N-乙酰半胱氨酸对尼古丁诱导的MC3T3-E1细胞凋亡的作用及其机制  

Effect of N-acetylcysteine on apoptosis of MC3T3-E1 cells induced by nicotine and its mechanism

作　　者：崔磊华 侯玉帛 苏畅 李明贺[3] 聂鑫 CUI Leihua;HOU Yubo;SU Chang;LI Minghe;NIE Xin(Department of Oral and Maxillofacial Surgery,Affiliated Stomatology Hospital,Wenzhou Medical University,Wenzhou 325000,China;Department of Periodontology,Affiliated Stomatology Hospital,Wenzhou Medical University,Wenzhou 325000,China;Department of Oral and Maxillofacial Surgery,Stomatology Hospital,Jilin University,Changchun 130021,China)

机构地区：[1]温州医科大学附属口腔医院口腔颌面外科,浙江温州325000 [2]温州医科大学附属口腔医院牙周科,浙江温州325000 [3]吉林大学口腔医院口腔颌面外科,吉林长春130021

出　　处：《吉林大学学报（医学版）》2022年第1期26-32,共7页Journal of Jilin University：Medicine Edition

基　　金：国家自然科学基金面上项目(51972240);浙江省教育厅一般科研项目(Y201942009);浙江省温州市科技局基础性科研项目(Y20180700,Y20180699)。

摘　　要：目的:探讨N-乙酰半胱氨酸(NAC)对尼古丁诱导的小鼠前成骨细胞系MC3T3-E1细胞凋亡的影响,并阐明其作用机制。方法:体外培养MC3T3-E1细胞,采用不同浓度(0、1.0、2.5、5.0和7.5 mmol·L^(-1))尼古丁诱导MC3T3-E1细胞损伤,采用MTT法检测各组MC3T3-E1细胞增殖活性,并确定尼古丁半数抑制浓度(IC_(50))。根据IC_(50)值将MC3T3-E1细胞分为对照组、尼古丁组和尼古丁+不同浓度(2.5、5.0和7.5 mmol·L^(-1))NAC组,采用MTT法检测各组细胞增殖活性,以确定NAC最适作用浓度。将细胞分为对照组、尼古丁(4.25 mmol·L^(-1))组、NAC(5.0 mmol·L^(-1))组和尼古丁(4.25 mmol·L^(-1))+NAC(5.0 mmol·L^(-1))组,采用MitoSOX荧光染色法检测各组细胞中线粒体来源活性氧簇(mitoROS)水平,采用流式细胞术检测各组细胞凋亡率,Western blotting法检测各组细胞中凋亡相关蛋白B细胞淋巴瘤2(Bcl-2)和Bcl-2相关X蛋白(Bax)蛋白表达水平,并计算Bax/Bcl-2比值。结果:与0 mmol·L^(-1)尼古丁组比较,不同浓度尼古丁组细胞增殖活性明显降低(P<0.05),且呈浓度依赖性。尼古丁的IC_(50)值为(4.25±0.03)mmol·L^(-1)。与对照组比较,尼古丁组细胞增殖活性明显降低(P<0.05);与尼古丁组比较,尼古丁+不同浓度NAC组细胞增殖活性明显升高(P<0.05),尼古丁+5.0 mmol·L^(-1) NAC组细胞增殖活性升高最明显。与对照组比较,尼古丁组细胞中mitoROS水平明显升高(P<0.05),细胞凋亡率明显升高(P<0.05),细胞中Bax/Bcl-2比值明显升高(P<0.05);与尼古丁组比较,NAC组和尼古丁+NAC组细胞中mitoROS水平明显降低(P<0.05),细胞凋亡率明显降低(P<0.05),尼古丁+NAC组细胞中Bax/Bcl-2比值明显降低(P<0.05)。结论:NAC可缓解尼古丁诱导的MC3T3-E1细胞凋亡,其作用机制可能与线粒体氧化应激有关。Objective:To investigate the effect of N-acetylcysteine(NAC)on the apoptosis of mouse MC3T3-E1 preosteoblast cells induced by nicotine,and to clarify its mechanism.Methods:The MC3T3-E1 cells were cultured in vitro and treated with different concentrations(0,1.0,2.5,5.0 and 7.5 mmol·L^(-1))of nicotine to induce the MC3T3-E1 cell injury;MTT method was used to determine the proliferation activities of cells in various groups and the half inhibitory concentration(IC_(50))of nicotine was determined.The MC3T3-E1 cells were divided into control group,nicotine group,NAC group and nicotine+different concentrations(2.5,5.0,and 7.5 mmol·L^(-1))NAC groups according the IC_(50) values,and MTT method was used to determine the proliferation activities of cells in various groups;the optimun concentration of NAC was confirmed.The cells were divided into control group,nicotine(4.25 mmol·L^(-1))group,NAC(5.0 mmol·L^(-1))group and nicotine(4.25 mmol·L^(-1))+NAC(5.0 mmol·L^(-1))group.The levels of mitochondrial ROS(mitoROS)was assessed with MitoSOX fluorescence staining method;Flow cytometry was used to detect the apoptic rates of MC3T3-E1 cells in various groups;Western blotting method was used to detect the expression level of apoptosis-related proteins B-cell lymphoma-2(Bcl-2)and Bcl-2 associated X proteins(Bax)in the cells in various groups,and the ratios of Bax/Bcl-2 was calculated.Results:Compared with 0 mmol·L^(-1) nicotine group,the proliferation activities of MC3T3-E1 cells in different concentrations of nicotine groups were significantly decreased(P<0.05)in a dose-dependent manner.The IC_(50) of nicotine was(4.25±0.03)mmol·L^(-1).Compared with control group,the proliferation activity of MC3T3-E1 cells in 4.25 mmol·L^(-1) nicotine group was significantly decreased(P<0.05);compared with nicotine group,the proliferation activites of MC3T3-E1 cells in nicotine+different concentrations of NAC groups were increased(P<0.05);the proliferation activity of cells in nicotine+5.0 mmol·L^(-1) NAC group was the highest.Compare

关 键 词：MC3T3-E1细胞 尼古丁 N-乙酰半胱氨酸 细胞凋亡 氧化应激 

分 类 号：Q25[生物学—细胞生物学] Q813