| OBJECTIVE:Based on the emerging field of the brain-gut axis,the intestinal microecological disturbance after ischemic stroke is the starting point.The protective effect of Tongqiao Huoxue Decoction(TQHXD)on cerebral ischemia reperfusion(I / R)inflammatory injury rats was preliminary studied.METHODS:1.Protective effect of TQHXD on cerebral ischemia-reperfusion injury in rats(1)MCAO was used to establish a model of cerebral ischemia at 2h and reperfusion for 24 h,and Longa's five-point scoring system was used to score neurological deficits.Models 1-3 were selected for experiments.Rats in each group were scored after 7,7 and 14 days.(2)The effect of TQHXD on the volume of cerebral infarction in I / R rats was measured by triphenyltetrazolium chloride(2,3,5-three phenyl tetrazole chloride,TTC)staining.(3)Hematoxylin-eosin staining(HE)method was used to observe the effect of TQHXD on pathological sections of brain tissue in I / R rats.2.TQHXD regulates intestinal microecological disorders in cerebral ischemia reperfusion rats(1)Intestinal bacterial 16 Sr RNA high-throughput sequencing of rat feces was performed to observe the effect of TQHXD on microbial disturbances in the intestinal tract of I / R rats.(2)The effect of TQHXD on morphological changes of small intestine in I / R rats was observed by HE staining.(3)Observe the effect of TQHXD on the change of tight junction structure in the small intestine of I / R rats by transmission electron microscope(TEM).(4)Western Blotting(WB)was used to detect the effects of TQHXD on the expression of tight junction proteins ZO-1 and Claudin-2 in the small intestine of I / R rats.(5)Enzyme linked immunosorbent assay(ELISA)method to detect the concentration of diamine oxidase(DAO),D-lactic acid,and endotoxin(LPS)in the plasma of I / R rats by TQHXD The effect of size.3.TQHXD regulates inflammation injury induced by intestinal microecological disturbance in rats with cerebral ischemia-reperfusion(1)The effect of TQHXD on the ratio of Treg cells and ??T cells in peripheral blood of I / R rats was detected by flow cytometry.(2)Detection of TQHXD on small intestine,spleen and ischemic lateral brain of I / R rats by immunohistochemical technique Influence of the distribution of Treg cells and ??T cells in tissues.(3)The effect of TQHXD on the expression of IL-17 and IL-10 in brain tissue of I / R rats was detected by ELISA.4.Effect of fecal microbial transplantation on outcome of rats with cerebral ischemia-reperfusion injury(1)Preparation of rat fecal microbial supernatant.Fresh feces of rats are prepared daily.(2)The effect of fecal microbiota transfer(FMT)on cerebral infarct volume in I /R rats was detected by TTC staining.(3)The effects of FMT on the distribution of Treg cells and ??T cells in the small intestine,spleen and ischemic lateral brain tissue of I / R rats were detected by immunohistochemical techniques.(4)The effect of FMT on the expression levels of IL-17 and IL-10 in brain tissueof I / R rats was detected by ELISA.RESULT:1.Protective effect of TQHXD on rats with I / R injury(1)Neurobehavioral defect score results show that after 7 days and 14 days of TQHXD administration,the neurological deficit score is significantly reduced.(2)TTC cerebral infarction staining results: Compared with the model group,the TQHXD group can significantly reduce the cerebral infarction volume at different time points,which has statistical significance.(3)The results of HE pathological staining showed that the nucleus of the sham operation group was intact and the cells were arranged tightly.The ischemic lateral brain tissue of the model group had loose cellular structure,reduced neuronal cells,and swollen cells,showing obvious ischemic focus.After 7 days and 14 days of administration,the TQHXD group was able to improve cell pathological damage to varying degrees.2.TQHXD regulates intestinal microecological disorders of cerebral ischemia reperfusion rats(1)High-throughput sequencing of rat feces 16 Sr RNA showed that the TQHXD group can improve the change of the main components of the intestinal flora of the model group,reduce the excessive increase of the proportion of Bacteroides,regulate the disturbance of Lactobacillus,and increase the number of Bifidobacterium Genus ratio.(2)HE small intestine pathological section staining results: compared with the sham operation group,the microvilli were sparsely arranged in the model group,with varying lengths;goblet cells were reduced.Compared with the model group,the TQHXD group improved at different time points.(3)The TEM results showed that the tight junction structure and demyelocytic cells were clearly visible in the sham operation group,and the tight junction bands inthe model group were dark or even disappeared at 7 and 14 days,and the demyeloblasts disappeared.The TQHXD group had different degrees of improvement at different time points.(4)The WB results showed that compared with the model group,the TQHXD group can increase the expression of the tight junction protein ZO-1 and decrease the expression of Claudin-2.(5)ELISA test showed that compared with the sham operation group,the concentrations of DAO,D-lactic acid and LPS in the plasma of the model group were significantly increased.Compared with the model group,the concentrations of DAO,D-lactic acid,and LPS in the TQHXD group were significantly reduced at different time points.3.TQHXD regulates inflammation injury induced by intestinal microecological disturbance in rats with cerebral ischemia-reperfusion(1)Flow cytometry results showed that compared with the sham operation group,the number of ??T cells in the model group increased significantly,compared with the model group,Treg cells in the TQHXD group increased significantly,and the number of ??T cells decreased significantly.(2)Immunohistochemistry showed that compared with the sham operation group,the distribution of ??T cells in the small intestinal epithelium and ischemic side brain tissue was significantly increased in the model group.Compared with the model group,the distribution of ??T cells in the small intestine epithelium and ischemic brain tissue was significantly reduced in the TQHXD group,and Treg cells in the small intestine epithelium,spleen tissue and ischemic brain tissue were significantly increased.(3)ELISA test showed that compared with the sham operation group,the IL-17 in the ischemic lateral brain tissue of the model group was significantly increased.Compared with the model group,the IL-10 in the ischemic lateral brain tissue of the TQHXD group was significantly increased,and the IL-17 was significantly decreased.4.Effect of fecal microbial transplantation on outcome of rats with cerebral ischemia-reperfusion injury(1)TTC staining results showed that compared with the model group,the volume of cerebral infarction in the Sham(FMT)group and the Sham + TQHXD(FMT)group was significantly reduced.(2)Immunohistochemical technique showed that the small intestine tissue and ischemic lateral brain tissue of the model group ??T cells increased significantly.Compared with the model group,the ?? T cells in the small intestine and ischemic lateral brain tissue of the Sham(FMT)group and Sham + TQHXD(FMT)group were significantly reduced,and Treg cells were significantly increased.(3)The ELISA test results showed that IL-17 in the ischemic lateral brain tissue of the model group increased significantly.IL-17 in the Sham(FMT)group and Sham+ TQHXD(FMT)group was significantly lower than that in the model group,while IL-10 obviously increase.CONCLUSIONS:Based on the above results,TQHXD has obvious protective effect on rats with cerebral ischemia-reperfusion injury,and this protective effect may be through regulating the disturbance of intestinal microecology of rats after ischemic stroke,thereby inhibiting Inflammation induced by intestinal microecological disorders. |
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