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

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 25μmol·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200μmol·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(Cyt c)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200μmol·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<0.05,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24h,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。

Protection of tenuigenin against apoptosis of PC12 cells induced by amyloid beta-protein fragment 1-40

OBJECTIVE To investigate the protective mechanism of tenuigenin (TEN) on the apoptosis of PC12 cells induced by amyloid beta-protein 1-40(Aβ_1-40) in vitro. METHODS Aggregated Aβ_1-40 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 Aβ_1-40 model group and TEN 50, 100 and 200 μmol·L^-1 groups. At the same time, normal cell control group was established without Aβ_1-40 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 Aβ_1-40 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 Aβ_1-40. 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.