【作者】 李相军；

【导师】 李才；

【作者基本信息】 吉林大学 ， 病理学与病理生理学， 2007， 博士

【摘要】 单侧输尿管阻塞(UUO)动物模型是经典的肾间质纤维化模型,因而成为国内外学者首选的动物模型。目前,银杏叶制剂主要用于心、脑血管疾病的防治,但对肾脏纤维化的防治作用尚未见报道。TGF-β是肾质纤维化过程中最重要的生长因子,CTGF被认为是TGF-β的下游递质,具有较特异的促组织纤维化效应。组织型谷氨酰转移酶(tTG)能够使细胞外基质(ECM)蛋白成分之间发生交联,抵抗胶原降解,因此,有关其与肾间质纤维化关系的研究逐渐引起学者的重视。UⅡ除具有调节血管舒缩作用外,还有许多非血流动力学效应,但有关UⅡ及其受体与肾纤维化关系的研究迄今尚未见报道。本研究应用免疫组化、RT-PCR、Western blot等方法,从体内、体外两方面探讨银杏叶提取物对大鼠肾间质纤维化的防治作用,并对其抑制肾间质纤维化的机制作一初步探讨。本研究结果证明,GBE可延缓UUO大鼠肾脏功能减退,抑制ECM成分在肾间质中积聚,从而减轻肾间质纤维化程度;tTG在UUO大鼠肾间质中表达显著增加,通过促进UUO大鼠肾间质中ECM成分交联,抵抗降解而促进肾间质纤维化;在肾间质纤维化中,间质成纤维细胞合成能力增加,并通过自分泌和或旁分泌作用,刺激TGF-β、CTGF等致纤维化因子介导肾间质(主要是成纤维细胞)ECM合成和分泌,从而加速肾间质纤维化过程;UⅡ及其受体(GPR14)在肾间质纤维化中发挥重要作用,GBE可通过降低肾间质中二者mRNA及蛋白表达水平而延缓肾间质纤维化进程。更多还原

【Abstract】 Renal interstitial fibrosis (RIF), which is the common pathway and main pathology foundation for almost all kidney diseases, is the common turnover of various kinds of chronic renopathy and its primary pathological changes are renal tubule atrophy and interstitial fibrosis. UUO is one of the most comman methods for researching renal interstitial fibrosis.Ginkgobioba, of which essential components are flavonoids and terpene lactone, is a kind of traditional Chinese medicine and has been utilized for thousand years and possess important medicine value. At present, ginkgobiobaextract (GBE) is mainly used on cardial and cerebrovascular diseases. Recent research showed that GBE had some preventive and therapeutic effects on hepatic fibrosis. However, the effects of GBE on renal fibrosis have not been reported.Transforming growth factor-beta (TGF-β) is presumed one of the most important cytokines in the course of renal interstitial fibrosis. As a superactive fibrosis factor, TGF-βplays a part in many link of origination and development of renal interstitial fibrosis. connective tissue growth factor (CTGF), having a close relationship with TGF-β, is considered downstream transmitter of TGF-βand participates the fibrosis effects of TGF-β. Due to its specific effects, CTGF is hopeful to become a new treatment target of fibrosis process.Tissue transglutaminase (tTG) is a Ca2+ dependent transamidation enzyme. After activated, tTG may cause a majority of ECM protein cross-linking including fibronectin, laminin and collagen, which form fast structure to resist degradation. At present, the relationship between tTG and renal interstitial fibrosis is evoking researchers’attention, but whether GBE can restrain or alleviate renal interstitial fibrosis through depressing tTG expression is unknown.Urotensin II (U II) is considered as constantly the most strong vasoconstriction peptides, nevertheless U II still possesses vasodilator function. Following the penetrating research, researchers discovered that U II still have much non-hemodynamics effects besides blood vessel regulating effects, such as promoting cell proliferation, regulating insuline release. The research about the relationship between U II as well as its acceptor and renal fibrosis and the inhibition effects of GBE on U II expression in interstitial fibrosis kidney still is not reported.This study investigated the preventive and therapeutic effects of GBE on rat renal interstitial fibrosis both in vivo and in vitro. In addition, the mechanisms involved in origination and development of renal interstitial fibrosis also been approached. The main research contents is composed of①to investigate the protection of GBE on renal function and structure in UUO rats;②to observe the influence of GBE on synthesis and secretion of renal ECM in UUO rats;③to approach the inhibition of GBE on synthesis and secretion of renal TGF-βas well as CTGF in UUO rats;④to identify the role of tTG in renal interstitial fibrosis and the inffluence of GBE on it;⑤to identify initially the relationship between U II and renal interstitial fibrosis and the inhibition of GBE on synthesis and secretion of U II.Main results were as follows:1. Comparing with the pathological changes of UUO group rats, GBE and benazepril group rats present alleviated renal lesions, in which interstitial fibrosis is not obvious and there is no obvious change in renal tubular epithelial cell.2. After rats were administered 2 weeks, NAG enzyme activity, urine creatinine, serum creatinine, urea nitrogen and 24 h total urinary protein of GBE group rats were lower than those of UUO group rats (P<0.05). After rats were administered 4 weeks, NAG enzyme activity, serum creatinine, urea nitrogen and 24 h total urinary protein of GBE group rats were lower than those of UUO group rats (P<0.05); urine creatinine of benazepril group rats is lower obviously than that of UUO group rats (P<0.05); and creatinine clearance rate of GBE group rats were higher obviously than that of UUO group rats (P<0.05).3. After rats were treated 2 weeks, Col I, Col IV, FN,α-SMA, TGF-β, CTGF, GPR14, and tTG expression in renal interstitium of GBE and benazepril group rats were lower than those of UUO group rats (P<0.05), but there was no obvious difference on renal U II expression between GBE and UUO group rats. After rats were treated 4 weeks, Col I, col IV, FN,α-SMA, TGF-β, and GPR14 expression in renal interstitium of GBE and benazepril group rats were lower than those of UUO group rats (P<0.01), but there was no obvious difference on renal tTG and CTGF expression between GBE and UUO group rats.4. After rats were treated 2 weeks, TGF-β, CTGF, GPR14, and tTG mRNA expression in renal interstitium of GBE and benazepril group rats were lower than those of UUO group rats (P<0.05). After rats were treated 4 weeks, TGF-β, CTGF, GPR14, and tTG mRNA expression in renal interstitium of GBE and benazepril group rats were lower than those of UUO group rats (P<0.05).5. Fibroblasts from renal interstitium were cultured and treated with AngⅡand GBE.①Comparing with C group (normal nutrient medium group), M group (containing 10-6 mol/L AngⅡin culture medium) had reinforced cell proliferation activity at 24 h and 48 h (P<0.05).Treated for 24 h, GBE3 group (containing 50 mg/L GBE in culture medium) and GBE4 group (containing 25 mg/L GBE in culture medium) had lower cell proliferation activity than M group at 24 h (P<0.05). Treated for 48 h, GBE2 group (containing 100 mg/L GBE in culture medium) also showed obvious inhibition activity on fibroblast proliferative (Comparing with M group, P<0.05). ②Treated by Ang II for 24 h and 48 h, increased Col I, CTGF, TGF-β, U II and tTG contents were found in M group compared with normal control group (P<0.05). However, TGF-βand U II contents were increased only at 24 h compared with normal control group (P<0.05).Treated by GBE for 24 h, GBE1, GBE2 and GBE3 had lower Col I, CTGF, TGF-β, and U II contents than M group (P<0.05). Treated fibroblasts by GBE for 48 h, Col I and TGF-βcontents in each GBE concentration group were all lower than M group (P<0.01), CTGF contents in each GBE concentration group (except GBE4 groups) were lower than M group (P<0.01), and U II as well as tTG contents in each GBE concentration group had no significant difference with M group.③Treated by different conditioned medium for 48 h, fibroblasts in M group had increased TGF-β, CTGF, U II, GPR14 and tTG mRNA expression (Comparing with normal control group, P<0.05), fibroblasts in different concentration of GBE had restrained TGF-β, CTGF, GPR14 and tTG mRNA expression (Comparing with M group, P<0.05) and the fibroblasts in GBE2 and GBE3 groups had lower U II mRNA expression than M group.④Treated by different conditioned medium for 48 h, fibroblasts in M group had increased U II, GPR14 and tTG protein expression (Comparing with normal control group, P<0.05) and fibroblasts in GBE2 and GBE3 groups had lower U II mRNA expression than M group. Comparing with M group fibroblasts, fibroblasts in different concentration GBE groups had lower GPR14 and tTG protein expression (P<0.05).Conclusions were as follows:1. For UUO rats, GBE can delay renal function deterioration and lessen renal interstitium fibrosis through inhibiting ECM accumulation in renal interstitium. Therefore, GBE can protect to renal function and structure of obstructive nephropathy.2. tTG expression increases significantly in renal interstitium of UUO rats, which resist ECM degradation through collogen cross-linking and promote renal interstitium fibrosis. GBE can blockade tTG expression and its activity transglutaminase in some measure, which may provide new methods for prevention and treatment of renal interstitium fibrosis.3. U II as well as its receptor interferes in the expression of TGF-βand CTGF in renal interstitium of UUO rats, which indicate U II as well as its acceptor can induce renal interstitium ECM synthesis and secretion through stimulating fibrosis factors such as TGF-βand CTGF and then accelerate renal interstitium fibrosis. GBE can blockade UⅡas well as its acceptor expression in some measure and then alleviate renal interstitium fibrosis, which may be the new drug for prevention and treatment of renal interstitium fibrosis.4. GBE can implement its renal protection through multiple mechanisms to delay renal interstitium fibrosis.更多还原