牡丹皮对AGEs诱导的系膜细胞增殖及基底膜增厚的影响

Effect of Moutan Cortex on AGEs-induced mesangial cell proliferation and basement membrane thickening

目的:探讨牡丹皮对晚期糖基化终产物(advanced glycation end products,AGEs)诱导的系膜细胞增生及基底膜增厚的影响。方法:建立AGEs诱导的肾小球系膜细胞(MC)损伤模型,设置空白组[牛血清白蛋白(bovine serum albumin,BSA),200 mg·L-1],模型组(AGEs,200 mg·L-1),氨基胍(aminoguanidine,AG)阳性组(10 mmol·L-1),牡丹皮高、中、低剂量组(2×10-4,1×10-4,0.5×10-4g·mL-1)。采用四甲基偶氮唑蓝(MTT)法观察牡丹皮对损伤的MC的增殖影响;采用酶联免疫吸附测定法(ELISA)测定细胞上清液中纤维连接蛋白(fibronectin,FN)和Ⅳ胶原蛋白(typeⅣcollagen secretion,ColⅣ)的含量;采用Western blotting观察FN的表达;Real-time PCR检测ColⅣ的mRNA表达水平。结果:牡丹皮能抑制AGEs刺激的MC增殖以及FN和ColⅣ分泌;Western blotting表明牡丹皮下调MC分泌的FN表达;Real-time PCR结果说明牡丹皮对AGEs刺激下的MC分泌ColⅣ的mRNA表达有下调作用。结论:牡丹皮对AGEs培养条件下的MC具有一定的保护作用,牡丹皮能明显抑制基质中ColⅣ及FN的合成与分泌,抑制基底膜增厚,为糖尿病肾病的治疗提供实验依据。

Objective： To investigate the effect of Moutan Cortex on mesangial proliferation and basement membrane thickening induced by advanced glycation end products （AGEs）. Method： The glomerular mesangial ceils （MC） injury model was established by inducing by AGEs. The cell were divided into 6 groups ： the blank group （ BSA, 200 mg . L- 1 ）, the model group （ AGEs, 200 mg . L-1 ）, the positive control group （AG, 10 mmol . L-1）, and drug administration groups, namely the Moutan Cortex-treated high-dose group （2 × 10 ^-4 g . mL^-1 ）, the Moutan Cortex-treated medium-dose group （ 1 × 10^ -4 g . mL-1 ）, and the Moutan Cortex-treated lowdose group （0. 5 × 10 ^-4 g . mL-1 ）. The MTT method was performed to observe the effect of Moutan Cortex on the proliferation of MC. The content of fibronectin （FN） and collagen secretion IV （ Col IV ） in cell supernatant were detected by ELISA kits. The western blot analysis was carried out to observe the FN expression. The Real-time PCR analysis was applied to examine the Col IV mRNA expression. Result： AGEs significantly increased AGEs-induced MC proliferation and FN and Col IV secretion. The western blot analysis showed that MC could down-regulate the FN expression of MC secretion. According to the results of the real-time PCR assay, MC could down-regulate AGEs-induced MC secretion Col IV mRNA expression. Conclusion： MC had a certain protective effect on MC cultured under AGEs conditions. MC could remarkably inhibit the composition and secretion of Col IV and FN in matrix and the basement membrane thickening, and provide an experimental basis for the treatment of diabetic nephropathy.