健脾补土方对脑缺血再灌注大鼠缺血侧皮质Claudin-5、Occludin、p-BAD、Bcl-2、Caspase-3蛋白表达的影响

The Effect of Spleen-strengthening Decoction on Claudin-5、Occludin、p-Bad,Bcl-2,Caspase-3 protein expression in ischemic cortex of cerebral ischemia/reperfusion rats

目的探讨健脾补土方减轻脑缺血再灌注大鼠神经元损伤的可能作用机制。方法将80只SD大鼠随机分为假手术组10只、手术组70只,手术组经模型制备后评价,剔除不成功的及死亡大鼠,二次分组,分为模型组、依达拉奉组、健脾补土方高、中、低剂量组,每组各13只。手术组采用大脑中动脉栓塞法制备脑缺血再灌注模型(middle cerebral artery occlusion,MCAO),干预7天,取材。取材前每组选取10只,采用国际公认的大鼠脑卒中后神经功能缺损评分法(mNss评分)进行神经功能缺损评分。取材部位为缺血侧脑皮质组织。每组随机选取3只采用苏木素—伊红(hematoxylin-eosin,HE)染色法观察病理形态改变;每组随机选取其中5只采用Western blot法检测紧密连接蛋白-5(Claudin-5)、闭合蛋白(Occludin)表达量;剩余5只采用免疫组化法检测磷酸化相关死亡启动因子(phospho-Bcl-xL/Bcl-2 asociated death promoter,p-BAD)、B淋巴细胞瘤-2基因(B-cell lymphoma-2,Bcl-2)和半胱天冬氨酸蛋白酶-3(Caspase-3)蛋白表达量。结果(1)造模后,与假手术组比较,模型组大鼠神经功能缺损评分明显上升,经药物干预后,各组神经功能缺损评分均有所下降(P<0.05),其中以依达拉奉组与健脾补土方高剂量组下降最为明显;(2)造模后,模型组大鼠较假手术组病理改变严重,出现组织大片坏死,经药物干预后,各组病理改变均有所改善,依达拉奉组和健脾补土方高剂量组尤为明显;(3)与模型组比较,各药物干预组脑组织缺血皮质区Occludin、Claudin-5蛋白表达均有所上调,差异有统计学意义(P<0.05),其中以依达拉奉组和健脾补土方高剂量组最为显著;(4)与模型组比较,各药物干预组脑缺血皮质组织p-BAD、Bcl-2蛋白表达均明显增加,Caspase-3蛋白表达有所降低(P<0.05),其中依达拉奉组与健脾补土方高剂量组最为显著。结论健脾补土方可能通过上调p-BAD、Bcl-2、Claudin-5、Occludin蛋白表达,下调Caspase-3蛋白表达,从而改善脑缺血再灌注大鼠神经功能缺损症状及脑组织病理改变,进而促进缺血脑组织损伤修复。

Objective To explore the possible mechanism of Spleen-strengthening Decoction in alleviating neuronal injury in rats with cerebral ischemia-reperfusion.Methods A total of 80 SD rats were randomly divided into sham operation group(n=10)and operation group(n=70).The operation group was evaluated after model preparation.After excluding unsuccessful and dead rats,the rats were divided into model group,edaravone group,high,medium and low dose groups of Spleen-strengthening Decoction,with 13 rats in each group.The middle cerebral artery occlusion(MCAO)model was prepared by middle cerebral artery embolization in the operation group,and the intervention lasted for 7 days.Before sampling,the internationally recognized neurological deficit score(mNss score)after stroke was used to score the neurological deficit.The tissue was taken from the ischemic cerebral cortex,and 3 rats in each group were randomly selected to observe the pathological changes by HE staining.Five of each group were randomly selected to detect the expression of Claudin-5 and Occludin by Western Blot.The expression of phosphorylation-associated death promoter(p-BAD),B-cell lymphoma-2(Bcl-2)and Caspase-3 were detected by immunohistochemistry in the remaining 5 rats.Results(1)Compared with the sham operation group,the neurological deficit scores of rats in the model group were increased significantly.After drug intervention,the neurological deficit scores of each group were decreased(P<0.05),among which the edaravone group and the high-dose Spleen-strengthening Decoction group were decreased most significantly.(2)Compared with the sham group,the pathological changes of rats in the model group were more serious,and large areas of tissue necrosis occurred.After drug intervention,the pathological changes of each group were improved,especially in the edaravone group and the high dose group of Spleen-strengthening Decoction.(3)Compared with the model group,the expression of Occludin and Claudin-5 protein in the ischemic cortex of each drug intervention group was up-regulated,and the difference was statistically significant(P<0.05),among which the edaravone group and the high-dose group were the most significant;(4)Compared with the model group,the expression of p-BAD and Bcl-2 protein in the cerebral ischemic cortex of each drug intervention group was significantly increased,and the expression of Caspase-3 protein was decreased(P<0.05).among which the edaravone group and the high-dose group were the most significant.Conclusion Spleen-strengthening Decoction may improve the symptoms of neurological deficits and pathological changes of brain tissue in rats with cerebral ischemia-reperfusion by up-regulating the expression of p-BAD,Bcl-2,Claudin-5 and Occludin protein and down-regulating the expression of Caspase-3 protein,thus promoting the repair of ischemic brain tissue injury.