Effects Of Exercise Preconditioning On Intestinal Flora And Oxidative Stress In Rats With Cerebral Ischemia/Reperfusion

Objective:To explore the effects of exercise preconditioning on intestinal flora and oxidative stress in rats with cerebral ischemia/reperfusion.Method:Forty-eight SD male rats were randomly divided into four groups: sham surgery group and exercise conditions + sham surgery group,model group,motor preconditioning + model group,12 rats in each group(n = 12).A modified suture method was used to block the middle cerebral artery blood flow to construct a middle cerebral artery occlusion cerebral ischemia/reperfusion model.Exercise pretreatment + sham operation group and exercise pretreatment + model group were given 3 weeks of electric treadmill exercise intervention.Pseudosurgery and model groups received the same joint seizures and feeding without intervention edition.The neurological defect score(m NSS)of rats in each group was evaluated 12 hours,1 day,3days,and 7 days after ischemia / reperfusion;HE staining was used to observe the degree of pathological damage of the brain tissue and intestinal tissue of each group of rats;enzyme-linked immunosorbent assay Detection of reduced glutathione(GSH)content in the serum of rats in each group by ELISA;high-throughput 16 s rDNA V3-V4 sequencing to analyze the composition of the intestinal flora of rats in each group;Western Blot method to detect each group Keap1,Nrf2,HO-1 protein expression in the ischemic brain tissue and intestinal tissue of the rat.Result:1.Neurological impairment score:Seven days after cerebral ischemia/reperfusion,compered with the sham operation group,there were no neurological deficits in the exercise + sham operation group.The model group had higher scores compared to the sham surgery group,and the difference was significant.(P<0.05);Compared with the group,the score of the exercise pretreatment + model group was significantly reduced,and the difference was significant(P<0.05).2.Histopathological changes:There was no obvious pathological damage in the brain tissue of rats in the sham operation group and exercise preconditioning + sham operation group.The neuronal cells with normal morphology and structure were clearly visible,the nucleus was centered,and the cytoplasm was lightly stained;the rats in the model group had neuronal loss and There are more dead cells,sparse cell structure,enlarged gaps,shrinkage of nuclei,deep staining of cytoplasm,showing a large number of vacuole-like changes;compared with the model group,the pathological damage of the brain tissue of the rats in the exercise preconditioning + model group is reduced,Vacuole-like changes are significantly reduced,the cells are arranged more neatly,and nuclear pyknosis is relatively rare.The colon tissue of rats in the sham operation group and exercise preconditioning + sham operation group showed no obvious pathological damage,the mucosal structure was intact,no inflammatory cell infiltration was seen,and the muscle layer was intact and smooth;the colon tissue of the model group showed disordered cilia arrangement.Mucosal epithelial cells were shed and necrotic,and some crypts were lost.Compared with the model group,rats in the model motor preconditioning + model group were able to see part of the complete tissue structure and had a relatively reduced degree of inflammatory cell infiltration and mucosal damage.3.GSH content in serum:Seven days after cerebral ischemia / reperfusion,serum levels in rats in the sham-surgery group and the motor preconditioning + sham-surgery group were baseline expression.Compared to the sham surgery group,the preconditioning exercise + sham surgery group had a higher GSH content(P>0.05)and the rat serum GSH content in the model group was lower(P <0.05).The serum GSH content of rats in the exercise preconditioning + model group was significantly increased compared to the model group(P <0.05).4.Changes in the intestinal microbial community:(1)Sequencing data results: In this experiment,each group of rats randomly selected 3 stool samples(cecal contents)for sequencing analysis.There were 12 cases in total.99.9% of the sequence length was valid,which met the analysis requirements.(2)The number of microbial groups at each classification level:After 7 days of cerebral ischemia/reperfusion,compared with the sham operation group,the OTU number of the fecal samples of the exercise preconditioning + sham operation group increased slightly,and the OTU number of the fecal samples of the model group was significantly reduced;compared with the model group Compared with the exercise preconditioning+ model group,the OTU value of rat stool samples increased significantly.(3)Alpha diversity:1)Sparse curve: The experimental results show that the current sample size is sufficient to reflect species diversity and can meet the technical requirements of subsequent bioanalysis.2)Species accumulation curve: The curve changes from steep to flat,which proves that the sample size in this experiment is sufficient to reflect the abundance of the community.3)Abundance grade curve: Compared with the sham operation group,the species abundance of the exercise pretreatment + sham operation group is closer,and the model group has a lower species abundance;compared with the model group,the exercise pretreatment + model group is rich in species Degree and uniformity have been improved.4)Alpha diversity index: Compared to the sham surgery group,the pre-exercise + sham surgery group has a higher alpha diversity index,but the difference is not statistically significant(P> 0.05).The model group diversity index decreased significantly,and the difference was significant(P <0.05).The alpha diversity index of the pre-exercise + model group was significantly higher than that of the model group,and the difference was significant(P<0.05).(4)Beta diversityThere is no significant difference in the composition of the intestinal flora in the pre-exercise + sham surgery group compared to the sham surgery group,but there is a significant difference in the model group;compared with the model group,the exercise pretreatment + model group has no significant difference in the flora structure However,it can be seen that after electroacupuncture pretreatment,the structure of the intestinal flora has a tendency to revert to the sham-operated flora.5.Keap1,Nrf2,HO-1 protein expression in the ischemic brain tissue and intestinal tissue:After 7 days of cerebral ischemia/reperfusion,Keap1,Nrf2,and HO-1proteins in the ischemic side brain tissue of the sham operation group and the exercise + sham operation group were all basic expressions,and the difference was not statistically significant(P>0.05);Compared with the operation group,the expression of Keap1 protein in the ischemic side brain tissue and intestinal tissue of the model group increased,while the expression of Nrf2 and HO-1 protein decreased,and the difference was statistically significant(P<0.05);compared with the model group,exercise prognosis In the treatment + model group,the expression of Keap1 protein in the ischemic side brain tissue and intestinal tissue of rats was down-regulated,and the expression of Nrf2 and HO-1 protein was up-regulated,and the difference was statistically significant(P<0.05).Conclusion:1.Exercise preconditioning can reduce the scores of neurological deficits in rats with cerebral ischemia/reperfusion,reduce the pathological damage of the ischemic brain tissue and intestinal tissue,and up-regulate the content of reduced glutathione in the serum.2.Exercise preconditioning can improve the number and structure of intestinal flora in rats with cerebral ischemia/reperfusion,and restore the diversity of intestinal flora.3.Exercise preconditioning may play a role in antagonizing the oxidative stress response after ischemic brain injury by regulating the expression of Keap1-Nrf2/ARE pathway related proteins in brain tissue and intestinal tissue of rats with cerebral ischemia/reperfusion.