but well-regulated control. With the development of high-quality preclinical research, the scattered advancements are gradually converging into the systemic body of knowledge, contributing to the identification of numerous promising therapeutic targets. Herein, we summarize current achievements in related molecular mechanisms to present a more complete network of pain modulation (Fig. 4).

Fig. 4

The schematic illustration of molecular mechanisms underlying pain modulation. The molecular mechanisms are generally categorized into six aspects, including gene mutation, epigenetic modification, posttranslational modification, inflammasome, signaling pathways and microbiota. They orchestrate pain perception and modulation

Gene mutation

Most gene mutations are neutral, but a small minority may cause diseases, including pain disorders. Various mutations can lead to totally different clinical outcomes, ranging from pain insensitivity to extreme pain sensation. Erythromelalgia, familial episodic pain syndrome, congenital insensitivity to pain with anhidrosis and Fabry disease are the representative inherited diseases with specific gene mutations. Due to the individual differences, mutation patterns associated with pain disorders are sporadic and most data have been presented as case reports. Mutations in ion channel-encoding genes account for a large portion of existing investigations.

Mutations of multiple sites of Scn9a gene cause truncation or function loss of Nav1.7, leading to congenital insensitivity to pain. Some cases are complicated with anosmia, while other patients have normal olfactory sensation,^159,160,161 suggesting that mechanisms by which Nav1.7 modulates pain and olfaction partially overlap. Common missense mutants of Scn9a are correlated with pain severity of clinical patients with symptomatic disc herniation.¹⁶² Mutations in introns, which do not directly encode Nav1.7 protein, can also affect pain sensitivity. A novel homozygous substitution in Scn9a intron 3 interferes with mRNA splicing and leads to Nav1.7 inactivation. Furthermore, mutations are not confined to channel function deficiency. A1632E is a type of gain‐of‐function mutation. Nav1.7/A1632E mutants can form dimers and maintain persistent currents, exempt from the effects of inactivation particles targeting VGSCs.¹⁶³ Such non-canonical mechanisms greatly expand the understanding in gene mutation functions.

Mutations in other VGSC-encoding genes also contribute to the dysregulation of pain perception. Two missense mutations in Scn11a (c.673 C > T and c.2423 C > G) facilitate channel activity and promote hyperexcitability of Nav1.9 in DRG sensory neurons, which is a critical reason for familial episodic pain syndrome.¹⁶⁴ The mutation at the R222S site of Scn11a has also been identified in patients with mechanical hyperalgesia sensitive to cold exposure.¹⁶⁵ Conversely, a heterozygous nonsynonymous mutation in exon 15 of Scn11a causes excessive activation at resting potential and sustained depolarization of nociceptors in individuals with the congenital inability to experience pain. The resultant action potential and excitatory transmission are impaired, leading to a loss of pain perception. This mechanism of overactivation-induced inactivation is similar to pain relief by capsaicin.¹⁶⁶ Additionally, the Nav1.1 channel with L263V missense mutation enhances spike activity induced by P2X3 and 5-HT3 receptors, increasing the excitability of peripheral trigeminal neurons and contributing to migraine pain .¹⁶⁷

One mutation pattern of the Cav3.1 channel in trigeminal neuralgia has been recently identified. The missense mutation of Cacna1g gene, encoding α1 subunit of Cav3.1, leads to the replacement of arginine with glutamine at position 706. Current density is enhanced and neuron excitability is significantly elevated.¹⁶⁸ Intriguingly, an α2δ1 mutant with arginine at position 217 does not change pain sensitivity but blocks the analgesic efficacy of pregabalin for neuropathic pain.¹⁶⁹ This finding clearly demonstrates that the analgesic action of pregabalin relies on α2δ1 subunit blockade.

In addition to VGSC and VGCC mutations, different VGPC variants have distinct impacts on pain sensitivity. A frameshift mutation in Kcnk18 gene, encoding the two-pore potassium channel, causes its loss of functions. Neuronal excitability is significantly increased, exaggerating mechanical and thermal hypersensitivity during migraine progression.¹⁷⁰ A recent study focusing on gene mutations in women requiring no analgesia during childbirth has identified Kcng4 with excessive heterozygotes carrying the rare allele of SNP rs140124801. The product, Kv6.4 mutant, loses the capability of regulating Kv2.1 activity. The potassium outflux and sensory neuron hyperpolarization in uterus are promoted, attenuating childbirth pain.¹⁷¹

The roles of mutations in representative members of the TRP family in pain modulation have been unveiled. TRPV1 with N331K mutation directly causes functional deficiency.¹⁷² The G564S mutant is a gain-of-function variant. Nevertheless, the overactivation-induced inactivation is also observed in this mutation pattern. This membrane transport of G564S mutant is simultaneously inhibited.¹⁷³ Notably, in addition to natural mutation, Trpv1 gene can be chemically edited by an alkylating agent to produce a loss-of-function product.¹⁷⁴ For the research on TRPA1, the N855S mutant exhibits a fivefold increase in inward current in activated nociceptors, resulting in the development of familial episodic pain syndrome.^175,176 A nonsense mutation in Trpa1 gene causes TRPV1 protein truncation, which can further assemble with wildtype TRPA1. The complex lowers energetic barriers and alters pore architecture, leading to neuronal hyperactivation.¹⁷⁷

Additionally, mutations in genes regulating neuron development and axon outgrowth have been found to modulate pain sensation loss or sensitivity, including transcription factors, structural proteins, membrane channels and receptors.^{178,179,180,181,182} Taken together, a great number of genes and mutated sites have been identified to have associations and causalities with pain. However, we have to acknowledge current research limitations: i) The concrete mechanisms by which these mutated proteins gain or lose functions are largely unknown. High-resolution structures and interactions may be promising research directions. ii) The typical individual differences in gene mutations mean that current achievements have lower universality, limiting their further clinical translation. iii) Few studies have investigated potential drugs targeting the mutants, leading to the dreadful scarcity of clinical therapies against congenital pain disorders. Robert et al. found a peptide with properties of blocking P2X7 receptor mutants without restraining normal channels, which is associated with nerve injury and inflammatory allodynia.¹⁸³

Epigenetic modification

Despite differences in hereditary information, there are extremely high similarities in gene sequences between individuals with significantly different characteristics. Environment, behavior and age can produce apparent and persistent influences on humans. These phenomena cannot be forcefully explained by inherent genetic information alone. Epigenetics refers to alterations in gene expression not rooted in DNA sequences. Rapid advancements in epigenetics knowledge have unveiled novel mechanisms underlying physiological and pathological processes. It primarily includes DNA methylation, histone modification and non-coding RNAs. These three molecular mechanisms play essential roles in pain modulation (Fig. 5).

Fig. 5

The mechanisms of epigenetic modification in pain modulation. The mechanisms are categorized into three aspects: DNA methylation, non-coding RNA and histone acetylation. a For DNA methylation, DNMTs and TETs are responsible for DNA methylation and demethylation, respectively. They regulate expression of various genes associated with pain perception. The expression of KCNA2, BDNF and OPRM1 are simultaneously under the control of DNMTs and TETs. b Non-coding RNAs, comprising miRNAs, lncRNAs and circRNAs, play various roles. miRNAs can bind to 3’UTR of mRNAs associated with pain, negatively regulating their expression. Some lncRNAs and circRNAs act as miRNA sponges to counteract the functions of downstream targets. Certain lncRNAs and circRNAs directly interact with proteins to enhance their stabilization, thereby affecting pain sensitivity. Several non-coding RNAs, like lncRNA NEAT1 and circVOPP1, have been shown to stabilize the mRNAs of their parental genes related to pain to promote their expression. c HDACs and HATs collaboratively maintain the balance in histone acetylation. Specific HDACs, including HDAC2, HDAC4, HDAC5, SIRT1 and SIRT3, along with HAT p300, regulate expression of genes involved in pain modulation. Notably, non-coding RNAs regulate expression of enzymes associated with DNA methylation and histone acetylation. The expression of non-coding RNAs are, in turn, regulated by the other two mechanisms. circRNA circular RNA, DNMT DNA methyltransferase, HAT histone acetyltransferase, HDAC histone deacetylase, lncRNA long non-coding RNA, miRNA microRNA, TET ten-eleven-translocation protein, UTR untranslated region

DNA methylation pertains to forms of DNA chemical modification. Catalysis of DNA methyltransferases (DNMTs) can transfer methyl groups derived from S-adenosylmethionine to specific bases. Most DNA methylation sites exhibit aggregated distributions, known as CpG islands. DNA methylation changes chromatin structure, DNA conformation, DNA stability and interactions with proteins, precisely regulating gene expression without editing base sequences. Studies have shown close associations between DNA methylation and pain perception.

Patients suffering from chronic pain universally undergo significant changes in DNA methylation states, particularly in promoter regions.^184,185 Global methylation data have been used to investigate pain-associated mechanisms with the support of bioinformatic analysis, such as G-protein coupled cholinergic signaling, neuron development and immunomodulation.^184,186,187 DNA methylation has quick responses to pain. Its alterations can be detected at the early phase of neuropathic pain and persist chronically.¹⁸⁸ DNA methylation has disease and organ specificities. For example, there are huge differences between DNA methylation induced by diabetes neuropathy, nerve injury and chemotherapy, although they are all typical neuropathic pain. The CpG sites present prevailing hypomethylation in DRG, whereas the CNS, such as spinal cord and PFC, gains more DNA methylation.^187,188,189 The methylation levels of genes encoding classical positive regulators of neuropathic and nociplastic pain sensation, like TRPA1, CGRP, and BDNF, are significantly altered in patients with pain disorders. The methylation levels negatively regulate their expression, potentially causing hyperalgesia or pain insensitivity.^{190,191,192,193}

The DNMT family mainly consists of three enzymes with catalytic activity, including DNMT1, DNMT3A, and DNMT3B, responsible for adding methyl to specific gene regions. They generally present hypomethylation and participate in neuropathic pain modulation in both the CNS and PNS.¹⁹⁴ DNMT1 and DNMT3A upregulate methylation of promoter and 5’-untranslated region of Kcna2 gene, decreasing membrane densities of VGPCs and Kv current, leading to central sensitization and neuropathic pain.^194,195 They also methylate promoters of genes encoding non-coding RNAs, with dysfunctions in these downstream non-coding RNAs contributing to various pain disorders, ranging from pain hypersensitivity to insensitivity.^196,197 Systemic inhibition of DNMT activity results in alleviation of neuropathic pain.¹⁸⁵ Therefore, it can be concluded that despite manifold targets of DNA methylation, its overall effects are pain hypersensitivity.

Ten-eleven-translocation proteins (TETs) mediate DNA demethylation, dramatically maintaining DNA methylation stability and sha** epigenome landscape along with the DNMT family. TET1, TET2, and TET3 are the main members. The double-sided nature of TET1 has been revealed. On the one hand, it can remove restrictions on gene expression induced by DNA methylation during the progression of nociceptive and neuropathic pain, involving membrane receptors (mGluR5), ion channels (TRPV4), transcription factors (SOX10), and signal transduction factors (STAT3 and BDNF).^{198,199,200,201} On the other hand, some reports have shown the analgesic properties of TET1. It can rescue suppression of VGPC functions by regulating methylation of Kcna2 and K2p1.1 promoters in the neuropathic pain models.^202,203 Restoration of PROX1 levels following TET1 overexpression attenuates depression comorbidity through neurogenesis enhancement.²⁰⁴ Some studies have claimed the opposite roles of TET1 in the same therapy.^198,204 More strangely, the contradictory data are based on investigations into the similar pain types and model generation methods, reflecting the complexity of epigenetic modification in pain sensation. Some factors not easily perceived, such as pain inducer doses, disease courses and experimental environments, may affect DNA methylation and require more attention in subsequent research.

The Oprm1 gene encodes μ-opioid receptor and its hypermethylation positively correlates with pain severity and opioid tolerance. Long-term exposure to opioids further enhances Oprm1 methylation levels.^205,206 These vicious cycles via epigenomics are critical mechanisms underlying the opioid tolerance development. Moreover, molecules with properties of neuropathic and nociplastic pain modulation, such as stress-related protein FKBP5, peptide hormone leptin, CDK5 regulatory subunit-associated protein CDK5RAP1 are under strict control of DNA methylation.^207,208,209

Histone is a key component of chromatin, with five types of core histones, including H1, H2A, H2B, H3, and H4. Histone acetylation, primarily occurring at lysine sites of H3 and H4, is an essential mechanism controlling histone activity. Unlike DNA methylation, acute pain has no evident impact on histone acetylation, which only responds to pain chronicity.⁴² Global alterations in histone acetylation are identified in both CNS and PNS.^39,210,211 During nociceptive and neuropathic pain development, H3 and H4 acetylation is upregulated in DRG and spinal dorsal horn.²¹² Key brain regions, such as the CeA, PFC and hippocampus, exhibit significant changes in histone acetylation, which are involved in visceral hypersensitivity, neuropathic pain sensation and its comorbidities.^213,214,215 In the descending pain modulation pathways, persistent enhancement of H3 acetylation occurs in the RVM, while this molecular event is short-lived, fading after long-term stress in the locus coeruleus.^211,216 These findings suggest distinct regulatory effects of histone acetylation in different brain regions. Inflammatory mediators like IL-6 and TNF-α promote hyperacetylation of H3 and H4, enhancing neuron excitability in neuropathic models.^215,217

The dynamic balance of histone acetylation is maintained by histone acetyltransferases (HATs) and histone deacetylases (HDACs). HDACs have 18 kinds of members, some of which are closely associated with pain perception. The reductions in HDAC1 and HDAC2 expression lead to the abnormal synaptic transmission, followed by somatic and visceral hypersensitivity.^75,213,218 However, nuclear recruitment of HDAC2 driven by transcription factor Sp1 conversely aggravates neuronal dysregulation and microglial inflammation,²¹⁹ suggesting that the cellular distribution of epigenetic regulators is another factor in pain modulation. Existing negative results concerning HDAC3 indicate its weak associations with pain modulation.^75,213 Inhibition of HDAC4 translocation into the cytoplasm epigenetically decreases HMGB1 expression and functions as an analgesic approach for neuropathic pain.²²⁰ Accumulation of HDAC5 in the nucleus inhibits H3 acetylation of Gad1 and Gad2 promoters, impairing GABAergic neuron activity and contributing to aberrant activation of astrocytes through direct interaction with STAT3. These mechanisms can lead to the development of peripheral neuropathic pain.^221,222 The analgesic properties of SIRT1 and SIRT3, class III of HDACs, have also been revealed. Restoring their expression downregulation in nervous lesions mitigates ectopic discharge of sensory neurons and excessive oxidative stress,^223,224 alleviating emotional vulnerability of neuropathic pain.²¹⁴

p300 is a representative molecule for pain modulation among HATs. Neuropathy following chemotherapy, stress and diabetes results in the upregulation of p300 expression or enhancement in p300 activity. It epigenetically modifies the hypothalamic–pituitary–adrenal (HPA) axis and promotes responses to norepinephrine.^225,226,227 p300 is also involved in inflammatory pain through activating macrophages and elevating expression of TNF-α, IL-1β, CCL2, and CXCL10.²²⁸ Regretfully, other HATs’ roles in modulating pain are rarely investigated. Future research should pay attention to this shortcoming.

EZH2 is a histone methyltransferase catalyzing histone H3 methylation on K27 site.²²⁹ In the rodent models suffering from nerve injury and cancer pain, the expression of EZH2 can be significantly upregulated in the CNS. The microglia are subsequently activated, accompanied with the abrupt release of proinflammatory factors. These mechanisms contribute to the development of mechanical and thermal hyperalgesia. Downregulation of EZH2 expression or topical injection of EZH2 inhibitors have been found to alleviate neuropathic and cancer pain.^230,231,232 Although several investigations have verified the pain-induced role of EZH2, its regulatory network of molecular mechanisms is still largely unclear. One study shows that mTOR signaling pathway-mediated autophagy may be a functional target of EZH2.²³³ The expression and activity of EZH2 are also under rigorous control of non-coding RNAs, including lncenc1, miR-124-3p, and miR-378.^234,235,236 Moreover, EZH2 has been selected as a biomarker of evaluating efficacy of analgesic methods for neuropathic pain.²³⁷

Non-coding RNAs are multiple kinds of RNAs mostly incapable of encoding proteins, but their functions are not secondary to proteins. MicroRNAs (miRNAs), long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) are key molecules. Their remarkable mechanisms in pain modulation have been demonstrated by numerous studies.

The primary function of miRNAs is binding to the 3′ untranslated regions of mRNA, blocking mRNA translation and promoting mRNA degradation. Patients with pain experience have evidently altered miRNA profiles, consequently dysregulating the expression of downstream targets.²³⁸ A large number of target genes have been identified, including but not limited to ion channels, inflammatory mediators, signaling molecules and transcription factors.^{239,240,241,242,243} Importantly, the miRNA regulatory network on pain is intricate, although most studies focused on their one-to-one relationships with target genes. One miRNA can regulate many downstream mRNAs. miR-183 cluster controls expression of over 80% of recognized pain-regulated genes.²⁴⁴ One target gene is likewise under regulation by multiple miRNAs, such as TRPV1.^245,246

LncRNAs and circRNAs can suppress miRNAs through complementary base pairing, described as miRNA sponges. This mechanism is the main research direction of current studies on pain modulation.^247,248 Additionally, some lncRNAs and circRNAs interact with transcription factors, changing their activity and nuclear localization. These molecular events lead to alterations in neuropathic pain-related gene expression, such as KCNN1, G9A, and VEGFB.^249,250,251 Certain lncRNAs and circRNAs have capabilities in regulating parental gene expression and modulating neuropathic pain, like lncRNA NEAT1 and circVOPP1.^252,253

Non-coding RNAs as exosomal cargos play important roles in intercellular communications. Hyperactivated neurons release exosomes loaded with non-coding RNAs with immunomodulation properties, like miR-21-5p. The exosomes are phagocytosed by macrophages, initiating the proinflammatory phenotype.²⁵⁴ This evidence demonstrates that sensory neurons are not only victims, but also accomplices in the progression of hyperalgesia triggered by neuroinflammation. The interactions among astrocytes, microglia and macrophages via exosomal non-coding RNAs exquisitely regulate inflammatory pain degrees.^255,256 Altogether, the ectopic levels of non-coding RNAs have great potential in pain evaluation.²⁵⁷ Correction of abnormal non-coding RNA networks using gene editing and chemical treatment has achieved favorable outcomes for nociceptive, nociplastic and neuropathic pain as shown in preclinical research.^258,259,260 Future clinical trials are eagerly required to promote the translational application of non-coding RNAs.

Notably, there exists crosstalk between the above three aspects of epigenetic modification. For example, DNA methylation and histone acetylation jointly regulate the expression of neuropathic pain-related genes.^225,261 miRNAs directly suppress the expression of key enzymes of the other epigenetic aspects.²³⁹ Noncoding RNA expression is under control of DNA methylation and histone acetylation.^225,243,262 Overall, investigations into epigenetic modification have unveiled a new landscape of mechanisms underlying pain modulation. The achievements may lay the foundations for progress in pain management.

Posttranslational modification (PTM)

The activity, structure, cellular localization and interactions of proteins are critically regulated by PTMs. PTMs refer to the chemical modifications involving the addition or removal of specific groups in amino acid residues. To date, more than 600 kinds of PTMs have been identified. Common PTMs include phosphorylation, ubiquitination, glycosylation, methylation, etc. Histone acetylation, as mentioned in the previous section of epigenetic modification, also belong to PTMs. Novel PTMs, such as crotonylation, succinylation and lactylation, are continuously being discovered with advancements in biotechnology.²⁶³ The uncovered mechanisms concerning PTMs in pain modulation are concentrated on several PTMs.

The associations between phosphorylation and pain have received the most attention among PTMs. Fyn, a member of the Src family protein kinases, phosphorylates downstream targets. Its regulatory functions on pain perception have been extensively revealed. In responses to nerve injury and inflammation, IL-33 and BDNF enhance phosphorylation and catalytic action of Fyn in a PKA-dependent manner.²⁶⁴ GluN2B, a subunit of NMDAR, at Tyr1472 is phosphorylated by Fyn. This molecular event inhibits GluN2B endocytosis, increasing its membrane densities and synaptic currents mediated by NMDAR.^264,265 The molecular functions of SHP-1 are opposite to Fyn, mediating target protein dephosphorylation. The DRG produces PD-L1 in response to acute and chronic pain. It further phosphorylates SHP-1, downregulating expression and phosphorylation of TRPV1.²⁷⁷ However, one study showed that glial glutamate transporter can be upregulated by p38 MAPK. This mechanism prevents long-lasting ongoing spontaneous pain. These results suggest that the branches of MAPK signaling may have opposite effects.³⁵³

There have been various regimens concerning MAPK signaling interference in preclinical research. Antisense oligonucleotides targeting p38 have been synthesized. They effectively inhibit microglia and astrocyte activation through suppressing MAPK signaling, thereby functioning as an analgesic method for inflammatory and neuropathic pain relief.^354,355 Some clinically applied drugs, such as tetrahydropalmatine, lidocaine and opioids, have been demonstrated to achieve analgesic effects for nociceptive and neuropathic pain, at least partially, through blocking MAPK signaling.^356,357,358 A large number of natural compounds may serve as MAPK signaling inhibitors for treating neuropathic pain.³⁵⁹

PI3K/Akt/mTOR signaling functions as a regulator of cell survival, proliferation, angiogenesis, metabolism, autophagy, etc. Different with previous introduced pathways, PI3K/Akt/mTOR signaling seems to play a two-sided role in pain development and management. On the one hand, overactivation of PI3K/Akt/mTOR signaling has been detected in models with nociceptive, neuropathic pain and opioid tolerance.^360,361 Mechanistically, Akt phosphorylates ASIC1a at the Ser25 site, which promotes its forward trafficking and membrane expression.³⁶² mTOR activation has been proved to facilitate reconstruction of nociceptive terminals following inflammation, diminishment of ACC synaptic protein involved in neuropathic pain perception. These mechanisms collectively underpin the pain hypersensitivity progression.^360,363 Furthermore, NALP1 inflammasome activation can be elicited by PI3K/Akt signaling, accelerating the formation of opioid tolerance.³⁶⁴ Topical injection or systemic administration of PI3K/Akt/mTOR signaling inhibitors have shown good performance in reversing hyperalgesia and opioid tolerance.^365,366

On the other hand, however, activation of this signaling may exert analgesic effects. Neurotrophic factor derived from bone marrow mesenchymal stem cells can enhance PI3K/Akt signaling activity, transforming destructive M1 phenotype into regenerative M2 phenotype of microglia. The autophagy is meanwhile enhanced.³⁶⁷ These mechanisms restore the abnormal discharging C-fiber neurons.³⁶⁸ During the progression of chronic postoperative pain, microglia can downregulate activity of PI3K/Akt signaling in astrocytes, which induces astrocyte transformation into A1 phenotype and promotes the chronicity of pain.³⁶⁹ Additionally, PI3K/Akt signaling has been proved to participate in nerve regeneration and alleviate neuropathic pain.³⁷⁰ The above evidence has suggested that the functions of PI3K/Akt/mTOR signaling may depend on cell types, pain types and development stages. Crude inhibition or activation of this signaling may be ineffective and bring about potential side effects. It is eagerly required to explore more precise targeted therapies for PI3K/Akt/mTOR signaling interference.

AMPK is a hub regulator of biological energy metabolism. The dysregulation of AMPK signaling contributes to various metabolism-related diseases. AMPK signaling-mediated metabolic disorders have been proved as an important factor of hyperalgesia. For instance, HSP22, a kind of heat shock proteins, is downregulated in the spinal cord neurons of models with nerve injury. Restoration of HSP22 expression improves mitochondrial biogenesis and reduces oxidative stress through activating AMPK/PGC-1α pathway, attenuating neuropathic pain.³⁷¹ Meanwhile, this mechanism underpins osteoarthritis pain relief caused by Sestrin2 overexpression.³⁷² AMPK signaling serves as a sensor of intracellular glucose concentrations. AMPK signaling hyperactivity can rapidly reduce TRPA1 membrane expression and its channel activity. High-glucose exposure significantly inhibits AMPK signaling in DRG neurons and potentiates TRPA1-mediated hyperalgesia, which is a critical mechanism underlying painful diabetic neuropathy.³⁷³ Suppressing NLRP3 inflammasome is another identified mechanism of AMPK-induced analgesia.³⁷⁴

AMPK signaling exerts huge impact on non-neuronal cells. Its activation promotes M2-type polarization of microglia and reduced the release of proinflammatory factors.³⁷⁵ AMPK signaling has also been found to participate in the functions of endocannabinoid-induced analgesia through reprogramming of the phosphoproteome and bioenergetics of macrophages.³⁷⁶ The autophagic flux of Schwann cells is also enhanced by AMPK hyperphosphorylation, attenuating peripheral neuropathic pain.^Opioids

Opioids are analgesic drugs usually used for treating many types of moderate and severe pain at all ages, based on extensive evidence-based medicine. They play a vital role in clinical symptomatic and palliative therapies. Emerging clinical trials have explored novel opioid regimens. For instance, an international, open-label trial gives a strong recommendation for two-step cancer pain management, which refers to bypassing weak opioids in the pathway from non-opioid therapies to strong opioids. This regimen can decrease the cost of two-step approach and achieve the comparable efficacy.590 The combinational use of CBD effectively improves patients’ quality of life receiving opioid treatment.^591,592 The sustained-release and topically administered forms have entered clinical trials for improving analgesic efficacy and reducing adverse event risks.^593,594

Opioids are agonists of μ, κ, and δ receptors, with diminishing effects across these subtypes. Opioid-induced analgesia involves multiple mechanisms. The canonical manner is the suppression of adenylyl cyclase and high-threshold VGCCs through G protein coupling pathways. The inwardly rectifying potassium channels are meanwhile activated, accompanied with inhibition of TRP family members, VGSCs and ASICs.^{595,596,597,598} These events jointly decrease neuronal excitability and excitatory neurotransmitter levels. In the brain’s reward circuits, opioids mitigate GABA-driven inhibitory neurotransmission. The suppression on dopaminergic neurons in the striatum and PFC is reversed.^599,600 The proinflammatory neuropeptide release is also downregulated, further promoting the analgesic effects of opioids.⁶⁰¹

The side effects of opioids are the vital reasons for a series of clinical and social problems, mainly including tolerance, hyperalgesia, respiratory depression and gastrointestinal reaction. They are driven by complicated mechanisms, but some crucial cross nodes have been identified. β-arrestin 2 is a negative regulator of GPCR signaling, implicated in opioid tolerance, addiction and respiratory depression through coupling with intracellular and cytoplasmic regions of phosphorylated μ receptors.⁶⁰² Biased agonists with reduced β-arrestin 2 recruitment, like 2S-LP2 and EM-2, are being developed to alleviate these effects.^603,604 However, there are dissenting opinions that β-arrestin 2 as a scaffolding protein is unlikely to be an ideal pharmacological target. Evidences have shown that severe side effects are not observed in mice with ablation of β-arrestin 2 functions,^605,606 implying that unknown mechanisms independent of β-arrestin 2 may contribute to chronic opioid tolerance.⁶⁰⁷ Instead, targeting recruited molecules of β-arrestin 2, like vasopressin 1b receptor, may be a promising approach.⁶⁰⁸

Neuroglia cells are involved in opioid tolerance and hyperalgesia. MAPK/NF-κB signaling activation in microglia promotes release of proinflammatory factors and upregulates expression of TLR4.⁶⁰⁹ Inhibitors of MAPK/NF-κB signaling can attenuate opioid-associated side effects in rodent models.^610,611,612 CR4056, an imidazoline I2 receptor ligand, suppresses microglia activation and enhances analgesic effects of morphine.⁶¹³ NMDAR in astrocytes is another potent target. The inhibitors targeting NMDAR effectively block intercellular communications between astrocytes and neurons, alleviating opioid tolerance.⁶¹⁴ Antagonizing IL-33-mediated crosstalk between astrocytes and oligodendrocytes also prolongs morphine’s analgesic effects.⁶¹⁵ Therefore, neuroglia may serve as promising targets in mitigating opioid side effects.

Tramadol is a weak opioid agonist widely used for pain relief. Tramadol is primarily used for postoperative pain and chronic musculoskeletal pain management. It has also been recommended as the non-first-line drug for the treatment of neuropathic pain by CPS and EFNS guidelines.^616,617 Additionally, it can be safely and effectively used for delivery analgesia without affecting the newborn’s respiration. Recently, a chewable tablet has been invented and used for children, further proving its safety.⁶¹⁸ For the basic mechanistic research, tramadol exhibits a dual mechanism, primarily through the activation of opioid receptors in the CNS presynaptic membrane and the inhibition of 5-HT and norepinephrine reuptake in the presynaptic membrane of the descending inhibitory system of spinal cord. This dual mechanism allows tramadol to achieve analgesic intensity comparable to opioids at appropriate dosages.

Otherwise, nitric oxide, a gasotransmitter, activates presynaptic and postsynaptic guanylate cyclase, leading to the production of cGMP. This signaling further promotes opioid tolerance proved by basic studies. NOS inhibitors, like L-NAME and aminoguanidine, attenuate morphine tolerance. Repeated administration can further alleviate the withdrawal symptoms of opioids.^619,620 Despite promising preclinical results, the translational speed has slowed markedly in recent years. The clinical application of NOS inhibitors remains cautious, pending further evidence.

Psychological and exercise therapies

Restoration of psychological and behavioral disorders has demonstrated significant value in pain management. CBT, which integrates behavior modification with psychotherapy, is a gold-standard approach for treating mental diseases. Its analgesic effects are applicable to adults at all ages with chronic pain, such as chronic low back pain, osteoarthritis and IBS.^621,622 Notably, a recent meta-analysis based on 153 trials and 8713 participants has strongly recommended CBT for management of chronic pain associated with temporomandibular disorders.⁶²³ To further promote application of CBT, clinical trials began to investigate the efficacy of online CBT. The results showed its comparable competences with traditional psychotherapies at the dramatically lower cost,^624,625 providing new directions of CBT development.

For the mechanistic investigations, CBT induces global alterations in brain region activities. Prevention of pain catastrophizing is an important mechanism of CBT, which relies on the regulation of the ventral posterior cingulate cortex, a hub of the DMN. CBT impairs the connectivity between the somatomotor and salience network regions in fibromyalgia patients.⁶²⁶ The connectivity strength involving the ventral posterior cingulate cortex is negatively correlated with CBT efficacy.⁶²⁷ The mPFC is another key node in the DMN. CBT facilitates new long-term potentiation connections in the mPFC with other critical regions, like the amygdala and insula, underpinning significant correction of chronic nociplastic pain.^628,629 The enhanced crosstalk of the amygdala with the ACC, frontal and precentral gyrus is also related to the responsiveness to CBT.⁶³⁰ Although most evidences only demonstrate its correlations with brain region activities, these extensive and profound influences suggest the substantial potency of CBT.

Exercise is another cost-effective therapy against pain, which has been strongly recommended by recent guidelines for the clinical management of pain associated with motor system, including fibromyalgia, osteoarthritis, low back pain, chronic musculoskeletal pain and temporomandibular disorders.^{549,623,631,632,633} It has also been evaluated as an important component of therapies against cancer and neuropathic pain.^634,635 Inflammation mitigation is a major mechanism of exercise, which is mentioned above in the section of pain sexual dimorphism.^457,458 The descending regulatory pathway is an important target of exercise. Regular exercise increases the concentrations of endogenous opioids (β-endorphin and enkephalin) in the PAG and RVM.⁶³⁶ The activities of regions, such as the anterior insula, left dorsolateral PFC, locus coeruleus and midbrain reticular formation, are globally altered in patients with nociplastic pain.^637,638 However, due to various methods and intensities of exercise therapy, the optimal prescription and delivery for specific diseases should be extensively discussed based on more high-level basic and clinical research.

Placebo effect refers to the phenomenon that symptoms are alleviated through psychological functions produced by patients’ belief after receiving dummy treatment. It is especially common in the pain research. The forms of placebos are various, such as tablets, pseudostimulus and sham operation. Their efficacy rivals some classical modalities.⁶³⁹ Recent investigations using virtual reality technology have proved that physical entities are unnecessary for pain management.⁶⁴⁰ The adjuvant functions in synergistically enhancing other therapies have also been demonstrated.⁶⁴¹ On the other hand, the reliability of existing analgesic methods is questioned because it is hard to tell whether placebo effect is involved. It is great pleasure to see that project designers of clinical trials have recently been aware of this confounding factor, leading to recalibration of specific therapy effectiveness.

Activation of opioid and endocannabinoid systems is the dominant mechanism of placebo-induced analgesia, which hints that placebos may become alternative therapies of opioids and cannabinoids.⁶⁴² The PFC, insula and somatosensory cortex have been found to be engaged in this top-down effect, together with processing pain anticipation and perception by the thalamus and brainstem.^643,644 Some researchers hold the view that functional connectivity may be more sensitive for manifesting placebo effect than isolated brain regions. The decreased connection between the left medial PFC and bilateral insula, responsible for cognition modulation, is correlated with placebo effect in patients with chronic back pain.⁶⁴⁵ The circuits from prefrontal cognitive to pain processing regions also serve as indicators of responsiveness to placebos.⁶⁴⁴ Furthermore, nocebo has properties of hyperalgesia induction. Existing basic studies have revealed totally different neural networks subserving placebo and nocebo effects,⁶⁴⁶ further validating the complexity of pain perception.

In addition to the above methods, mindfulness, short-term dynamic psychotherapy and hypnosis are other psychotherapies for pain management. The research on psychological and exercise therapies has thrived. However, a disparity exists between the abundance of clinical trials and the scarcity of basic research, partly due to challenges in replicating psychological or voluntary exercise in animal models. Future studies should address this imbalance and develop more foundational experimental methods.

Acupuncture

Acupuncture originates in ancient China and its effectiveness has been confirmed through clinical practice. According to the guidelines, acupuncture can serve as an alternative and complementary therapy for pain management, especially for cancer, low back pain and postoperative and osteoarthritis pain.^{546,632,647,648} Additionally, clinical trials stimulation of pain-specific acupoints produces inhibitory effects of comorbidities of depression, anxiety and sleep disturbance.^649,650 The underlying principles of acupuncture, based on traditional Chinese medicine, initially led to skepticism within modern medicine. In the last century, Jisheng Han et al. clarified the spatial and temporal events and related mechanisms of acupuncture-induced analgesia. This is a historic milestone of utilizing biomedical technologies to elucidate acupuncture-induced analgesia mechanisms.

Early preclinical studies indicated that acupuncture stimulates Aδ and C afferent nerves and promotes secretion of endogenous opioid peptide, as well as the reduction in activities of norepinephrine and serotonin systems. With the deepening of basic research on clinical patients, the nodes in default mode and frontoparietal networks have been identified.⁶⁵¹ The connectivity between the amygdala, right middle cingulate cortex and temporal gyrus is enhanced in patients with nociplastic pain.⁶⁵² Activities of the thalamus, caudate, claustrum and lentiform are likewise modulated.⁶⁵³ Importantly, there are typical differences between acupuncture and sham control groups, allaying the concerns of the placebo effect.⁶⁵² Molecular mechanisms underlying acupuncture are considerably diverse, including epigenetic modification, PTMs, non-coding RNAs, inflammasome and microbiota.^{41,204,574,654,655} However, most studies merely reveal their changes following acupuncture, which may be accompanying effects, rather than action mechanisms. The causal studies using rescuing experiments are needed. Taken together, acupuncture is a promising therapy for pain management, but requires further exploration to develop individualized regimens.

Neuromodulation and brain–computer interface (BCI)

Neuromodulation refers to the approaches that directly or indirectly implant electrodes into innervation regions to improve pathological changes and clinical symptoms. Brain, spinal cord, vagus, sacral nerve, auditory nerve, etc. are all the interventional targets of invasive neuromodulation. Deep brain stimulation has garnered the most attention with the deepening understanding in brain region functions. The sensory thalamus, PAG, ACC and periventricular gray matter are main anatomic regions of deep brain stimulation.⁶⁵⁶ In contrast, transcranial alternating current stimulation represents noninvasive neuromodulation. Though less potent, its safety and convenience may propel it to the forefront of future research. In summary, neuromodulation has proven effective in clinical trials for chronic pain management.^657,658

Spinal cord stimulation refers to a pain management technique that involves implanting electrodes into the epidural space in the spinal cord for modulating neural electrophysiology. The non-nociceptive electrical signals can inhibit the transmission of nociceptive signals by stimulating the large diameter Aβ-fibers. The most common indications of spinal cord stimulation are complex regional pain syndrome, failed back surgery syndrome and peripheral neuropathy induced by ischemia, herpes zoster or diabetes. Many clinical trials and meta-analysis have verified the efficacy of spinal cord stimulation as a supplementary approach in attenuating neuropathic pain, especially for complex regional pain syndrome and failed back surgery syndrome.^659,660 It has also been recommended as a third-line therapy for patients who have failed to respond to gabapentinoids and antidepressants.⁶⁶¹ In addition to the tonic stimulation, some novel stimulation waveforms have been proposed, including burst, high frequency and close-loop stimulation. These further enhance pain relief or reduce the risks associated with paresthesia perception.⁶⁶²

Although spinal cord stimulation was initiated based on Gate control theory, the extensive regulatory mechanisms have been gradually uncovered by many preclinical studies. Opioid system is involved in the effects of spinal cord stimulation and the stimulation at different frequencies rely on different endorphins and opioid receptors.⁶⁶³ The expression of CB receptors is upregulated following spinal cord stimulation treatment. Nociceptive-evoked activation of supraspinal areas, such as the locus coeruleus, RVM, reticular formation and PAG, can be inhibited by spinal cord stimulation.^664,665 The descending inhibitory system is activated, leading to the release of 5-HT and attenuation of chronic neuropathic pain.⁶⁶⁶ The above mechanisms collectively contribute to the analgesic effects of spinal cord stimulation.

Despite significant achievements in spinal cord stimulation, there are difficulties in positioning specific pain regions, such as low back, knee and groin. The complex anatomy of spinal cord, shunting of electrical stimulation through cerebrospinal fluid and relative displacement of spinal cord in the canalis spinalis all impair the application of spinal cord stimulation.⁶⁶⁷ DRG stimulation can overcome these shortcomings. It simultaneously activates Aβ, Aδ, and C fibers. The cerebrospinal fluid around DRG forms a groove, attenuating the dispersion of electrical currents and avoiding the side effects of paresthesia within peripheral regions. Moreover, it can produce stable currents in the regions that spinal cord stimulation hardly achieves. According to the existing evidences from clinical trials, DRG stimulation has been selected as the primary treatment of lower limb type I or II complex regional pain syndrome.^668,669 Patients suffering from chronic postsurgical inguinal pain, knee pain and types of chronic intractable pain can gain typical benefits from DRG stimulation.^670,671,672 Some studies have found that stimulation frequencies are a determinant factor of DRG stimulation efficacy and 20 Hz might become the best choice.^673,674 There are limitations in clinical investigations into either spinal cord stimulation or DRG stimulation. Although the effectiveness of electrical stimulation has been proved, the concrete regimens, such as stimulation frequencies, treatment interval, best indications, need further exploration. Their surgical characteristics make it difficult to set standard sham groups and adhere to blinding principles, affecting the reliability of current clinical data. More high-quality real-world studies should be conducted to compensate for these shortcomings.

The innovation of BCI has ushered neuromodulation into a new era. Preclinical and clinical research has shown its potential in alleviating neuropathic pain. Hence, in this section, we highlight this cutting-edge technique. Pain perception drives fluctuations in the brain network. Extracting features of this process may provide the sources of decoding pain perception with great accuracy.⁶⁷⁵ BCI can analyze the data supported by AI and produce real-time neuromodulation on multiple regions to mitigate pain. The S1, ACC, and PFC are the crucial targets of BCI. More importantly, the regulatory effects are not unilateral. Brain has been found to actively communicate with BCI and change its responses to pain, embodied by enhanced activities of the ACC and PAG and modulation of pain attention.⁶⁷⁶

The studies aiming at prompting more extensive use are ongoing. Patients with phantom limb pain have been trained with BCI to control a phantom hand.^677,678 A three-day training session can alleviate pain perception for more than 1 week.⁶⁷⁹ In contrast, another study indicated that despite the enhanced discriminability for movement and prosthetic control, overconcentration on the phantom hand driven by BCI intensifies neuropathic pain. Dissociation between prosthetic and phantom hands is a more feasible way for analgesia.⁶⁸⁰ A multisensory intervention strategy consisting of BCI, virtual reality, and transcutaneous electrical nerve stimulation sharply increases the efficiency of decoding pain memory and attenuating neuropathic pain.⁶⁸¹ The invention of a home-use, patient-managed BCI device has further accelerated the translation of BCI.⁶⁸² More importantly, another application of BCI is assisting movement for paralytic patients. Pain perception has been found to damage the performance of BCI on motor system control.⁶⁸³ These findings underscore the broader significance of these advancements, extending beyond mere pain relief.

Microbial intervention

The breadth of research on microbiota and pain has guided the development of related approaches to analgesics. Investigations into associations between microbiota dysbiosis and pain progression have promoted the translation of novel interventional regimens, mainly including probiotics supplementation and fecal microbiota transplantation (FMT). Notably, given the natural associations between microbiota and gastrointestinal tract, most studies focus on therapies against abdominal pain, particularly IBS.

Probiotic supplementation has exhibited translational value. In the preclinical research, administration of Saccharomyces boulardii reduces colonic TRPV1 expression and alleviates pain sensation in an IBS model.⁶⁸⁴ Lactobacillus paracasei and butyrate-producing Roseburia hominis can respectively attenuate visceral hypersensitivity through mitigating dysfunctions of gut homeostasis.^685,686 Bifidobacterium dentium and Lactococcus lactis both have properties of enzymatic decarboxylation of glutamate. Their analgesic effectiveness by GABA production has been detected in visceral hypersensitivity models.^397,687 The nociceptive perception induced by 5-HT is ameliorated by Lactobacillus plantarum through downregulating responses of the HPA axis.⁶⁸⁸ The efficacy of probiotic supplementation have also been confirmed by clinical trials.⁷³⁵ However, clinical requirement for pain relief is far from satisfaction, again implying the complexity of pain sensation. Multidisciplinary cooperation, supported by high-quality preclinical and clinical research, may be a key solution to this dilemma.

Shortcomings of the current pain research

The strength of evidence in existing research, particularly regarding brain regions, is generally weak. Studies often detected activity alterations in brain regions after pain induction or intervention, along with synergistic regional reactions. On the basis of these data, they inferred that some brain regions and functional connectivity might participate in pain modulation. Similar situations were also observed in the research on specific inflammatory mediators, neurotransmitters and molecular regulators. These findings are useful for develo** novel diagnostic biomarkers and screening out potential targets. Nevertheless, they fall short in supporting concrete conclusions about pain etiology and treatment. Researchers should exercise caution in drawing conclusions based on assumptions from previous reports and established knowledge. Such weak evidence may obscure the true nature of pain modulation. For instance, the accepted view that TRPV1 activation serves as a marker of hypersensitivity, used in pain perception assessment. However, CB1-dependent TRPV1 overactivation is an important mechanism in dipyrone-induced analgesia,⁵⁷² overturning the stereotype regarding TRPV1. β-arrestin 2 has been recognized as a critical mediator of opioid-induced respiratory depression based on previous studies. Its role is now questioned, as β-arrestin 2 knockout does not affect respiratory rhythms.⁶⁰⁶ Otherwise, GABA is a negative regulator of hyperalgesia, but its depression comorbidity-inducing effects⁴⁷³ are often overlooked. Therefore, the lack of studies related to causal and mechanistic data increases difficulties in identifying more valuable targets.

The experimental methods require significant improvement. The reliance on mouse and rat models for human disease studies has been long debated due to substantial differences in nervous systems.⁷³⁶ This species gap may introduce biases in clinical translation. Additionally, gene editing and controlling certain substance levels in in-vivo models are relatively challenging compared to in-vitro models, slowing progress in exploring mechanisms underlying pain modulation. As mentioned previously, pain-related disorders are multifaceted. A single modeling approach cannot fully simulate human pain sensation, further affecting the credibility of current research. Fortunately, scientists are addressing this by develo** composite modeling strategies. Moreover, commonly used pain indicators, such as c-fos expression, ion channel activity and hormone levels, may not accurately reflect pain severity in experimental animals. The representativeness of these indicators warrants scrutiny and validation. The above problems, of course, are universal flaws of basic experiments and are not unique to pain research.

The potential of multi-omics and high-resolution approaches remain underexploited. The authors only find that microbiome combined with metabolomics is widely used in the existing studies on microbiota and pain. While the findings have deepened our understanding and advanced pain diagnosis and treatment, it risks forming research stereotypes of the multi-omics pattern of microbiome plus metabolomics. Furthermore, several recent studies analyzed basic structures and mechanisms underlying responses to nociception using single-cell transcriptome. However, other omics approaches, like transcriptomes, proteomics and spatial omics, are overlooked, especially the combinational application. The multi-omics approaches and high-resolution have led to significant discoveries in other nervous system diseases, such as Alzheimer’s disease, depression and autism spectrum disorder.^737,738,739 By contrast, few pioneer investigations have preliminarily shown the potential of transcriptomes and proteomics in exploring pain modulation,^740,741 implying that pain research lags due to underutilization of multi-omics analysis. On the other hand, the efficiencies of omics data analysis integration are relatively low. The depth of omics data analysis is limited, especially for the basic research for investigating pain causality. In summary, the small data pool and rough analytical tools of omics both hamper the clinical progress in pain relief.

Data on pain modulation mechanisms and therapeutic regimens often present contradictions. The prime example is that the performances of specific therapeutic approaches are different across clinical trials, particularly in alternative and complementary therapies for pain management. Such discrepancies, which are influenced by numerous unpredictable confounding factors, are common in clinical research. However, similar inconsistencies are also observed in preclinical research. For instance, 17β-estradiol is reported as both a promoter of pain in females and a protective agent against hyperalgesia.⁷⁴² Moreover, the changes in expression of HDACs in response to pain modeling are controversial.^213,743 These opposing conclusions without reasonable explanations create barriers to deeper studies. Thus, monism is unsuitable for estimating the roles of pain regulators, which may function distinctly under various conditions.

The progress in clinical trials for pain management is slow. The scarcity of large-sample, multicenter clinical trials hamper clinical translation of novel pain management approaches. Limited patient inclusion reduces the practicality of subgroup analysis in identifying potential beneficiaries. Long-shot clinical investigations for high-level evidence-based support are lacking. Moreover, potential bias of publication, favoring studies with positive results, is a serious yet neglected problem. The clinical trials that display negative data or vigilance about the side effects have more difficulties in gaining extensive attention. Although this phenomenon has been improved to some extent recently, the follow-up impact following the previous phenomenon will persistently exist, probably causing more waste of basic and clinical resources.

Some mechanisms remain underexplored, especially in burgeoning research areas. For instance, current studies primarily focus on a few PTMs, including phosphorylation, ubiquitination, SUMOylation and glycosylation. This does not mean the unimportant roles of other PTMs. Instead, sporadic studies have reported their potentials in regulating nociceptor sensitivity and molecular activity, implying the unidentified PTM networks in pain modulation. Similarly, NLRP3 inflammasome is undisputedly a key mediator in hyperalgesia. This research trend may make other inflammasomes with properties in pain modulation, like NLRP2, ignorable. Otherwise, in the research on microbiota and pain, nonbacterial microbiota, like fungi, which regulate pain sensation, receive minimal attention. Basic knowledge about pain and biofilms, an important accessory structure of microbial community, is also limited. The sluggish paces with research frontiers may result in missing out on many diagnostic and therapeutic methods.

Future perspectives of the pain research

Improvement in human-based in-vitro systems as research models. To surpass the inherent limitations of experimental animals as in-vivo models and in-vitro two-dimensional cells models (cell lines and induced pluripotent stem cells), the utilization of organoid and organs-on-a-chip technologies is essential in the follow-up studies. Organoids are the three-dimensional culture systems derived from self-organizing stem cells. Organs-on-a-chip systems are in-vitro microfluidic devices containing the cell types of interest in close recapitulation of the original tissue structure, function, and physiology. The advent of these two in-vitro culture biotechnologies provides more opportunities for narrowing the gap in mechanistic insights into pain. Organoids and organs-on-a-chip systems possess both the maneuverability of in-vitro cell line models and integrity of in-vivo animal models. They have been widely adopted in various research areas, establishing a robust foundation for data production. Nevertheless, few studies on pain perception chose them as experimental models, likely due to technological and financial barriers. The application of new biotechnologies always requires powerful supports of experimental technique and funding. To this end, just as develo** trends of other biotechnologies, like high-throughput sequencing and antibody preparation, extensive commercialization of organoids and organs-on-a-chip systems may sharply reduce their application thresholds, fully unlocking research potential. Moreover, improvement in extracellular matrix structures and functions is important likewise. Three-dimensional bioprinting, microfluidics device and biomaterials should be further developed to generate organs with fine and complex structures incorporated with vasculature and innervation networks. Furthermore, cryoelectron microscopy (cryo-EM) is an emerging technique for analyzing molecular structures, providing in-depth evidences for investigating mechanisms and exploring new analgesic drugs. We are glad to see that cryo-EM has been extensively used in pain research (Table 4). More studies are required to illustrate functions of critical regulators in pain modulation.

Table 4 The research progress in structure analysis of pain-associated molecules using cryo-EM

Comprehensive use of omics profiling. Single-cell and spatial omics technologies have become landmark achievements in the technological revolution. Pain research has begun employing single-cell and spatial omics to unravel new mechanisms, such as compiling DRG atlas, identifying mechanistic networks and discovering new cell subtypes.⁷⁴⁴ More focus is needed in areas like analgesic therapy responses, opioid tolerance and pain sensation variations among populations. Furthermore, the integration of multi-omics detection with these high-resolution technologies is absent but greatly needed. The joint analysis of microbiome and metabolomics has already laid a foundation. Expanding omics types and data volumes should be further encouraged. The rapid advancement of AI offers robust data analysis capabilities for multi-omics technologies.⁷⁴⁵ Future studies should try to widen the scope of multi-omics profiling and deepen omics data mining. Recently, the most comprehensive human brain cell map has been disclosed. This work set a benchmark for future omics studies. Although introduction of new omics approaches may disclose more unexplored zones and put more demand on researchers, it will tremendously broaden the horizons concerning pain modulation. The profits in better understanding mechanisms and identifying more related targets can boost the development of pain diagnosis and treatment.

Development of noninvasive methods for pain diagnosis. Due to the subjectivity and heterogeneity of pain perception, clinical pain evaluation typically depends on scales and assessment by doctors, which are not always reliable. Novel approaches like gene biomarkers and brain imaging have yet to effectively address these limitations. Based on the fact, the future directions can be concluded into four aspects. i) Collection of more high-level evidence-based medical data. Since existing data have shown their optimal performance, trials with larger samples and subsequent commercialization processes should be advocated. This measure may allow patients to benefit from these achievements at an earlier time. The differences of pain perception in various subpopulations can be better excavated. ii) Rapid diagnosis based on molecular targets and easily accessible samples. Current approaches have difficulties in balancing efficiency and accuracy. Novel bioengineering techniques are favorable for overcoming this dilemma. Lin and colleagues set a good example. They develop a nanochip detecting saliva CGRP concentrations for migraine diagnosis. The results can be obtained within 10 min.⁷⁴⁶ More investigations into rapid diagnosis need to be encouraged. iii) Streamlining detected indicators. Some studies used ten more biomarkers or global high-throughput data to train pain models. Despite theoretically good performances, they may not conform to clinical reality due to their high cost and complexity. Herein, the authors call for investigations into applications of smaller-scale indicators. iv) Multimodal diagnostic methods. Although we encourage decreases in included indicators of one diagnostic methods, the advantages of multimodal approaches should be highlighted. Multimodal approaches can mutually compensate for shortcomings of single method, like BCI combined with skin conductance.⁶⁸¹ The integration of AI in multimodal approaches could offer the most efficient ways to meet realistic conditions.

Extension of novel strategies for pain management. i) Activation of immunoreaction against pain. Immune disorders significantly contribute to hyperalgesia, and most approaches pertain to the passively mitigation. The success of cancer immunotherapy hints that the immune system may be another key to pain relief. For instance, Sara et al. creatively employed microbiota antigens to activate specific immunity, obliterating visceral hypersensitivity-associated microbiota.⁷⁴⁷ This research is opening new possibilities in this field. On the basis of the reported advantages of immunotherapy, responders are likely to obtain more benefits from it, embodied by lasting medical effects, mild side effects and good tolerance.⁷⁴⁸ ii) Leveraging biomaterial superiority. Interdisciplinary investigation is a promising approach to promote translation, mainly including efficacy enhancement, potency prolongation, side effect mitigation and cost minimization. Despite its promise, collaborations involving biomaterials remain scarce. The need for more high-quality studies is evident. iii) Formation of comprehensive treatment strategies. The characteristics of pain, as a multifactorial disease, necessitates multifaceted interventions, including, analgesics, emotional management, social relations improvement, together with alternative and complementary therapies. Research is shifting towards examining the synergistic effects of combined therapies, such as analgesic drugs with CBT and cell stem therapy with probiotics.^749,750 More research investment should be conducted to make one plus one larger than two. iv) Learning from nature. Many breakthroughs in pain research are inspired by natural phenomena. Certain natural compounds are found to have analgesic efficacy, potentially more effective than synthetic drugs.^751,752 The natural compounds and endogenous substances, such as melatonin, curcumin and peppermint, with a history of safe use, offer wide regulatory mechanisms. The advantages can effectively decrease risks associated with new drug development.

Conclusions

Pain, a kind of universal experience, brings about overwhelming physical and mental distress. Attenuating pain is a fundamental right of patients. Extensive preclinical and clinical studies have delved into pain pathology and the molecular mechanisms of pain modulation. Key brain regions and critical molecules have been identified involved in pain perception. The research achievements regarding TRPV1, TRPM8 and Piezo even garner recognition of the Nobel prize. The significant progress has facilitated a shift from empirical management to personalized interventions. A range of promising diagnostic and therapeutic approaches have emerged. Some Chinese traditional therapies are rehabilitated by modern medical evidence. Multidisciplinary collaborations have further advanced the field and integrated cutting-edge technologies, such as virtual reality, biomaterials, and high-resolution omics technologies. However, current research limitations are impeding further advancements in pain management. This review suggests several potential directions for future research (Fig. 10). The authors believe that the unpleasure experience of pain will be eradicated as neuroscience continues to evolve.

Fig. 10

The status quo, limitations and future perspectives of the pain research. Most current preclinical and clinical studies in the pain field focused on its mechanisms, assessment and therapy. However, there are some limitations as follows. i) Current research evidence is relatively weak and the underlying mechanisms remain largely unknown. ii) The in-vitro and in-vivo experimental models cannot thoroughly mimic the clinical conditions of pain. iii) The research value of omics techniques is not fully exploited. iv) There are contradictions in the results from some studies. v) Current progress in clinical translation of pain research achievements is far from clinical requirements. vi) Some potential mechanisms underlying pain modulation should be emphasized, like the regulatory role of fungi. Herein, we propose four future perspectives for pain research, including development of advanced experimental models, comprehensive application of omics, emphasis on noninvasive pain diagnosis and optimization of strategies for pain relief