摘要
目的探讨脂联素对氯化钴诱导的肾小管上皮细胞凋亡及炎症反应的影响。方法将人肾小管上皮细胞株HK-2细胞分入4组:正常对照组,用DME/F-12培养液培养细胞24 h;氯化钴诱导缺氧模型组,用含600.0μM氯化钴的培养液培养细胞24 h;单独脂联素处理组,用含2.5μg/ml脂联素的培养液培养细胞24 h;氯化钴+脂联素联合处理组,用600.0μM氯化钴处理细胞,再加入2.5μg/ml脂联素联合处理细胞24 h。光镜观察HK-2细胞的形态学变化,全光谱激光共聚焦显微镜观察活性氧表达,流式细胞仪检测凋亡率,Western Blot检测炎症因子白细胞介素(IL)-1β、IL-6、肿瘤坏死因子(TNF)-α的蛋白表达水平。结果光镜下,正常对照组细胞呈梭形,胞膜完整,生长良好。氯化钴诱导缺氧模型组细胞形态明显变圆,细胞皱缩,出现细胞碎片,即细胞发生了凋亡和坏死。单独脂联素处理组细胞形态无变化。氯化钴+脂联素联合处理组大部分细胞形态由圆形恢复至梭形,但仍有少量细胞碎片。正常对照组和单独脂联素处理组的HK-2细胞呈现微弱的红色MitoSOX荧光;氯化钴诱导缺氧模型组的HK-2细胞呈现明亮的红色荧光,而氯化钴+脂联素联合处理组红色荧光的强度明显弱于氯化钴诱导缺氧模型组。与正常对照组比较,氯化钴诱导缺氧模型组、氯化钴+脂联素联合处理组的细胞凋亡率均明显上升,差异有统计学意义(P<0.05)。与氯化钴诱导缺氧模型组比较,氯化钴+脂联素联合处理组的细胞凋亡率明显下降,差异有统计学意义(P<0.05)。与正常对照组比较,氯化钴诱导缺氧模型组IL-1β、IL-6、TNF-α蛋白表达水平上升,差异有统计学意义(P<0.05)。与氯化钴诱导缺氧模型组比较,氯化钴+脂联素联合处理组IL-1β、IL-6、TNF-α蛋白表达水平下降,差异有统计学意义(P<0.05)。结论脂联素可能通过减少活性氧的产生抑制氯化钴诱导的肾小管上皮细胞凋亡及炎症反应。
Objective To investigate the effect of adiponectin(APN) on the apoptosis and inflammation of renal tubular epithelial cells induced by cobalt chloride(CoCl_(2)).Methods Human renal tubular epithelial cell line HK-2 cells were divided into 4 groups:normal control group,cultured in DME/F-12 medium for 24 hours;in the CoCl_(2) induced hypoxia model group,the cells were cultured in the medium containing 600.0 μM CoCl_(2) for 24 hours;in the single APN treatment group,the cells were cultured in the medium containing 2.5 μg/ml APN for 24 hours;in the CoCl_(2)+APN combined treatment group,the cells were treated with 600.0 μM CoCl_(2) and 2.5 μg/ml APN for 24 hours.The morphological changes of HK-2 cells were observed by light microscope,the expression of reactive oxygen species was observed by full spectrum laser confocal microscope,the apoptosis rate was detected by flow cytometry,and the protein expression levels of inflammatory factors such as interleukin(IL)-1β,IL-6 and tumor necrosis factor(TNF)-α were detected by Western blot.Results Under light microscope,the cells in normal control group were fusiform with intact cell membrane and good growth.In the hypoxia model group induced by CoCl_(2),cell morphology became round,cell shrinkage and cell fragmentation appeared,cell apoptosis and necrosis occurred.There was no change in cell morphology in single APN treatment group.Most of the cells in the CoCl_(2)+APN treatment group recovered from round to spindle shape,but there were still a few cell fragments.HK-2 cells in normal control group and single APN treatment group showed weak red MitoSox fluorescence.HK-2 cells in the CoCl_(2) induced hypoxia model group showed bright red fluorescence,while the intensity of red fluorescence in the CoCl_(2)+APN combined treatment group was significantly weaker than that in the CoCl_(2) induced hypoxia model group.Compared with the normal control group,the apoptosis rate of CoCl_(2) induced hypoxia model group and CoCl_(2)+APN combined treatment group were significantly increased,with statistical significance(P<0.05).Compared with the hypoxia model group induced by CoCl_(2),the apoptosis rate of CoCl_(2)+APN combined treatment group was significantly reduced(P<0.05).Compared with the normal control group,the protein expression levels of IL-1β,IL-6 and TNF-α in the hypoxia model group induced by CoCl_(2) increased,P<0.05.Compared with the hypoxia model group induced by CoCl_(2),the protein expression levels of IL-1β,IL-6 and TNF-α in the combined treatment group were reduced(P<0.05).Conclusion Adiponectin may inhibit the apoptosis and inflammation of renal tubular epithelial cells induced by cobalt chloride by reducing the production of reactive oxygen species.
作者
郑晓彤
刘大军
ZHENG Xiao-tong;LIU Da-jun(Department of Nephrology,Shengjing Hospital of China Medical University,Shenyang 110004,China)
出处
《临床军医杂志》
CAS
2021年第1期10-14,共5页
Clinical Journal of Medical Officers
基金
辽宁省自然科技基金(20170541057)。
关键词
脂联素
氯化钴
肾小管上皮细胞
凋亡
炎症反应
Adiponectin
Cobalt chloride
Renal tubular epithelial cells
Apoptosis
Inflammatory response
作者简介
第一作者:郑晓彤(1994-),女,辽宁辽阳人;通信作者:刘大军,E-mail:liudj@sj-hospital.org。
