基于PI3K/Akt通路探讨首乌丸对D-半乳糖衰老大鼠海马神经元凋亡的影响  

Effect of Shouwuwan on Apoptosis of Hippocampal Neurons in Aging Rats Induced by D-galactose Based on PI3K/Akt Pathway

作　　者：张超 加秀凤 黄万凌 周勇[3] 汤琪[1] 陈刚 ZHANG Chao;JIA Xiufeng;HUANG Wanling;ZHOU Yong;TANG Qi;CHEN Gang(School of Basic Medicine,Hubei University of Chinese Medicine,Wuhan 430065,China;Hubei College of Chinese Medicine,Jingzhou 434020,China;Wuhan Hospital of Traditional Chinese Medicine(TCM),Wuhan 430017,China;Hubei Provincial Hospital of TCM,Wuhan 430061,China)

机构地区：[1]湖北中医药大学基础医学院,武汉430065 [2]湖北中医药高等专科学校,湖北荆州434020 [3]武汉市中医医院,武汉430017 [4]湖北省中医院,武汉430061

出　　处：《中国实验方剂学杂志》2022年第22期76-84,共9页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：湖北省第二届医学领军人才工程培养对象暨湖北名医工作室负责人项目[鄂卫通(2019)47号];湖北省卫生计生委中西医结合重点科研项目[鄂卫生计生通(2017)20号];湖北省卫生健康委员会中医药科研项目青年人才项目(ZY2021Q031);湖北省卫健委面上重点项目(WJ2019H422)。

摘　　要：目的:探讨首乌丸通过磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)通路对D-半乳糖衰老大鼠海马神经元凋亡的影响。方法:将SPF级SD雄性大50只随机分为正常组、模型组、维生素E组、首乌丸中剂量组及首乌丸高剂量组,除正常组外,其余4组均用D-半乳糖(120 mg·kg^(-1))制造衰老模型,同时首乌丸中、高剂量组用首乌丸(1.08、2.16 g·kg^(-1))进行灌胃,维生素E组予以维生素E(0.018 g·kg^(-1))灌胃。造模6周后取全脑、海马组织,尼氏染色观察海马神经元形态学变化,原位末端标记法(TUNEL)检测海马细胞凋亡,蛋白免疫印迹法(Western blot)检测海马组织中磷脂酰肌醇3-激酶(PI3K),磷酸化(p)-PI3K,蛋白激酶B(Akt),p-Akt、胱天蛋白酶-3(Caspase-3)、叉头框蛋白O3a(FoxO3a)、p-FoxO3a、B细胞淋巴瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)的蛋白表达水平,实时荧光定量聚合酶链式反应(Real-time PCR)检测海马FoxO3a mRNA表达水平。结果:与正常组比较,模型组大鼠海马神经元凋亡阳性细胞明显增多,凋亡指数显著升高(P<0.01),海马CA1区尼氏染色显示海马神经元丢失、排列稀疏,尼氏体数量减少,存在尼氏体固缩和深染,p-PI3K、p-Akt、Caspase-3蛋白相对表达、p-PI3K/PI3K、p-Akt/Akt均显著升高(P<0.01),FoxO3a、p-FoxO3a蛋白相对表达、p-FoxO3a/FoxO3a显著降低(P<0.01),Bcl-2蛋白表达显著降低(P<0.01),Bax蛋白表达显著升高(P<0.01),FoxO3a mRNA表达显著降低(P<0.01);与模型组比较,经首乌丸治疗后,凋亡阳性细胞明显减少,细胞凋亡指数显著降低(P<0.01),海马CA1区尼氏染色显示各治疗组神经元丢失好转,尼氏体增多,排列较密集,大鼠PI3K、Akt蛋白表达差异无统计学意义,首乌丸中、高剂量组的p-PI3K、p-Akt、Caspase-3蛋白表达、p-PI3K/PI3K、p-Akt/Akt显著降低,FoxO3a、p-FoxO3a蛋白表达、p-FoxO3a/FoxO3a显著升高(P<0.01),Bcl-2蛋白表达显著升高(P<0.01),Bax蛋白表达显著降低(P<0.01),Foxo3a mRNA表达显著�Objective:To investigate the effect of Shouwuwan on apoptosis of hippocampal neurons in D-galactose-induced aging rats through phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)pathway.Method:Fifty male SD rats of SPF grade were randomly divided into normal group,model group,vitamin E group,and Shouwuwan medium-and high-dose groups.Except the normal group,the other four groups were given D-galactose(120 mg·kg^(-1))to prepare aging model.Additionally,Shouwuwan was used to intervene in the Shouwuwan medium-and high-dose groups(1.08 and 2.16 g·kg^(-1),respectively),and vitamin E group(0.018 g·kg^(-1))was given vitamin E by gavage.After 6 weeks of modeling,the whole brain and hippocampal tissue were taken and the morphological changes of hippocampal neurons were observed by Nissl staining.The apoptosis of hippocampal cells was detected by in situ end labeling[TdT-mediated dUTP nick-end labeling(TUNEL)].The protein expression levels of PI3K,phosphorylated(p)-PI3K,Akt,p-Akt,Caspase-3,forkhead box protein O3a(FoxO3a),p-FoxO3a,B-cell lymphoma-2(Bcl-2)and Bcl-2-associated X protein(Bax)were detected by Western blot.Real-time fluorescence quantitative PCR(Real-time PCR)was performed to determine the mRNA expression level of FoxO3a in hippocampus.Result:Compared with the conditions in the normal group,the apoptotic cells of hippocampal neurons in the model group were significantly increased,and the apoptosis index was elevated(P<0.01).Nissl staining of the hippocampal CA1 region showed that hippocampal neurons were lost and sparse,and the number of Nissl bodies was reduced,with pyknosis and deep staining.The relative protein expression of p-PI3K,p-Akt,Caspase-3 and Bax,p-PI3K/PI3K ratio and p-Akt/Akt ratio were all increased(P<0.01),while the relative protein expression of FoxO3a,p-FoxO3a and Bcl-2,and p-FoxO3a/FoxO3a ratio were decreased(P<0.01).The mRNA expression of FoxO3a was lowered(P<0.01).Compared with the conditions in the model group,after treatment with Shouwuwan,the apoptotic cells were markedly reduced

关 键 词：脑衰老 首乌丸 神经元凋亡 磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)通路 叉头框蛋白O3a(FoxO3a) 

分 类 号：R2-0[医药卫生—中医学] R33R289R745.1