【作者】 吴林；

【导师】 陶立坚；

【作者基本信息】 中南大学 ， 临床医学， 2013， 博士

【摘要】 第一章ZHC116抗UUO大鼠肾间质纤维化的疗效观察研究背景肾间质纤维化(Renal interstitial fibrosis, RIF)是指由多种原因引起的细胞外基质(Extracellular matrix, ECM)在肾间质的过度沉积,是多种慢性肾脏病(Chronic Kidney Disease, CKD)进展至终末期肾病(End-stage renal disease,ESRD)的病理基础和共同通路。探讨肾间质纤维化的发生机制、研发抗肾间质纤维化的药物对慢性肾脏疾病的控制具有重要意义。ZHC-116是本课题组设计的新一代抗纤维化新药。前期体外研究表明,ZHC116具有抗肾小管上皮细胞转分化(Epithelial mesenchymal transition,EMT)的作用,具有抗纤维化的潜能。目的观察ZHC116对单侧输尿管结扎(Unilateral Ureteral Obstruction, UUO)大鼠肾间质纤维化动物模型间质损伤、纤维化、Ⅲ型胶原表达的影响。方法建立UUO肾间质纤维化模型。SD大鼠(20只,每组5只)随机分为假手术组、UUO模型14天组、ZHC116(100mg/d)治疗组、吡非尼酮(500mg/d)治疗组；于UUO术后14天留取各组大鼠梗阻侧肾脏标本,用HE和Masson染色方法观察大鼠肾间质纤维化成模情况及药物疗效,免疫组化方法观察Ⅲ型胶原表达。结果UUO术后14天,模型组大鼠梗阻侧肾脏肾间质损伤指数、Masson染色评分以及Ⅲ型胶原表达比假手术组显著升高(p<0.05)；与模型组相比,ZHC116和吡非尼酮治疗均能显著降低UUO大鼠梗阻侧肾间质损伤评分、Masson染色评分和Ⅲ型胶原的表达(p<0.05)；与毗非尼酮治疗组相比,ZHC116治疗组肾间质Ⅲ型胶原阳性表达面积百分比显著减少(p<0.05)。结论ZHC116具有抗肾间质纤维化的作用,100mg/kg ZHC116疗效优于500mg/kg吡非尼酮。第二章ZHC116对AngⅡ刺激肾小管上皮细胞MAPK通路的影响研究背景肾素-血管紧张素-醛固酮系统(RAAS)的激活,尤其是血管紧张素Ⅱ (Angiotensin Ⅱ)的产生是肾间质纤维化和慢性肾脏病进展的重要介质。丝裂原活化蛋白激酶(Mitogen-activated protein kinase, MAPK)是参与多种细胞过程如分化、增殖、炎症、纤维化的重要信号转导通路。Ang Ⅱ可以诱导肾小管上皮细胞MAPK通路的活化,参与肾间质纤维化中的炎症、氧化应激、细胞增殖、致纤维化因子产生等发病机制。初步研究显示ZHC116具有抗肾间质纤维化的疗效,本部分将初步探讨ZHC116抗肾间质纤维化的作用机制。目的观察ZHC116对Ang Ⅱ刺激大鼠肾小管上皮细胞(NRK-52E) MAPK通路中细胞外信号调节激酶1/2(Extracellular signal-regulated protein kinase1/2, ERK1/2)通路的影响。方法培养NRK-52E细胞,设立正常对照组、Ang Ⅱ刺激模型组、AngⅡ+ZHC116治疗组、Ang Ⅱ+p-ERK抑制剂组。模型组予以Ang Ⅱ(10-7M)刺激15min; ZHC116治疗组予以ZHC116(50ug/ml)预处理24小时,并用Ang Ⅱ (10-7M)刺激15min；抑制剂组p-ERK抑制剂(PD98059,10μM)处1h,并用Ang Ⅱ(10-7M)刺激15min。同时收集各组细胞总蛋白,Western Blot方法检测p-ERK、ERK、β-actin的表达。结果Ang Ⅱ刺激NRK-52E细胞15min后,p-ERK/ERK蛋白表达比值(p<0.05)增高；ZHC116和p-ERK抑制剂能够显著降低p-ERK/ERK蛋白表达比值(p<0.05)。结论ZHC116可能通过抑制Ang Ⅱ诱导的ERK1/2磷酸化起到抗肾间质纤维化的作用。更多还原

【Abstract】 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.更多还原