中风胶囊对脑缺血/再灌注损伤模型鼠脑组织自噬相关蛋白表达的影响

Effects of Zhongfeng capsule on autophagy related proteins expressions of brain tissue in rats with cerebral ischemia/reperfusion injury

目的:观察中风胶囊对脑缺血/再灌注损伤(CIRI)模型鼠脑组织自噬相关蛋白表达的影响,初步揭示其对神经元损伤保护的分子机制。方法:采用改良线栓法构建大鼠脑缺血/再灌注损伤模型,随机将60只雄性SD大鼠分为假手术组、模型组、丁苯酞组(0.054 g/kg)、中风胶囊高剂量组(1.08 g/kg)、中风胶囊中剂量组(0.54 g/kg)、中风胶囊低剂量组(0.27 g/kg),每组10只。造模结束后灌胃给药10 d,每天1次,实验结束后处死各组大鼠,摘取脑组织。各组大鼠末次给药24 h后进行神经功能评分;HE染色法观察各组大鼠脑组织病理形态;ELISA法检测各组大鼠血清雌二醇(E2)和卵泡刺激素(FSH);RT-PCR法与Western blot法分别测定各组大鼠脑组织PI3K/Akt/Beclin1信号通路关键基因及蛋白的表达。结果:与假手术组比较,模型组大鼠体重及脑组织中p-PI3K、p-Akt等蛋白表达均显著降低,脑指数、神经功能缺损评分及脑组织Beclin1、LC3基因和蛋白表达均显著升高(P<0.05或P<0.01),脑组织结构排列疏松,间质水肿,神经细胞呈三角形,核固缩深染。与模型组相比,中风胶囊高剂量组大鼠体重显著升高,神经功能缺损评分显著下降(P<0.05),脑组织病理损伤较模型组明显改善;中风胶囊各剂量组的脑指数及脑组织Beclin1、LC3的基因和蛋白表达均显著降低,脑组织中p-PI3K、p-Akt等蛋白表达均显著升高(P<0.05或P<0.01)。结论:中风胶囊通过调控PI3K/Akt/Beclin1信号通路中Beclin1和LC3的表达来抑制CIRI模型鼠的自噬反应,从而发挥保护其脑神经元损伤的作用。

Objective:To investigate the effects of Zhongfeng capsule on the autophagy-related proteins expression in rats with cerebral ischemia/reperfusion injury(CI/RI),and to explore its neural protection mechanisms of the decoction.Methods:Rat middle cerebral artery ischemia/reperfusion injury model(ischemia for 2 h,reperfusion for 24 h)was prepared by the improved line plug method.Sixty male SD rats were randomly divided into sham operation group,model group,butylphthalide group(0.054 g/kg),Zhongfeng capsule high-dose groups(1.08 g/kg),Zhongfeng capsule middle-dose groups(0.54 g/kg),Zhongfeng capsule low-dose groups(0.27 g/kg),with 10 rats in each group.Rats were treated with Zhongfeng capsule by gavage once a day for 10 days.The rats were sacrificed and the brain tissue was obtained after the experiment in each group.Score neurological deficit was evaluated after 24 h of the last intervention in rat of each group.The pathological changes of brain tissue were observed by HE staining.The serum levels of estradiol(E2)and follicle stimulating hormone(FSH)were determined by ELISA.The expressions of key genes and proteins of PI3K/Akt/Beclin1 signaling pathway in brain tissue were detected by qRT-PCR and Western blot respectively.Results:Compared with the sham operation group,the body weight and protein expressions of p-PI3k and p-Akt in brain tissue of rats were decreased significantly in the model group,while the brain index,neurological deficit score,gene and protein expressions of Beclin1 and LC3 were increased markedly in the model group(P<0.05 or P<0.01).In the model group,nerve cells of brain tissue were loosely packed,interstitial edema,triangular in shape,nuclear pyknosis and dark-blue staining were observed.Compared with the model group,the body weight of rats was increased obviously,the neurological deficit score was decreased significantly and the pathological injury of brain tissue was alleviated evidently in high-dose of Zhongfeng capsule group(P<0.05).The brain index,the gene and protein expressions of Beclin1 and LC3 were decreased apparently in Zhongfeng capsule treatment groups(P<0.05 or P<0.01),while the expressions of p-PI3k and p-Akt in brain tissue were increased evidently in Zhongfeng capsule treatment groups(P<0.05 or P<0.01).Conclusion:Zhongfeng capsule can inhibit autophagy and improve brain neurons lesion of CIRI rats,the mechanism may be related to regulate the expression of Beclin1 and LC3 in PI3K/Akt/Beclin1 signaling pathway.