| Part I The effect of ZHC116on renal interstitial fibrosis in a rat model of UUOBackground:Renal interstitial fibrosis is the common pathway of virtually all progressive kidney diseases. As the pathologic basis for the progression of CKD to end-stage renal diseases, RIF is characterized by the deposition of massive extracellular matrix in the renal interstitial. With the purpose of controlling the progression of chronic kidney disease, it is of great value to elucidate the mechanisms of renal fibrosis and develop new drugs with anti-fibrotic effect. Our team has recently discovered a new drug—ZHC116, with potential anti-fibrotic effect. Previous research has shown that ZHC116has the effect of anti-EMT and anti-fibrotic effect in vitro.Objective:To investigate whether ZHC116can ameliorate interstitial injury, interstitial fibrosis, and expression of collagen III in order to study the effect of ZHC116on renal interstitial fibrosis.Methods:20Sprague-Dawley rats were randomly divided into four groups:Sham operation group,14days of UUO model group, ZHC116(100mg/kg) treated group and pirfenidone (500mg/kg) treated group. SD rats were submitted to unilateral ureteral obstruction (UUO) and sacrificed at day14. The obstructed kidney tissue after ureteral obstruction at day14was stained by HE and Masson methods to observe the extend of interstitial fibrosis and the anti-fibrotic effect of ZHC116, and by immunochemistry of Collagen type III to examine the collagen content.Result:HE and MASSON staining revealed that the tubulointerstitial damage index and relative area of renal interstitial collagen III significantly increased in UUO models as compared with the sham-operated group (p<0.05). Compared with UUO group, treatment of ZHC116and pirfenidone significantly ameliorated interstitial damage and fibrosis, as well as the deposition of collage type Ⅲ(p<0.05)100mg/kg of ZHC116shows better effect of reducing the area of collagen type III than500mg/kg of pirfenidone.Conclusion:ZHC116significantly ameliorates renal interstitial damage and fibrosis.100mg/kg of ZHC116shows more anti fibrotic effect than500mg/kg of pirfenidone.Part II The effect of ZHC116on MAPK signaling ERK in Ang Ⅱ-stimulated NRK-52E cellsBackground:The activated renin-angiotensin-aldosterone system (RAAS), especially the production of angiotensin Ⅱ, is a key mediator in the progression of renal interstitial fibrosis and chronic kidney diseases. Mitogen-activated protein kinase is an important signal transduction pathway related to multiple cellular processes such as differentiation, proliferation, inflammation and fibrosis. Ang Ⅱ induce the activation of MAPK pathway in tubular cells, leading to its effect on mechanisms of renal interstitial fibrosis, such as inflammation, oxidative stress, proliferation of interstitial fibroblast and the production of pro-fibrotic cytokines. Previous research has shown the anti-fibrotic effect of ZHC116. The present study is objected to examine the mechanism of ZHC116’s anti-fibrotic effect in renal epithelial cells.Objective:Observing the effect of ZHC116on the expression of extracellular signal-regulated protein kinasel/2(ERK1/2) in normal rat tubular cell line (NRK-52E cells) stimulated with Ang II.Methods:In vitro experiments, NRK-52E cells were divided into normal control group, Ang II stimulated model group, ZHC116treatment group, and p-ERK inhibitor (PD98059,10μM) treatment group. ZHC116(50ug/ml) were given for24hours, p-ERK inhibitor were given for1hour. All group except normal control were stimulated with Ang Ⅱ (10-7M) for15min. western blot is used for the detection of the expression of p-ERK, ERK and β-actin.Result:In Ang-II stimulated NRK-52E cell, expression of p-ERK were significantly increased, as shown by the expression ratio of p-ERK to ERK (p<0.05). ZHC116treatment markedly attenuated the elevation of p-ERK/ERK (P<0.05). These responses were also attenuated by ZHC116and p-ERK inhibitor (PD98059).Conclusion:The anti-fibrotic effect of ZHC116probably depends on its inhibition of phosphorylation of ERK1/2. |
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