基于PI3K/AKT信号通路探讨白藜芦醇保护APP-NSCs的作用机制  

Mechanism Study of Resveratrol on Protecting APP-NSCs Based on PI3K/Akt Signaling Pathway

作　　者：殷紫 张二飞[2] 丁旭 陈桂娟 朱星宇 谭东明 陈二华 徐秉忠 张建华 韩永红 Yin Zi;Zhang Erfei;Ding Xu;Chen Guijuan;Zhu Xingyu;Tan Dongming;Chen Erhua;Xu Bingzhong;Zhang Jianhua;Han Yonghong(College of Pharmacy and Chinese Medicine,Jiangsu College of Nursing,Huai'an 223008;School of Pharmaceutical Engineering,Jiangsu Food&Pharmaceutical Science College,Huai'an 223003)

机构地区：[1]江苏护理职业技术学院药学与中药学院,淮安223008 [2]江苏食品药品职业技术学院制药工程学院,淮安223003

出　　处：《中药药理与临床》2021年第3期61-66,共6页Pharmacology and Clinics of Chinese Materia Medica

基　　金：淮安市科技局课题(编号:HAB201843);江苏省高等学校自然科学研究面上项目(编号:19KJD360001);江苏省高校青蓝工程项目[苏教师(2019)3号];江苏省高校“青蓝工程”项目。

摘　　要：目的:探讨白藜芦醇对高表达淀粉样前体蛋白(Amyloid precusor protein,App)基因的神经干细胞(APP-NSCs)的保护作用及其可能的作用机制。方法:从出生72 h内的C57BL/6小鼠脑室下区及海马区分离、培养原代NSCs,通过转染APP基因构建阿尔茨海默病(Alzheimer’s disease,AD)NSCs模型,利用免疫荧光染色检测NSCs标志物神经巢蛋白(Nestin)与胚胎干细胞关键蛋白(Sox-2)表达以鉴定NSCs,并通过免疫荧光染色评价NSCs向星形胶质细胞、神经元及少突胶质细胞分化的能力。以白藜芦醇1、5、10、20μmol/mL或LY29400210μmol/mL孵育细胞。检测NSCs活力及乳酸脱氢酶(Lactate dehydrogenase,LDH)释放量;测量NSCs球半径并绘制生长曲线;5-溴脱氧尿嘧啶核苷(5-Bromo-2-deoxyUridine,BrdU)染色检测NSCs增殖能力;原位末端转移酶标记(Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling,TUNEL)染色检测NSCs凋亡率;Real-time PCR检测细胞中Bax、Bcl-2及Caspase-3 mRNA表达水平;蛋白质印迹法(Western blot)检测磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase,PI3K)、蛋白激酶B(Protein kinase B,AKT)及p-AKT蛋白表达情况。结果:与空白对照组比较,模型对照组细胞活力明显降低,LDH释放量明显升高,NSCs的增殖能力明显降低,细胞凋亡明显增多,Caspase-3 mRNA明显上调,Bax/Bal-2比值明显升高,且PI3K与p-AKT蛋白表达明显下调(P<0.05);与模型对照组比较,白藜芦醇5、10、20μmol/mL能明显提高APP-NSCs活力,降低LDH释放量(P<0.05);白藜芦醇10μmol/mL明显促进NSCs的增殖,并明显抑制其凋亡(P<0.05);下调Caspase-3 mRNA表达,增加Bax/Bal-2比值,还能够上调PI3K与p-AKT蛋白表达(P<0.05);白藜芦醇的上述作用可被LY29400210μmol/mL所抑制。结论:白藜芦醇对APP-NSCs具有保护作用,能促进APP-NSCs的增殖并抑制其凋亡,该作用机制可能与激活PI3K/AKT信号通路有关。Objective:To explore the protective effect of resveratrol(Res)on neural stem cells with high expression of APP gene(APP-NSCs)and its possible mechanism.Methods:The primary NSCs were isolated from the subventricular area and hippocampus of C57 BL/6 mice within 72 hours of birth and cultured.The Alzheimer’s disease(AD)NSCs model was established by transfection of APP gene.The expression of NSCs marker Nestin and the embryonic stem cell key protein(Sox-2)were determined by immunofluorescence staining to identify NSCs.The abilities of NSCs to differentiate into astrocytes,neurons and oligodendrocytes were evaluated by immunofluorescence staining.The cells were incubated with different concentrations of resveratrol(1μmol/mL,5μmol/mL,10μmol/mL and 20μmol/mL)or LY294002(10μmol/mL).The activities of NSCs and the release of lactate dehydrogenase(LDH)were detected,the sphere radius of NSCs was measured and the growth curve was drawn.BrdU staining was used to detect its proliferation ability.TUNEL staining was used to detect the apoptosis rate of NSCs.Real-time PCR was used to detect the mRNA expression levels of Bax,Bcl-2 and Caspase-3 of cells.Western blot was used to detect the protein expression of PI3 K,Akt and p-Akt.Results:Compared with the control group,the cell viability in the model group was significantly reduced,the release of LDH was significantly increased,the proliferation ability of NSCs was significantly reduced,the apoptosis was significantly increased,the mRNA level of Caspase-3 was up regulated,the Bax/Bal-2 ratio was increased,and the expressions of PI3 K and p-Akt protein were significantly reduced(P<0.05).Compared with the model group,resveratrol(5μmol/mL,10μmol/mL and 20μmol/mL)significantly increased the activity of APP-NSCs and reduced the release of LDH(P<0.05),resveratrol(10μmol/mL)significantly promoted the proliferation of NSCs and significantly inhibited the apoptosis(P<0.05).Resveratrol(10μmol/mL)down regulated the mRNA level of Caspase-3,increased the ratio of Bax/Bal-2,it also i

关 键 词：白藜芦醇 阿尔茨海默病 神经干细胞 增殖 凋亡 磷脂酰肌醇3-激酶/蛋白激酶B信号通路 

分 类 号：R285.5[医药卫生—中药学]