| Topic basis: Focal cerebral ischemia is one of the ischemic stroke,which is a common disease with high morbidity,mortality and disability.Usually,cerebral ischemia is caused by interruption of blood supply to the brain caused by vascular occlusion,which further causes ischemia,hypoxia and related nerve damage in local brain tissue.Huangqi-Danshen herbal pair is a commonly used drug for invigorating qi and promoting blood circulation in clinic.A large number of studies have shown that Huangqi-Danshen has unique advantages in the treatment of stroke.However,its mechanism of action in the treatment of focal brain damage caused by ischemic stroke has not yet been fully elucidated.With the proposal and continuous development of the “brain-gut” axis theory,the important role of gut microbiota in cerebrovascular diseases has been confirmed in large numbers.Therefore,to explore the role of the “microbiota-brain-gut” axis in the pathogenesis of focal cerebral ischemia,and to study the regulation of HuangqiDanshen herbal pair on focal cerebral ischemia through the “brain-gut” axis,is helpful for understanding how traditional Chinese medicine treats focal cerebral ischemia.Objective: From the perspective of the “microbiota-brain-gut” axis,this study explores the therapeutic mechanism of Huangqi-Danshen herbal pair on rats with focal cerebral ischemia.In addition,traditional pharmacodynamics,metabolomics and microbiomics were combined to systematically study the interaction between functional intestinal microbiota and body metabolism.So as to provide a new perspective for in-depth study of the relationship between intestinal microbiota,body metabolism and the effect of traditional Chinese medicine.Methods:(1)The rats were divided into control group,model group,low-dose Huangqi-Danshen group,high-dose Huangqi-Danshen group and Nimodipine group.All rats except the control group were modeled with middle cerebral artery occlusion(MACO).The traditional pharmacodynamic indicators include Longa and Bederson neurological score,brain water content,TTC brain section staining and cerebral infarct size.These indicators were compared to evaluate the difference in drug efficacy among the drug administration groups.Subsequently,UHPLC-Q Exactive MS was used to conduct metabolomics research on the brain tissue and cecal content samples of rats in each group.The differences in metabolic profiles of each group were systematically compared,and the pharmacodynamic basis of Huangqi-Danshen intervention in cerebral ischemia was clarified.(2)16S r RNA high-throughput sequencing technology was used to conduct microbiome research on rat cecal content samples to clarify the composition and structural changes of the intestinal microbiota of rats in each group,and to determine the differential microbiota and pharmacological microbiota.The role of intestinal microbiota in the pathogenesis of focal cerebral ischemia and its contribution to the anti-ischemic efficacy of Huangqi-Danshen were clearly revealed.(3)Spearman correlation analysis was performed on traditional pharmacodynamic indicators,metabolomics data,and microbiomics data to clarify the significant correlation pathways of the interaction between gut microbiota and the “brain-gut” axis.So as to further clarify how the Huangqi-Danshen herbal pair regulates the two-way communication between the intestinal microbiota and the brain to exert an anti-cerebral ischemia effect.Results:(1)Traditional pharmacodynamic indicators and metabolomics research results show that: Compared with the control group,the rats in the model group were unable to walk spontaneously,and could not fully stretch the front paw on the opposite side of the ischemic brain area.The symptoms of the rats in each treatment group were alleviated,and the symptoms of the high-dose Huangqi-Danshen group were the mildest.The results of TTC staining showed that the brain slices of the rats in the model group were mostly pale,and the calculation results of cerebral ischemic area and brain water content were significantly different from those of the control group.HuangqiDanshen high-dose group had the best improvement effect.The above results indicated that the MCAO model was replicated successfully,and the pharmacodynamic effect of the high-dose Huangqi-Danshen group was the best.LC-MS metabolomics results showed that the metabolic profile of the model group was changed in both brain tissue and cecal contents.Through the screening strategy of differential metabolites,40 differential metabolites in brain tissue and 29 differential metabolites in cecal content were obtained,among which the Huangqi-Danshen high-dose group could recall 27 metabolites in brain tissue and 19 metabolites in cecal content.The results of metabolomics showed that Huangqi-Danshen herbal pair had a significant effect on focal cerebral ischemia.(2)Microbiome research results show that: According to 16 S r RNA sequencing analysis,the intestinal microbiota structure of MCAO rats had significant changed in bacterial abundance at 14 phylum levels,15 family levels,11 genera levels and 10 species levels,while the Huangqi-Danshen herbal pair can significantly adjust the abundance of 5 phyla,8 families,11 genera and 5 species.The results of this study preliminarily indicate that the disorder of intestinal microbiota is an important role in the pathogenesis of focal cerebral ischemia,and Huangqi-Danshen herbal pair can regulate the disorder of intestinal microbiota to exert its medicinal effect.(3)Spearman correlation analysis was used to comprehensively analyze the correlation between traditional pharmacodynamic indicators,metabolomics and gut microbiota.The results showed that 16 brain tissue metabolites and 16 cecal content metabolites were closely related to traditional pharmacodynamic indicators and intestinal microbiota.The Huangqi-Danshen herbal pair mainly affects the pathways of tryptophan metabolism,lysine degradation,arginine biosynthesis,arachidonic acid metabolism,unsaturated fatty acid biosynthesis and butyrate metabolism by regulating the abundance of Lactobacillus,Bifidobacterium,Allobaculum,Jeotgalicoccus,Clostridiaceae＿Clostridium,Bacteroidetes,Prevotella,[Thermicanaceae]＿Bacillus and [Ruminococcus],further realizing the interconversion between intestinal and brain metabolites.Conclusion: From the perspective of the “microbiota-brain-gut” axis,this study used traditional pharmacodynamic evaluation,metabolomics and microbiomics research to preliminarily expound the pharmacodynamic mechanism of HuangqiDanshen against focal cerebral ischemia in rats.Preliminary recognition of the important role of intestinal microbiota in the “brain-gut” axis,and clarification that Huangqi-Danshen can exert curative effect by regulating the structure of intestinal microbiota.Furthermore,based on the association analysis of “traditional pharmacodynamic evaluation-intestinal microbiota-metabolism”,it was revealed that the Huangqi-Danshen herbal pair has an overall regulatory effect on the “differential metabolites-gut microbiota” in focal cerebral ischemia,and provided a new perspective for explaining the intervention of Huangqi-Danshen in focal cerebral ischemia. |
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