Study On The Empirical Mechanism Of Xinglou Chengqi Decoction In The Treatment Of Acute Ischemic Stroke With Phlegm-heat And Fu-organs

Acute ischemic stroke(AIS)is one of the most common types of stroke in clinic.At present,thrombolysis is the fastest and most effective method in the treatment of acute stage of IS,but the clinical effect of thrombolysis is limited due to strict indications,short time window,high risk of bleeding and reperfusion injury.A large number of experimental and clinical observation have proved that TCM has a unique advantage in the treatment of stroke.Phlegm-heat and Fu-organ Excess Syndrome is the most common syndrome type in acute stage of ischemic stroke.Xinglou Chengqi Decoction(XCD)is a representative prescription for removing phlegm and clearing Fu organs.The TCM theory of "treating intestine with brain disease" and " treating the diseases in the upper portion by managing the lower portion " of XCD is similar to that of " bacteria-gut-brain axis" proposed by modern medicine.A large number of clinical and basic studies have shown that XCD has a definite effect on IS,but the mechanism of its effect is still insufficient.Therefore,based on the theory of network pharmacology and microecology,this paper discusses the mechanism of intestinal microecology of ischemic stroke and the effect mechanism of XCD in treating IS.Object:Based on the theory of "treating the diseases in the upper portion by managing the lower portion " and "bacteria-gut-brain axis",(1)through the network pharmacology and molecular docking technology,the multi-component,multi-target and multi-channel mechanism of XCD in the treatment of IS was comprehensively explored;(2)the brain protection effect of XCD with the function of Huatan Tongfu method on the model of acute ischemic stroke with Phlegm-heat and Fu-organ Excess Syndrome was clarified(3)The effect of XCD on intestinal flora of acute ischemic stroke model mice with Phlegm-heat and Fu-organ Excess Syndrom,and the intestinal microecological mechanism of the difference between "Tongfu" and "Tongbian" in the treatment of Phlegm-heat and Fu-organ Excess Syndrome were both explored;(4)To verify the hypothesis that XCD can improve the prognosis of stroke by directly affecting the intestinal bacteria,the effect of XCD on intestinal flora of pseudoaseptic acute ischemic stroke model mice was observed.Methods:(1)The active components and targets of XCD were collected by TCMSP、BATMAN-TCM、ETCM and TCMID,and the target prediction of the compounds without target from databases above was performed by STITCH、SwissTarget Prediction and SEA.The targets of IS were mined through six databases.Go and KEGG enrichment were used to analyze the high-level function of the intersection targets.Finally,the PPI network,compound-target network and drug-target-pathway network were constructed by using Cytoscape 3.6.0.Finally,the molecular docking was verified by AutoDock.(2)Male C57BL/6 mice were randomly divided into sham operation(Sham)group,model(MCAO)group,Xinglou Chengqi Decoction(XCD)group,nimodipine(Nim)group and ShuTaiQing(PGE)group.To establish the model of acute ischemic stroke mice with Phlegm-heat and Fu-organ Excess Syndrom by high-fat and low fiber diet combined with thread suppository method,the neuroprotective effect of XCD was comprehensively evaluated by neurological function score,behavioral testing,TTC and TUNEL staining,and the key targets and pathways of network pharmacology were verified by ELISA,Western blot and other molecular biological techniques.(3)Male C57BL/6 mice were randomly divided into Sham,MCAO,XCD,Nim and PGE groups.The acute ischemic stroke model of Phlegm-heat and Fu-organ Excess Syndrom in mice was established by high-fat and low fiber diet.The intestinal flora,short chain fatty acids(SCFAs)and their receptor GPR43,neuroendocrine immune pathway(The cecal norepinephrine and tyrosine hydroxylase,serum MTL,brain IBA-1 GFAP,serum inflammatory factors)were analyzed by high-throughput 16SrDNA gene sequencing,metabonomics,immunehistochemistry,immunofluorescence,ELISA and flow cytometry respectively.(4)After the male C57BL/6 mice were treated with antibiotic cocktail 14 days before operation,they were randomly divided into:ABX-Sham group,ABX-MCAO group and ABX-XCD group.Neurological function score,behavioral testing,TTC staining,high-throughput 16SrDNA gene sequencing,metabolomics,immunohistochemistry,immunofluorescence,ELISA and flow multi-factor technique were used to observe the effect of XCD on the related indexes of bacteria-gut-brain axis in pseudosterile mice.Results(1)The results of network pharmacology and molecular docking showed that XCD contained 51 active components and 44 overlapping targets for the treatment of IS.Advanced functional analysis showed that the mechanism of XCD on IS may be related to lipopolysaccharide mediated signaling pathway,regulation of apoptosis,inflammatory response,establishment of endothelial barrier and response to fatty acids.In addition,10 key KEGG signaling pathways of XCD were obtained.PPI network analysis showed AKT1,PTGS2,TNF,TP53,CASP3,IL1B and other 10 key targets.Molecular docking further verified that the active components of XCD had good docking ability with key targets.(2)The neuroprotective effect of XCD and its influence on the key targets and pathways of network pharmacology:①Neurological function score:compared with MCAO group,the neurological function score of XCD group and Nim group decreased 48 hours and 72h after operation(P<0.05),and PGE group had no significant change(P>0.05).②Behavioral testing:the contact time and removal time of 48 and 72h after operation in XCD group and Nim group were significantly shortened(P<0.05),and PGE group had no significant change(P>0.05).③TTC staining:the infarct area of XCD group decreased,and no significant change was found in Nim group and PGE group.④TUNEL:apoptosis in infarcted area of MCAO group increased significantly,and TUNEL positive staining in XCD group and Nim group was significantly reduced(P<0.05).⑤The results showed that TNF-α in XCD group and Nim group decreased(P>0.05),and p-AKT,PI3K and NF-kappa B protein expression in XCD group were higher.There was no significant change in AKT protein expression group(P>0.05).(3)Effects of XCD on bacteria-gut-brain axis of mice with Phlegm-heat and Fu-organ Excess Syndrome in acute stage of stroke:① Intestinal flora:compared with MCAO group,α-diversity of XCD group increased,but there was no significant change in Nim and PGE group.β-diversity showed that there was significant difference in PCoA curve between MCAO and Sham group(P<0.05).In MCAO group,Bacteroidetes and Proteobacteria increased significantly,but Firmicutes decreased significantly;Bacteroidetes decreased significantly and Verrucomicrobia increased significantly in XCD group;Proteobacteria increased significantly and Firmicutes decreased in Nim group;the composition of flora in PGE group was similar to MCAO,but Proteobacteria decreased slightly.In addition,Akkermansia ratio increased significantly in XCD group.Klebsiella increased most significantly in Nim group,while Parabacterioides and Escherichia/Shigella increased more significantly in PGE group.②SCFAs and GPR43:the content of SCFAs in MCAO group was significantly decreased(P<0.05),and the content of butyric acid in XCD group was significantly increased(P<0.05).The expression of GPR43 in XCD group and Nim group was significantly decreased(P<0.05).③Autonomic nerve pathway:NE decreased in XCD group and increased in PGE group(P>0.05).The expression of TH protein increased in MCAO group and PGE group(P<0.05),but there was no significant change in XCD group and Nim group(P>0.05).④ Neuroendocrine pathway:the MTL of MCAO group increased significantly(P<0.05),PGE group increased(P>0.05),XCD and Nim group decreased significantly(P<0.05).⑤Immune pathway:In MCAO group,astrocytes proliferated,hypertrophied and activated significantly.Microglia rapidly changed from "resting state" to "activated state",and were recruited from the infarct surrounding area to the lesion site,while the activation of astrocytes and microglia in XCD group was lower than that in MCAO group.(4)The effect of XCD on the bacteria-gut-brain Axis of the mice with pseudosterile Phlegm-heat and Fu-organ Excess Syndrome in acute stroke:①Neurological function score:compared with ABX-MCAO group,there was no significant change in the Neurological function score,behavioral testing and TTC staining in ABX-XCD group(P>0.05).②Intestinal flora:the α-diversity of ABX-MCAO and ABX-XCD group was lower than that of ABX-Sham.PCoA analysis showed that the β-diversity and composition of intestinal flora in ABX-XCD group were similar to that of ABX-MCAO group.The results of relative abundance showed that the microbial flora of mice in ABX group was characterized by Proteobacteria,which was significantly different from that of mice without antibiotics.LEfSe analysis showed that there were more pathogens or opportunistic pathogens in MCAO group,including Bacteroidetes,Escherichia_Shigella and Helicobacter,while XCD enriched beneficial bacteria such as Verrucomicrobia and Akkermansia.Opportunistic pathogens such as Streptococcus,Lactococcus,Morganella,Klebsiella,Proteobacteria and Enterobacteriaceae were enriched significantly in ABX-XCD group.In PGE group,the enriched bacteria were mainly o Selenomonadales、c_Negativicutes、g_Romboutsia and f_Peptostreptococcaceae.③The propionic acid of SCFAs and GPR43:ABX-XCD decreased significantly(P<0.05).The expression of GPR43 in ABX-MCAO group increased significantly(P<0.05),while that of ABX-XCD group was significantly lower(P<0.05).④Autonomic nerve pathway:NE content in ABX-XCD group was not significantly changed(P>0.05),and TH protein expression was not significantly changed(P>0.05).⑤Neuroendocrine pathway:the MTL of ABX-MCAO group increased(P>0.05),and the MTL of ABX-XCD group had no significant change(P>0.05).⑥ Immune pathway:IL-22 increased significantly in ABX-XCD group(P<0.05),IL-17A decreased(P>0.05),and no significant change was found in the rest(P>0.05).The expression of astrocytes in ABX-MCAO group increased significantly and microglia was activated significantly.Astrocytes and microglia activation in ABX-XCD group were lower than that in ABX-MCAO group.ConclusionXCD has a neuroprotective effect on IS mice with Phlegm-heat and Fu-organ Excess Syndrome,and its mechanism has the characteristics of multi-component,multi-target and multi-channel.However,no definite brain protective effect was found in the pseudoaseptic mouse model of stroke,indicating that part of the brain protective mechanism was achieved by improving the intestinal microecology.The mechanisms include increasing the content of SCFAs,especially butyric acid,increasing the expression of GPR43,increasing blood IL10,reducing the levels of inflammatory factors such as TNF-α and MTL,and ultimately reducing the activation of glial cells and apoptosis of nerve cells in the infarcted area,so as to achieve the effect of stroke brain protection.The results of this study have important guiding significance for the clinical treatment of stroke for patients with constipation after stroke,only"defecation" treatment is not enough.We need to consider the characteristics of the patient’s syndrome "Huatan Tongfu" treatment,in order to achieve good clinical effect.