骆驼蓬总碱通过诱导细胞自噬促进Tau蛋白和α-突触核蛋白的降解

Total Alkaloid of harmaline promotes Tau and α-synuclein degradation by regulating autophagy

目的:探讨骆驼蓬总碱(TAH)能否诱导神经细胞自噬并促进神经毒性蛋白Tau蛋白和α-突触核蛋白(α-Syn)的降解。方法:(1)将体外培养的PC12细胞分为空白对照组及1、2.5、5、10、20、50μg/mL TAH组,分别加入0、1、2.5、5、10、20、50μg/mL TAH作用24 h,观察细胞形态及数目的变化,采用MTT法检测细胞存活率;(2)将PC12细胞分为空白对照组、雷帕霉素组及1、2.5、5、10、20μg/mL TAH组,分别加入等量溶剂、50 nmol/L自噬激活剂雷帕霉素及1、2.5、5、10、20μg/mL TAH作用4 h,采用免疫荧光染色检测细胞自噬体数;(3)将PC12细胞分为空白对照组、雷帕霉素组、10μg/mL TAH组,分别加入等量溶剂、50 nmol/L雷帕霉素、10μg/mL TAH作用4 h,Western blotting检测细胞p62和微管相关蛋白1轻链3(LC3)-Ⅱ蛋白的表达;(4)将PC12细胞分为空白对照组、氯喹组、TAH组、TAH+氯喹组,分别加入等量溶剂、50 nmol/L自噬抑制剂氯喹、10μg/mL TAH、10μg/mL TAH+50 nmol/L氯喹作用4 h,Western blotting检测细胞LC3-Ⅱ蛋白的表达;(5)将PC12细胞分为TAH组和空白对照组,分别加入10μg/mL TAH和等量溶剂处理4 h后,Western blotting检测细胞磷酸化哺乳动物雷帕霉素靶蛋白(p-mTOR)、磷酸化p70核糖体蛋白S6激酶(p-p70S6K)的表达;(6)将过表达Tau-绿色荧光蛋白(GFP)的Tet on HEK293细胞分为空白对照组、TAH组、强力霉素组、强力霉素+TAH组、强力霉素+TAH+3-甲基腺嘌呤(3-MA)组、强力霉素+TAH+氯喹组,后4组细胞加入200 ng/mL强力霉素处理24 h后,空白对照组加入等量溶剂,TAH组加入10μg/mL TAH,后3组细胞分别加入10μg/mL TAH、10μg/mL TAH+5 mmol/L 3-MA、10μg/mL TAH+50 nmol/L氯喹,作用24 h后在激光共聚焦显微镜下观察细胞绿色荧光强度的变化,Western blotting检测细胞Tau-GFP和LC3-Ⅱ蛋白的表达;(7)将稳定表达α-Syn的HEK293细胞分为空白对照组、氯喹组、TAH组、TAH+氯喹组,分别加入等量溶剂、50 nmol/L氯喹、10μg/mL TAH、10μg/mL TAH+50 nmol/L氯喹作用24 h,Western blotting检测细胞α-Syn和LC3-Ⅱ蛋白的表达。结果:(1)与空白对照组比较,20、50μg/mL TAH组细胞存活率降低,且50μg/mL TAH组细胞存活率低于20μg/mL TAH组,差异均有统计学意义(P<0.05);(2)与空白对照组比较,雷帕霉素组、10、20μg/mL TAH组细胞自噬体数增多,且10μg/mL TAH组细胞自噬体数多于20μg/mL TAH组,差异均有统计学意义(P<0.05),因此,选择10μg/mL TAH用于后续实验;(3)与空白对照组比较,雷帕霉素组、TAH组细胞p62蛋白的表达均降低,LC3-Ⅱ蛋白的表达均增加,差异均有统计学意义(P<0.05);(4)与空白对照组比较,氯喹组、TAH组、TAH+氯喹组细胞LC3-Ⅱ蛋白的表达均增加,且TAH+氯喹组细胞LC3-Ⅱ蛋白的表达高于氯喹组,差异均有统计学意义(P<0.05);(5)与空白对照组比较,TAH组细胞p-mTOR、p-p70S6K的表达均降低,差异均有统计学意义(P<0.05);(6)强力霉素组细胞Tau-GFP蛋白的表达较空白对照组增加,强力霉素+TAH组细胞Tau-GFP蛋白的表达较强力霉素组降低,强力霉素+TAH+3-MA组、强力霉素+TAH+氯喹组细胞Tau-GFP蛋白的表达较强力霉素+TAH组增高,差异均有统计学意义(P<0.05)。TAH组细胞LC3-Ⅱ蛋白的表达较空白对照组增加,强力霉素+TAH组细胞LC3-Ⅱ蛋白的表达较强力霉素组增加,强力霉素+TAH+3-MA组细胞LC3-Ⅱ蛋白的表达较强力霉素+TAH组降低,强力霉素+TAH+氯喹组细胞LC3-Ⅱ蛋白的表达较强力霉素+TAH组增高,差异均有统计学意义(P<0.05);(7)与空白对照组比较,TAH组HEK293细胞α-Syn蛋白的表达降低,LC3-Ⅱ蛋白的表达升高,差异均有统计学意义(P<0.05);TAH+氯喹组细胞LC3-Ⅱ蛋白的表达高于氯喹组,TAH+氯喹组细胞α-Syn蛋白的表达高于TAH组,差异均有统计学意义(P<0.05)。结论:TAH可能通过抑制mTOR/p70S6K信号通路激活神经细胞自噬,从而促进Tau和α-Syn的降解。

Objective To investigate the effect of total alkaloid of harmaline(TAH)on inducing cellular autophagy and degradating of neurotoxic proteins Tau andα-synuclein(α-Syn).Methods(1)The in vitro cultured PC12 cells were divided into blank control group,and 1,2.5,5,10,20 and 50μg/mL TAH groups,respectively;and they were treated with 0,1,2.5,5,10,20 and 50μg/mL TAH for 24 h;cell morphology and number were observed,and cell survival rate was determined by MTT assay.(2)PC12 cells were divided into blank control group,rapamycin group,and 1,2.5,5,10 and 20μg/mL TAH groups;these cells were treated with same amount of solvent,50 nmol/L autophagy activator rapamycin,and 1,2.5,5,10 and 20μg/mL TAH for 4 h,respectively,and the number of autophagosomes was detected by immunofluorescent staining.(3)PC12 cells were divided into blank control group,rapamycin group,and 10μg/mL TAH group;these cells were treated with same amount of solvent,50 nmol/L rapamycin,and 10μg/mL TAH for 4 h;the protein expression levels of p62 and microtubule-associated protein 1 light chain 3 II(LC3-II)was detected by Western blotting.(4)PC12 cells were divided into blank control group,chloroquine group,TAH group,and TAH+chloroquine group;these PC12 cells were treated with 50 nmol/L autophagy inhibitor chloroquine,10μg/mL TAH,and 10μg/mL TAH+50 nmol/L chloroquine for 4 h,respectively;the LC3-II protein expression was detected by Western blotting.(5)PC12 cells were divided into TAH group and blank control group;10μg/mL TAH and same amount of solvent were given to each group for 4 h,and then,the phosphorylated mammalian target of rapamycin(p-mTOR)and phosphorylated 70-KD ribosomal protein S6 kinase(p-P70S6K)protein expression levels were detected by Western blotting.(6)Tet on HEK293 cells with Tau-green fluorescent protein(GFP)overexpression were divided into blank control group,TAH group,doxycycline group,doxycycline+TAH group,doxycycline+TAH+3-MA group,and doxycycline+TAH+chloroquine group.Cells in the later 4 groups were treated with 200 ng/mL Tet system inducer doxycycline for 24 h;cells in the blank control group were treated with same amount of solvent,those in the TAH group were treated with 10μg/mL TAH,and cells in the latter 3 groups were treated with 10μg/mL TAH,10μg/mL TAH+5 mmol/L 3-MA,and 10μg/mL TAH+50 nmol/L chloroquine,respectively,for 24 h;the changes of green fluorescence intensity of these cells were observed under laser confocal microscope.The Tau-GFP and LC3-II protein expression levels were detected by Western blotting.(7)HEK293 cells with stableα-Syn expression were divided into blank control group,chloroquine group,TAH group and TAH+chloroquine group;these cells were treated with same amount of solvent,50 nmol/L chloroquine,10μg/mL TAH and 10μg/mL TAH+50 nmol/L chloroquine for 24 h,respectively;theα-Syn and LC3-II protein expression levels were detected by Western blotting.Results(1)As compared with that in the blank control group,the cell survival rate in 20 and 50μg/mL TAH groups was significantly lower,and that in the 50μg/mL TAH group was statistically lower than that in 20μg/mL TAH group(P<0.05).(2)As compared with that in the blank control group,the number of autophagosomes in rapamycin group,and 10 and 20μg/mL TAH groups was significantly increased,and that in 10μg/mL TAH group was statistically higher than that in 20μg/mL TAH group(P<0.05);10μg/mL TAH group was selected for subsequent experiments.(3)As compared with the blank control group,the rapamycin group and TAH group had significantly decreased P62 protein expression and significantly increased LC3-II protein expression(P<0.05).(4)As compared with that in the blank control group,the LC3-II protein expression in the chloroquine group,TAH group and TAH+chloroquine group was significantly increased,and LC3-II protein expression in TAH+chloroquine group was statistically higher than that in chloroquine group(P<0.05).(5)The p-mTOR and p-p70S6K expression levels in the TAH group were significantly decreased as compared with those in the blank control group(P<0.05).(6)The Tau-GFP protein expression in doxycycline group was significantly increased as compared with that in the blank control group(P<0.05);that in doxycycline+TAH group was significantly decreased as compared with that in the doxycycline group(P<0.05);that in the doxycycline+TAH+3-MA group and doxycycline+TAH+chloroquine group was statistically increased as compared with that in doxycycline+TAH group(P<0.05).The LC3-II protein expression in the TAH group was significantly increased as compared with that in the control group,that in the doxycycline+TAH group was significantly increased as compared with that in the doxycycline group,that in the doxycycline+TAH+3-MA group was significantly decreased as compared with that in the doxycycline+TAH group,and that in doxycycline+TAH+chloroquine group was significantly increased as compared with that in the doxycycline+TAH group(P<0.05).Conclusion TAH may activate autophagy by inhibiting the mTOR/p70S6K signaling pathway,which in turn promotes the degradation of neurotoxic proteins Tau andα-Syn.