| Objective:Breviscapine is flavonoids extracted from Erigeron breviscapus(Vant.)Hand.-Mazz.,possessing the functions of improving microcirculation,dilating blood vessels,reducing blood viscosity,increasing cerebral blood flow and cardiac coronary flow.Clinically,it is mainly used to treat heart,cerebrovascular and other diseases with definite curative effect.Although breviscapine has a protective effect on cerebral ischemia-reperfusion injury,its mechanisms involve many factors and have not been fully elucidated.In this study,we demonstrated the effects of breviscapine on cerebral ischemia-reperfusion injury in rats and its mechanisms through gut microbiota,cerebral TLR4/MYD88/NF-κB signaling pathway and intestinal CYP3A4.This experiment can search a new target for the treatment of cerebral ischemiareperfusion injury,and open up a new idea for the use of breviscapine.Methods:Two-month-old male SD rats were randomly divided into 7 groups(n = 16 each group): control group,sham operation group,model group,high,medium and low dose of breviscapine groups,positive control group.Breviscapine groups were pretreated with high,medium and low dose of breviscapine(60,30,15 mg/kg),the positive control group was given 12 mg/kg nimodipine,and the blank control group,sham operation group and model group were given equal amount of saline.The model of cerebral ischemia-reperfusion injury was established by occlusion of middle cerebral artery with suture.After successful modeling,the neurological function of rats was evaluated by Bederson score table,the cerebral infarction volume ratio and serum NES level were calculated by TTC staining method.16 S r RNA sequencing was used to detect the intestinal flora of rats in each group;The expression levels of cerebral TLR4 / My D88 / NF-κ B and CYP3A4 m RNA and protein in different intestinal segments were detected by RT-q PCR and Western blot;Spearman correlation analysis was used to analyze the correlation between intestinal flora and NSE,neurological deficit score,TLR4/My D88/NF-κB and intestinal CYP3A4.Results:1.Compared with the blank control group,the neurologic behavior score of the model group was increased(P < 0.001),the cerebral infarct volume ratio was significantly increased(P < 0.001),and the serum NSE activity was significantly increased(P < 0.001).There was no obvious difference between the sham operation group and the blank control group.Compared with model group,neurologic behavioral scores of rats in high,medium and low doses of breviscapine in positive control group were significantly decreased(P<0.05).The cerebral infarct volume ratio of rats with cerebral ischemia reperfusion injury was decreased by 65.5%,44.9% and 25.6% in high,medium and low doses of breviscapine,respectively(P<0.05).The positive control group decreased by 33.7%,P<0.01),presenting a significant dose-dependent;Breviscapine can effectively reduce serum NSE activity in rats with cerebral ischemia reperfusion injury(P<0.05).2.Compared with the normal group,the number of OTU in model group was significantly decreased,the span of Out Rank curve was decreased,and Alpha diversity was significantly decreased.Compared with model group,the number of OTU and the span of abundance curve increased in breviscapine low-dose,medium-dose and highdose groups,and the up-regulation of Alpha diversity tended to be higher than that in normal group.NMDS analysis showed that there were significant differences in the composition of microflora among the normal control group,the cerebral ischemiareperfusion injury model group and the drug intervention group.Compared with the control group,the abundance of Firmicutes in the model group was lower than that in the control group at the phylum level.Compared with the model group,the abundance of Firmicutes increased gradually in breviscapine low-dose,medium-dose and highdose groups,while that of Proteobacteria was the opposite.At class level,the abundance of Clostridium in model group was lower than that in normal group.Compared with model group,the abundance of Clostridium increased gradually in breviscapine low-dose,medium-dose and high-dose groups.Compared with the normal group,the abundance of Clostridium in the model group was lower than that in the blank control group.At order level,the abundance of Clostridium in model group was lower than that in normal group.Compared with model group,the abundance of Clostridium increased gradually in breviscapine low-dose,medium-dose and high-dose groups.At the family level,the abundance of Lactobacillaceae was lower than that of normal group.Compared with model group,the abundance of Lactobacillaceae in breviscapine low-dose,medium-dose and high-dose groups increased gradually.At the genus level,compared with the normal group,the abundance of Clostridium and Bifidobacterium was lower than that of the normal group.Compared with the model group,the abundance of Clostridium and Bifidobacterium were increased in breviscapine low-dose,medium-dose and high-dose groups3.Compared with normal group,TLR4/MyD88/NF-κB transcription and protein expression were significantly up-regulated in a dose-dependent manner in cerebral ischemia-reperfusion injury rats.Compared with the model group,each administration intervention group had a different degree of decline.4.Compared with blank control group,CYP3A4 was significantly increased in different intestinal segments(duodenum,jejunum,ileum and colon)of rats in model group.Compared with model group,CYP3A4 in different intestinal segments of breviscapine high-dose,medium-dose and low-dose groups was significantly decreased in positive control group.5.The correlation analysis of intestinal flora with brain injury related indexes NSE,neurological deficit score,cerebral infarct volume ratio,TLR4/My D88/NF-κB in brain tissue and CYP3A4 in different intestinal segments showed that: NSE,neurological deficit score and cerebral infarct volume ratio were negatively correlated with Desulfovibrio,Ruminococcus and Oscillo Spira.TLR4/My D88/NF-κB was negatively correlated with Oscillospira.Ingredient CYP3A4 has shown a negative correlation with Oscillo Spira and Desulfovibrio.Conclusion:Breviscapine showed potent protective effects on cerebral ischemia-reperfusion injury in rats,which may be achieved through the regulation of intestinal flora,inhibition of cerebral TLR4 /My D88 / NF-κB pathway and intestinal CYP3A4. |
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