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Journal of Traditional Chinese Medicine logoLink to Journal of Traditional Chinese Medicine
. 2024 May 15;44(4):794–803. doi: 10.19852/j.cnki.jtcm.20240515.001

Sanhua Tang (三化汤) protects against ischemic stroke by preventing blood-brain barrier injury: a network pharmacology and in vivo experiments

Shan LUO 1, Fan YANG 2, Yuanchun CHEN 2, Ruoxi ZHAO 2, Haiye LIU 1, Fei GAO 3, Wencan MA 3, Weijuan GAO 1,4,✉, Wentao YU 3,4,5,✉
PMCID: PMC11337263  PMID: 39066540

Abstract

OBJECTIVE:

To assess the effect and mechanism of Sanhua Tang (三化汤, SHT) in treating ischemic stroke (IS) through the "Kaitong Xuanfu" theory by using network pharmacology and animal experiments.

METHODS:

The active ingredients and targets of SHT and IS were screened by public databases such as Traditional Chinese Medicine systems pharmacology, GeneCards, and online mendelian inheritance in man. Visual network topographies were constructed using R, Cytoscape 3.6.0, AutoDockTools, a user-sponsored molecular visualization system on an open-source foundation, and other software to analyze the correlation between targets and active ingredients. The middle cerebral artery occlusion (MCAO) model was established by operation. Animals were divided into the Sham group, MCAO group (M group), aloe-emodin (AE) group (MCAO rats treated with aloe-emodin), SHT at low dosage (SL group) (MCAO rats treated with SL), SHT at medium dosage (SM group), and SHT at high dosage (SH group). 2,3,5-triphenyl tetrazolium chloride staining was used to detect the volume of cerebral infarction; Nissl staining was used to observe the morphology of neuronal cells; transmission electron microscopy was used to observe the integrity of the blood-brain barrier (BBB); enzyme-linked immunosorbent assay was used to detect the content of interleukin-6 (IL-6), IL-10, tumor necrosis factor α (TNF-α) in serum. Western blot was used to detect the expression of vascular endothelial growth factor A (VEGFA) protein in the cerebral ischemic penumbra.

RESULTS:

Using network pharmacology and molecular docking validation, four active ingredients (lignan, naringenin, aloe-rhodopsin, and β-sitosterol), seven target proteins (protein kinase b 1, IL-6, TNF, VEGFA, TP53, jun proto-oncogene, and cysteinyl aspartate specific proteinase 3), and inflammatory signaling pathways were identified. Animal experiments showed that the SH and AE groups had fewer neurological deficits, reduced brain infarct volumes, decreased serum inflammatory factor levels, increased expression of VEGFA protein, and less structural damage to neurons and BBB.

CONCLUSION:

The present study found that the therapeutic mechanism of SHT against IS may be related to the inhibition of BBB inflammatory damage, which is also the mechanism of "Kaitong Xuanfu." The high-dose group of SHT was relatively effective in regulating inflammatory factors, improving BBB permeability, and protecting neuronal cells from damage.

Keywords: network pharmacology, ischemic stroke, blood-brain barrier, inflammatory reactions, Sanhua Tang

1. INTRODUCTION

Ischemic stroke (IS) is a cerebrovascular disease caused by focal occlusion or stenosis of the artery.1 According to epidemiological surveys, more than 11 million people worldwide suffer from IS every year.2 In addition, stroke has become the leading cause of death in China, with 70% of strokes being ischemic stroke.3 Excitatory amino acid toxicity, oxidative stress, energy metabolism disorder, inflammatory injury, and apoptosis are the essential pathological basis of IS.4 Among those, inflammatory injury is the critical pathological link leading to the blood-brain barrier (BBB) and neuronal damage.5

In 1999, professor Li6 first put forward the hypothesis that there is a relationship between Traditional Chinese Medicine (TCM) and biomolecule network. In 2007, professor Hopkins7 proposed a concept of network pharmacology to describe diseases' occurrence, development, and evolution from the perspective of system biology and biological network balance. The research paradigm of network pharmacology has realized the transformation of TCM from empirical therapy to evidence-based medicine.8 For example, the study of the treatment mechanism of IS found that TCM can inhibit the inflammatory reaction, regulate the level of oxidative stress, reduce the apoptosis of neurons in brain tissue, and exert a protective role in the brain.9

Sanhua Tang (三化汤, SHT) is the representative prescription of "Kaitong Xuanfu" for treating IS created by Liu Wansu. Studies have shown that when acute stroke occurs, the prevalence of constipation is 41.9%.10 It is reported that the investigation of 503 patients with IS shows that constipation accounts for 47.9%. Clinical studies11 have confirmed that SHT can effectively relieve the clinical symptoms of stroke patients, but its exact mechanism is unclear.

The BBB damage is the key to "Xuanfu blockage". This study investigated the effect and mechanism of SHT in treating IS through the "Kaitong Xuanfu" theory by using network pharmacology and animal experiments.

2. MATERIALS AND METHODS

We used different databases and software sources to perform this work, as shown in supplementary Table 1.

2.1. Ingredient target prediction and disease target collection

Traditional Chinese Medicine Systems Pharmacology (TCMSP) database, UniProt database, GeneCards, and Online Mendelian Inheritance in Man (OMIM), were used for ingredient target prediction and disease target collection. SHT active ingredients and targets were collected from the TCMSP database by setting oral bioavailability (OB) ≥ 30% and drug-likeness (DL) ≥ 0.18.12 In the UniProt database,13 the species was limited to "Homo sapiens" to normalize the naming of the obtained components. In GeneCards,14 and OMIM,15 IS genes with high correlation were screened with the keyword "ischemic stroke." The R software and Venn diagram constructed the intersecting genes of drug components and diseases.

2.2. Construction of an SHT-compound-target-IS network

Crossover genes are the critical targets of SHT for IS treatment. A visualization network was constructed using Cytoscape 3.6.0 (National Resource for Network Biology, Santiago, CA, USA) to elucidate the relationship between compounds and targets.

2.3. Enrichment analysis

Enrichment analysis was performed using the Metascape online platform, setting the species as "H. sapiens" and "P < 0.05" to reduce the false positive rate of screening results, and the top 20 ranked bars and bubble plots were plotted using R language (Version 3.6.3, Lucent Techn-ologies, Murray Hill, NJ, USA) and Bioconductor (https: //www.bioconductor.org/developers/). The main potential biological processes and signaling pathways for IS treatment with SHT were clarified by enrichment analysis. Finally, we created a visual network using Cytoscape.

2.4. Protein-protein interaction (PPI) network construction and core gene screen

The PPI network can help mine core regulatory genes. Using the STRING database species was set as "Homo sapiens," "Mediumconfidence 0.4". Topology analysis was performed using the Cytoscape plugin centiscape 2.2 (National Resource for Network Biology, Santiago, CA, USA) to construct the PPI optical network.

2.5. Molecular docking

Compounds in the SHT-compound-target-IS network were sorted by degree using the cytoNCA plugin (National Resource for Network Biology, Santiago, CA, USA). The structural formulae of the top 10 compounds were retrieved using the TCMSP database, AutoDock-Tools (Molecular Graphics Laboratory, Santiago, CA, USA) verified potential targets and molecular docking maps were drawn using open-source foundation (PyMOL) (DeLano Scientific LLC, Boston, MA, USA).

2.6. Drugs and reagents

Aloe-emodin (high performance liquid chromato-graphy, HPLC ≥ 97%) was purchased from Yuanye Biotechnology Company (Shanghai, China). All 4 types of Chinese medicines of SHT were purchased from YiLing Pharmaceutical (Shijiazhuang, China): Dahuang (Radix Et Rhizoma Rhei Palmati, Product batch number: B2109001); Houpu (Cortex Magnoliae Officinalis, Product batch number: B2012001); Zhishi (Fructus Aurantii Immaturus, Product batch number: B2106001); Qianghuo (Rhizoma et Radix Notopterygii, Product batch number: B2001001). All herbs in SHT were processed conforming to the standards of Chinese Pharmacopeia 2015. Vascular endothelial growth factor A (VEGFA) (R pAb, dilution concentration 1∶3000) and β-actin (R pAb, dilution concentration 1∶10000), were purchased from Bioss Company (Beijing, China). Rat interleukin-6 (IL-6), IL-10, tumor necrosis factor α (TNF-α), enzyme-linked immunosorbent assay (ELISA) kit was purchased from QuantiCY Company (Shenzhen, China).

2.7. Animals

Male Sprague-Dawley rats, specific pathogen free grade, aged 8 weeks (230 ± 10) g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (license No. SCXK, Beijing 2021-0011). All the animals were housed in an environment with a temperature of (24 ± 1) ℃, relative humidity of 60% ± 10%, and a light/dark cycle of 12/12 h, and given water and food ad libitium. All animal studies (including the mice euthanasia procedure) were done in compliance with the regulations and guidelines of Hebei University of Chinese Medicine institutional animal care and conducted according to the Association for Assessment and Accreditation of Laboratory Animal Care A and the Institutional Animal Care and Use Committee guidelines (approval No. DWLL202206013).16,17

2.8. Animal grouping, model preparation, and drug administration

Animals were randomly divided into 6 groups (twelve rats per group): Sham group, Middle cerebral artery occlusion (MCAO) group (M group), Aloeemodin (AE) group (MCAO rats treated with Aloe-emodin), SHT at low dosage (SL group) (MCAO rats treated with SL), SHT at medium dosage (SM group), and SHT at high dosage (SH group). The MCAO model was performed, as explained previously.18 The procedure for the sham operation group was the same as those in the model group, except that the string was not inserted into the carotid artery.

Neurobehavioral scores were performed 24 h after modeling. SHT is a granule made of Dahuang (Radix Et Rhizoma Rhei Palmati), Houpu (Cortex Magnoliae Officinalis), Zhishi (Fructus Aurantii Immaturus), and Qianghuo (Rhizoma et Radix Notopterygii) according to the ratio 10∶10∶10∶10. The dose of intragastric administration in rats was 10 mL/kg. The dosing concentration for each group was (SH group 0.72 g/mL, SM 0.36 g/mL, SL 0.18 g/mL, and AE 5 mg/mL). The drug concentration is configured by dissolving in different volumes of 0.5% Carboxy-methylcellulose sodium separately and configuring into a suspension of corresponding con-centration. AE group and SHT groups were orally administered Aloe-emodin or SHT before stroke induction once daily for 4 days, and continued admin-istration for one day after MCAO surgery. M and sham groups were orally administered with saline (1 mL/100 g per day).

2.9. PeriCam Perfusion Speckle Imaging Zoom technology

We used a laser speckle blood flow meter to monitor the cerebral cortical blood perfusion before MCAO and 2 h after thrombolysis. Successful modeling was defined based on the following criteria:19 the blood flow of the right cerebral cortex decreased by at least 65% and 70% 2 h after embolization.

2.10. Neurobehavioral deficit evaluation

Animal neurological deficit was examined at 24 h after reperfusion using Modified Neurological Severity Score test,20 as follows: 13-18 was considered severe, 7-12 was moderate, and 1-6 was mild. The success evaluation criteria of the model were mild, moderate, and severe.

2.11. Infarct area measurement

At 24 h after reperfusion, five rats were randomly selected from each group. Animals were euthanized, after which triphenyltetrazolium chloride (TTC) staining was performed. The sections were photographed, and the infarct size was calculated using Image-Pro Plus software (version 6.0, Media Cybernetics, Rockville, MD, USA), expressed as infarct area percentage (%).

2.12. Nissl staining and transmission electron microscope observation (TEM)

Three rats were randomly taken from each group, after which Nissl staining and TEM were performed. Images were collected for analysis.

2.13. Validation of key targets and impact on inflammatory factors

ELISA kit was used to detect the contents of IL-6, TNF-α, and IL-10 in serum, following the manufacturer's instructions. Western Blot detected the protein expression of VEGFA in the ischemic penumbra.

2.14. Statistical analysis

The data were analyzed and plotted using IBM SPSS 23.0 statistical software (Statistical Product and Service Solutions, IL, USA) and GraphPad Prism (version 9.0, Prism scientific research drawing tool, CA, USA). Results are expressed as mean and standard deviation. One-way analysis of variance (ANOVA) was used to compare multiple groups with a normal distribution and homogenous variance. The comparison between groups was performed using the Bonferroni method, and the non-parametric test was used for data that did not conform to the normal distribution. A P value < 0.05 was considered statistically significant.

3. RESULTS

3.1. Results of bioactive compounds and targets in SHT and IS

Through database analysis, 55 active ingredients were identified. Target genes were transformed into standard gene names through the UniProt database, and finally, 122 genes, considered drug-related genes, were obtained. Details of the 55 drug ingredients are shown in supplementary Table 2. A total of 474 IS-related genes were collected by searching disease gene databases. Chemical targets were intersected with disease targets to obtain 80 common targets, which were retained as IS-related targets (supplementary Figure 1A).

3.2. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis

A total of 80 core targets were subjected to gene ontology (GO) enrichment analysis, using R software to generate a bar plot and bubble graph (supplementary Figures 1B1-1B2). The KEGG pathways related to this study included lipid and atherosclerosis, the phosphoinositide 3-kinase-protein kinase B signaling pathway, the IL-17 signaling pathway, and the TNF signaling pathway (supplementary Figures 1C1-1C2).

3.3. Visual network construction and analysis

The process of PPI network core target screening can be found in supplementary Figures 1D-1E. These core components were later used to construct the herbal pathway-target network (Figures 1A, 1B). The screening results of the core genes in the PPI network can be found in supplementary Tables 3-4.

Figure 1. Compound-targets network and Herb-pathway-targets network.

Figure 1

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A: the network had 121 nodes representing the potential active components of SHT and 353 edges, describing the relationship between them. The triangle represents IS, and the square represents the four herbs of SHT. DH (Dahuang), ZS (Zhishi), QH (Qianghuo), and HP (Houpu) stand for herbs' abbreviation. The hexagon represents effective chemical compounds, and the diamond represents targets. B: circles represent compounds, arrows represent pathways, squares represent the top 10 core targets, and triangles represent IS. SHT: Sanhua Tang; IS: ischemic stroke.

3.4. Molecular docking

Compounds associated with the ten core targets were luteolin, naringenin, nobiletin, aloe-emodin, and beta-sitosterol. We assessed the affinity between receptors and ligands by calculating the binding free energy (kcal/ mol), and compound-target interactions with binding en-ergies less than - 5.0 kcal/mol are shown in supplementary Table 5. According to the results, binding energy ≤ - 7.0 kcal/mol indicated a better ability to bind (Figure 2).

Figure 2. Conformations of some key compounds and core targets.

Figure 2

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A: AKT1 with luteolin; B: IL-6 with luteolin; C: TNF with luteolin; D: CASP3 with luteolin; E: AKT1 with naringenin; F: CASP3 with beta-sitosterol. GLN: glutamine; ASN: asparagine; LYS: lysine; ALA: alanine; MET: methionine; LEU: leucine; ARG: arginine; PHE: phenylalanine; TRP: tryptophan; AKT1: v-akt murine thymoma viral oncogene homolog 1; IL-6: Interleukin-6; TNF: tumor necrosis factor; CASP3: cysteinyl aspartate specific proteinase 3.

3.5. Identification of components in SHT formula by HPLC chromatography

Chromatographic conditions: COSMOSIL C18 (250 mm × 4.6 mm, 5 μm) was utilized. Gradient elution: acetonitrile (A), 0.1% trifluoroacetic acid (B) with a flow rate of 1 mL/min: 0-8min, 15% A; 8-23 min, 15%A→ 25%A; 23-29 min, 25%A→45%A; 29-45 min, 45%A→45%A; 45-66 min, 45%A→70%A; 66-75 min, 70%A→90%A ;75-80 min, 90%A→15%A. Ten components targeting hub genes were selected to perform HPLC; the information on the components and the concentration of the solution were shown in supplementary Table6, and the HPLC chromatograms of ten components were presented in previous studies.21

3.6. Effect of SHT on neurological dysfunction

The neuroprotective effect of SHT in treating IS was comprehensively assessed from neurological deficit score, cerebral infarct volume, and neuronal necrosis. There was no sign of neurological deficits in the sham group. Compared with the sham group, the neurological deficit score, the infarct area, and the number of degenerative and necrotic neurons in the M group were increased (all P < 0.05) (Table 1). The SHT and AE groups showed lower neurological deficit scores, cerebral infarct area, and lower number of degenerated and necrotic neurons compared with the M group, especially the SH and AE groups (all P < 0.05) (Figures 3A, 3B).

Table 1.

Statistical results of neurological score, infarct area and the number of positive cells between each group after ANOVA

Neurological dysfunction ANOVA Treatment (between columns) Residual (within columns) F (DFn, DFd) P value
Neurological score SS 924.4 447.4 F (5, 102) = 42.15 < 0.0001
DF 5 102
MS 184.9 4.387
Infarct area SS 3.165 0.6348 F (5, 23) = 22.93 < 0.0001
DF 5 23
MS 0.6329 0.02760
Number of positive cells SS 19256 5910 F (5, 30) = 19.55 < 0.0001
DF 5 30
MS 3851 197.0
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Notes: details of the comparisons between each group can be found in supplementary Tables 7-9. ANOVA: analysis of variance; DF: degree freedom; SS: stdev square; MS: mean square.

Figure 3. Effect of SHT on neurological dysfunction.

Figure 3

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A: TTC staining of cerebral slices of each group; B: Nissl staining observation of ischemic penumbra of rats in each group under × 400 magnification. B1: the sham group; B2: the M group; B3: the SL group; B4: the SM group; B5: the SH group; B6: the AE group. Microscopic observation showed that the green arrows were normal neurons, the neurons were regularly arranged, the whole blue staining and the Nissl corpuscles were dark blue, thick, and dense granules. The red arrows were degenerative and necrotic neurons, the arrangement of neurons was disordered, Nissl corpuscles decreased or even disappeared, and the whole was in a lightly stained state. M: middle cerebral artery occlusion; AE: aloeemodin; SL: Sanhua Tang at low dosage; SM: medium dosage; SH: high dosage; SHT: Sanhua Tang; TTC: triphenyltetrazolium chloride; MCAO: middle cerebral artery occlusion. M: model group were orally administered with saline 1 mL/100 g per day for 5 d; SL: this group were orally administered Sanhua Tang 0.18 g/mL before stroke induction once daily for 4 d, and continued administration for one day after MCAO surgery. As the same goes SM 0.36 g/mL, SH group 0.72 g/mL, and AE 5 mg/mL.

3.7. Effect of SHT on inflammatory damage of BBB

The effects of different SHT doses on BBB's inflammatory damage was evaluated in terms of serum inflammatory factors, VEGFA protein expression levels, and the ultrastructure of BBB. Compared with the sham group, the content of IL-6, TNF- α in the M group increased and IL-10 decreased (all P < 0.05). Compared with the M group, the SH and AE groups had lower levels of IL-6 and TNF- α and higher levels of IL-10 (all P < 0.05). Moreover, compared with the sham group, the expression levels of VEGFA in the M group were increased (P < 0.05). Compared with the M group, the expression of VEGFA in the SH group was higher (P < 0.05) (Figure 4A, Table 2).

Figure 4. Effect of SHT on inflammatory damage of BBB.

Figure 4

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A: comparison of the protein expression levels of VEGFA in rats. [Mean (SD), n = 4]. B: comparison of BBB ultrastructure under TEM in each group. Each group had two views, B1-6 at low power (× 3000) and B7-12 at high power (× 8000); B1, B7: the sham group; B2, B8: the M group; B3, B9: the AE group; B4, B10: the SL group; B5, B11: the SM group; B6, B12: the SH group. M: middle cerebral artery occlusion; AE: aloeemodin; SL: Sanhua Tang at low dosage; SM: medium dosage; SH: high dosage; SHT: Sanhua Tang; MCAO: middle cerebral artery occlusion; BBB: blood-brain barrier; TEM: transmission electron microscope. M: model group were orally administered with saline 1 mL/100 g per day for 5 d; SL: this group were orally administered Sanhua Tang 0.18 g/mL before stroke induction once daily for 4 d, and continued administration for one day after MCAO surgery. As the same goes SM 0.36 g/mL, SH group 0.72 g/mL, and AE 5 mg/mL.

Table 2.

Statistical results of inflammatory factors and VEGFA protein expression levels between each group after ANOVA

Inflammatory damage of BBB ANOVA Treatment
(between columns) 		Residual
(within columns) 		F (DFn, DFd) P value
Levels of IL-6 SS 966.0 954.9 F (5, 30) = 6.070 0.0005
DF 5 30
MS 193.2 31.83
Levels of IL-10 SS 109.4 172.7 F (5, 30) = 3.802 0.0087
DF 5 30
MS 21.89 5.757
Levels of TNF-α SS 99.52 171.7 F (5, 30) = 3.478 0.0135
DF 5 30
MS 19.90 5.723
Levels of VEGFA SS 3.438 0.6922 F (5, 18) = 17.88 < 0.0001
DF 5 18
MS 0.6876 0.03846
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Notes: details of the comparisons between each group can be found in supplementary Tables 10-13. ANOVA: analysis of variance; BBB: blood-brain barrier; IL-6; 10: interleukin-6; 10; TNF-α: tumor necrosis factor α; VEGFA: vascular endothelial growth factor A; DF: degree freedom; SS: stdev square; MS: mean square.

TEM observation revealed no obvious abnormalities in the structure of the BBB and no prominent edema of endothelial cells in the sham group. In the M group, the structure of the BBB was severely damaged, endothelial cells showed edema, the vascular lumen was narrowed locally, and the basement membrane was blurred; there was also an uneven thickness, discontinuous (red arrow), intercellular space of endothelial cells widened (black arrow), and the length of a dense area of tight junction shortened (white arrow). Compared with the M group, the integrity of BBB in the SH and AE groups improved (Figure 4B).

4. DISCUSSION

For patients with IS, timely thrombolysis or mechanical embolization can reduce neurological deficits.22 In China, unfortunately, due to the limitation of the treatment time window and the economic status of patients, some patients cannot carry out thrombolytic therapy in time, which increases disability or mortality. Recently, Chinese medicine has received more attention for its advantages in improving cerebral ischemic injury.23 Basic research shows that Tong fu therapy can increase levels of ACTH and CORT, decrease levels of MMP-9 and S100B proteins, inhibit the release of MIF and MMP-9 cytokines, reduce inflammatory reactions, improve neurological function scores, restore neuro-logical function to a certain extent, and improve quality of life.24,25 Pharmacological studies have also shown that the extract of Dahuang can improve the permeability of the BBB in rats with cerebral infarction.26 Houpu reduces the neuroinflammatory response after stroke in a mouse model of depression.27 Pathological changes occur in the gastric mucosa in rats with acute cerebral infarction, and Zhishi has a certain protective effect on gastrointestinal dysfunction in rats with acute cerebral infarction.28

Because of its systematic and holistic nature, network pharmacology provides a new approach to studying complex Chinese medicine components. " TCM network pharmacology "has undergone several stages of incubation-development-formation-perfection.29 The combination of network pharmacology and TCM can facilitate the discovery of new bioactive components and biomarkers, revealing mechanisms of TCM treatment.30,31 Zhang9 and Huang31 used the network pharmacology method to screen the pathway of SHT in treating ischemic stroke; they found that SHT may protect against IS injury by regulating APP, phosphatidylinositide 3-kinase/protein kinase b/cAMP responsive element binding protein 1, and TNF pathways.

Although there are similar studies, the indicators verified by the above two studies are not the same as those in this study. This study assessed the mechanism of SHT "Kaitong Xuanfu" in treating IS. The BBB comprises brain endothelial cells (BECs) embedded in the capi-llary basement membrane, pericytes and the terminal foot of astrocytes.32 Inflammatory injury after cerebral ischemia-reperfusion leads to the destruction of the BBB; on the contrary, the destruction of the BBB aggravates cerebral ischemia-reperfusion injury.33 Xuanfu is highly consistent with the BBB regarding structure and function, and BBB damage is the key to neuroinflammatory injury. This study concluded that SHT exerts its therapeutic effect through the inflammatory signaling pathway. Other studies have shown that luteolin may protect the brain from ischemic damage by reducing oxidative stress and apoptosis.34 Aloe-emodin has an anti-inflammatory and antioxidant effect and may act against angiogenesis,35,⇓-37 and the likely anti-inflammatory mechanism may involve a decrease in pro-inflammatory cytokine via inhibition of nuclear factor kappa-B, mitogen-activated protein kinases, and PI3K pathways.37 Naringenin may reduce the accumulation of intracellular ROS and improve cell survival by activating the mTOR/p70S6K signaling pathway.38 Tumor necrosis factor (TNF) is a kind of inflammatory cytokine with bidirectional regulation on the central nervous system.39 IL-6 is an inflammatory factor involved in vascular endothelial cell inflammation and atherosclerosis. In the state of cerebral ischemia, IL-6 participates in secondary brain injury as an important inflammatory mediator.40 IL-17A, a potent proinflammatory cytokine, is believed to have a specific role in the delayed phase of the postinfarct inflammatory response.41 IL-10 has potent anti-inflammatory and immunosuppressive activity.42 VEGFA is a double-edged sword that regulates vascular endothelial function, promoting vascular renewal and restoring cerebral blood flow, but that can also increase vascular permeability, cause cerebral edema, and aggravate BBB injury and neurological damage.43,44 This study showed severe neurological dysfunction, increased pro-inflammatory factors IL-6 and TNF-α, and decreased IL-10 levels in the M group. Also, the expression of VEGFA in brain tissue decreased, indicating that the inflammation of brain tissue after cerebral ischemia injury was aggravated. Compared with the M group, neurological dysfunction in the SH and AE groups were lighter, TNF-α was decreased, and the expression of IL-10 and VEGFA in brain tissue was increased suggesting that SHT could change the serum inflammatory factors and regulates vascular endothelial function, thus exerting neuroprotective effects. Nissl staining further showed that a large number of neurons in the M group were degenerative and necrotic. In contrast, the damage of neurons in the SH, SM, and AE groups was slighter compared to the M group. Furthermore, TCM showed that the damage degree of the BBB in the M group was the highest, indicating that cerebral ischemia could lead to ultrastructural damage to the BBB, while the ultrastructure of the BBB in the SH and AE groups was more intact. The SH group (7.2 g/kg) was considered the best treatment dose because it had the highest effect in improving neurological deficit scores, reducing infarct size, lowering serum inflammatory factors, and reducing structural damage in the BBB compared to other dose groups.

In this study, we investigated the SHT "Kaitong Xuanfu" mechanism for treating IS by combining network pharmacology, molecular docking, and animal experiments. The present study confirmed that BBB damage is the key to the "blockage of Xuanfu", and the efficacy of SHT in "Kaitong Xuanfu" is reflected in changing the serum inflammatory factors and BBB, reducing the inflammatory state of the body, alleviating the inflammatory damage of the BBB, and exerting neuroprotective effects.

5. SUPPORTING INFORMATION

Supporting data to this article can be found online at http://journaltcm.cn.

Footnotes

Supported by Hebei Province Natural Science Foundation Project: Based on The Xuan Fu Theory to Explore Sanhua Tang on Ischemic Stroke Blood-brain Barrier Protection Mechanism Research (No. H2022423327), Science and Technology Program of Hebei Provincial Administration of Traditional Chinese Medicine: based on The Changes in Gut Microbiota, Explore Sanhua Tang From The Perspective of "Opening the Xuan Fu" Protective Phlegm Heat Relieving Excess Syndrome of Ischemic Stroke Mechanism of Action of the Blood-brain Barrier (No. 2022090), Hebei Key Laboratory of Chinese Medicine Research on Cardio- cerebrovascular Disease in 2021 Project: Based on the Gut-brain Axis, Explore Sanhua Tang's Treatment of Ischemic Stroke Syndrome (Phlegm Heat Relieving Empirical Model) (No. 2021201), the Innovation Funding Program for Doctoral Students in Hebei Province: the Mechanism Research of Sanhua Tang Regulating Gut Microbiota, Improving the Blood-brain Barrier Damage of Ischemic Stroke (No. CXZZBS2022095), National College Students' Innovation and Entrepreneurship Training Program: Based on the Xuan Fu Theory to Explore Sanhua Tang on Ischemic stroke Blood-brain Barrier Protection Mechanism Research (No. 202114432005), and Special Project on the Cultivation of Scientific and Technological Innovation Ability of College and Middle School Students in Hebei Province: Mechanism Research of Bushen Huoxue Fang on Synaptic Plasticity in the Hippocampal Nerve After Vascular Dementia through Phosphatidylinositide 3-Kinase/Protein Kinase b/Mammalian Target of Rapamycin Signaling Pathways (22E50142D)

Contributor Information

Weijuan GAO, Email: gwj6088@163.com.

Wentao YU, Email: yuwentao@hebcm.edu.cn.

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