Objective To investigate the role of autophagy in MnC l2-induced apoptosis in human bronchial epithelial 16 HBE cells.Methods Cell proliferation was measured by MTT assay.Mitochondrial membrane potential（MMP） and ap...Objective To investigate the role of autophagy in MnC l2-induced apoptosis in human bronchial epithelial 16 HBE cells.Methods Cell proliferation was measured by MTT assay.Mitochondrial membrane potential（MMP） and apoptosis were measured by flow cytometry.Autophagic vacuoles were detected by fluorescence microscopy.Cellular levels of apoptosis and autophagy-related proteins were measured by western blotting.Results 16 HBE cell proliferation was inhibited by Mn Cl2 in a dose-and time-dependent manner.Mn Cl2-induced 16 HBE cell growth inhibition was related to MMP depolarization prior to the induction of apoptosis.Our data revealed that Mn Cl2-induced apoptosis in 16 HBE cells was mediated by decreased expression of Bcl-2 and increased levels of cleaved caspase-3.It was observed that when we exposed 16 HBE cells to MnCl2 in a dose-dependent manner,the formation of autophagic vacuoles and the levels of LC-3B-II were elevated.RNA interference of LC3 B in these Mn Cl2-exposed cells demonstrated that MMP loss and apoptosis were enhanced.Additionally,the pan-caspase inhibitor Z-VAD-FMK increased the cellular levels of Bcl-2 and decreased apoptosis,but did not affect the cellular levels of LC3 B in Mn Cl2-treated 16 HBE cells.Conclusion Mn Cl2 dose-and time-dependently inhibits 16 HBE cell proliferation and induces MMP loss and apoptosis.Autophagy acts in a protective role against Mn Cl2-induced apoptosis in 16 HBE cells.展开更多
目的研究鱼藤素致人支气管上皮细胞(16HBE)毒性与线粒体损伤和凋亡的相关性,为研究鱼藤素毒性提供参考。方法将1.56~100μmol·L^(-1)鱼藤素与16HBE细胞共孵育,培养24~72 h,CCK-8法测定细胞抑制率,得到半数抑制浓度(IC 50),确定鱼...目的研究鱼藤素致人支气管上皮细胞(16HBE)毒性与线粒体损伤和凋亡的相关性,为研究鱼藤素毒性提供参考。方法将1.56~100μmol·L^(-1)鱼藤素与16HBE细胞共孵育,培养24~72 h,CCK-8法测定细胞抑制率,得到半数抑制浓度(IC 50),确定鱼藤素中剂量。将16HBE细胞分为对照组(正常培养),鱼藤素小剂量组(15μmol·L^(-1)鱼藤素),鱼藤素中剂量组(30μmol·L^(-1)鱼藤素)及鱼藤素大剂量组(60μmol·L^(-1)鱼藤素),培养24 h,倒置荧光显微镜和透射电镜观察线粒体和细胞核超微结构形态,比色法测定谷胱甘肽过氧化物酶(GPx)和超氧化物歧化酶(SOD)活力,流式细胞术检测线粒体膜电位(MMP)和细胞凋亡率。结果鱼藤素对16HBE增殖具有抑制作用,且抑制率呈浓度和时间依赖关系(P<0.05),24,48和72 h IC 50分别为(32.95±2.39),(21.07±2.21)和(15.46±0.93)μmol·L^(-1)。与对照组比较,鱼藤素作用后细胞核凋亡,线粒体明显肿胀、空泡化和嵴脱落,GPx与SOD活力显著下降,MMP急剧下降;对照组荧光强度(57682±719.1),鱼藤素小剂量组、中剂量组、大剂量组分别为(51593±1525.4),(29427±578.4)和(23575±1366.8);鱼藤素各剂量组细胞凋亡率明显上升,且呈浓度依赖性(P<0.05)。结论鱼藤素致16HBE细胞毒性可能与线粒体损伤和细胞凋亡相关。展开更多
基金supported by National Natural Science Foundation of China(Nos.81370079 and 81001253)Beijing Natural Science Foundation(No.7132122)
文摘Objective To investigate the role of autophagy in MnC l2-induced apoptosis in human bronchial epithelial 16 HBE cells.Methods Cell proliferation was measured by MTT assay.Mitochondrial membrane potential（MMP） and apoptosis were measured by flow cytometry.Autophagic vacuoles were detected by fluorescence microscopy.Cellular levels of apoptosis and autophagy-related proteins were measured by western blotting.Results 16 HBE cell proliferation was inhibited by Mn Cl2 in a dose-and time-dependent manner.Mn Cl2-induced 16 HBE cell growth inhibition was related to MMP depolarization prior to the induction of apoptosis.Our data revealed that Mn Cl2-induced apoptosis in 16 HBE cells was mediated by decreased expression of Bcl-2 and increased levels of cleaved caspase-3.It was observed that when we exposed 16 HBE cells to MnCl2 in a dose-dependent manner,the formation of autophagic vacuoles and the levels of LC-3B-II were elevated.RNA interference of LC3 B in these Mn Cl2-exposed cells demonstrated that MMP loss and apoptosis were enhanced.Additionally,the pan-caspase inhibitor Z-VAD-FMK increased the cellular levels of Bcl-2 and decreased apoptosis,but did not affect the cellular levels of LC3 B in Mn Cl2-treated 16 HBE cells.Conclusion Mn Cl2 dose-and time-dependently inhibits 16 HBE cell proliferation and induces MMP loss and apoptosis.Autophagy acts in a protective role against Mn Cl2-induced apoptosis in 16 HBE cells.
文摘目的研究鱼藤素致人支气管上皮细胞(16HBE)毒性与线粒体损伤和凋亡的相关性,为研究鱼藤素毒性提供参考。方法将1.56~100μmol·L^(-1)鱼藤素与16HBE细胞共孵育,培养24~72 h,CCK-8法测定细胞抑制率,得到半数抑制浓度(IC 50),确定鱼藤素中剂量。将16HBE细胞分为对照组(正常培养),鱼藤素小剂量组(15μmol·L^(-1)鱼藤素),鱼藤素中剂量组(30μmol·L^(-1)鱼藤素)及鱼藤素大剂量组(60μmol·L^(-1)鱼藤素),培养24 h,倒置荧光显微镜和透射电镜观察线粒体和细胞核超微结构形态,比色法测定谷胱甘肽过氧化物酶(GPx)和超氧化物歧化酶(SOD)活力,流式细胞术检测线粒体膜电位(MMP)和细胞凋亡率。结果鱼藤素对16HBE增殖具有抑制作用,且抑制率呈浓度和时间依赖关系(P<0.05),24,48和72 h IC 50分别为(32.95±2.39),(21.07±2.21)和(15.46±0.93)μmol·L^(-1)。与对照组比较,鱼藤素作用后细胞核凋亡,线粒体明显肿胀、空泡化和嵴脱落,GPx与SOD活力显著下降,MMP急剧下降;对照组荧光强度(57682±719.1),鱼藤素小剂量组、中剂量组、大剂量组分别为(51593±1525.4),(29427±578.4)和(23575±1366.8);鱼藤素各剂量组细胞凋亡率明显上升,且呈浓度依赖性(P<0.05)。结论鱼藤素致16HBE细胞毒性可能与线粒体损伤和细胞凋亡相关。
