Introduction

Peripheral neuropathy is a complication with a high incidence of diabetes, often involving the nervous system, and has no obvious symptoms at the beginning of the disease. With the aggravation of neuropathy, abnormal sensory function, limb numbness, and local tingling gradually appear, and further development may lead to foot ulcers, and amputation is required in severe cases, with a certain disability rate. It can cause great damage to patients’ physical and mental health and quality of life. Timely and effective treatment can slow down the progression of diabetic peripheral neuropathy. At present, although drug treatment of diabetic peripheral neuropathy can be seen in clinic, including hypoglycemic and trophoneurotic drugs, the efficacy is still limited.

As an important part of Chinese medicine in China, Mongolian medicine plays an irreplaceable role in the treatment of diseases, which is a traditional medicine that has gradually formed and developed in long-term medical practice. Zhenzhu Tongluo pills is a Mongolian medicine composed of a variety of drugs, including Hyriopsis cumingii (Pearl), Gypsum Fibrosum (Gypsum), Carthamus tinctorius L.(Safflower), Fructus Gardeniae (gardenia), Syzygium aromaticum L. (Flos Caryophyllata), Myristica fragrans Houtt. (Semen Myristicae), Amomum tsaoko Crevost et Lemarie (amomum tsao-ko), Myristica fragrans Houtt. (Nutmeg), Syzygium aromaticum L. (Flos Caryophyllata), Aquilariae Lignum Resinatum (Rosewood Heart Wood), Amomun kravanh Pierre ex Gagnep (cardamom), Cinnamomum cassia Presl (Cassia Bark), the plant name has been checked with http://www.worldfloraonline.org) (2023.07.01). It has the effects of clearing heat, opening the orifice, and drying dampness. Gypsum clears heat and relieves fire, and stops bleeding; Safflower promotes blood circulation, and removes blood stasis; Flos Caryophyllata and Nutmeg dispel cold and relieve pain, and calm the mind; White cardamom promotes digestion, removes cold and dampness; amomum tsao-ko promotes digestion, relieves cold and dampness. Clinical treatment over the years has found that the use of Zhenzhu Tongluo pills alone or in combination with other treatments can significantly improve the symptoms of patients with cerebral infarction, contusion-induced bone diseases, ischemic stroke and chronic rotator cuff injuries [1,2,3,4,5,5]. Du et al. also found that combined Zhenzhu Tongluo pills with rotator cuff function exercise can improve the clinical efficacy of chronic rotator cuff injury, improve shoulder joint function, enhance shoulder joint motion, and relieve patients’ pain [6]. The results of these different clinical studies suggest that Zhenzhu Tongluo pills alone or combination with other therapeutic means plays an important role in neuroprotection in clinic. However, most clinical studies are observational studies, which only observe the effect without explaining the mechanism.

Although network pharmacological analysis of Zhenzhu Tongluo pills has found that PI3K–AKT, MAPK and NFκB signals may be important pathways for the function of Zhenzhu Tongluo pills, further study is missing. In this study, we constructed a rat model of diabetic peripheral neuropathy, combined with behavioral, morphological, functional and other molecular biological techniques. The possible mechanism of the neuroprotective function of Zhenzhu Tongluo pills was investigated. Our results suggest that the protective effect of Zhenzhu Tongluo pills on hyperalgesia in diabetic peripheral neuropathy rats is closely related to its anti-inflammatory and anti-apoptotic functions. In fact, many previous studies have found that the analgesic effects of proprietary Chinese medicines or components from plant extracts in rat models of diabetic peripheral neuropathy may be related to anti-inflammatory effects. For example, kaverol extracted from mustard seed reduced the expression of TNF-α, TGF-β and IL-1β in sciatic nerve [11], **mai Tong regulated the inflammasome in DRG [13], koumine and loganin regulate inflammation at the spinal cord level [10, 14] and treadmill training combined with insulin achieved anti-inflammatory effect [9, 15]. In summary, combined with the results of our study, the anti-inflammatory effect of Zhenzhu Tongluo pills is one of the mechanisms to exert its analgesic effect.

Our results in this study confirmed that Zhenzhu Tongluo pills can indeed regulate the expression of PI3K–AKT, MAPK, NFκB and other related molecular proteins, suggesting that the neuroprotective function of Zhenzhu Tongluo pills may be realized through the above signaling pathways. In fact, previous studies have shown that different drugs alleviate diabetic peripheral neuropathy through regulating these signaling pathways. For example, empagliflozin could regulate the expression of MAPK and NFκB pathway and finally reducing inflammatory response [16], Baimai cream has good efficacy in regulating the expression of PI3K/AKT and MAPK signaling pathways [7], and diosgenin could regulate the expression of NFκB pathway and thus alleviating inflammatory response [17]. Combined with our study, we have reason to speculate that multi-target action may be the dominant feature of Chinese medicine therapy.

Zhenzhu Tongluo pills can improve the structural and functional damage of sciatic nerve in diabetic peripheral neuropathy rats

Pathological lesion of sciatic nerve is a significant feature of diabetic peripheral neuropathy rats. Under a light microscope, the sciatic nerve often shows vacuol-like degeneration of medullated nerve fibers and axonal swelling. Under electron microscope, it showed serious demyelination. In this study, on one hand, the longitudinal and transverse sections of sciatic nerve were observed by HE staining, and it was found that the sciatic nerve of STZ group rats was characterized by loose nerve fiber arrangement and uneven myelin edema. On the other hand, the results of transmission electron microscopy also showed the separation of myelin lamella, axon atrophy and demyelination of myelinated nerve fibers in the STZ group. In addition, the results of sciatic nerve conduction velocity also showed that the sciatic nerve conduction velocity was significantly damaged in the STZ group. These results suggest the successful construction of STZ-induced diabetic peripheral neuropathy rat model both structurally and functionally.

Many previous studies have confirmed that different drugs can also improve the sciatic nerve structure injury when improving the peripheral injury of diabetic peripheral neuropathy rats. Abdelkader et al. found the improvement effect of empagliflozin on sciatic nerve structural damage in diabetic peripheral neuropathy rats by HE staining, toluidine blue staining and projective electron microscopy [16]. Li et al. also found that Baimai cream had a protective effect on the structural and functional damage of sciatic nerve in rats with diabetic peripheral neuropathy by electron microscope and evaluation of sciatic nerve conduction velocity [7]. Other studies have also found that extracts from Labisia pumila or Datura innoxa can reduce the structural damage of sciatic nerve [8, 18]. In this study, on one hand, we found that Zhenzhu Tongluo pills had a significant improvement on the structural damage of sciatic nerve in rats with STZ-induced diabetic peripheral neuropathy, which was specifically manifested in the structural effects of nerve fiber arrangement, axon injury and demyelination. On the other hand, Zhenzhu Tongluo pills can also relieve STZ-induced reduction of sciatic nerve conduction velocity functionally. These findings further confirm that Zhenzhu Tongluo pills has significant neuroprotective function from the structure and function of nerves.

In this study, although we preliminarily explored some mechanisms of the neuroprotective effect of Zhenzhu Tongluo pills in an STZ-induced diabetic peripheral neuropathy rat model, it is still insufficient. In fact, in the future, it is necessary to build relevant animal models of nervous system diseases, such as animal models of cerebral ischemia reperfusion injury and cerebral hemorrhage, etc., combined with behavioral tests of motor function, cognitive function, memory function, etc., to explore the central mechanism of neuroprotective effect of Zhenzhu Tongluo pills at the brain level.