Background

Spontaneous nontraumatic intracerebral hemorrhage(ICH) is a serious subtype that accounts for approximately 10–15% of all types of strokes [1]. The overall incidence of ICH is 24.6 per 100 000 person-years, with a mortality rate of 40% [2, 3]. Moreover, ICH presents acutely and progresses rapidly, resulting in hemiplegia and altered consciousness, thus imposing a substantial global clinical and economic burden [4, 5]. Current treatments for ICH involve surgical and conservative medical approaches [6]. However, the results of multiple clinical trials have indicated that surgical interventions did not significantly improve the adverse outcomes in ICH [7]. Furthermore, no specific therapeutic medication has been approved for ICH treatment, and supportive therapy remains the primary approach for patients. Despite increased attention to ICH management, the available treatment options and effective targets are currently limited [8].

Brain injuries resulting from ICH encompass intricate pathophysiological processes, categorised as either primary or secondary brain injury based on pathological patterns [9]. Primary injury refers to the local tissue damage caused by an intracerebral haematoma, primarily occurring within minutes to hours after the onset of the disease [9]. Secondary brain injury is primarily induced by inflammation, oxidative stress, and excitotoxicity, all of which can initiate substantial neuronal cell death [10]. In such instances, the extensive cellular and tissue debris cannot be timely cleared, leading to the accumulation of detrimental substances that can result in persistent neurological dysfunction [11,12,13,14,15,16,17]. The neurological deficit caused by brain injury may induce autophagy. Autophagy plays a critical role in the elimination of cellular debris and waste following injury, thereby facilitating neuronal repair in nervous system diseases [18,19,20,21,22,23].

Buyang Huanwu Decoction (BYHWD) is widely recognised as a clinically effective formula for treating various types of strokes. Previous systematic reviews and meta-analyses have consistently demonstrated the efficacy and safety of BYHWD for treating ICH. [24, 25]. As a neuroprotective agent, BYHWD modulates the non-classical NF-κB pathway, attenuating the inflammatory response following ICH [23]. Furthermore, previous experimental studies have indicated that BYHWD promotes lactate accumulation and activates the HIF-1α/VEGF signal pathway to induce angiogenesis post-ICH [24]. Additionally, BYHWD downregulates the expression of leukemia inhibitory factors, thereby reducing glial scar formation induced by ICH [33]. In this study, we combined transcriptomics and network pharmacology analyses to explore crucial target mRNAs and specific regulatory molecules associated with BYHWD in ICH treatment. This research is expected to provide deeper insights into the transcriptional-level effects of BYHWD in ICH treatment.

Methods

BYHWD preparation

The original prescription of BYHWD is available in the ‘TCM Prescriptions Dictionary’, which lists the following seven Chinese herbs: Astragalus mongholicus Bunge, Angelicae sinensis (Oliv.) Diels, Ligusticumi chuanxiong Hort, Paeonia lactiflora Pall, Prunus persica (L.) Batsch, Carthamus tinctorius L, and Pheretima aspergillum (E. Perrier) (Table 1). The raw herbs were sourced from the ** targets: cathepsin B (Ctsb), ATP binding cassette subfamily B member 1 (Abcb1b), toll-like receptor 4 (Tlr4), chemokine (C–C motif) ligand 12 (Ccl12), and baculoviral IAP repeat-containing 5 (Birc5). Disease-drug-target network analysis of BYHWD and ICH was performed to visualise the link between disease and drugs (Fig. 4b). The clustering heatmap (Fig. 4c) was used to observe trends among the three groups. To verify the results derived from bioinformatics analysis, haemojuvelin (Hfe2), scavenger receptor class A member 5 (Scara5), Ras-related protein Rab-7b, transferrin, PYD and CARD Domain Containing (Pycard), Ccl12, Abcb1b, Tlr4, Ctsb, and Birc5 were randomly selected for RT-qPCR (Fig. 4d–l). In particular, Abcb1b, Birc5, Ccl12, Trf, Tlr4, Rab7b, Pycard, and Ctsb expressions were upregulated after ICH; however, a significant decrease was observed after BYHWD treatment. The expression of Hfe2 and Scara5 demonstrated opposite trends in that they were downregulated after ICH but upregulated after BYHWD treatment. The results of the experiment were consistent with the microarray results. Among the five core targets, Ctsb showed the most significant change in mRNA expression (P < 0.001).

Fig. 4
figure 4

Differential expressed genes of Combined network pharmacology and Illumina sequencing display in Venn Diagram a. Disease-Drug-Targets network analysis of BYHWD and ICH b. Heat map illustrating target mRNAs c. RT-qPCR is performed to validate the results of Illumina sequencing d–m. n = 3, *P < 0.05 **P < 0.01 ***P < 0.001

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BYHWD enhances autophagy by regulating Ctsb in ICH

As shown in Fig. 4d, the significant change in mRNA expression of Ctsb was also visualised using immunofluorescence and western blot (Fig. 5a–c). The increase in Ctsb expression following ICH was reduced after BYHWD treatment. Due to its localisation on lysosomes, Ctsb may potentially influence the autophagic process [34]. Western blotting was used to assess changes in autophagy-related proteins to further investigate the potential mechanism of BYHWD regulation of Ctsb (Fig. 5b). After ICH on day 7, the expression of the autophagy substrate P62 (Fig. 5d) decreased in the ICH group than in the sham group, whereas the expression of autophagy-related proteins such as Beclin1, Atg5, and Lc3b increased (Fig. 5e–g). The trends were reinforced in the BYHWD group. Autophagosomes were observed by TEM, the gold standard for studying autophagy. The experimental results suggested that autophagic activity was active and autophagy levels were enhanced after BYHWD treatment (Fig. 6a). Ctsb cleaves the adhesive protein 1 (mucolipin 1, Mcoln1) in lysosomal calcium channels, inhibiting the synthesis of transcription factor EB (Tfeb) and reducing the expression of lysosomal and autophagy-related proteins, thus impacting cellular autophagy [35]. The mRNA expression of Tfeb (Fig. 6b) and Mcoln1 decreased in the ICH group but increased in the BYHWD group (Fig. 6c), which was negatively correlated with the mRNA expression of Ctsb. These experiments suggest that BYHWD exerts a neuroprotective effect by inhibiting Ctsb and enhancing the expression of Tfeb and Mcoln1 to increase autophagy.

Fig. 5
figure 5

Immunofluorescence was utilized to detect Ctsb-positive cells in different groups, the white solid line in the figure represents the hematoma area, and the magnified image in the top left white box shows a local enlargement a. The sample and pathological observation position of mouse brain in immunofluorescence b. Western blotting (WB) was conducted to assess the levels of Ctsb and autophagy-related molecules c. The quantitative analysis in WB of CTSB d, P62 e, BECLIN f, ATG5 g and LC3B h, n = 3–6, *P < 0.05 **P < 0.01

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Fig. 6
figure 6

A transmission electron microscopy image was obtained to visualize autophagosomes. The white arrows refer to autophagosomes. a. RT-qPCR analysis was used to examine the mRNA expression levels of Tfeb b, and Mcoln1 c. n = 3, *P < 0.05 **P < 0.01 ***P < 0.001

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Discussion

In this study, we identified five critical differentially expressed mRNAs following BYHWD intervention after ICH using a transcriptomic-combined network pharmacology strategy. Among these mRNAs, the most prominently altered mRNA was Ctsb, which acts therapeutically by enhancing autophagy. We have attempted to elucidate the possible mechanisms of the pharmacotherapeutic efficacy of BYHWD for ICH treatment and to achieve precise therapy at the transcriptional level.

Previous studies have shown that the administration of BYHWD can attenuate neuronal death and improve neurological deficits after ICH [27, 32]. In this study, BYHWD significantly enhanced the recovery of neurological function defects, as determined by several classical behavioural tests. Furthermore, BYHWD effectively reversed the neurofunctional and pathological impairments induced by ICH. These findings underscore the crucial role of BYHWD in exerting a neuroprotective response against ICH.

The mRNA alterations caused by BYHWD treatment are a complex process. Five differentially expressed mRNAs, namely Abcb1b, Ccl12, Birc5, Tlr4, and Ctsb, overlapped (Table 3). Our research confirmed an association between Abcb1b and BYHWD on ICH. Abcb1b is an ATP-dependent drug efflux pump that maintains intracellular homeostasis and belongs to subfamily B of the ATP-binding cassette transporter superfamily. In the nervous system, Abcb1b is present in the blood–brain barrier (BBB), where it restricts drug penetration into the brain [36, 37]. Recent clinical cross-sectional findings have suggested an association between two SNPs in Abcb1b and post-thrombolytic hemorrhagein ischaemic stroke [38]. However, the specific mechanism of action remains unclear. Our results indicated that the crucial role Abcb1b plays may be attributed to the modulation of drug penetration across the BBB by affecting proton pump activity. Inhibitor of apoptosis (IAP) repeat containing 5 (Birc5), also known as survivin, is a member of the IAP family and is highly expressed in most tumor cells [36]. Birc5 negatively regulates apoptosis by inhibiting the expression of caspase 3 [37]. The therapeutic effect of BYHWD on ICH may be attributed to its ability to induce apoptosis through Birc5 downregulation. Ccl12 belongs to the C–C (or beta) chemokine family, which modulates immune regulation and inflammation [38]. During ICH, Ccl12 induces inflammation and exacerbates brain damage by recruiting macrophages and T cells [39]. In this study, BYHWD intervention reversed the upregulation of Ccl12 after modelling.

Table 3 Differentially expressed genes regulated by BYHWD
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The neuroprotective effect of BYHWD may be achieved by reducing the expression of Ccl12, resulting in the inhibition of inflammation. Tlr4 is an innate immune receptor belonging to the toll-like receptor family [40, 41]. Tlr4 is a marker of inflammation and apoptosis in ICH and is increased following secondary brain injury. In turn, high Tlr4 expression exacerbates neurological defects, consistent with our results [42]. We speculate that BYHWD downregulates Tlr4 expression to alleviate neuroinflammation and improve the prognosis of patients with ICH. In conclusion, although we have confirmed the association between key genes such as Abcb1b, Birc5, Ccl12, Tlr4, and Ctsb and BYHWD treatment for ICH in our study. Further experimental validation is still required to elucidate their functional significance and mechanisms of action in ICH pathophysiology. The specific roles and interrelationships of these genes are crucial for understanding the pathogenesis of ICH and the therapeutic effects of BYHWD.

The most significant alteration in gene expression following BYHWD intervention in ICH was observed in Ctsb during the study. Cathepsin B, a member of the cysteine cathepsin family, is primarily located in lysosomes and endosomes. It plays a role in autophagy, antigen presentation, cellular stress signalling, and lysosome-dependent cell death [43]. Previous studies have reported that the deletion of the Ctsb gene has a significant positive impact on behavioural deficits in conditions such as ischaemic neuropathic damage, inflammatory pain, opioid tolerance, epilepsy, and Alzheimer's disease [44]. Additionally, research has indicated the involvement of Ctsb in neuronal cell death following ICH. Selective Ctsb inhibition improves neurological function in rats, potentially by promoting cell survival at the ICH border and restoring neuroplasticity and angiogenesis [45]. Ctsb directly activates several matrix metalloproteinases (MMPs), and its absence inhibits MMP-9 expression [35]. MMP-9 is associated with ICH enlargement and the development of perihaematomal oedema [46]. Interestingly, a previous study revealed that Ctsb plays a role in correct chromosome segregation, which is closely related to lysosomal function [47]. This finding aligns with the biological enrichment results of transcriptomics analysis, specifically highlighting the involvement of Ctsb in chromosomal maintenance. Collectively, these findings support the notion that Ctsb is involved in neural restoration following ICH through multiple pathways. This further underscores the potential of Ctsb as a critical target for BYHWD therapy. The localisation of Ctsb within lysosomes suggests its potential impact on the autophagy process [34]. Autophagy plays a vital role in degrading intracellular materials and maintaining cellular homeostasis by utilising lysosomal enzymes [48]. The production and transportation of autophagosomes primarily occur in axons, and proper axonal transport is crucial for clearing damaged mitochondria [19]. Ctsb is closely associated with autophagy and negatively regulates neurite outgrowth by modulating lysosomal trafficking in neurons [49]. In our study, autophagy levels were elevated following ICH injury. However, concomitant increases in both Ctsb mRNA and protein expressions suggest that Ctsb may not be the sole factor influencing autophagy levels, as the autophagy process is regulated by multiple factors [50]. Conversely, after intervention with BYHWD, a significant enhancement in autophagy levels was observed, accompanied by a decrease in Ctsb levels. This indicates that BYHWD can partially inhibit Ctsb expression and enhance autophagy.

Under normal homeostatic conditions, Ctsb cleaves Mcoln1 within the lysosome, inhibiting calcium efflux from the lysosomal lumen to the cytoplasm. This process restricts the expression of Tfeb and subsequently reduces the expression of lysosomal and autophagy-related proteins [34]. Tfeb is a key regulator of autophagy and lysosomal biogenesis [51], and its activation alleviates neurodegeneration by promoting lysosomal function and inducing autophagy [52, 53]. Mcoln1 is a ubiquitously expressed lysosomal calcium channel, which is activated during autophagy and is involved in lysosomal calcium transport [54]. Furthermore, the Mcoln1/calcineurin-dependent mechanism is implicated in autophagy initiation [52].

In our study, the mRNA expression levels of Mcoln1 and Tfeb decreased after BYHWD intervention. Thus, we hypothesised that BYHWD moderates the expression of Ctsb, Mcoln1, and Tfeb to regulate autophagy after ICH. Autophagy plays a bidirectional role in ICH. Some studies have demonstrated that autophagy is involved in neuronal death following brain injury [55]; others have revealed that enhancing autophagy 7 days after ICH improves disease prognosis [21]. However, the integrity of autophagic flux and the autophagy level are critical factors that influence the effectiveness of autophagy [56].

Basic experimental research has suggested that in the early stages after injury, many autophagic vesicles around the haematoma show impaired autophagic flux and production of cellular waste. At this point, high levels of autophagy and endoplasmic reticulum stress are in a positive feedback loop, exacerbating nerve damage. However, 7 days after ICH, the autophagic flux returns to normal, allowing autophagy to efficiently clear the metabolic waste and play a neuroprotective role [57]. These studies collectively demonstrate that cellular autophagy activation has a beneficial effect 7 days post-ICH. In summary, the therapeutic mechanism of BYHWD at 7 days after ICH likely involves enhancing cellular autophagy through the downregulation of Ctsb expression.

Conclusions

In summary, based on a combination of bioinformatics analyses and validation experiments, we have elucidated the key target proteins and specific mechanisms of BYHWD treatment for ICH from multiple perspectives, which are associated with apoptosis, signal pathways, and PI3K-Akt/mTOR biological processes. In addition, we confirmed that BYHWD improves the prognosis of ICH by inhibiting Ctsb and enhancing cell autophagy.