Treatment of Ischemic Stroke with Wenyang Huayu Formula: network pharmacology analysis and experimental validation

doi:10.21203/rs.3.rs-2076903/v1

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Research Article

Treatment of Ischemic Stroke with Wenyang Huayu Formula: network pharmacology analysis and experimental validation

https://doi.org/10.21203/rs.3.rs-2076903/v1

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Objective

To explore the mechanism of Wenyang Huayu Formula in the treatment of ischemic stroke based on network pharmacology, molecular docking and experimental verification.

Methods

The chemical components and potential targets in Wenyang Huayu Formula were obtained by network pharmacology, and the targets related to the pathological process of cerebral ischemia-reperfusion were obtained, and the "drug-component-target-disease" was drawn by Cytoscape software,protein interaction network map was drawn based on STRING database.Gene enrichment analysis was using the Metascape database, and the Autodock vina software was using for molecular docking to determine the pathways and targets of Wenyang Huayu Formula, which were verified by vivo experiments in rats.

Results

277 drug targets and 3777 disease targets of Wenyang Huayu Formula were retrieved. The core components were Quercetin, kaempferol, luteolin, etc., and the core targets were AKT1, TP53, IL6, CASP3, IL1β, etc.Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analysis yielded 222 entries, involving Cellular senescence, TNF signaling pathway, Toll-like receptor signaling pathway, etc. Molecular docking results showed that the core component and the core protein had good binding energy. Vivo experiments results shown that: Stigmasterol, C-Homoerythrinan, luteolin and other components in Wenyang Huayu Formula may act on targets such as IL-6, IL-1β and TNF-α through the TLR4/NF-κB signaling pathway, and play roles in neural protection, reduce reperfusion injury, inhibit apoptosis, anti-inflammatory and other effects.

Conclusion

The mechanism of ischemic stroke by Wenyang Huayu Formula was the result of multi-component, multi-target and multi-channel interaction, and its mean mechanism may inhibit cell apoptosis, reduce inflammation and promote blood vessels through the TLR4/NF-κB signaling pathway.

Wenyang Huayu Formula (WYHYF)

network analysis

ischemic stroke

molecular docking

animal experiment

Ischemic Stroke (IS) has the characteristics of high morbidity, high disability rate, high mortality and many complications. It is caused by cerebral blood circulation disorder which was caused by various reasons. A series of clinical neurological deficit syndromes caused by oxygen and even further necrosis(1)

Traditional Chinese medicine has certain advantages in the prevention and treatment of IS. The application of traditional Chinese medicine can not only improve the condition of stroke patients, but also adjust the general state of the body, effectively relieve accompanying or secondary symptoms, and improve patients’ life quality(2).The traditional Chinese medicine compound Wenyang Huayu Formula（WYHYF）has a significant clinical effect in the treatment of IS,which was composed of Aconitum Carmichaeli Debx,the dry root of Aconitum chinense Paxton is a plant of Araceae；Acorus Tatarinowii Schott,the dry root of Acorus tatarinowii Schott is a grass like perennial herb；Cinnamomum Cassia Presl,the dried twigs of Cinnamomum cassia Presl is Lauraceae plant；Atractylodes Macrocephala Koidz,the dry rhizome of Atractylodes macrocephala Koidz is a Compositae plant； Epimedium Brevicornu Maxim,the dry leaf of Epimedium brevicornu Maxim is a Berberidaceae plant；Panax Notoginseng,the dry root of Panax notoginseng is a ginseng plant of acanthopanaceae；Rheum Palmatum,the dry root of rhubarb belongs to many perennial plants of Rheum palmatum in Polygonaceae；and Glycyrrhiza Uralensis Fisch，the dried roots of Glycyrrhiza uralensis Fisch is bean plants.Relevant clinical studies were conducted on the treatment of IS with Wenyang Huayu Formula, and it was believed that this Formula had advantages in improving neurological function and carotid artery media thickness in patients with IS(3). In addition, our research team demonstrated that Wenyang Huayu Formula can inhibit the occurrence of autophagy by regulating the expression of related proteins, reduce the neurological function, reduce the infarct size, and protect the brain tissue in rats with ischemia-reperfusion brain injury. However, due to the characteristics of multi-component, multi-target and multi-channel treatment of traditional Chinese medicine compounds, those above researches were only a part of the mechanisms of Wenyang Huayu Formula.

Network pharmacology is the elucidation of possible mechanisms of drugs at the molecular level, explains the complex relationship between drugs and organisms, and analyzes the synergistic effects of multiple drugs. Molecular docking is a method for virtual prediction of molecular ligands and targets based on computer technology, and is one important method in the field of drug design(3). Therefore, in order to more comprehensively predict the mechanism of action of Wenyang Huayu Formula, the method of network pharmacology was used to predict the active ingredients, therapeutic targets, and signaling pathways of Wenyang Huayu Formula, and evaluated and confirmed by molecular docking technology.Futhure with experimental verification in this study, it provides a reference for subsequent clinical applications and basic researches.

1.1 Collection of Traditional Chinese Medicine and Disease Targets

Through Traditional Chinese Medicine systems pharmacology data-base and analysis platform(TCMSP), the components with oral bioavailability(OB)≥ 30% and drug-like properties(DL)≥ 0.18 were screened as active ingredients(6). Then, ETCM, TCMID, CNKI and PubMed database platform were used as the supplement to search for the active ingredients and corresponding targets of Aconite, Shichangpu, Guizhijian, Atractylodes, Epimedium, Panax notoginseng, Rhubarb, Zhigancao, and Bawei, the target name conversion is performed through the String platform. Disease targets were collected through the GeneCards and OMIM databases with "Ischemic stroke, cerebral infarction" as the search terms (7). Drawing the Venn diagram of the traditional Chinese medicine targets and the disease targets to obtain the intersection targets from the results of previous work..

1.1.1 Drawing of the drug-component-target-disease network diagram of Wenyang Huayu Formula

Use the Cytoscape 3.8.2 software to draw the drug-component-target-disease network diagram for the data collected in 1.1, use the Network Analyzer tool to calculate the network diagram, and screen out the core components according to the degree value.

1.1.2 The protein-protein interaction (PPI) network diagram was drawn through the STRING platform, and the PPI calculation was performed on the intersection targets, and the calculation results were imported into Cytoscape software for network topology analysis.

1.1.3 Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes（KEGG） enrichment analysis

Through the Metascape database, GO enrichment analysis and KEGG pathway analysis were performed on the intersection targets. Use software such as R language to visualize the data in bubble charts and histograms.

1.1.4 Molecular docking

The core components were selected for molecular docking with the core protein, and the compound structure was obtained by downloading from the PubChem database; the core protein was obtained by downloading from the PDB database (8). Use Pymol software to remove water molecules, separate proteins and visualize data. Molecular docking was performed using Autodock Vina, resulting in corresponding computational results (9).

1.2 Animal experimental verification

1.2.1 Drug

Wenyang Huayu Formula (Aconitum carmichaeli Debx 20g batch number A200029810, Acorus tatarinowii Schott 20g A210003710,Cinnamomum cassia Presl 15g A210084810, Atractylodes macrocephala Koidz 15g A210006210, Epimedium brevicornu Maxim 15g A200162510, Panax notoginseng 15g A210087101, Rheum palmatum 6g A210075410, Glycyrrhiza uralensis Fisch 6g A210165010) was produced by Jiangyin Tianjiang Pharmaceutical Co., Ltd, Jiangyin, Jiangsu Province.

1.2.2 Animal

SD rats（n=24）, male,body weight 200±20g with animal certificate number 430727211101291332, experimental animal license number: SCXK (Xiang) 2019-0004, provided by Hunan Slike Jingda Laboratory Animal Co., Ltd., reared in the SPF animal room of Guangxi University of Traditional Chinese Medicine, with temperature 18-26℃ and humidity 40-70%. The animals were adaptively fed for 1 week before the experiment. All experimental procedures were approved by the Ethics Committee of Guangxi University of Traditional Chinese Medicine (DW20200415-53).For euthanasia, rats were anesthetized by an intraperitoneal injection of 3% pentobarbital sodium (30 mg/kg body weight，SHBD9164V). Once fully anesthetized, rats were sacrificed by cervical dislocation and death was confirmed by cessation of breathing and faded eye color.The animals were monitored every day and the humane endpoint of this experiment was as follows: Marked reduction in food or water intake, labored breathing, inability to stand and no response to external stimuli. No abnormal signs that signified the humane endpoints of the experiment were observed from any of the rats during the experiment. Mortality was verified by the lack of heartbeat and a cold body.

1.2.3 Animal Experiment

The rats in the present study were anesthetized with 1% pentobarbital sodium (intraperitoneal; 30 mg/kg) and the rats were placed in a position such that their heads were fixed on a stereotactic frame in order for the posterior and anterior fontanelle to be on the same horizontal plane.Preparation of cerebral ischemia-reperfusion rat model according to reference (10). After modeling, the wounds were sutured layer by layer, treated with iodophor. After 2 hours of cerebral ischemia and reperfusion, the rats with neurological deficit score > 0 were randomly divided into model group (MCAO), nimodipine tablet 30 mg/kg control group, warm Yang Huayufang Granules treatment group. Each group was given the corresponding drugs by gavage at a volume of 30 g/kg/d. The normal control group and the model control group were given the same volume of normal saline by gavage for 6 consecutive days, twice a day.

1.2.4 Observation Indicator

1.2.4.1 Neurological function score

The neurological function test was performed on the 6th day after administration, and the five-point scoring method of Zea Longa was used for scoring. The specific scoring criteria are: grade 1 (0 points), no signs of neurological deficits, and the animal behaves normally; grade 2 (1 point), unable to fully flex the contralateral forepaw; grade 3 (2 points), turning to the side of the paralysis; 4 Grade 3 (3 points), dumping to the paralyzed side; Grade 5 (4 points), unable to walk spontaneously, loss of consciousness. The higher of the score means the more severe the rat cerebral ischemia-reperfusion injury.

1.2.4.2 The expression of related proteins was detected by Western Blot method. Rat brain tissue was extracted. Bicinchoninic acid assay (BCA) was used to calculate the protein concentration. After SDS gel electrophoresis, membrane transfer, antigen blocking and primary antibody incubation were performed [Anti-Caspase-3 antibody, ab184787, abcam), Anti-NF-kB p65 antibody (ab239882, abcam), Anti-TLR4 antibody (ab217274, abcam)], secondary antibody incubation [with peroxidase-conjugated goat anti rabbit IgG (HL) 1:10,000],display image.

1.2.4.3 ELISA detection

After the last administration of rats.Intraperitoneal injection of 1% sodium pentobarbital at a dosage of 30 mg/kg，5 ml of blood was collected from the abdominal aorta, Rats were euthanized after blood collection.Centrifuged at 3000 r·min^-1 for 10 min (centrifugation radius 8.58 cm), serum was collected, stored in a -80℃ refrigerator, and the protein concentration of the samples was determined. Follow the procedure described in the ELISA assay kit [Rat Tumor necrosis factor alpha (TNF-alpha) ELISA Kit (RA20035, Bioswamp), Rat interleukin6 (IL-6) ELISA Kit (RA20607, Bioswamp), Rat Interleukin 1β (IL-6) -1β) ELISA Kit (RA20020, Bioswamp)].

1.2.4.4 Tunel staining

After the last administration of rats.Intraperitoneal injection of 1% sodium pentobarbital at a dosage of 30 mg/kg，the rats were killed by decapitation, and the brain were taken out,fixed in formaldehyde, embedded in paraffin, and then sectioned. After the solution, hydrogen peroxide, Streptavidin HP solution, DAB chromogenic solution, hematoxylin counterstaining, dehydrate with alcohol and clear with xylene; finally mount the slides and observe with a microscope.

1.2.4.5 Transmission electron microscope observation

After 6 days of treatment,intraperitoneal injection of 1% sodium pentobarbital at a dosage of 30 mg/kg，the rats were sacrificed by decapitation, and the samples were collected and fixed: the brain tissue was collected with the volume of 1 mm×1 mm×1 mm, and was quickly put into the electron microscope fixative solution at 4℃ for 2-4 hours, then rinsed 3 times with 0.1M phosphate buffer PB (pH7.4) for 15min each time. 1% osmic acid·0.1M phosphate buffer PB (PH7.4)was used to post-fixated the brain for 2 hours at room temperature (20℃). Rinse 3 times with 0.1M phosphate buffer PB (pH7.4) for 15min each time. Dehydration, infiltration, embedding, ultra-thin sectioning were performed in sequence, double-stained with uranium and lead, and the sections were dried overnight at room temperature. Observed under a transmission electron microscope, collected images for analysis.

2. Statistical analysis

Statistical Product and Service Solutions (SPSS) 20.0 (IBM, Armonk, NY, USA) were used to analyze the data of this study and Graphpad Prism 9.0 (La Jolla, CA, USA) was used to make graphs.The measurement data were represented by the mean ± standard deviation. Comparison of data between multiple groups was performed using one-way analysis of variance followed by post-hoc test (Bonferroni); p<0.05 was considered as statistically significant.

3.1 Through the treatment of traditional Chinese medicine targets and disease targets

According to the database, a total of 147 active ingredients of traditional Chinese medicine were collected, including 6 aconite, 4 calamus, 6 cassia twig, 5 atractylodes, 7 notoginseng, 23 epimedium, 10 rhubarb, and 86 licorice. A total of 2846 TCM targets were obtained by standardizing the targets and removing duplicate targets through the STRING platform. Finally, a total of 277 TCM targets were obtained. A total of 3777 IS disease targets were collected through the disease database. Taking the intersection of traditional Chinese medicine targets and disease targets, 212 intersection targets were obtained (Figure 1).

3.2 Drug-ingredient-target-disease network graph analysis

The drug-ingredient-target-disease (IS) network of Wenyang Huayu Formula was constructed by Cytoscape 3.7.2 software (Figure 2). There were 2667 edges and 421 nodes in the whole network, including 8 traditional Chinese medicines, 52 active ingredients, 277 potential targets, and 1 disease. The Triangle represents the name of Wenyang Huayufa Formula, the Diamond node represents the name of the traditional Chinese medicine, the Hexagon node represents the ingredient, the Ellipse node represents the potential target, the V-shaped node represents the disease, and the edge represents the interaction relationship between the nodes. It can be seen that Wenyang Huayu Formula may treat IS in the form of multi-component, multi-target and multi-channel intervention. PTGS2, ESR1, CALM2, HSP90AA1, AR and other genes may be important potential targets. Quercetin, kaempferol, luteolin, wogonin,7-Methoxy-2-methylisoflavone,beta-sitosterol,formononetin,C-Homoerythrinan,1,6-didehydro-3,15,16-trimethoxy-, (3.beta.)-, Isorhamnetin, naringenin, etc. may be important potential active ingredients.

3.3 PPI network graph analysis

Using the STRING platform, PPI processing was performed on the intersection targets, and the network topology analysis was performed on the obtained data with Cytoscape, and 212 nodes and 4537 edges were obtained, with an average degree value of 42.8 (Figure 3). The interaction information was imported into Cytoscape 3.7.1 software, the median degree value was 43,88 targets with all indicators greater than the median were screened out as core targets..The larger the construction degree value, the darker the color and the larger the graph, the more closely it interacted with other proteins. The top ten targets were AKT1, TP53, IL6, CASP3, VEGFA, JUN, IL1β, EGFR, MAPK3, STAT3.These targets may be the key targets for the treatment of IS with Huoxue Huayu Formula, and only the first 36 core targets with larger degree values were listed in Figure 3.

3.4 GO function enrichment analysis and KEGG pathway enrichment analysis of potential target genes of Wenyang Huayu Formula for IS

Through the Metascape database, GO enrichment analysis obtained 4475 biological process (BP) entries, 367 cell composition (CC) entries and 502 Molecular Function (MF) entries. In bioengineering, the target functions focus on response to inorganic substance, response to cytokine, positive regulation of cell migration, etc.; in cell composition, targets mainly involve transcription regulator complex, membrane raft, vehicle lumen, cyclin-dependent, protein Kinase holoenzyme complex, endoplasmic reticulum lumen, perinuclear region of cytoplasm, etc.; in terms of molecular functions, the targets mainly involve kinase binding, RNA polymerase II-specific DNA-binding transcription factor binding, NF-kappaB binding, etc., see Figure 4. KEGG pathway analysis showed that these key targets were involved in signaling pathways, mainly involving Cellular senescence, FoxO signaling pathway, TNF signaling pathway, Toll-like receptor signaling pathway, Non-alcoholic fatty liver disease, Th1 and Th2 cell differentiation, Pathways of neurodegeneration - multiple diseases, Epithelial cell signaling in Helicobacter pylori infection, etc. The top 20 pathways were shown in Figure 5.

3.5 Molecular docking results

The key targets selected from the core targets of Wenyang Huayu Formula in the treatment of IS were molecularly docked with the main active ingredients. The molecular docking verification showed that 88% with the docking score <-5kcal·mol^-1, that means most of the targets were combined with the ingredients. The heat map of the docking score was shown in Figure 6, and the schematic diagram of part of the docking with the active ingredient molecule was shown in Figure 7. The smaller the docking binding force value, the more stable the binding between the ligand and the receptor, and the more likely the interaction occurs. Among them, the lowest binding force of Stigmasterol to IL-1β was -9.6 kcal·mol (-1 1 kcal≈4.186 kJ).

3.6 Animal experimental verification

3.6.1 The effect of Wenyang Huayu Formula on the neurological function of IS

Compare with the normal control group, the neurological function damage of the model control group was severe, and the Zea Longa score was significantly increased (P<0.01); Compared with nimodipine 30 mg/kg and Wenyang Huayu Formula group, the symptoms of neurological damage were improved, and the Zea Longa score was significantly decreased (P<0.05), as shown in Figure 8.

3.6.2 The protein expressions of TLR4, NF-κB p65 and Caspase-3

Compared with the normal group, the protein expressions of TLR4, NF-κB p65 and Caspase-3 in the rat brain tissue were significantly up-regulated in the model group (P<0.01). Compared with rats, the expressions of TLR4, Caspase-3 and NF-κB p65 in Nimodipine Tablets control group and Wenyang Huayu Formula group were significantly decreased (P<0.05). The expressions of TLR4 and NF-κB p65 proteins in the prescription group were decreased (P<0.05), and the expression of Caspase-3 protein was not statistically significant, as shown in Figure 9.

3.6.3 Effects of Wenyang Huayu Formula on serum TNF-α, IL-6 and IL-1β in mice with IS

The levels of TNF-α, IL-6 and IL-1βof sham group were significantly increased when compared with the model group, the nimodipine control group and the Wenyang Huayu Formula group could significantly reduce the serum levels of TNF-α, IL-6, and IL-1β in mice. IL-1β content, compared with the control group, Wenyang Huayu Formula can significantly reduce the serum TNF-α, IL-6, IL-1β content of rats, as shown in Figure 10.

3.6.4 Tunel staining

It was found that compared with the sham operation group, the apoptosis rate of the model group increased significantly, indicating that the model group induced acute brain injury modeling successfully, and the model group would induce brain cell apoptosis; The apoptosis rate of nimodipine group decreased significantly, and the apoptosis rate of rats gradually decreased (P<0.01). Compared with the control group, the Wenyang Huayu Formula group showed that the apoptosis rate was significantly reduced, and the apoptosis rate of the rats was reduced. The results were shown in Figure 11.

3.6.5 Transmission electron microscope observation

In the sham operation group, the blood-brain barrier structure was acceptable, the perivascular glial cell pedicle structure was acceptable, the glial filaments were abundant, the coupling was good, the endothelial cell morphology was acceptable, the vascular lumen was not atrophied, and the vascular basement membrane was intact. The damage to the blood-brain barrier in the model group was relatively the most serious, mainly in the peripheral blood vessels, with extensive edema and dissolution, slight compression of blood vessels, mild edema of endothelial cells, and blurred structure of the basement membrane. Astrocyte foot plate edema, disintegration. In the nimodipine group, blood-brain barrier damage was second, moderate damage, extravascular matrix edema, tissue dissolution in some areas, disappearance of structure, partial blurring and dissolution of basement membrane, obvious edema of astrocyte foot plate, matrix dissolution, and massive collapse untie. The pictures of the Wenyang Huayu Formula group showed mild damage to the blood-brain barrier, the endothelial cell structure was fair or slightly edema, mainly mild edema of the extravascular matrix, intact astrocyte foot plate membrane, mild edema, and local matrix dissolution.(Figure 12).

The etiology, pathological process, and related mechanisms of ischemic stroke are complex and diverse. In addition to intravenous thrombolysis and mechanical thrombectomy, medical conservative treatment is the mainstay, and there is a lack of evidence-based effective treatment measures (11). Traditional Chinese medicine has the characteristics of multi-component and multi-target action, and the treatment of IS is not limited by the time window. Therefore, it is of great significance to explore the mechanism of traditional Chinese medicine in the treatment of stroke based on the experience of famous experts (11).

The network pharmacology analysis was based on 277 compounds with potential biological activity, 2846 corresponding protein targets and 3777 disease targets in Wenyang Huayu Formula to construct a "drug-ingredient-target-disease" network . The results of pathway enrichment analysis indicated that inflammation is closely related to cerebral ischemia-reperfusion, and the inflammatory signaling pathway may play an important role in the treatment of cerebral ischemia-reperfusion with Wenyang Huayu Formula. The KEGG results showed that there were 150 pathways in total and Toll like receptor signaling pathway pathway was more enriched in genes which was most commonly studied in the role of toll-like receptor 4(TLR4) (14). It has been suggested that activated TLR4 can promote neurogenesis and angiogenesis(15, 16). The researchers found that activation of TLR4 can initiate MyD88-dependent and TRIF-dependent signaling pathways, and MyD88-dependent signaling cascades activate nuclear factor-κB light chain enhancer (NF-κB) or mitogen-activated protein kinase (MAPK) signaling pathways in activated B cells(17, 18). By mediating the activation of the nuclear transcription factor NF-κB, it promotes the expression of downstream inflammatory factors such as IL-6, IL-1β and TNF-α, thereby amplifying the inflammatory signal.After the occurrence of IS, microglia exert their pro-inflammatory effects by activating NF-kB. Due to the neurotoxic effect of activated microglia, TNF-α can mediate neuronal necrosis through apoptotic mechanism, which in turn aggravates brain damage(19, 20). In the molecular docking, the key components of Wenyang Huayu Formula have relatively stable binding activity to IL-6, IL-1β and TNF-α. In the molecular docking, the key components of Wenyang Huayu Formula have relatively stable binding activity to IL-6, IL-1β and TNF-α. Therefore, the TLR4 pathway, which is closely related to the pathogenesis of ischemic stroke, was detected in animal experiments, and the expression levels of IL-6, IL-1β, and TNF-α were verified.

According to the results of network pharmacology, animal experiments confirmed that Wenyang Huayu Formula could improve neurological function and effectively reduce cerebral ischemia-reperfusion injury after the 6th day of administration. Electron microscopy results confirmed that Wenyang Huayu Formula could significantly reduce the proliferation and migration of endothelial cells, increase vascular permeability, and promote the formation of new blood vessels. The results of TUNEL staining showed that the number of TUNEL-positive cells in the Wenyang Huayu Formula group decreased significantly, which indicated that Wenyang Huayu Formula could play a protective effect on cerebral ischemia injury by reducing the apoptosis of neurons in and around the injured area.After cerebral ischemia-reperfusion, the increased levels of IL-1β, IL-6 and TNF-α in the brain were a reliable basis for brain tissue damage. IL-1β is highly expressed in the brain and acts by regulating neurotropism and ion channel expression and activity(21). After ischemic stroke, the levels of TNF-α and IL-1β increase dramatically due to the activation of microglia and macrophages(22). This study shown that Wenyang Huayu Formula could significantly reduce the levels of inflammatory factors IL-1β, IL-6 and TNF-α in brain tissue of rats with cerebral ischemia-reperfusion injury, indicating that Wenyang Huayu Formula could reduce cerebral ischemia-reperfusion injury and futhure reduce the inflammatory response mediated by cerebral ischemia-reperfusion injury.Western Blot and ELISA results showed that Wenyang Huayu Formula could down-regulate the expression of inflammation-related proteins TLR4, NF-κB p65, and caspase-3 in brain tissue, and reduce the levels of IL-6, IL-1β, and TNF-α inflammatory factors in brain tissue.All the resuolts indicated that Wenyang Huayu Formula might play an important role in improving cerebral ischemia-reperfusion injury by down-regulating TLR4/NF-κB signaling pathway and inhibiting IL-1β, IL-6, TNF-α and other inflammatory factors.

In summary, the mechanism of action of Wenyang Huayu Formula for IS treatment was analyzed and discussed through network pharmacology, and the molecular docking analysis and experimental verification were combined on the basis of the initially screened target targets. The results show that Wenyang Huayu Formula may act on IL-6, IL-1β and TNF-α and other targets through the TLR4/NF-κB signaling pathway to exert neuroprotection and reduce reperfusion injury, inhibition of apoptosis, anti-inflammatory and other effects. From the holistic view of traditional Chinese medicine, the synergistic therapeutic effect of multiple components and multiple targets of traditional Chinese medicine is revealed, which has certain guiding significance for the further determination of key active components and the development of new drugs in the future researches(23). This study only verified some of the core targets and individual pathways. Whether the above-mentioned core targets and pathways were involved in the actual treatment process needs to be verified by further animal experiments and cell experiments. Therefore, further research needs to be carried out on the basis of continuous supplementation to clarify the specific mechanism of Wenyang Huayu Formula in the treatment of IS, and to provide a clear theoretical basis for clinical treatment of IS.

Author Contributions

XM and XL designed this research; WM analyzed the network pharmacology data, conducted the experiments, and wrote the manuscript; YY,TX performed the preparation of herbal prescriptions; YX contributed to data analysis and interpretation of the results; XM and XL reviewed the manuscript. All authors have read and approved the manuscript.

Conflicts of Interest

The authors declare no conflflicts of interest, fifinancial, or

otherwise.

Funding

This work was supported by the National Natural Science Foundation of China (81874453),the Natural Science Foundation of Guangxi Province, China (2020GXNSFAA297270).

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No competing interests reported.

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Treatment of Ischemic Stroke with Wenyang Huayu Formula: network pharmacology analysis and experimental validation

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Abstract

Objective

Methods

Results

Conclusion

Figures

Introduction

Materials And Methods

1.1 Collection of Traditional Chinese Medicine and Disease Targets

1.2 Animal experimental verification

2. Statistical analysis

Result

3.1 Through the treatment of traditional Chinese medicine targets and disease targets

3.2 Drug-ingredient-target-disease network graph analysis

3.3 PPI network graph analysis

3.4 GO function enrichment analysis and KEGG pathway enrichment analysis of potential target genes of Wenyang Huayu Formula for IS

3.5 Molecular docking results

3.6 Animal experimental verification

3.6.1 The effect of Wenyang Huayu Formula on the neurological function of IS

3.6.2 The protein expressions of TLR4, NF-κB p65 and Caspase-3

3.6.3 Effects of Wenyang Huayu Formula on serum TNF-α, IL-6 and IL-1β in mice with IS

3.6.4 Tunel staining

3.6.5 Transmission electron microscope observation

Discussion

Declarations

References

Additional Declarations

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