顺铂诱导氧化应激引起耳蜗血管纹周细胞线粒体途径的细胞凋亡  

Cisplatin induces oxidative stress and apoptosis in pericytes of cochlear stria vascularis via mitochondria-mediated pathway

作　　者：黄天兰 柴荣奎 施添凤 马靖雯 余蒙 余苗 司军强[1] 李丽 Huang Tianlan;Chai Rongkui;Shi Tianfeng;Ma Jingwen;Yu Meng;Yu Miao;Si Junqiang;Li Li(Department of Physiology,Shihezi University,Faculty of Medicine,Shihezi 832002,China;Department of Basic Medicine,Jiaxing University,Jiaxing 314000,China)

机构地区：[1]石河子大学医学院生理教研室,石河子832002 [2]嘉兴学院医学院基础医学部,嘉兴314000

出　　处：《中华耳鼻咽喉头颈外科杂志》2023年第11期1093-1101,共9页Chinese Journal of Otorhinolaryngology Head and Neck Surgery

基　　金：国家自然科学基金(81960188);浙江省自然科学基金探索项目(LY21H130001)。

摘　　要：目的探讨顺铂是否通过诱导氧化应激水平升高引起耳蜗血管纹毛细血管周细胞线粒体途径的细胞凋亡。方法将20只6~8周的雄性C57BL/6J小鼠分为对照组和顺铂组;同时原代培养小鼠耳蜗血管纹周细胞并鉴定,分为对照组、顺铂组和N-乙酰半胱氨酸(N-acetylcysteine,NAC)+顺铂组。听性脑干反应(ABR)检测小鼠听力变化,苏木精-伊红(HE)染色观察小鼠耳蜗血管纹形态学变化,总超氧化物歧化酶(superoxide dismutase,SOD)活性检测试剂盒(WST-1法)和硫代巴比妥酸(thiobarbituric acid,TBA)法分别检测SOD活性和丙二醛(malondialdehyde,MDA)含量,DCFH-DA荧光探针检测周细胞上活性氧的含量,Hoechst 33342和流式细胞术检测周细胞的凋亡率,免疫荧光技术检测耳蜗血管纹上周细胞的凋亡相关蛋白分布表达,免疫组化和蛋白免疫印迹法(WB)检测凋亡相关蛋白、线粒体相关蛋白的表达,Mito SOXTM-red和JC-1检测周细胞线粒体功能,伊文思蓝染色观察血迷路屏障(blood labyrinth barrier,BLB)的通透性。采用SPSS 18.0软件进行统计分析。结果与对照组相比,顺铂组可升高小鼠听力阈值和Ⅰ波潜伏期(t值为4.72和12.25,P值均<0.05),升高耳蜗和周细胞内氧化应激水平(t=38.34,P<0.01),使耳蜗血管纹结构紊乱皱缩,BLB通透性增加[伊文思蓝渗漏(1.08±0.42)AU比(0.55±0.23)AU,t=4.64,P<0.05],差异均有统计学意义。顺铂组小鼠耳蜗血管纹细胞凋亡蛋白c-Caspase-3和Bax的表达增加(t值为5.01和6.33,P值均<0.01),且顺铂可使原代培养的周细胞凋亡并上调c-Caspase-3、Bax的表达(P值均<0.05),NAC+顺铂组可逆转顺铂诱导的周细胞凋亡(P<0.05)。顺铂使周细胞线粒体功能损伤,诱导线粒体向胞浆释放凋亡诱导因子(apoptosis-inducing factor,AIF)和细胞色素C(cytochrome-c,Cyt-c),NAC+顺铂组可逆转顺铂诱导的线粒体损伤(P值均<0.05)。结论顺铂可升高耳蜗内氧化应激水平引起C57BL/6J小鼠耳蜗血管纹周细胞Objective To study whether cisplatin may induce apoptosis in the pericytes of cochlear stria vascularis and underlying mechanisms.Methods Twenty male C57BL/6J mice aged 6-8 weeks were divided into control group and a cisplatin group.Primary cultured mouse cochlear vascular peristriatal cells were identified and divided into control group,cisplatin group,and N-acetylcysteine+cisplatin group.Auditory brainstem response(ABR)was used to detect hearing in mice.Hematoxylin eosin(HE)staining was used to observe morphological changes in the stria vascularis of the cochlea.The total superoxide dismutase(SOD)activity test kit(WST-1 method)and thiobarbituric acid(TBA)method were used to detect SOD activity and Malondialdehyde(MDA)content,respectively.DCFH-DA fluorescence probe was used to detect the content of reactive oxygen species in peripheral cells.Hoechst 33342 and flow cytometry were used to detect the apoptosis rate of pericytes.Immunofluorescence technology was used to detect the distribution and expression of apoptosis related proteins in pericytes of cochlear stria vascularis.Immunohistochemistry and Western blotting(WB)were used to detect the expression of apoptosis-and mitochondrial-related proteins.Mito SOXTM-red and JC-1 were used to detect the mitochondrial function of pericytes.Evans blue staining was used to observe the permeability of the blood labyrinth barrier(BLB).Statistical analysis was conducted using SPSS 18.0 software.Results Compared with the control group,the cisplatin group significantly increased in the hearing threshold(t=4.72,P<0.01),Ⅰ-wave latency(t=12.25,P<0.05),and the levels of oxidative stress in the cochlea and pericytes(t=38.34,P<0.01),and also cisplatin caused disorder and contraction of the cochlear stria vascularis structure,increased BLB permeability[Evans blue leakage(1.08±0.42)AU vs(0.55±0.23)AU,t=4.64,P<0.05],with a statistically significant difference,enhanced the expressions of apoptotic proteins c-Caspase-3(t=5.01,P<0.01)and Bax(t=6.33,P<0.01)in the peristriatal cells of

关 键 词：顺铂 耳毒性 周细胞 氧化应激 细胞凋亡 线粒体 

分 类 号：R764[医药卫生—耳鼻咽喉科]