| Objective: In this study,the pathophysiology process of cerebral ischemia reperfusion injury was simulated by Middle cerebral artery occlusion model(MCAO)in rats,and biochanin A,a typical phytoestrogen,was selected for intervention treatment.By observing the expression of the key molecules in the endoplasmic reticulum stress and the p38 MAPK signaling pathway,the neuroprotective effects of biochanin A against cerebral ischemia reperfusion injury,as well as related probable mechanisms,was investigated.Methods: Healthy adult male Sprague–Dawley rats weighing 250 ± 20 g were selected.Totally 50 rats were randomly divided into 5 groups with each 10 rats: the sham-operated group;the ischemia/reperfusion model group;and the biochanin A-treated groups with different doses(intraperitoneal injection with biochanin A 10,20,40 mg/kg/d for 2 weeks).The sham-operated group and the model group were given corresponding amount of saline injection.After the final injection,a cerebral ischemic and reperfusion injury model was established with the right middle cerebral artery suture occlusion method.The blood flow was blocked for 2 hours before reperfusion for 24 hours.The neurological deficit was evaluated according to Longa’s method at 2 hours of block and 24 hours of reperfusion,respectively.Then rats were decapitated and the brain was taken immediately for TTC(2,3,5,5-triphenyltetrazolium chloride)staining.The infarction area of cerebral ischemia reperfusion injury was observed by staining.The expression of endoplasmic reticulum stress-related proteins GRP78(Glucose-regulated protein 78,GRP78)and CHOP(C/EBP homologous protein 10,CHOP),p38MAPK-related proteins p38 and phosphorylated p38,Apoptosis-related proteins bcl-2(B-cell lymphoma-2,Bcl-2)、bax and caspase-3 was determined by Western blot assay.GRP78 and CHOP gene expression were detected by Reverse transcription PCR(Reverse transcription PCR,RT-PCR)and real-time fluorescent Quantitative PCR(Real-time policy PCR,qPCR).Results: In the neurologic deficit grade at 2 hours of block and 24 hours of reperfusion,the score of the model group was significantly higher than that of the sham-operated group.The score of the biochanin A-treated groups was lower than that of the model group and decreased with increasing drug concentration.In the brain TTC staining results,large area infarction occurred in the ischemic zone in the model group.After biochanin A treatment,the infarction area decreased significantly and it becomes more obvious with the increase of drug concentration.The Western blotting results showed that the protein expressions of p-p38,CHOP,GRP78,bax and caspase-3 significantly increased while bcl-2 expression decreased in the model group compared with those in the sham-operated group.After biochanin A treatment,the protein expressions of p-p38,CHOP,bax and caspase-3 decreased in a dose-dependent manner,and the expression of GRP78 and bcl-2 increased with the increase of drug concentration.The RT-PCR and qPCR detection results showed that the mRNA expression of CHOP and GRP78 in the model group significantly increased compared with the sham-operation group.After biochanin A treatment,the mRNA expression of CHOP decreased with the increase of drug concentration,while the mRNA expression of GRP78 increased in a dose-dependent manner.Conclusions: Biochanin A exerts a neuroprotective effect on cerebral ischemia reperfusion rats,and the mechanisms maybe associated with its involvement in regulation of endoplasmic reticulum stress,its influence on the p38 MAPK signaling pathway and its anti-apoptosis effect. |
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