高糖状态下肾小管上皮细胞肌肌醇加氧酶表达变化对TGF-β1表达的影响

Effects of the abnormal expression of myo-inositol oxygenase (MIOX) on the expression of TGF-β1 in rat renal tubular epithelial cells under high glucose

目的:探讨高糖状态下肌肌醇加氧酶(MIOX)表达变化对肾小管上皮细胞TGF-β1表达的影响。方法:①在体外培养大鼠肾小管上皮细胞NRK-52E,应用不同浓度高糖刺激NRK-52E细胞48h,观察MIOX、TGF-β1 mRNA和蛋白表达的改变;②观测不同浓度TGF-β1刺激NRK-52E细胞48h后MIOX mRNA和蛋白表达的变化;③应用针对MIOX基因的ASODN干涉NRK-52E细胞抑制MIOX基因表达后再用高糖刺激,观测其对TGF-β1 mRNA和蛋白表达的影响。结果:①不同浓度(15、30mmol/L)高糖刺激NRK-52E细胞48h后MIOX、TGF-β1 mRNA和蛋白表达增加;②不同浓度(1、5、10、25mmol/L)TGF-β1刺激NRK-52E细胞48h后MIOX mRNA和蛋白表达无明显变化;③针对MIOX基因的ASODN干涉NRK-52E细胞使MIOX基因表达下降,同时NRK-52E细胞TGF-β1 mRNA和蛋白的表达也下降。结论:本研究结果提示,高糖刺激肾小管上皮细胞能使MIOX、TGF-β1 mRNA和蛋白表达增加,而且MIOX表达异常可能通过影响细胞因子TGF-β1的表达在糖尿病肾病中起作用。

Objective：To investigate the effects of the abnormal expression of myo-inositol oxygenase （MIOX） on the expression of TGF-β1 in rat renal tubular epithelial cells under high glucose. Methods： ① In vitro, rat renal tubular epithelial cell line NRK-52E was maintained in dulbecco＇s modified eagle＇s medium （DMEM） containing 5.6 mmol/L glucose and 10% fetal bovine serum （FBS）. Then the cells were treated with D-glucose at different concentrations （ranging from 15 to 30 mmol/L） for 48 h, D-mannitol served as control. Gene expressions were analyzed at mRNA level by semi-quantitative RT-PCR and at protein level by Western blot. ② The expressions of MIOX mRNA and protein were observed in NRK-52E cells treated with TGF-β1 at different concentrations （ranging from 1 to 25 mmol/L） for 48 h. ③ Transient transfection of the normal NRK-52E cells with anti-sense oligodeoxynucleotide （ASODN） for 8 h was performed to knockdown MIOX gene using LipofectamineTM 2000. The cells were then treated with D-glucose at concentration of 30 mmoL/L for 48 h. Gene expressions were analyzed at mRNA level by RT-PCR and at protein level by Western blot. Results： ① In vitro, the expression of MIOX mRNA and protein in NRK-52E cells was induced by high D-glucose in a dose-dependent manner. Paralleled increases were observed with respect to TGF-β1 （P 〈 0.05）. ② There was no significant difference of the MIOX mRNA and protein levels in NRK-52E cells between each group after the stimulation of TGF-β1 at different concentrations （ranging from 1 to 25 mmol/L,P 〉 0.05）. ③ Transient transfection of the normal NRK-52E cells with ASODN significantly attenuated the increase of TGF-β1 expression in normal NRK-52E cells induced by high D-glucose （P 〈 0.05）. Conclusion：High glucose can induce the expression of MIOX and TGF-β1 gene in NRK-52E cells in a dose-dependent manner. MIOX might play a role in the progression of diabetic nephropathy in rat renal tubular epithelial cells through the activation of cytokine TGF-β1.