盐酸去氢骆驼蓬碱对人神经母细胞瘤SH-SY5Y细胞凋亡及自噬的影响  

Effect of harmine hydrochloride on apoptosis and autophagy of SH-SY5Y cells

作　　者：西仁阿依·西热甫 雷秀英 张瑜 伊力亚斯·艾萨 木塔力甫·艾买提 陈倩 冯学召 李飞 米娜[2] Xi-re-na-yi XIREFU;LEI Xiu-ying;ZHANG Yu;Yi-li-ya-si AISA;Mu-ta-li-fu AIMAITI;CHEN qian;FENG Xue-zhao;LI Fei;MI Na(School of Pharmacy,Xinjiang Medical University,Urumqi 830011,China;Clinical Medical Research Institute,the First Affiliated Hospital,Xinjiang Medical University,Urumqi 830011,China;School of Basic Medical Science,Xinjiang Medical University,Urumqi 830011,China;State Key Laboratory,China Pharmaceutical University,Nanjing 210009,China;Central Laboratory,Xinjiang Medical University,Urumqi 830011,China)

机构地区：[1]新疆医科大学药学院,新疆乌鲁木齐830011 [2]新疆医科大学第一附属医院临床医学研究院,新疆乌鲁木齐830011 [3]新疆医科大学基础医学院,新疆乌鲁木齐830011 [4]中国药科大学国家重点实验室,江苏南京210009 [5]新疆医科大学中心实验室,新疆乌鲁木齐830011

出　　处：《中国药理学与毒理学杂志》2020年第11期825-831,共7页Chinese Journal of Pharmacology and Toxicology

基　　金：新疆地产中药民族药新药研发培育项目(2017-02-04)。

摘　　要：目的探讨盐酸去氢骆驼蓬碱(HMH)对人神经母细胞瘤SH-SY5Y细胞凋亡及自噬的影响。方法①SH-SY5Y细胞分别加入HMH 10,20,40,80和160μmol·L^(-1)或DMSO(细胞对照组)孵育24 h,通过MTT实验检测SH-SY5Y细胞存活率;Hoechst33342/PI染色法观察细胞凋亡;Western印迹法检测凋亡相关蛋白Bax、Bcl-2、胱天蛋白酶3、活化的胱天蛋白酶3及自噬相关蛋白微管相关蛋白轻链3B-Ⅱ(LC3B-Ⅱ)和P62的表达。②SH-SY5Y细胞分别加入HMH(2和4μmol·L-1)或DMSO孵育14 d,检测SH-SY5Y细胞克隆形成率。③HMH 40μmol·L^(-1)单独或与3-甲基腺嘌呤(3-MA)5 mmol·L^(-1)同时加入SH-SY5Y细胞,分别培养4或24 h后,Western印迹法检测LC3B-Ⅱ蛋白的表达。④将SH-SY5Y细胞分别加入HMH 40μmol·L^(-1)或溶剂孵育24 h后,Western印迹法检测蛋白激酶B(Akt)、磷酸化Akt(p-Akt)、哺乳动物雷帕霉素靶蛋白(mTOR)和p-mTOR的表达。结果①与细胞对照组相比,药物作用24 h后HMH 20~160μmol·L^(-1)组细胞存活率降低(P<0.01),凋亡细胞增多,LC3B-Ⅱ蛋白的相对表达量及Bax/Bcl-2的比值增加(P<0.05,P<0.01),P62蛋白的相对表达量减少(P<0.01)。HMH 40~160μmol·L^(-1)组活化的胱天蛋白酶3/胱天蛋白酶3的比值增加(P<0.05)。②与细胞对照组相比,药物作用14 d后,HMH 2和4μmol·L^(-1)组细胞克隆形成率降低(P<0.01)。③HMH与3-MA同时孵育24 h后,细胞LC3B-Ⅱ的相对表达量比HMH 40μmol·L^(-1)单独处理组降低(P<0.05)。④与细胞对照组相比,HMH 40μmol·L^(-1)组Akt和mTOR的磷酸化水平均降低(P<0.05)。结论HMH通过抑制Akt/mTOR通路抑制人神经母细胞瘤SH-SY5Y细胞增殖,诱导凋亡与自噬。OBJECTIVE To investigate the effect of harmine hydrochloride(HMH)on apoptosis and autophagy to neuroblastoma SH-SY5Y cells.METHODS The SH-SY5Y cells were treated with HMH 10,20,40,80 and 160μmol·L^(-1) or DMSO(cell control group)for 24 h.The cell viability was detected by MTT assay.Hoechst/PI staining was used to observe the apoptotic activity.Western blotting was used to detect the expression levels of Bax,Bcl-2,cleaved-caspase 3,caspase 3,microtubulerassociated protein light chain 3B-Ⅱ(LC3B-Ⅱ)and P62.The SH-SY5Y cells were treated with HMH 2 and 4μmol·L^(-1) or DMSO for 14 d,while the cloning formation rate was observed.The SH-SY5Y cells were treated with HMH 40μmol·L^(-1) or HMH 40μmol·L^(-1) in combination with autophagy inhibitor 3-Meth⁃yladenine(3-MA)5 mmol·L^(-1) for 4 or 24 h.The expression level of LC3B-Ⅱwas detected by Western blotting.The SH-SY5Y cells were treated with HMH 40μmol·L^(-1) or DMSO for 24 h,and the expression levels of protein kinase B(Akt),phosphorylated-Akt(p-Akt),mammalian target of Rapamycin(mTOR),p-mTOR were detected by Western blotting.RESULTS Compared with the cell control group,after 24 h treatment with HMH,the cell viability of SH-SY5Y cells was significantly decreased(P<0.01),the apop⁃tosis rate was increased,the expression levels of LC3B-Ⅱand Bax/Bcl-2 were significantly increased(P<0.05,P<0.01),and the expression level of P62 was significantly decreased(P<0.01)in the HMH 20-160μmol·L^(-1) group,and the expression level of cleaved-caspase 3/caspase 3 was significantly increased in the HMH 40-160μmol·L^(-1) group(P<0.05).Compared with the control group,after 14 d treatment with HMH 2 and 4μmol·L^(-1),the cloning formation rate was significantly decreased(P<0.01).When SH-SY5Y cells were treated with HMH 40μmol·L^(-1) in combination with 3-MA 5 mmol·L^(-1) for 24 h,the expression level of LC3B-Ⅱwas decreased(P<0.05)compared to SH-SY5Y cells treated with HMH 40μmol·L^(-1) alone.Compared with the control group,after 24 h treatment with HMH 40μmol

关 键 词：盐酸去氢骆驼蓬碱 SH-SY5Y细胞 细胞凋亡 自噬 

分 类 号：R964[医药卫生—药理学]