醋酸铅暴露对PC12细胞凋亡与自噬的影响及可能机制  

Effect of lead acetate exposure on apoptosis and autophagy in PC12 cells and possible mechanism

作　　者：张谊 王立晖[1] 陈月芳 李永金[2] 柳浦青[3] ZHANG Yi;WANG Lihui;CHEN Yuefang;LI Yongjin;LIU Puqing(Danyang Hospital Affiliated to Nantong University,People′s Hospital of Danyang City,Jiangsu Province,Danyang 212300,China;Department of Pharmacology,School of Medicine,Jiangsu University,Zhenjiang 212013,China;The Second Affiliated Hospital of Zhejiang University of Traditional Chinese Medicine,Hangzhou 310005,China)

机构地区：[1]南通大学附属丹阳医院,江苏省丹阳市人民医院,江苏丹阳212300 [2]江苏大学医学院药理学教研室,江苏镇江212013 [3]浙江中医药大学附属第二医院,浙江杭州310005

出　　处：《中国药理学与毒理学杂志》2023年第10期774-780,共7页Chinese Journal of Pharmacology and Toxicology

基　　金：国家自然科学基金(31700444);丹阳市社会发展科技支撑计划(SF201804);镇江“金山英才”高层次领军人才培养计划(第六期“169工程”)(2021);浙江省自然科学基金(LYY22H280001);浙江省中医药现代化专项(2022ZB161)。

摘　　要：目的探讨醋酸铅〔Pb(Ac)_(2)〕暴露对大鼠肾上腺嗜铬细胞瘤PC12细胞凋亡与自噬的影响及相关分子机制。方法体外培养的PC12细胞用Pb(Ac)_(2)0(细胞对照),100,200和400μmol·L^(-1)处理24 h后,采用MTT法检测细胞存活率,乳酸脱氢酶(LDH)试剂盒检测细胞培养液中LDH含量,吖啶橙/溴化乙锭(AO/EB)染色观察细胞凋亡,流式细胞术检测细胞凋亡率,单丹磺酰尸胺(MDC)试剂盒检测细胞自噬,免疫荧光法观察Bcl-2相互作用蛋白1(Beclin1)在细胞内的表达,Western印迹法检测细胞内自噬相关蛋白沉默信息调节因子1(Sirt1)、叉头框蛋白转录因子3a(FoxO3a)、叉头蛋白M1(FoxM1),Beclin1、微管相关蛋白1轻链3(LC3)以及P62蛋白表达。结果与细胞对照组相比,Pb(Ac)_(2)100,200和400μmol·L^(-1)处理24 h后,PC12细胞存活率显著降低(P<0.05,P<0.01),细胞外液LDH含量升高(P<0.05,P<0.01)。AO/EB染色结果显示,随着Pb(Ac)_(2)浓度的提高,PC12细胞颜色逐渐加深为橙红色,出现明显的凋亡现象;与细胞对照组相比,PC12细胞凋亡率随Pb(Ac)_(2)浓度的提高而显著增加(P<0.01),PC12细胞内自噬体数量增加(P<0.01)。免疫荧光结果显示,Pb(Ac)_(2)暴露后,PC12细胞胞质内Beclin1呈现大量点状聚集且荧光强度加强,表明细胞发生了自噬。Western印迹法结果显示,与细胞对照组相比,Pb(Ac)_(2)各浓度组Sirt1,FoxO3a和Beclin1蛋白表达水平及LC3-Ⅱ/LC3-Ⅰ比值显著增加(P<0.05,P<0.01),FoxM1和P62蛋白表达水平显著下降(P<0.05,P<0.01)。结论Pb(Ac)_(2)暴露可诱导PC12细胞发生凋亡与自噬,其机制可能与Sirt1/FoxO3a信号通路激活有关。OBJECTIVE To investigate the effects of lead acetate(Pb(Ac)_(2))exposure on apoptosis and autophagy in adrenal pheochromocytoma PC12 cells of rats and related signaling pathway.METHODS PC12 cells were exposed to Pb(Ac)_(2)at concentrations of 100,200,and 400μmol·L^(-1)for 24 h.The cell survival rate was measured by MTT assay,and the lactate dehydrogenase(LDH)leakage rate was measured by LDH kit.Acridine orange/ethidium bromide(AO/EB)staining and flow cytometry were used to detect cell apoptosis.Monodansylcadaverine staining kit was used to detect autophagy.Immunofluorescence was performed to examine the expression of Bcl-2 interacting protein 1(Beclin1).The expressions of silent information regulator 1(Sirt1),forkhead box protein transcription factor 3a(FoxO3a),forkhead protein M1(FoxM1),Beclin1,microtubule-associated protein 1 light chain 3(LC3)and P62 proteins in the cells were detected by Western blotting.RESULTS After 24 hours of exposure to Pb(Ac)_(2)at concentrations of 100,200,and 400μmol·L^(-1),PC12 cell proliferation was inhibited(P<0.05,P<0.01)and the leakage of intracellular LDH was increased(P<0.05,P<0.01)compared with the cell control group.The results of AO/EB staining showed that with the increase of Pb(Ac)_(2)concentrations,more cells became EB-positive and developed orange red color,indicating the induction of apoptosis.Flow cytometry analysis showed that compared with the control group,the apoptosis rate of PC12 cells increased significantly in a concentration-dependent manner(P<0.01).At the same time,autophagy analysis showed that compared with the control group,the number of autophagosomes in PC12 cells increased(P<0.01).Immunostaining also showed that after PC12 cells were exposed to different concentrations of Pb(Ac)_(2),Beclin1 aggregation in the cytoplasm was enhanced,indicating the promotion of autophagy.The results of Western blotting showed that with the increase of Pb(Ac)_(2)concentration,the protein expressions of Sirt1,FoxO3a,Beclin1 and the ratio of LC3-Ⅱ/LC3-Ⅰincreased(P<0.

关 键 词：醋酸铅 PC12细胞 细胞凋亡 细胞自噬 Sirt1/FoxO3a信号通路 

分 类 号：R99[医药卫生—毒理学] R963[医药卫生—药学]