黄芪甲苷通过调控p38 MAPK信号通路改善肾小管间质纤维化

Astragaloside Ⅳ attenuates renal tubulointerstitial fibrosis by inhibiting p38 MAPK signaling

目的 探讨黄芪甲苷（AS-Ⅳ）改善肾小管间质纤维化的作用和p38丝裂原活化蛋白激酶（MAPK）信号通路对其的调控作用.方法 体内建立小鼠单侧输尿管梗阻（UUO）模型,治疗组（AS-Ⅳ,n=10）连续7d给予AS-Ⅳ （20 mg·kg-1 ·d-1）灌胃,假手术组（Sham,n=8）及模型组（UUO,n=10）小鼠同时给予等量溶剂灌胃.分别于术后第7、14天取双侧肾组织,观察小鼠一般情况及病理改变.体外采用转化生长因子β1 （TGF-β1）（10 ng/ml）刺激人近端小管上皮细胞（HK-2）,分别加入不同浓度的AS-Ⅳ（0、50、100、200 μg/ml）或SB203580（10 μmol/L）进行干预,24 h后收集细胞.采用Western印迹及实时定量PCR法分别检测TGF-β1、纤维连接蛋白（FN）、Ⅳ型胶原蛋白（ColⅣ）、α平滑肌肌动蛋白（α-SMA）的表达,并观察p38 MAPK信号蛋白磷酸化的情况.结果 体内实验发现：AS-Ⅳ治疗组小鼠肾小管间质纤维化改善,肾组织TGF-β1、FN、Col Ⅳ、α-SMA mRNA和蛋白的表达下降（均P＜0.05）.体外实验结果显示：TGF-β1刺激后HK-2细胞FN、Col Ⅳ、α-SMA的表达较正常组增多（均P＜0.05）;AS-Ⅳ干预后可致其表达下降（均P＜ 0.05）;其抑制作用与SB203580作用效果相近.体内和体外实验结果均提示：AS-Ⅳ可抑制p38 MAPK的磷酸化水平（均P＜0.05）.结论 AS-Ⅳ可改善肾小管间质纤维化,减少TGF-β1分泌,抑制TGF-β1诱导的FN、Col Ⅳ和α-SMA的表达,其作用机制可能与AS-Ⅳ抑制p38 MAPK信号活化有关.

Objective To investigate the effect of astragaloside Ⅳ （AS-Ⅳ） on renal tubulointerstitial fibrosis and its regulation on p38 MAPK signaling.Methods In vivo,UUO model with renal tubulointerstitial injury was constructed.Mice in AS-Ⅳ group were orally administrated AS-Ⅳ 20 mg· kg 1 · d-1 for 7 days after operation,and mice in other groups were administrated the equal volume vehicle.Bilateral kidneys were collected in 7 and 14 days after operation.Transverse kidney slices were stained with Masson trichrome to evaluate the severity of renal tubule injury.In vitro,normal human renal tubular epithelial cells （HK-2） were stimulated with recombinant TGF-β1 （10 ng/ml） and simultaneously treated with different concentrations of AS-Ⅳ （0,50,100,200 μg/ml） for 24 h.SB203580 （10 μmol/L） was also ultilized to pre-treat HK-2 cells for 1 h to inhibit phosphorylation of p38 MAPK signaling.The expression of FN,Col Ⅳ,and α-SMA were investigated by western blotting and real-time PCR.The expression of p-p38 MAPKs were also observed by Western Blotting.Results Astragaloside Ⅳ morphologically ameliorated renal tubulointerstitial fibrosis.The proteins and mRNA expression of FN,Col Ⅳ,α-SMA,and TGF-β1 were also increased significantly in UUO kidney tissues （all P ＜ 0.05）,which could be reversed by AS-Ⅳ administration （all P ＜ 0.05）.In vitro,the expression of FN,Col Ⅳ,and α-SMA were up-regulated by TGF-β1 after stimulating for 24 h （all P＜0.05）,which were decreased by AS-Ⅳ.The inhibition effect on FN and α-SMA were similar between AS-Ⅳ and MAPK inhibitor SB203580.AS-Ⅳ inhibited p-p38 MAPK signals both in vivo and in vitro.Conclusions AS-Ⅳ could attenuate renal tubulointerstitial fibrosis induced by UUO and TGF-β1 through reducing FN、Col Ⅳ、α-SMA expression in renal tubular cells.The mechanism of AS-Ⅳ protective effect might be associated with inhibition of p38 MAPK phosphorylation.