| Stroke is one of the most common causes of death and disability,involving the interaction of multiple injury mechanisms and 80%of stroke is ischemic origin.Its clinical manifestations are dizziness headache,limb numbness,xinclear articulation and hemiplegia.Currently,once a stroke has occurred,treatment options are limited and only available for a short time after the symptom onset.Due to the narrow therapeutic time window and severe adverse reactions,the majority of patients with acute ischemic stroke can not receive effective treatment.Xiaoxuming decoction(XXMD)is a classical traditional Chinese medicine(TCM)prescription with the effect of "Wen jing Tong yang and Fu zheng fang feng" for treatment of ischemia stroke.The efficacy of the TCM compound depends on its material basis.Active ingredients of XXMD can give full play to the efficacy via being absorbed by various biofilms into the blood and distributing to the target organ with the systemic circulation.Therefore,it is very necessary to study the material basis of XXMD in blood and target organs.It is reported that intestinal flora is closely related to stroke.Intestinal flora disorder can lead to metabolic disorder and enhance inflammatory response and oxidative stress in the host.As an important participant in metabolism and immune regulation,the study of the relationship between intestinal flora and stroke is of great significance for the treatment of stroke.In this paper,the active fraction of XXMD(AF-XXMD)in rat plasma and cerebral cortex were studied by UPLC-HRMS technology at first.And then the Illumina MiSeq high-throughput sequencing technique was used to analyze the V3-V4 region of 16S rDNA gene of fecal bacteria of rats in order to study the regulatory effect of AF-XXMD on intestinal flora in MCAO rats.Finally,the proteomics technique was used to explore the biological targets of AF-XXMD on the cerebral cortex.The experiments are as follows in detail:1.Study on chemical constituents of AF-XXMD in rat plasma and cerebral cortex·The MCAO animal model was established by nylon wire.Reperfusion was performed after 1.5 h of ischemia,and then the Zea-Longa's grade standard scoring method and TTC staining method were used to evaluate Evaluation of brain protective effect of AF-XXMD at 24 h after reperfusion.After then,UPLC-HRMS was used to quickly analyze and identify the effective components of AF-XXMD in rat plasna and cerebral cortex.It was found that a total of 29 compounds were identified in plasma of Model and XXMD groups,including flavonoids,alkaloids,chromones,terpenoids and cyanosides.Among them,amygdalin and tetrandrine were only detected in the plasma of XXMD group.However,aconitine,5,7-dioxomethyl-4',6-dihydroxyflavone,5,7-dimethoxy-4',6-dihydroxy flavone glucuronidation,chrysin-7-O-?-D-glucoronide and ginsenoside Rc were only detected in the plasma of Model group.At the same time,a total of 14 compoxmds were identified in rat cerebral cortex of the two groups,including flavonoids,alkaloids and terpenoids.Among them,5-O-methylvisammioside,paeoniflorin,aconite,ursolic acid,baicalin,wogonoside and apiganin-7-O-glucronide were only detected in rat cerebral cortex of Model group.The results showed that there were differences in the chemical components of plasma and cerebral cortex between normal and pathological rats,which provided a reference for the study of substance metabolism in vivo and pharmacological action of AF-XXMD in the state of disease.2.The effect of AF-XXMD on intestinal barrier function and intestinal flora on MC AO rats.HE(Hematein-Eosin)staining,TEM(transmission electron microscopy)and concentrations of LPS(lipopolysaccharide)in plasma were used to observed intestinal barrier function in rat ileum.And the Illumina MiSeq high-throughput sequencing was used to analyze the diversity of intestinal flora of rats.The result showed that the ileal mucosa edema and lymphatic capillary dilatation were serious and the morphological structure of epithelial cells,goblet cells,mitochondrial and microvilli showed serious pathological changes,meanwhile,the concentration of LPS in plasma increased significantly of MCAO rats.In XXMD group,the tissue structure of ileum was restored to a certain extent and the concentration of LPS in rat plasma decreased significantly.The result of Illumina MiSeq high-throughput sequencing showed that the diversity and richness of intestinal flora decreased significantly of MCAO rats.After administration of AF-XXMD,its diversity and richness could be significantly reversed.Meanwhile,the conditional pathogenic bacteria increased and the beneficial bacteria decreased in the homeostasis by increasing the beneficial bacteria and reducing the pathogenic bacteria.These results suggested that AF-XXMD can alleviate the damage of intestinal barrier function caused by acute cerebral ischemia-reperintestinal tract of MCAO rats,while AF-XXMD could regulate the intestinal flora fusion and regulate intestinal flora homeostasis by increasing metabolism-related beneficial bacteria and reducing inflammation-related pathogenic bacteria.At the same time,it laid a foundation for the pharmacological effect of XXMD on MCAO rats based on intestinal flora.3.Proteome data mining and verification of AF-XXMD in the treatment of MCAO rats.Proteomics and Western Blot technique were used to analyze the total protein of cerebral cortex in rats to explore the biological basis of the therapeutic effect of AF-XXMD on MCAO rats.The results of proteomics showed that 522 differential proteins were obtained between Sham group and Model group,and 464 differential proteins were obtained between Model group and XXMD group.And these differential proteins were mainly involved in cell transformation,biological regulation,metabolic processes and other biological processes.And then,the differential proteins were annotated by Pathway.It was found that these differential proteins involved inflammatory and apoptotic pathways,such as PI3K/AkT/NF-?B and MAPK/JNK signaling pathways.The result of Western Blot showed AF-XXMD could induce a significant decrease in the level of p-AKT/AKT,p-NF-?B P65/NF-?B P65 and p-JNK in cerebral cortex of MCAO rats.These results suggest that AF-XXMD may play its anti-inflammatory and anti-apoptotic effects by inhibiting PI3K/AkT/NF-?B and MAPK/JNK signaling pathways. |
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