氯化锂通过JNK信号通路诱导HEI-OC1毛细胞样细胞的凋亡  

Lithium Chloride Induce the Apoptosis of HEI-OC1 Cells via the JNK Signaling Pathway

作　　者：孙宇轩 唐晓敏 徐晨宇 郭小涛 赵轶 梁越 潘春晨 孙家强 孙敬武 Sun Yuxuan;Tang Xiaomin;Xu Chenyu;Guo Xiaotao;Zhao Yi;Liang Yue;Pan Chunchen;Sun Jiaqiang;Sun Jingwu(Department of Otolaryngology-Head and Neck Surgery,The Affiliated Provincial Hospital of Anhui Medical University,Hefei,230001,China)

机构地区：[1]安徽医科大学附属省立医院耳鼻咽喉头颈外科,合肥230001

出　　处：《听力学及言语疾病杂志》2021年第4期429-433,共5页Journal of Audiology and Speech Pathology

基　　金：国家自然科学基金(81470699,81600793,81800911);安徽省自然科学基金(18085QH248);中国科学技术大学新医学联合基金(WK9110000053)。

摘　　要：目的探讨氯化锂对HEI-OC1毛细胞样细胞的毒性作用及可能作用机制。方法首先采用CCK-8试剂盒检测0、25、50、75、100、125、150 mM氯化锂浓度处理12、24、48、72小时对HEI-OC1毛细胞样细胞的抑制作用,计算出IC50的浓度值。然后将对数生长期的HEI-OC1毛细胞样细胞分为对照组(不进行任何干预)和实验组(IC50浓度处理24小时)、DMSO组(溶剂组)和JNK抑制组(这两组仅进行Western blot实验以验证氯化锂是否通过JNK信号通路诱导细胞凋亡);采用TUNEL法和流式细胞术检测对照组和实验组细胞的凋亡率,采用RT-PCR检测Caspase-3、Caspase-9、JNK、BAX、Bcl-2的mRNA的表达,使用Western blot检测Cleaved-Caspase-3、Cleaved-Caspase-9、JNK、p-JNK、BAX、Bcl-2蛋白的表达。结果与对照组相比,随着氯化锂浓度和处理时间增加,氯化锂对HEI-OC1毛细胞样细胞的抑制率逐渐增高(P<0.01),拟合出氯化锂在24小时的IC50值为69.53 mM,故实验组氯化锂处理浓度取70 mM。TUNEL法显示实验组凋亡率(36.00%±2.65%)较对照组(2.33%±0.58%)明显提高(t=21.53,P<0.01);流式细胞术检测结果显示,与对照组(0.42%±0.02%)相比,实验组凋亡率(42.40%±0.82%)明显上升(t=88.33,P<0.01)。RT-PCR实验结果表明,与对照组相比,实验组Caspase-3、Caspase-9、JNK、BAX mRNA表达增加,Bcl-2 mRNA表达减少(均为P<0.01)。Western blot实验结果表明,与对照组相比,实验组Cleaved-Caspase-3、Cleaved-Caspase-9、p-JNK、BAX蛋白表达增加,Bcl-2、Bcl-2/BAX蛋白表达减少(均为P<0.01);与实验组相比,JNK抑制组Cleaved-Caspase-3、Cleaved-Caspase-9、p-JNK、BAX蛋白表达减少,Bcl-2、Bcl-2/BAX蛋白表达增加(均为P<0.01)。结论氯化锂通过JNK信号通路诱导HEI-OC1毛细胞样细胞的凋亡。Objective To study the toxic effect and possible mechanism of lithium chloride on HEI-OC1 cells.Methods Firstly,CCK-8 kit was used to detect the inhibitory effect of 0,25,50,75,100,125,150 mM lithium chloride concentrations and 12,24,48,72 hours treatment time on HEI-OC1 cells,and the concentration value of IC50 was calculated.Then the HEI-OC1 cells in logarithmic phase were divided into control group(without any intervention)and experimental group(treated with IC50 concentration for 24 hours).TUNEL assay and flow cytometry were used to analyze apoptosis rates.Expression of Caspase-3,Caspase-9,JNK,BAX,Bcl-2 mRNA were detected by RT-PCR.Expression of Cleaved-Caspase-3,Cleaved-Caspase-9,JNK,p-JNK,BAX,Bcl-2 protein were detected by Western blot.Results The results of the CCK-8 experiment showed that with the increase of lithium chloride concentration and treatment time,the inhibition rate of lithium chloride on HEI-OC1 cells gradually increased compared with the control group(P<0.01).The IC50 value of lithium chloride in 24 hours was 69.53 mM,so the concentration of lithium chloride in the experimental group was 70 mM.The results of TUNEL showed that the apoptosis rate of the experiment group(36.00%±2.65%)was significantly increased than the control group(2.33%±0.58%)(t=21.53,P<0.01).Flow cytometry indicated that the rate of apoptosis in the experiment group(42.40%±0.82%)was significantly increased than the control group(0.42%±0.02%)(P<0.01).The results of RT-PCR showed that,compared with the control group,the mRNA expression of Caspase-3,Caspase-9,JNK,and BAX increased in the experimental group,but Bcl-2 decreased(P<0.01).The results of Western blot showed that,compared with the control group,the protein expression of Cleaved-Caspase-3,Cleaved-Caspase-9,p-JNK,and BAX increased in the experimental group,while Bcl-2,Bcl-2/BAX decreased(P<0.01).Compared with the experimental group,the protein expression of Cleaved-caspase-3,Cleaved-caspase-9,p-JNK,and BAX decreased in the JNK inhibited group,while Bcl-2 and Bcl-2/

关 键 词：氯化锂 HEI-OC1毛细胞样细胞 凋亡 JNK 

分 类 号：R764.43[医药卫生—耳鼻咽喉科]