The Molecular Mechanism Of Nocantharidin Inhibits Renal Interstitial Fibrosis Through Down Regulating PP2Ac

ObjectiveTo investigate whether NCTD inhibiting renal interstitial fibrosis is related to its role of reduction of PP2A expression. To study the relationship between the antifibrotic effect of NCTD and its role of PP2A inhibition regulating the dephosphorylation of Smad3linker region.Methods1. A proximal tubular cell line, HK-2cells, were stimulated by5ng/ml TGF-β1and treated with2.5μg/ml NCTD for24h after transfected with or without plasmid pEGFP-N1-PP2Ac or SD11-PP2Ac. Real-time RT-PCR and Western blot analysis were used to detect the mRNA and protein expression of PP2Ac, fibronectin, collagen I, a-SMA and E-cadherin.2. HK-2cells were pretreated with2.5μg/ml NCTD for24h and stimulated by5ng/ml TGF-β1for1h. Indirect immunofluorescence techical were used to analysis the expression of pSmad3-L(Ser204) and pSmad3-L(Ser208). Western blot analysis were used to detect the protein expression of PP2Ac、pSmad3-L(Ser204) and pSmad3-L(Ser208).Results1. Real-time RT-PCR and Western blot analysis showed that in TGF-β1stimulated HK-2cells, while the expression of PP2Ac mRNA and protein were significantly increased, fibronectin, collagen I and a-SMA mRNA and protein expression were also increased, and the E-cadherin mRNA and protein expression was decreased. And a further increase of PP2Ac expression caused by incubation with TGF-β1after transfection of pEGFP-N1-PP2Ac led to an aggravation of interstitial fibrosis. However, NCTD effectively downregulated the expression of PP2Ac, moreover, blocked the PP2Ac-mediated fibronectin, collagen I and a-SMA induction and E-cadherin suppression.2. Plasmid SD11-PP2Ac inhibited PP2Ac mRNA and protein expression in HK-2cells, and accompanied with which, fibronectin, collagen I and a-SMA mRNA and protein expression were also downregulated, while the E-cadherin mRNA and protein expression was upregulated, thus abrogated the fibrotic lesions induced by TGF-β1, as detected by real-time RT-PCR and Western blot analysis. However, compared with simple SD11-PP2Ac shRNA transfection, cells transfecting with SD11-PP2Ac shRNA and incubated with NCTD showed no further inhibition of fibronectin, collagen I and a-SMA mRNA and protein level or further increasing of E-cadherin, indicating that NCTD had no significant anti-fibrosis effect after PP2Ac was knocked down.3. Immunofluorescense showed that in normal HK-2cells, there was little expression of pSmad3-L(Ser204) and pSmad3-L(Ser208). And after stimulated by TGF-β1for1h, the expression of pSmad3-L(Ser204) and pSmad3-L(Ser208) in nucleus were elevated. However, NCTD could promote the nucleoprotein expression of pSmad3-L(Ser204) and pSmad3-L(Ser208). Western blot analysis showed that in TGF-β1stimulated HK-2cells, the expression of PP2Ac protein was upregulated, meanwhile, the expression of pSmad3-L(Ser204) and pSmad3-L(Ser208) protein in the nucleus were elevated. Moreover, both of which were further upregulated when treated with NCTD.Conclusion1. NCTD inhibiting renal interstitial fibrosis was related to its role of reduction of PP2Ac expression.2. The antifibrotic effect of NCTD may be related to its role of PP2Ac inhibition regulating the dephosphorylation of Smad3linker region.