Study On Protective Effects And Mechanisms Of Huai Qi Huang Against Renal Fibrosis

Part ? Curative Effect Observation of Traditional Chinese Medicine Huai Qi Huang in the Treatment of Renal Fibrosis on Unilateral Ureteral Obstrction RatsObjective To evaluate the protective effects of Huai Qi Huang on patholpgical changes in obstructed kidney of UUO rats.Methods We used a well-established rat model of renal fibrosis, unilateral ureteral obstruction (UUO). Totally 50 rats were randomlly assigned into sham operation group(n=10),UUO group(n=10) and the treatment group (1.5 g/kg/d,2.25 g/kg/d and 3.0 g/kg/d; n=10 in each dose group). HQH was intragastrically given to UUO rats from day 3 after obstruction at dosages of 1.5,2.25 or 3.0 g/kg/d respectively, but rats in sham and UUO groups were administered by gavage with normal saline. Half of each group was sacrificed on day 7 post-ligation, and the other half on day 14. The obstructed kidney was removed and the cortex was spliced into several parts. Some of them were fixed in 4% paraformaldehyde for histological studies, and others were stored in liquid nitrogen for protein and RNA extractions. HE and Masson's staining method was performed to detect the pathological changes in renal interstitium. Immunohistochemical assay for ?-SMA on paraffin-embeded sections was used to visualize the accumulated myofibroblasts in tubular interstitial areas. Western blot analysis of ?-SMA and TGF?1 protein level was carried out, and quantitative PCR analysis showed levels of CTGF and fibronectin mRNA.Results Fibrous deposition in interstitium was observed by HE and Masson's staining after tubular obstruction. Some fibrosis was evident on day 7 after obstruction, while severe fibrosis was shown on day 14. Immunohistochemical results showed that renal fibrosis is accompanied by amounts of ?-SMA+ cells. Obstruction time encouraged more and more aggressive such cells. High dose and medium dose Huai Qi Huang exerted anti-fibrosis and inhibitory effect on ?-SMA+ cells accumulation induced in UUO rats on day 7 and 14. Western Blot revealed that both high and medium dose Huai Qi Huang could down-regulate ?-SMA protein levels. Huai Qi Huang suppressed mRNA increase of CTGF and FN in obstructed kidney tissue of UUO rats.Conclusions Huai Qi Huang attenuates UUO renal fibrosis in rats with good inhibitory effect on myofibroblasts accumulation.Part ? Effect of Huai Qi Huang on Epithelial-Mesenchymal transition in renal tubular epithelial cellsObjective To study the effect of Huai Qi Huang on the phenotypic transition of NRK-52E cells from epithelium to myofibroblast.Methods E-cadherin protein in obstructed kidneys of UUO rats was detected using IHC and Western Blot assay. NRK-52E cells were sub-cultured on cover slips. The cells were stimulated with TGF-?1 (10 ng/ml) at gradient concentration (60,120,180 ug/ml) of Huai Qi Huang, subsequent to starvation treatment with serum-free medium for 12h. Protein abundance of E-cadherin and ?-SMA was determined at 6h,12h,24h and 48h using Western Blot assay. Antibodies for proteins E-cadherin and ?-SMA were used to localize the protein by indirect immunofluorescence in NRE-52E cells at 24h.Results In sham rats, E-cadherin was highly expressed in renal tubular epithelial cells. While in UUO rats, part of renal tubular epithelial cells lost more E-cadherin on day 14 than on day 7 after obstruction. Treatment of NRK-52E cells with 10 ng/ml TGF-?1 showed down-regulated expression of E-cadherin but up-regulated expression of ?-SMA. In vivo experiment of UUO rats revealed that Huai Qi Huang sustained E-cadherin protein expression in obstructed kidneys. In vitro experiment demonstrated that Huai Qi Huang could directly suppress TGF-?1-induced phenotype changes of NRK-52E cells and maintain their epithelial phenotype. Huai Qi Huang increased E-cadherin and decreased ?-SMA in time and dose dependent manner in NRK-52E cells simulated with TGF-?1.Conclusions Huai Qi Huang inhibits EMT of renal tubular cells in UUO rats, and directly suppresses EMT of NRK-52E cellsPart ? Molecular mechanism of Huai Qi Huang in Epithelial-Mesenchy mal transition of renal epithelial cellsObjective To further explore the molecular regulation mechanism of Huai Qi Huang on miR-200a/ZEBs signal axis in renal tubular epithelial cells.Methods Using quantitative real-time PCR to detect miR-200a, ZEB1 and ZEB2 mRNA expression levels in kidneys of all rats, we analyzed and compared the relative expression of each detecting parameter. Similarly, miR-200a, ZEB1 and ZEB2 mRNA were examined in 10 ng/ml TGF-?1-induced NRK-52E cells at 3h.Results The qPCR results showed that ZEB1 mRNA expression was almost undetectable and ZEB2 mRNA expression very low in sham operation rats, but their expression were significantly increased in obstructed kidneys on days 7 and 14. The miR-200a expression was significantly down-regulated by three quarters in obstructed kidney in UUO rats compared to sham operation rats on day 14. Different dosages of Huai Qi Huang up-regulated miRNA-200a expression on days 7 and 14 after obstruction. UUO rats treated with Huai Qi Huang showed a significant decline in ZEBs mRNA expression. The miRNA-200a was down-regulated at 3h in NRK-52E cells stimulated with TGF-?1, while ZEBs up-regulated. Huai Qi Huang at concentration of 60,120,180 ug/ml restored miR-200a expression in TGF-?1-stimulated NRK-52E cells. However, Huai Qi Huang dramatically inhibited ZEBs mRNA expression in dose dependent manner.Conclusions Huai Qi Huang inhibits EMT of renal tubular epithelial cells through miR200a/ZEBs axis.