Intestinal Flora And Formation Of Syndromes Of Ischemic Stroke With Qi Deficiency And Blood Stasis And The Intervention Mechanism Of Buyang Huanwu Decoction

Objective:1.To explain the development of ischemic stroke and the relationship between TCM syndromes of ischemic stroke and gut microbiota and metabolites.2.To explore the interaction and mechanism of Buyang Huanwu Decoction and gut microbiota of qi deficiency and blood stasis of ischemic stroke.Methods:Sprague-Dawley?SD?rats were used to establish three rat models.Specifically,MCAO rat models were established by the suture-occluded method.Exhausted swimming combining with the suture-occluded method were used to make the rat model of qi deficiency and blood stasis syndrome of ischemic stroke.The rat model with qi stagnation and blood stasis syndrome of ischemic stroke was made by subcutaneous injection of adrenalin combining with multiple mixed stimulation and suture-occluded method.1.Rats cecum contents and plasma of MCAO group and sham operation group were collected 72 hours after operation,then 16 S r RNA gene sequencing and non-targeted metabolomics were performed to detect gut microbiota and plasma metabolites,respectively.Bioinformatics analysis and correlation analysis were carried out on the omics data.2.All of rats cecum contents including the MCAO group,qi deficiency and blood stasis syndrome ischemic stroke group,qi stagnation and blood stasis syndrome ischemic stroke group and sham operation group were collected 72 hours after operation.Performed16 S r RNA gene sequencing to detect gut microbiota and bioinformatics analysis.3.Both rats of qi deficiency and blood stasis syndrome of ischemic stroke and sham operation group were gavaged Buyang Huanwu decoction?12 m L/Kg · d?at 1h,24 h and 48 h when we have established models successfully.The cecum feces and plasma of all rats were taken respectively for 16 S r RNA gene sequencing and metabolites by non-targeted metabolomics at 72 hours after surgery.Analyzed the differences of drug-related metabolites of Buyang Huanwu decoction in two groups.4.Rats with qi deficiency and blood stasis syndrome of ischemic stroke were randomly divided into three groups,including the Buyang Huanwu decoction group,Bifidobacterium quadruplex viable tablet group and Saline control group.All rats were gavaged with Buyang Huanwu decoction?12 m L/ Kg · d?,Bifidobacterium quadruplex viable tablet?0.81 g/ Kg · d?,and Saline?4 m L/d?at 1h,24 h,and 48 h respectively.The rats were sacrificed after neurobehavioral scoring at 72 hours after operation.The whole brain of rats was taken for TTC staining to detect the volume of cerebral infarction,the cecum contents were taken for 16 S r RNA gene sequencing to detect the gut microbiota,the plasma was separated,then the plasma metabolites were detected by metabolomics.Modeling and analysis of associations between gut microbiota and metabolites.Evaluated the neurobehavioral score and sacrificed all rats after 72 hours.Rats whole brains were used for TTC staining to detect the volume of cerebral infarction,the cecum contents and plasma were taken for 16 S r RNA gene sequencing and metabolomics to detect the gut microbiota and plasma metabolites.Modeled and analyzed the relationship between gut microbiota and metabolites.Results:1.The changes of gut microbiota and plasma metabolites of ischemic stroke:?1?There were significantly changes about the composition and diversities of gut microbiota in the MCAO group,in which firmicutes were significantly down-regulated while the proteobacteria and deferribacteres were significantly up-regulated.According to the genus,escherichia-Shigella was up-regulated,and lactobacillus and bifidobacteria were downregulated.?2?There were significant diversities in the profiles of rats plasma metabolism in MCAO group.A total of 308 metabolites altered significantly,of which 155 were up-regulated and 153 were down-regulated.The KEGG pathway of disordered metabolite was significantly enriched in amino acid metabolism,lipid metabolism,cholinergic synapse,m TOR signaling pathway,2-oxycyclopentane methyl carboxylate metabolism,etc.?3?Some abnormal metabolites in the MCAO group were closely related to the diversities of gut microbiota.In which that eicosapentaenoic acid,daidzein,glycitein and progesterone which with neuroprotective effects were significantly down-regulated,and all were highly correlated with gut microbiota.2.The specific changes of gut microbiota in rats with qi-deficiency and blood-stasis syndrome of ischemic stroke:?1?Compared to the sham operation group,the abundance of species in the qi deficiency and blood stasis syndrome of IS group decreased significantly?P <0.05?,the abundance of Firmicutes decreased significantly,while the abundance of Proteobacteria increased significantly.On the other hand,although there were no significant differences,both abundance of Proteobacteria and Verrucobacteria increased slightly,and Firmicutes decreased slightly in the qi stagnation and blood stasis syndrome of IS group compared with the sham operation group.The Firmicutes decreased significantly,and proteobacteria and Deferribacteres increased significantly in the MCAO group comparing with the sham operation group.?2?Compared to MCAO group,the abundance of Firmicutes and Deferribacteres decreased significantly in the qi deficiency and blood stasis syndrome IS group,similarly,the Deferribacteres decreased significantly in qi stagnation and blood stasis syndrome of IS group.?3?A total of 10 genera displayed significantly higher levels,including Escherichia-Shigella,Phascolarctobacterium,Alistipes,Faecalibacterium,Odoribacter,Sutterella,etc.while the abundance of Parasutterella and Lachnospiraceae_UCG-001 decreased significantly in the qi stagnation and blood stasis syndrome of IS group comparing to the MCAO group.Interestingly,Sutterella was the main genus of gut microbiota in qi deficiency and blood stasis syndrome IS group by LEFSE analysis.3.Gut microbiota affected the absorption of active ingredients of Buyang Huanwu Decoction:?1?Compared to the sham operation group,a total of 10 genera upregulated significantly and 10 genera downregulated significantly in qi deficiency and blood stasis syndrome of IS group.Specifically,Bacteroides,Prevotella,Parabacteroides,Phascolarctobacterium,Butyricimonas increased significantly,while Lactobacillus,Helicobacter,Tyzzerella,Lachnospiraceae_NK4A136_group,Ruminiclostridium₉,etc decreased significantly.?2?The components of Buyang Huanwu decoction changed significantly in qi deficiency and blood stasis syndrome of IS group.Betaine,Alpha-ionone,Methylgallic acid-O-sulphate,Arabinose,Palmitic acid,and Decursinol were significantly up-regulated,while Adenosine and 3-O-methyl gallic acid were significantly down-regulated.Interestingly,many components were related to oxidative stress and anti-inflammatory response.Except for 3-O-methylgallic acid,other 7 ingredients were associated with intestinal flora by correlation analysis.4.Buyang Huanwu Decoction affects the rats gut microbiota and plasma metabolites of IS:?1?Buyang Huanwu Decoction could improve the neurobehavioral score and reduce the volume of cerebral infarction of ischemic stroke in rats models.?2?Compared with Bifidobacterium quadruplex viable tablet group,Buyang Huanwu decoction significantly down-regulated Proteobacteria and up-regulated the Bacteroidetes in rats with qi deficiency and blood stasis of IS.On the other hand,the Actinobacteria was significantly down-regulated by Buyang Huanwu decoction comparing with saline control.?3?A total of 58 plasma metabolisms showed significant differences in Buyang Huanwu decoction group compared with the saline control.The significant metabolisms involved in amino acid metabolism such as glycine,threonine,tyrosine,arginine,etc.Moreover,the oleamide and isoliquiritin correlating with antioxidant effects and the warfarin,which could down-regulate the hypercoagulability were increased significantly.?4?The profiles of plasma metabolites were correlated with gut microbiota in Buyang Huanwu decoction group.Conclusion:?1?Both the significantly changed of gut microbiota and plasma metabolism profile correlated with each other in ischemic stroke.The differences of complex metabolic phenotype may play an important role in affecting the occurrence and development of ischemic stroke.?2?There were significant differences of gut microbiota between qi deficiency and blood stasis and other syndromes of ischemic stroke,thus suggesting that gut microbiota might be involved in the formation of TCM syndromes of qi deficiency and blood stasis.?3?The specific changes of gut microbiota therefore resulted to the components of antiinflammatory and antioxidant stress upregulated and the components promoting oxidation downregulated by Buyang Huanwu decoction in rats of ischemic stroke with qi deficiency and blood stasis syndrome.Consequently,it suggested that the gut microbiota affected the intestinal absorption of pharmaceutical components in Buyang Huanwu decoction.?4?Buyang huanwu decoction regulated the rats gut microbiota of IS,affecting the plasma metabolism profile,some of which were correlated with gut microbiota.This revealed that it may be an important biological basis on the therapeutic effect of Buyang Huanwu decoction.