高糖对肾小管上皮细胞Toll样受体4表达的影响及安体舒通的干预作用

Effect of spironolactone on the expression of Toll-like receptor 4 in renal tubular epithelia cells exposed to high glucose

摘要:

目的：旨在探讨高糖对肾小管上皮细胞Toll样受体4（TLR4）表达的影响及安体舒通（spironolactone，SP）对其表达的干预作用。方法：体外培养肾小管上皮细胞（NRK-52E），分成低糖组（5 mmol/L葡萄糖的DMEM）、高糖组(25 mmol/L葡萄糖的DMEM)，SP组(25 mmol/L葡萄糖的DMEM+10-7mol/L安体舒通)。采用细胞免疫化学、RT-PCR、Western blot检测各组细胞TLR4蛋白和mRNA表达情况，同时取细胞培养上清液用ELISA法检测白介素-6（IL-6）、肿瘤坏死因子-α（TNF-α）水平。结果：高糖组培养6 h TLR4 mRNA出现表达增高，24 h仍高于低糖组；SP组TLR4 mRNA明显低于同时期高糖组的表达，但仍高于低糖组。高糖组培养24 h TLR4蛋白较低糖组升高，48 h TLR4蛋白表达进一步升高；SP组TLR4蛋白表达明显低于同时期高糖组表达，但仍高于低糖组。高糖组IL-6、TNF-α较低糖组明显升高；而SP组IL-6、TNF-α高于低糖组，但明显低于高糖组。结论：高糖促使NRK-52E细胞的TLR4的表达上调，TNF-α、IL-6等炎性因子表达升高，TLR4参与了糖尿病肾病的微炎症反应；安体舒通可通过下调TLR4表达水平，减少相关炎症介质的生成，这可能是其在糖尿病肾病中具有肾脏保护作用的机制之一。［中国当代儿科杂志，2010，12（4）：280-283］

Abstract:

OBJECTIVE: To study the expression of Toll-like receptor 4 (TLR4) in renal tubular epithelial cells exposed to high glucose and the effect of spironolactone on the TLR4 expression. METHODS: In vitro renal tubular epithelial cells (NRK-52E) were randomly exposed to DMEM culture solution with low glucose (5 mmol /L), high glucose (25 mmol/L) or 10-7 mol/L spironolactone plus 25 mmol/L glucose. Immunohistochemistry, RT-PCR and Western blot were used to determine TLR4 protein and mRNA expression. The levels of IL-6 and TNF-α in the cell culture supernatant were determined using ELISA. RESULTS: The expression of TLR4 mRNA in the high glucose group began to increase 6 hrs and remained at a higher level up to 24 hrs after exposure as compared with the low glucose group. The TLR4 mRNA expression in the spironolactone treatment group was significantly lower than that in the high glucose group, although it was higher than that in the low glucose group between 6 and 24 hrs after exposure. TLR4 protein expression increased significantly in the high glucose group 24 and 48 hrs after exposure compared with that in the low glucose group. The TLR4 protein expression in the spironolactone treatment group was lower than that in the high glucose group, but higher than that in the low glucose group. IL-6 and TNF-α expression in the supernatant from the NRK-52E cells in the high glucose groups increased significantly as compared with the low glucose group. The spironolactone treatment group had significantly reduced IL-6 and TNF-αexpression compared with the high glucose group. CONCLUSIONS: High glucose triggers an increase in the expression of TLR4 and inflammatory factors in NRK-52E cells. TLR4 may participate in the progress of diabetic nephropathy. Spironolactone can reduce expression of TLR4 and inflammatory factors, which might be attributed to one of the mechanisms of protection by spironolactone against diabetic nephropathy.［Chin J Contemp Pediatr, 2010, 12 (4):280-283］