远志皂苷对β淀粉样蛋白片段1-40诱导PC12细胞凋亡的抑制作用

摘要

目的 探讨远志皂苷抑制β淀粉样蛋白片段1-40(Aβ1-40)诱导的PC12细胞凋亡的作用机制.方法 采用聚集状的Aβ1-40 25 μmol·L-1诱导PC12细胞凋亡,然后将处理后的PC12细胞分为Aβ1-40模型组和远志皂苷50,100和200 μmol·L-1组,同时设正常细胞对照组.采用MTT比色法检测细胞存活率；膜联蛋白-Ⅴ和PI双染法检测细胞凋亡率；免疫细胞化学法检测细胞凋亡基因Bcl-2和Bax及细胞色素c(Cytc)表达阳性的细胞百分率；Western印迹法检测PC12细胞中Cyt c的表达水平.结果 与正常对照组比较,Aβ1-40模型组PC12细胞的存活率明显降低(P＜0.01),为(31±7)％；Bcl-2阳性表达细胞率降低(P＜0.01),为(23.9±1.9)％；Bax和Cyt c阳性表达细胞率升高(P＜0.01),分别为(79.0±3.7)％和(49.2±3.6)％,Bcl-2/Bax阳性表达细胞比值为0.30.与模型对照组比较,远志皂苷50,100和200 μmol·L-1作用24 h后,细胞存活率分别升高至(51±13)％,(64±7)％和(84±10)％(P＜0.01)；Bcl-2阳性率升高至(38.7±0.9)％,(53.7±1.6)％和(60.3±0.8)％(P＜0.01),Bax阳性率分别降低为(60.8±1.9)％,(41.5±2.2)％和(32.7±1.4)％(P＜0.01),Bcl-2/Bax比值亦分别上升为0.64,1.29和1.84；Cyt c阳性率分别降低至(45.4±3.4)％,(30.2±2.2)％和(27.5±1.0)％(P＜O.05,P＜0.01).与正常对照组比较,模型组PC12细胞凋亡率和Cyt c蛋白表达水平亦明显升高(P＜0.01)；远志皂苷50,100和200μmol·L-1作用24 h,PC12细胞凋亡率和Cyt c表达水平较模型组均降低(P＜0.01).结论 远志皂苷对Aβ1-40诱导的PC12细胞凋亡具有明显的抑制作用,其作用机制可能是抑制Bax和Cyt c表达,增加Bcl-2表达和Bcl-2/Bax比值,从而阻断内源性细胞凋亡通路.%OBJECTIVE To investigate the protective mechanism of tenuigenin (TEN) on the apoptosis of PC12 cells induced by amyloid beta-protein 1-40(Aβ1-40) in vitro.METHODS Aggregated Aβ1-40 25 μmol·L-1 which induced the apoptosis of PC12 cells was used to establish Alzheimer's disease neuronal cell model.These model neurons were divided into Aβ1-40 model group and TEN 50,100 and 200 μmol·L-1 groups.At the same time,normal cell control group was established without Aβ1-40 pretreatment.The survival rate of PC12 cells was detected by MTT assay.The apoptosis rate of PC12 cells was detected by flow cytometery with Annexin-Ⅴ/PI double staining.The rates of positive cells expressed Bcl-2,Bax and cytochrome c (Cyt c) were observed by immunocytochemical method.The expression level of Cyt c was detected through Western blotting analysis.RESULTS Compared with normal control group,survival rate of PC12 cell decreased to (31 ± 7)％ (P ＜ 0.01),the positive rate of Bcl-2 was declined to (23.9 ± 1.9) ％ (P ＜ 0.01),the positive rate of Bax and Cyt c increased to (79.0 ±3.7) ％ and (49.2 ±3.6) ％ (P ＜ 0.01),respectively,and the ratio of Bcl-2/Bax was 0.30 in Aβ1-40 model group.Compared with model group,after 24 h incubation of with PC12 cells TEN 50,100 and 200 μmol·L-1,PC12 cell survival rate increased to (51 ±13)％,(64 ±7)％ and(84 ±10)％ (P＜0.01),respectively;the positive rate of Bcl-2 increased to (38.7 ±0.9)％,(53.7 ±1.6)％ and(60.3 ±0.8)％ (P ＜ 0.01),respectively; the positive rate of Bax was declined to (60.8 ± 1.9)％,(41.5 ±2.2)％ and (32.7 ±1.4)％(P＜0.01),and the ratio of Bcl-2/Bax increased to 0.64,1.29 and 1.84; the positive rate of Cyt c decreased to (45.4 ± 3.4) ％,(30.2 ± 2.2) ％ and (27.5 ± 1.0) ％ (P ＜0.05,P ＜0.01).Compared with normal control group,the apoptosis rate in PC12 cells and expression level of Cyt c protein were also obviously elevated (P＜0.01) ;and after 24 h incubation of TEN 50,100 and 200 μmol· L-1 with PC12 cells,the apoptosis rate in PC12 cells and expression level of Cyt c protein were lower than those in model group (P ＜ 0.01).CONCLUSION TEN has an obvious protecting effect against PC12 cell apoptosis induced by Aβ1-40.TEN may block the endogenous apoptosis pathway of PC12 cells by inhibiting the expression of Bax and Cyt c and increasing the expression of Bcl-2 and the ratio of Bcl-2/Bax.