抑制自噬对艾塞那肽诱导的AR42J细胞凋亡的影响

Antophagy inhibition can decrease AR42J cell apoptosis induced by exendin-4

目的探讨艾塞那肽对AR42J细胞株的损伤及其可能机制。方法1、5、10pm0I/L艾塞那肽处理AR42J细胞株24、48、72、96、120h,噻唑蓝(Mrrll)法检测细胞活力,筛选最佳浓度和时间作为后续实验条件。设置空白对照组(Nc)、艾塞那肽组(Ex一4)和Ex一4+z—VAD.fmk组,检测艾塞那肽是否可直接导致AR42J细胞凋亡;设置NC组、Ex一4组、3一MA组和Ex-4+3-MA组,探究3一MA能否抑制艾塞那肽诱导的AR42J细胞凋亡。3一MA为自噬抑制剂,z—VAD—fmk为凋亡抑制剂。细胞活力检测采用M1Tr法。流式细胞术检测细胞凋亡率,westemblot检测caspase.3等凋亡相关蛋白以及微管相关蛋白轻链3(Lc3)自噬相关蛋白表达。结果以10pmoL/L艾塞那肽作用72h为后续实验条件。z—VAD—fmk预处理可明显抑制艾塞那肽的细胞毒性,两组细胞活力为(49.4±3.0)%比(81.2±3.3)%,差异有统计学意义(P<0.05)。Ex一4组细胞凋亡率(28.2±1.4)%,高于Nc组的(3.6±0.8)%,差异有统计学意义(P<0.05)。westemblot也显示caspase一3表达明显上调,而z—VAD—fmk预处理则可明显抑制艾塞那肽引起的细胞凋亡和caspase一3表达增加。3-MA预处理可明显抑制艾塞那肽的细胞毒性,处理72h两组细胞活力为(49_8±2.5)%比(79.1±2-3)%,差异有统计学意义(P<O.05)。Ex一4组细胞凋亡率为(29.2±3.2)%,高于Ex一4+3一MA组的(14.5±2.1)%,差异有统计学意义(P<0.05)。westemb10t结果表明艾塞那肽可上调LC3B一Ⅱ、p62和caspase一3的表达,而3一MA预处理则可明显抑制艾塞那肽引起的Lc3B一Ⅱ和caspase一3上调,但p62的表达却进一步上调。结论艾塞那肽可诱导AR42J细胞凋亡,3一MA可通过抑制自噬而抑制艾塞那肽诱导的凋亡。使用3一MA可作为减轻艾塞那肽对胰腺腺泡细胞毒性的一种潜在方法。

Objective To explore whether exendin-4inhibits AR42J cells and its mechanism. Methods AR42J cells were treated with exendin-4under multiple concentrations(1,5,10pmol/L)at 24, 48,72,96,120h to assess its cell viability by MTI"assay and got the IC-50and time points.Then checking whether exendin-4could induce the AR42Jcells apoptosis by setting normal control (NC)group,exendin-4 (Ex-4)group and Ex-4±z-VAD-fmk (apoptosis inhibitor)group,and exploring whether 3-MA which is au- tophagy inhibitor could inhibit the AR42J cells apoptosis induced by exendin-4by setting NC group,Ex-4 group and Ex-4±3-MA group.Cell viability was analyzed by MTT and the cells apoptosis was detected by flow cytometry and the protein levels of caspase-3,LC3and p62were studied by Western blot.Results Concentration of 10pmol/L exendin-4and and time point 72h were selected for the further study .z-VAD- fmk pretreatment can significantly inhibit the cell viability of exendin-4by (81.2±3.3)%vs.(49.4±3.0)% (P<0.05).Flow cytometry showed that exendin-4could induce the AR42J cells apoptosis by (28.2±1.4)% vs.(3.6±0.8)%,and increased the caspase-3level by Western blot,which both can be reversed by (79.1± 2.3)%vs.(49.8±2.5)%(P<0.05)when the cells were treated for 72h,as was apoptosis ratio by (14.5±2.1)%vs.(29.2±3.2)%.Westem blot showed that exendin-4can upregulate protein levels of LC3B-II,p62 125I caspase-3and 3-MA,and pretreatment can inhibit the upregulation of LC3B-II and caspase-3 but further increased the upregulation of p62induced by exendin-4.Conclusions Exendin-4can induce AR42J cells apoptosis and 3-MA pretreating can inhibit exendin-4 cytotoxieity through downregulating autophagy.So autophagy inhibitor 3-MA could potentially extenuate the cytotoxicity of exendin-4in pancreatic acinar cells.