Aim: To investigate the mechanism of action of Fuzheng Huayu recipe (FZHY) and vitamin E (Vit E) against renal interstitial fibrosis related to transforming growth factor-beta1 (TGF-beta1) mediated tubular epithelial-to-mesenchymal transition.
Materials and methods: Renal interstitial fibrosis was induced by administration of HgCl(2) at a dose of 8 mg/kg body weight once a day for 9 weeks. Rats were randomly divided into four groups: normal, model, FZHY, and Vit E group. Rats in the latter two groups were treated with the FZHY recipe and Vit E respectively. HK-2 cells were treated with TGF-beta1 for 24h, followed by incubation with either SB-431542 (a potent and specific inhibitor of TbetaR-I kinase) or FZHY drug-containing serum for another 24h. Hyp content in rat kidney tissue was assayed with Jamall's method and collagen deposition in kidney was visualized using Masson stain. Protein expression of TGF-beta1, TbetaR-I, Smad2, p-Smad2, Smad3, and p-Smad3 was analyzed by Western blotting. Protein expression and the location of Smad3 in kidney was assayed by immunohistochemistry, E-cadherin, cytokeratin 18 (CK-18), alpha-SMA and TGF-beta1 by immunofluorescent stain.
Results: FZHY and Vit E inhibited renal collagen deposition and reduced Hyp content significantly. They upregulated E-cadherin protein expression and down-regulated the protein expression of alpha-SMA, TGF-beta1, p-Smad2, p-Smad3, and TbetaR-I. Lastly, they inhibited the nuclear translocation of Smad3 in fibrotic kidney tissue. FZHY drug-containing serum significantly upregulated the expression of CK-18 and down-regulated the expression of alpha-SMA, TbetaR-I, p-Smad2/3 in TGF-beta1 stimulated HK-2 cells.
Conclusion: The mechanism of action of FZHY and Vit E against renal interstitial fibrosis is related to the reversal of tubular EMT induced by TGF-beta1.
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