糖原合成酶激酶3β在高糖诱导人肾小管上皮细胞损伤中的作用及可能机制

The role and possible mechanism of glycogen synthase kinase 3β in the high glucose induced injury of renal tubular epithelial cells

目的研究糖原合成酶激酶3β(GSK-3β)与高糖诱导人肾小管上皮细胞(HK-2)损伤和炎症反应的关系,探讨其参与糖尿病肾病进展的可能机制。方法培养HK-2细胞于37℃、5%CO2培养箱,按随机数表法分为4组:(1)正常糖浓度组(D-葡萄糖5.6 mmol/L);(2)高渗对照组(D-葡萄糖5.6 mmol/L+D-甘露醇24.4 mmol/L);(3)高糖组(D-葡萄糖30 mmol/L);(4)高糖+SB216763组(D-葡萄糖30 mmol/L+10μmol/L SB216763)。采用Western印迹法和实时荧光定量PCR法(real-time PCR)检测标志蛋白E-钙黏连蛋白(E-cadherin)、肾损伤分子(kidney injury molecule-1,KIM-1)、肿瘤坏死因子(tumor necrosis factor,TNF-α)和GSK-3β和Tristetraprolin(TTP)的m RNA和蛋白的表达;给予HK-2细胞SB216763以下调GSK-3β的表达,并对比上述指标的变化;采用免疫荧光法观察GSK-3β和TTP,E-cadherin和KIM-1在HK-2细胞的表达和定位。结果与正常糖浓度组相比,高糖组肾小管上皮细胞损伤明显,表现为E-cadherin m RNA表达减少,TNF-α和KIM1 m RNA表达增加。与正常糖浓度组相比,GSK-3β蛋白表达增加,高糖组TTP m RNA及蛋白表达减少(P<0.05)。高糖条件下抑制GSK-3β的表达,细胞损伤减轻(P<0.05)。结论高糖环境下人肾小管上皮细胞的损伤与GSK-3β表达增多有关,其机制可能是高糖环境下GSK-3β通过下调肾小管上皮细胞TTP的表达促进细胞炎症反应。

Objective To investigate the role of glycogen synthase kinase 3β（ GSK-3β） in the injury of renal tubular epithelial cells（ HK-2） under high glucose conditions and to explore the possible mechanism of its involvement in diabetic nephropathy. Methods HK-2 cells were cultured in 37 ℃ and 5% CO2 incubator and randomly divided into four groups：（1）normal glucose concentration（ 5. 6 mmol/L D-glucose）;（2）mannitol control（ 5. 6 mmol/L D-glucose ＋ 24. 4 mmol/L mannitol）;（3） high glucose group（ 30 mmol/L D-glucose）;（4） high glucose ＋ SB216763（ 30 mmol/L D-glucose ＋ 10 μmol/L SB216763）. Western bolt and real-time quantitative PCR（ real-time PCR） were used to detect the expression of HK-2 cells marker,E-cadherin,kidney injury molecule-1（ KIM-1）,tumor necrosis factor（ TNF-α）,GSK-3β and tristetraprolin（ TTP）. To reduce the expression of GSK-3β,SB216763 were used and the changes of the above indicators were detected. Besides,the expression and localization of GSK-3β and TTP,E-cadherin and KIM-1 were observed by immunofluorescence. Results HK-2 cells obviously damaged with down-regulated E-cadherin,elevated KIM-1 and TNF-α after exposed to high glucose. The differences were statistically significant（ P〈0. 05）. Compared with normal glucose group,The expression of GSK-3β increased,accompanied with the level of TTP m RNA and protein decreased in HK-2 cells treated with high glucose（ P〈0. 05）. In addition,inhibition of GSK-3β expression could alleviate the injury and inflammatory response of HK-2 cells caused by high glucose（ P〈0. 05）.Conclusion GSK-3β plays a crucial role in high glucose induced injury via down-regulating the expression of TTP of HK-2 cells,which resulted in the inflammatory response of cells.