高糖诱导下人肾小球系膜细胞AMPK、MPO、α-SMA的变化以及α-硫辛酸的干预作用

High glucose-induced AMPK and MPO and α-SMA changes of human mesangial cells and the effect of α-lipoic acid

目的探讨高糖诱导下人肾小球系膜细胞(HMCs)中腺苷酸活化蛋白激酶(AMPK)、炎症反应指标、细胞外基质的变化及α-硫辛酸的干预作用。方法传代培养的HMCs同步化后分组:正常对照组、高糖组、甘露醇对照组、高糖+α-硫辛酸处理组(α-硫辛酸浓度分别为50、100和200μmol/L),于实验0、12、24、48和72 h收集各组细胞及上清液,采用RT-PCR法和ELISA法检测各组细胞中AMPK mRNA及上清液中AMPK、髓过氧化物酶(MPO)、α-平滑肌肌动蛋白(α-SMA)的含量。结果高糖组人肾小球系膜细胞AMPK mRNA及AMPK蛋白的表达较正常对照组明显下降(P<0.01),MPO、α-SMA蛋白的表达明显增加(P<0.01)。α-硫辛酸各处理组AMPK mRNA及AMPK蛋白表达比高糖组有明显增加(P<0.01),MPO、α-SMA蛋白的分泌较处理前明显降低(P<0.05)。结论高糖能够抑制人肾小球系膜细胞中AMPK的表达,诱导MPO(炎症反应指标)、α-SMA(系膜细胞活化从而分泌细胞外基质增多的指标)的表达,抗氧化剂α-硫辛酸可以上调高糖抑制的AMPK的表达,减少高糖刺激下HMCs中MPO、α-SMA蛋白的分泌,为糖尿病肾病的防治提供新的理论依据。

[ Objective] To investigate the changes of AMPK and inflammation index and extracellular ma- trix in the process of human mesangial cells （HMCs） induced by high glucose and the effect of α--lipoic acid. [Methods] Synchronized HMCs were divided into following groups： normal control group （5.5 mmol/L G）, High glucose group （30 mmol/L G）, mannitol group （5.5 mmol/L G with 24.5 mmol/L mannitol）, high glucose ＋ α-lipoic acid groups （30 mmol/L G with α-lipoic acid 50 μmol/L, 100 μmol/L, 200 μmol/L）. The supernatant and cells were collected at 0 h, 12 h, 24 h, 48 h and 72 h respectively, the concentration changes of the AMPK mRNA were determined by Real-time PCR method in the cells, then the ELISA method was used to check the protein expression of the MPO, α-SMA in the supernatant. [Results] AMPK mRNA and AMPK protein of the HMCs cultured under the high glucose expressed significantly lower than the ones of the HMCs in the normal group （P〈0.01）, and the MPO, α-SMA protein secretion was increased （P〈0.01）. After the α- lipoic acid pretreatment, compared with without treatment groups, AMPK mRNA expression and AMPK proteinsecretion were significantly increased （P〈0.01）, the MPO, α-SMA protein secretion was significantly reduced compared with that before （P 〈0.05）. [ Conclusion] High glucose can inhibit the expression of AMPK in HM- Cs, up-regulate the expression of MPO, α--SMA. A-lipoic acid can stimulate the expression of AMPK protein and reduce the expression of MPO,α-SMA induced by high glucose in HMCs, supporting a potential role in treatment of diabetic kidney disease.