前蛋白转化酶枯草溶菌素9在肾脏缺血再灌注损伤中对HK-2细胞氧化炎性反应和细胞焦亡的影响及作用机制  

Effects of proprotein convertase subtilisin/kexin type 9 on oxidative inflammation and cell pyroptosis in HK-2 cells during renal ischemia-reperfusion injury and the mechanisms

作　　者：伍骏锋 吴宇波 沈巧琳 贾本忠 杨长庆 Wu Junfeng;Wu Yubo;Shen Qiaolin;Jia Benzhong;Yang Changqing(Department of Urology,Qiandongnan People's Hospital,Kaili 556000,China;Department of Urology,Guizhou Medical University,Guiyang 550000,China)

机构地区：[1]贵州省黔东南州人民医院泌尿外科,凯里556000 [2]贵州省医科大学附属医院泌尿外科,贵阳550004

出　　处：《中华实验外科杂志》2024年第9期2033-2036,共4页Chinese Journal of Experimental Surgery

摘　　要：目的 探讨前蛋白转化酶枯草溶菌素9(PCSK9)在肾脏缺血再灌注损伤(IRI)中对HK-2细胞氧化炎性反应和细胞焦亡的作用及其机制.方法 建立肾脏HK-2细胞缺氧复氧模型,将HK-2细胞随机分为缺氧复氧组、对照组、沉默PCSK9组、沉默PCSK9的对照组.采用定量实时聚合酶链反应(qRT-PCR)检测不同复氧损伤时间点PCSK9的表达变化;使用商业试剂盒检测转染PCSK9后对HK-2细胞中超氧化物歧化酶(SOD)和丙二醛(MDA)含量的影响;使用酶联免疫吸附试验(ELISA)试剂盒检测转染PCSK9后对HK-2细胞的炎性因子白细胞介素(IL)-1β和IL-18的影响;采用蛋白质印迹法(Western blot)实验检测转染PCSK9后对HK-2细胞焦亡相关蛋白Nod样受体家族包含pyrin结构域蛋白3(NLRP3)和半胱氨酰天冬氨酸特异性蛋白酶(Caspase)-1表达水平的影响.组间比较采用配对样本t检验,多组间比较采用单因素方差分析.结果 qRT-PCR结果显示对照组及缺氧复氧组(0、2、4、6、8 h)PCSK9的mRNA表达水平分别为(1.00±0.18、3.24±0.31、3.61±0.36、3.79±0.39、4.26±0.43、4.08±0.39)高于未缺氧 HK-2 细胞的对照组(1.00±0.18、1.02±0.11、1.01±0.15、1.00±0.12、1.05±0.11、0.99±0.12),随着复氧损伤时间的增加,PCSK9表达逐渐增高,并在复氧6 h达到峰值(F=34.47,P<0.05).SOD和MDA检测结果显示缺氧复氧组的SOD含量低于对照组,而MDA含量高于对照组[SOD:(5.23±0.46)U/mg比(13.18±1.02)U/mg,t=12.306,P<0.05;MDA:(3.53±0.27)nmol/mg 比(1.66±0.18)nmol/mg,t=-9.981,P<0.05].沉默PCSK9 组中 SOD 含量高于其对照组[(10.12±0.98)U/mg 比(5.19±0.50)U/mg,t=-7.762,P<0.05].沉默 PCSK9 组中 MDA 含量低于对照组[(2.09±0.18)nmol/mg 比(3.54±0.29)nmol/mg,t=7.358,P<0.05].ELISA 试剂盒检测结果显示缺氧复氧组的 IL-1β 和 IL-18 含量均高于对照组[IL-1β:(35.12±3.21)pg/ml 比(15.64±1.88)pg/ml,t=-9.070,P<0.05;IL-18:(98.76±9.71)pg/ml 比(52.17±5.09)pg/ml,t=-7.361,P<0.05].沉默PCSK9组中IL-1β 和IL-18 含Objective To investigate the role and mechanisms of proprotein convertase subtilisin/kexin type 9(PCSK9)in oxidative inflammation and cell pyroptosis in HK-2 cells during renal ischemia-reperfusion injury(IRI).Methods A hypoxia-reoxygenation model was established using HK-2 cells(American Type Culture Collection).HK-2 cells were randomly divided into hypoxia reoxygenation group,control group,PCSK9 silenced group and PCSK9 silenced control group.Quantitative real-time polymerase chain reaction(qRT-PCR)was employed to detect PCSK9 expression at different time points of reoxygen-ation injury.Commercial assay kits were used to assess the impact of PCSK9 transfection on superoxide dis-mutase(SOD)and malondialdehyde(MDA)levels in HK-2 cells.Enzyme-linked immunosorbent assay(ELISA)kits were utilized to measure the effects of PCSK9 transfection on the inflammatory factors[inter-leukin(IL)-1βand IL-18]in HK-2 cells.Western blotting experiments were conducted to evaluate the in-fluence of PCSK9 transfection on the expression levels of pyroptosis-related proteins NLR family,pyrin do-main containing 3(NLRP3)and cysteinyl aspartate-specific protease(Caspase)-1 in HK-2 cells.The paired sample t test was used for comparison between groups and one-way analysis of variance for multiple group comparisons.Results The results of qRT-PCR showed that the mRNA expression levels of PCSK9 in control group and hypoxia reoxygenation group(0,2,4,6,8 h)(1.00±0.18,3.24±0.31,3.61±0.36,3.79±0.39,4.26±0.43,4.08±0.39)were higher than those in the control group of non-hypoxic HK-2 cells(1.00±0.18,1.02±0.11,1.01±0.15,1.00±0.12,1.05±0.11,0.99±0.12).Com-pared to the control group of non-hypoxic HK-2 cells,PCSK9 expression significantly increased with pro-longed reoxygenation injury,peaking at 6 h(F=34.47,P<0.05).SOD and MDA assays revealed that SOD levels in the hypoxia-reoxygenation group were lower than those in the control group[(5.23±0.46)U/mgvs.(13.18±1.02)U/mg,t=12.306,P<0.05],and MDA levels were higher than those in the control

关 键 词：肾脏缺血再灌注损伤 前蛋白转化酶枯草溶菌素9 炎性反应 细胞焦亡 

分 类 号：R692[医药卫生—泌尿科学]