抑制着丝粒蛋白M通过抑制细胞炎症及凋亡缓解转化生长因子-β诱导的胶原蛋白沉积  

Inhibition of centromere protein M alleviates transforming growth factor-β-induced collagen deposition via inhibiting cell inflammation and apoptosis

作　　者：杨康 余伟民[1] 阮远[1] 程帆[1] 葛名欢[1] Yang Kang;Yu Weimin;Ruan Yuan;Cheng Fan;Ge Minghuan(Department of Urology,Renmin Hospital of Wuhan University,Wuhan 430060,China)

机构地区：[1]武汉大学人民医院泌尿外科,430060

出　　处：《中华实验外科杂志》2021年第9期1728-1732,共5页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金面上项目(811870471);中央高校青年教师资助项目(2042021kf0099)。

摘　　要：目的利用体外模型探讨着丝粒蛋白M(CENPM)对肾纤维化的影响及其机制。方法体外培养人源肾小管上皮细胞(HK-2),先用0、10、20、50 ng/ml的转化生长因子-β(TGF-β) HK-2 24 h,利用蛋白质印迹法(Western blot)和细胞免疫荧光检测CENPM蛋白表达;倒置显微镜观察HK-2细胞形态变化。随后利用腺病毒载体构建sh-CENPM病毒转染HK-2细胞,将细胞分为3组:短发卡RNA(shRNA)空白载体组;shRNA+TGF-β组(TGF-β 50 ng/ml处理24 h);sh-CENPM+TGF-β组(sh-CENPM腺病毒转染+TGF-β处理)。利用细胞免疫荧光检测Fibronectin(Fn)表达,Western blot检测胶原蛋白、核因子-κB(NF-κB)通路相关蛋白及凋亡蛋白表达;同时酶联免疫吸附试验(ELISA)检测NF-κB下游白细胞介素(IL)-1β、IL-6、肿瘤坏死因子-α(TNF-α)浓度变化;流式细胞术检测3组细胞内活性氧(ROS)水平。两组间比较采用t检验。结果 50 ng/ml TGF-β处理24 h后CENPM表达量显著升高(2.85±0.32比1.00±0.01,t=9.803,P<0.01),且HK-2细胞形态由卵圆形变为长梭形。与shRNA+TGF-β组比较,利用sh-CENPM腺病毒抑制CENPM表达后,能明显减少TGF-β诱导的Fn及COL-I的表达(1.74±0.27比2.37±0.21,t=2.635,P<0.05;1.98±0.34比3.23±0.26,t=2.477,P<0.05),且抑制NF-κB信号通路的激活,减少下游IL-1β(144.23±18比297.45±41.63,t=6.278,P<0.01)、IL-6(197.46±27.51比378.43±41.22,t=7.954,P<0.01)和TNF-α(311.42±31.57比557.38±49.34,t=10.290,P<0.05)的生成,缓解细胞内ROS的产生及细胞凋亡。结论抑制CENPM能够通过反向调节肾小管上皮细胞内的炎性反应及细胞凋亡,减缓肾纤维化的发展。Objective To explore the effect of centromere protein M(CENPM)on renal fibrosis and its mechanism in vitro.Methods Human proximal tubular epithelial cells(HK-2)were treated with 0,10,20,50 ng/ml transforming growth factor-β(TGF-β)for 24 h.The expression of CENPM protein was detected by Western blotting and immunocytochemistry.The morphological changes of HK-2 cells were observed by inverted microscope.HK-2 cells transfected with or without sh-CENPM adenovirus were cultured in vitro and divided into three groups:short hairpin RNA(shRNA)blank group,shRNA+TGF-βgroup(TGF-β50 ng/ml treatment for 24 h),sh-CENPM+TGF-βgroup(sh-CENPM adenovirus transfection+TGF-βtreatment).Cellular immunofluorescence was used to detect the fibronectin(Fn)expression.The expression of fibrosis-related proteins,nuclear factor-κB(NF-κB)pathway related proteins and apoptosis-related proteins was detect by Western blotting.The downstream molecules including interleukin(IL)-1β,IL-6,tumor necrosis factor-α(TNF-α)were detected by enzyme linked immunosorbent assay(ELISA).Ros kit was used to detect the level of reactive oxygen species(ROS)in cells.T test was used for comparison between two different groups.Results At the cellular level,the expression of CENPM increased significantly after treatment with 50 ng/ml TGF-βfor 24 h(2.85±0.32 vs.1.00±0.01,t=9.803,P<0.01).The morphology of HK-2 cells changed from oval to long spindle.Compared with the shRNA+TGF-βgroup,the use of sh-CENPM adenovirus to inhibit the expression of CENPM could significantly reduce the expression of Fn and COL-I induced by TGF-β(1.74±0.27 vs.2.37±0.21,t=2.635,P<0.05;1.98±0.34 vs.3.23±0.26,t=2.477,P<0.05),inhibit the activation of NF-κB signaling pathway,decrease the production of IL-1β(144.23±18 vs.297.45±41.63,t=6.278,P<0.01),IL-6(197.46±27.51 vs.378.43±41.22,t=7.954,P<0.01)and TNF-α(311.42±31.57 vs.557.38±49.34,t=10.290,P<0.05),and reduce the production of intracellular ROS and cell apoptosis.Conclusion Inhibition of CENPM can slow down the developm

关 键 词：肾纤维化 核因子-ΚB 凋亡 

分 类 号：R692[医药卫生—泌尿科学]