己酮可可碱联合骨髓间充质干细胞降低高糖诱导的肾小球系膜细胞纤维化倾向及糖基化终末产物聚集的体外研究

Pentoxifylline combined with mesenchymal stem cells inhibits TGF-β/SMAD signaling pathway and reduces advanced glycation end products aggregation in glomerular mesangial cells induced by high glucose

为探讨己酮可可碱(pentoxifylline,PTX)联合骨髓间充质干细胞(mesenchymal stem cell,MSC)对高糖条件下肾小球系膜细胞纤维化及糖基化终末产物(advanced glycation end product,AGE)聚集的影响,将体外高糖培养的小鼠肾小球系膜细胞分为6个组:正常组、损伤组、MSC组、MSC+PTX 0.1mmol/L组、MSC+PTX 0.3mmol/L组和MSC+PTX 1mmol/L组。MSC组将MSC和肾小球系膜细胞共培养。MSC+PTX 3组在MSC和肾小球系膜细胞共培养时分别加入0.1、0.3和1mmol/L的PTX。采用ELISA检测各组肾小球系膜细胞AGE水平,Western blotting检测各组肾小球系膜细胞中结缔组织生长因子(connective tissue growth factor,CTGF)、果蝇抗同源序列蛋白3(drosophila mothers against decapentaplegic 3,SMAD3)、SMAD7及转化生长因子β(transforming growth factorβ,TGF-β)的表达。结果显示,与正常组比较,损伤组肾小球系膜细胞的AGE水平显著升高(P<0.01)。与损伤组比较,MSC组、MSC+PTX 0.1 mmol/L组、MSC+PTX0.3mmol/L组和MSC+PTX 1mmol/L组肾小球系膜细胞的AGE水平显著降低(P<0.01),加入PTX后肾小球系膜细胞的AGE水平降低得更为显著,且呈现一定的浓度依赖性。与正常组比较,损伤组CTGF、SMAD3及TGF-β的蛋白表达量显著升高(P<0.01),SMAD7表达量显著降低(P<0.01)。MSC+PTX 0.1mmol/L组、MSC+PTX 0.3mmol/L组和MSC+PTX 1mmol/L组的CTGF、SMAD3和TGF-β蛋白表达量显著低于损伤组(P<0.05),但SMAD7蛋白表达量显著高于损伤组(P<0.01),且呈现PTX浓度依赖性。提示PTX联合MSC可能具有通过降低共培养肾小球系膜细胞中AGE的聚集以及多靶点抑制TGF-β/SMAD信号通路来抑制细胞纤维化的潜能,且呈现PTX浓度依赖性。

To investigate the effects of pentoxifylline(PTX)in combination with mesenchymal stem cells(MSC)on the fibrosis and aggregation of advanced glycation end products(AGE)in glomerular mesangial cells induced by high glucose,mouse mesangial cells were cultured in high glucose mediumin vitro and divided into 6groups:normal group,injury group,MSC group,MSC+PTX 0.1mmol/L group,MSC+PTX 0.3mmol/L group and MSC+PTX 1mmol/L group.In the three MSC+PTX groups,mesangial cells were co-cultured with MSC in medium containing different concentration of PTX.The levels of AGE in mesangial cells were detected by ELISA.The expressions of connective tissue growth factor(CTGF),drosophila mothers against decapentaplegic 3(SMAD3),SMAD7and transforming growth factorβ(TGF-β)in mesangial cells were detected by Western blotting.The results showed that the AGE levels of mesangial cells in the injured group were significantly higher than those in the normal group(P<0.01).Compared with the injured group,the AGE levels in mesangial cells of the MSC group,the MSC+PTX 0.1mmol/L group,the MSC+PTX 0.3mmol/L group and the MSC+PTX 1mmol/L group were significantly lower(P<0.01).The levels of AGE in mesangial cells were decreased more significantly after the addition of PTX and showed a concentra tion-dependent effect.Compared with the normal group,the protein expression levels of CTGF,SMAD3 and TGFβin the injured group were sigmificantly increased(P<0.01),and the expression of SMAD7 was significantly decreased(P<0.01).The expressions of CTGF,SMAD3 and TGFβin the MSC+PTX 0.1 mmol/L group,the MSC+PTX 0.3 mmol/L group and the MSC+PTX 1 mmol/L group were significantly lower than those in the injured group(P<0.05),and the expressions of SMAD7 were significantly higher than those in the injured group(P<0.01),all in a PTX concentration-dependent manner.These results suggest that PTX combined with MSC could reduce AGE aggregation in cocultured glomerular mesangial cells and potentially inhibit fibrosis by inhibiting TGFβ/SMAD signaling pathway in a concentration-d ependent manner.