激活素A在高糖诱导的人肾小管上皮细胞中的表达变化

Expression of activin A in high glucose-induced human kidney tubular epithelial cells

目的 通过体外高糖刺激的人近端肾小管上皮细胞株（HK-2）,探讨激活素A（ACT A）的表达变化及卵泡抑素（FS）的干预作用.方法 HK-2细胞生长于DMEM培养基,待细胞生长至亚融合状态时,更换为无血清DMEM培养基使细胞生长同步化24h,然后将细胞分为以下5组：正常对照组（NG,5.5 mmol/L葡萄糖）、甘露醇对照组（MG,5.5 mmol/L葡萄糖＋25 mmol/L甘露醇）、高糖组（HG,25 mmol/L葡萄糖）、HG＋FS100组（25 mmol/L葡萄糖＋100 μg/L FS）、HG＋FS500组（25 mmol/L葡萄糖＋500 μg/L FS）.12、24、48 h后收集各组细胞及细胞上清液.Western印迹法检测各组细胞ACT A和p-Smad2/3的表达；ELISA检测各组细胞上清液转化生长因子β（TGF-β）和纤连蛋白（FN）的含量.结果 NG组细胞ACT A有微量表达,而HG组表达显著增加（P＜0.05）,呈时间依赖性.与HG组相比,FS干预组ACT A表达显著减少（P＜0.05）,呈剂量依赖性.与NG组相比,p-Smad2/3在HG组培养24 h后表达显著增加（P＜0.05）；FS可抑制p-Smad2/3的高表达（P＜0.05）.ELISA检测结果显示,与NG组相比,HG组培养12 h后TGF-β表达即显著增加（P＜0.05）,呈时间依赖性,FS干预对高糖诱导的TGF-3高表达没有影响.与NG组相比,HG组培养24h后FN表达显著增高（P＜0.01）,FS对此有明显抑制作用,呈剂量依赖性（P＜0.01）.结论 高糖能刺激HK-2细胞ACT A表达增加,从而促进肾小管上皮细胞FN的合成；FS可通过阻断ACT A的活化,减轻肾小管上皮细胞FN的合成.

Objective To investigate the expression of activin A and the intervention effect of follistatin on high glucose-cultured human proximal tubular epithelial cells （HK-2） in vitro.Methods HK-2 cells were maintained in Dulbecco＇s modified Eagle＇s medium （DMEM）.By 80% confluent,the cells were incubated in a serum-free medium for 24 h and then exposed to the following experimental conditions for different time periods （12,24 and 48 h）：5 mmol/L glucose （normal glucose group,NG）,5 mmol/L glucose plus 25 mmol/L mannitol （osmotic control,mannitol group,MG）,25 mmol/L glucose （high glucose group,HG）,25 mmol/L glucose plus 100 μg/L rhfollistatin （100 μg/L rh-follistatin intervention group,HG＋FS100） or 25 mmol/L glucose plus 500μg/L rh-follistatin （500 μg/L rh-follistatin intervention group,HG＋FS500）.Then,the cells and cellular supernatant were collected for further detection.Activin A and p-Smad2/3 expression were determined by Western blotting.TGF-β and fibronectin released into media were quantitatively analyzed by enzyme-linked immunosorbent assay （ELISA）. Results Activin A had very slight activation in the NG group,but its production was enhanced in the HG group after 12 h culture （P〈0.05） and increased gradually until study completion.Follistatin blocked its production in a dose-dependent manner.p-Smad2/3 was significantly increased after 24 h culture in the HG group compared with the NG group and follistatin treatment decreased its overexpression in the HG group （P〈0.05）.TGF-β was markedly higher in the HG group than that in the NG group after 12 h culture （P〈0.05） and increased in a time-dependent manner,but both doses of follistatin failed to modify the overexpression of TGF-β in HG.Compared with the NG group,fibronectin in the HG group was obviously upregulated after 24 h culture （P〈0.01）.Follistatin reduced its overexpression in a dose-dependent manner. Conclusions High glucose can induce activin A activation and then promote FN production in HK-2 cells.Decreased fibronectin production inhibited by follistatin in HK-2 cells in high glucose condition is associated with blocking of activin A activation.