中文摘要：

目的 证实去甲斑蝥素（NCTD）具抗肾间质纤维化的作用,其抗肾间质纤维化的机制与靶向抑制蛋白磷酸酶2Ac（PP2Ac）对Smad3中间连接区（Smad3-L）的去磷酸化修饰有关.方法 常规培养人肾小管上皮细胞（HK-2）,转染PP2Ac shRNA质粒后,细胞分为5组：（1）空白组;（2）TGF-β1（5μg/L）组;（3） TGF-β1 ＋NCTD （2.5 mg/L）组;（4）TGF-β1＋PP2Ac shRNA组;（5）TGF-β1＋PP2Ac shRNA＋NCTD组.采用实时荧光定量PCR和Western印迹检测PP2Ac、纤维连接蛋白（FN）、胶原蛋白Ⅰ （Col-Ⅰ）、α-平滑肌肌动蛋白（α-SMA）和E-钙黏蛋白（Ecadherin） mRNA及蛋白的表达.然后将HK-2细胞随机分为3组：（1）空白组;（2）TGF-β1组;（3）TGF-β1＋NCTD组,采用免疫荧光检测pSmad3-L（Ser204）和pSmad3-L（Ser208）在HK-2细胞的分布,Western印迹检测HK-2细胞核蛋白pSmad3-L（Se204）和pSmad3-L（Ser208）的表达.结果 （1）TGF-β1促进HK-2细胞PP2Ac表达,同时上调FN、Col-Ⅰ和α-SMA的表达,下调E-cadherin 的表达.NCTD和PP2Ac shRNA均抑制PP2Ac的表达,下调FN、Col-Ⅰ和α-SMA表达,上调E-cadherin的表达.但NCTD与小干扰RNA共同作用对PP2Ac表达的抑制,以及对TGF-β1诱导的上述指标的改变程度同单纯PP2Ac小干扰RNA干预组无明显差异.（2）TGF-β1刺激HK-2细胞核内pSmad3-L（Ser204）、pSmad3-L（Ser208）表达明显增多,NCTD干预使pSmad3-L（Ser204）、pSmad3-L （Ser208）在细胞核内表达进一步增多.结论 NCTD通过抑制PP2Ac介导的Smad3-L区去磷酸化,发挥抗肾间质纤维化作用.

英文摘要：

Objective To investigate the inhibition of interstitial fibrosis by NCTD is related to the dephosphorylation of Smad3 linker region mediated by the inhibition of PP2Ac.Methods HK -2 cells were cultured and devided into 5 groups：（1） normal control group;（2） TGF-β1 group （5 μg/L）;（3） TGF-β1 ＋NCTD group （2.5 mg/L）;（4） TGF-β1 ＋PP2Ac shRNA group;（5）TGF-β1 ＋PP2Ac shRNA＋ NCTD group.Real-time PCR and Western blot were used to detect the expression of PP2Ac,FN,Col-Ⅰ,α-SMA and E-cadherin.Additionally,the HK-2 cells were assigned to three groups：（1） normal control group;（2） TGF-β1 group;（3） TGF-β1 ＋NCTD group.Immunofluorescence were used to analysis the distribution of pSmad3-L（Ser204） and pSmad3-L（Ser208）.Western blot analysis were used to detect the protein expression of pSmad3-L（Ser204） and pSmad3-L（Ser208）.Results （1） TGF-β1 stimulated the expression of PP2Ac in HK-2 cells,increased the expression of FN,Col-Ⅰ and α-SMA,and decreased the expression of E-cadherin.Both NCTD and PP2Ac shRNA could inhibit PP2Ac expression accompanied with the downregulation of FN,Col-Ⅰ and α-SMA,and upregulation of E-cadherin.However,compared with PP2Ac shRNA transfected group,cells transfecting with PP2Ac shRNA and incubated with NCTD showed no obvious differences on the relief of the above indicators induced by TGF-β1 in HK2 cells.（2）The expression of pSmad3-L（Ser204） and pSmad3-L（Ser208） in the nucleus of HK2 cells stimulated by TGF-β1 was significantly elevated.The expression of pSmad3-L（Ser204） and pSmad3-L（Ser208） in the nucleus was further upregulated when treated with NCTD.Conclusions NCTD has anti-fibrosis effect and it may be due to the inhibition the dephosphorylation of Smad3 linker region mediated by the inhibition of PP2Ac.