【作者】 陈斌；

【导师】 黄培志；

【作者基本信息】 复旦大学 ， 内科学， 2010， 硕士

【摘要】 [目的]本研究旨在通过观察还原型谷胱甘肽(reduced glutathione, GSH)对盲肠结扎和穿刺术(cecal ligation and puncture, CLP)诱导的脓毒症大鼠急性肝损伤时肝组织热休克蛋白70 (heat shock protein 70, HSP70)、toll样受体4(toll like receptor 4, TLR4)和肿瘤坏死因子a (tumor necrosis factor-a,TNF-α)水平的影响,探讨GSH对脓毒症大鼠急性肝损伤的保护作用及其可能的机制。[方法]清洁级雄性Sprague Dawley (SD)大鼠采用盲肠结扎和穿刺术制备大鼠脓毒症急性肝损伤模型。实验大鼠随机分成假手术组、模型组、GSH干预组(每组各24只),每组大鼠再随机分为0h、2h、6h、24h 4个亚组(每组各6只)。假手术组(Sham组)：行剖腹术但不结扎和穿刺,术后立即经尾静脉注射0.1mL生理盐水；模型组(CLP组)：行盲肠结扎和穿刺术,术后立即经尾静脉注射0.1mL生理盐水；GSH干预组(GSH+CLP组)：行盲肠结扎和穿刺术,术后立即经尾静脉注射GSH 300 mg/kg (0. 1mL)。每组大鼠在CLP术后0h、2h、6h、24h分别采集血标本和肝组织标本,标本用于检测血清肝功能和肝组织HSP70、TLR4、TNF-α水平。术后6h、24h肝组织标本经HE染色观察病理改变。[结果]1、大鼠血清肝功能(TBIL DBIL、ALT、AST)水平：术后6h和24h CLP组明显升高,术后6h分别为3.82±0.48μmol/L、2.05±0.36μmmol/L、59.3±18.1U/L、129.5±21.1U/L,与Sham组相比(分别为2.45±0.29 p mol/L、1.05±0.21μmol/L、27.5±4.2U/L、47.2±7.8U/L),CLP组明显高于Sham组,差异有统计学意义,P<0.05；GSH干预组术后6h分别为2.82±0.52μmol/L、1.48±0.31μmol/L、39.0±13.8U/L、84.5±17.9U/L,明显低于CLP组,差异有统计学意义,P<0.05。2、大鼠肝组织TLR4和TNF-α水平：CLP组术后2h即显著升高,6h达高峰(分别为22.14±3.04ng/mL、98.84±13.57pg/mL),明显高于Sham组(分别为7.40±0.65ng/mL、12.65±1.96pg/mL),差异有统计学意义,P<0.05：GSH干预组术后6h分别为15.23±2.06ng/mL、54.24±9.53pg/mL,明显低于CLP组,差异有统计学意义,P<0.05。3、大鼠肝组织HSP70水平：CLP组术后2h、6h、24h分别为44.17±5.72ng/mL、51.71±7.03ng/mL、36.45±7.13ng/mL,均轻度高于Sham组(分别为17.09±2.11ng/mL、16.36±2.39ng/mL、16.33±1.84ng/mL),差异有统计学意义,P<0.05；GSH干预组术后2h、6h、24h分别为68.37±8.21ng/mL、82.52±10.61ng/mL、77.05±14.85ng/mL,均明显高于CLP组,差异有统计学意义,P<0.05。4、大鼠肝组织病理改变：CLP组术后6h和24h HE染色显示肝窦充血、肝细胞肿胀、炎症细胞浸润、细胞变性坏死等损伤性改变。GSH干预组显示肝组织损伤性改变较CLP组明显减轻。[结论]脓毒症早期应用GSH对脓毒症致急性肝损伤有保护作用,其机制可能与通过降低肝组织TLR4水平和提高肝组织HSP70水平,继而使肝组织TNF-α产生减少,从而减轻炎症反应对肝组织的损伤有关。更多还原

【Abstract】 ObjectiveBased on the potent antioxidant effects of reduced glutathione (GSH), we investigated the putative protective role of GSH by the level of toll-like receptor 4(TLR4), heat shock protein 70 (HSP70), and that of tumor necrosis factor-α(TNF-a) in liver against sepsis-induced hepatic injury in rats.MethodsSepsis-induced hepatic injury was induced by cecal ligation and puncture (CLP) in male Sprague-Dawley rats. Rats were randomly divided into 3 groups (n=24), which were sham group, CLP group and GSH treatment group. Rats of GSH treatment group were intravenously administered with GSH (300mg/kg) after the operation and the rats of other two groups were treated with same dose saline. Rats of each group were sacrificed at the time point of Oh,2h,6h and 24h after the operation (n=6). The samples of blood and liver tissues were harvested at each time point. TLR4, HSP70 and TNF-a in liver tissues were examined by ELISA kits and histological alterations by HE staining. The serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), direct bilirubin (DBIL) and total bilirubin (TBIL) activities were determined by auto analyzer.Results1、The serum levels of TBIL, DBIL, ALT and AST in rats:Compared with sham group, the levels of TBIL, DBIL, ALT and AST in CLP group were highly increased at 6h and 24h after operation (the serum levels of TBIL, DBIL, ALT and AST at 6h after operation were 3.82±0.48μmol/L, 2.05±0.36μmol/L,59.3±18.1U/L,129.5±21.1U/L in CLP group vs. 2.45±0.29μmol/L,1.05±0.21μmol/L,27.5±4.2U/L,47.2±7.8U/L in sham group, P<0.05). Compared with CLP group, the levels of TBIL, DBIL, ALT and AST in GSH treatment group (6h) were dominantly decreased (the serum levels of TBIL, DBIL, ALT and AST at 6h after operation were 2.82+0.52μmol/L,1.48μ0.31μmol/L,39.0±13.8U/L,84.5±17.9U/L in GSH treatment group vs.3.82±0.48μmol/L,2.05±0.36μmol/L, 59.3±18.1U/L,129.5+21.1U/L in CLP group, P<0.05).2、The levels of TLR4 and TNF-a in liver tissues: Compared with sham group, the levels of TLR4 and TNF-αin liver tissues in CLP group were highly increased at 2h and got the peak at 6h after operation (the levels of TLR4 and TNF-a at 6h after operation were 22.14±3.04ng/mL, 98.84±13.57pg/mL in CLP group vs.7.40±0.65ng/mL,12.65±1.96pg/mL in sham group, P<0.05). After GSH intervention, the levels of TLR4 and TNF-a in liver tissues were highly decreased at 6h after operation compared with CLP group (the levels of TLR4 and TNF-a at 6h after operation were 15.23±2.06ng/mL,54.24±9.53pg/mL in GSH treatment group vs.22.14±3.04ng/mL,98.84±13.57pg/mL in CLP group, P<0.05).3、The levels of HSP70 in liver tissues:Compared with sham group, the levels of HSP70 in liver tissues in CLP group were mildly increased at 2h,6h and 24h after operation (44.17±5.72ng/mL,51.71±7.03ng/mL, 36.45±7.13ng/mLinCLPgroupvs.17.09±2. 11ng/mL,16.36±2.39ng/mL, 16.33±1.84ng/mL in sham group, P<0.05). After GSH intervention, the levels of HSP70 in liver tissues were highly increased at 2h,6h and 24h after operation compared with CLP group (68.37±8.21ng/mL, 82.52±10.61ng/mL,77.05±14.85ng/mL in GSH treatment group vs. 44.17±5.72ng/mL,51.71±7.03ng/mL,36.45±7.13ng/mL in CLP group, P <0.05).4、Histopathological injuries were observed significantly in liver tissues in CLP group at 6h and 24h, characterized by inflammatory cells infiltration, profound cellular degeneration and severe vascular congestion. The histopathological changes of liver tissues in GSH treatment group were much milder than that of the CLP group.ConclusionThese data revealed that GSH could exert protective effects on sepsis-induced hepatic injury. And these beneficial effects appeared partly due to decrease TLR4 levels and increase HSP70 levels in liver tissues and thereby attenuating the production of TNF-a in liver tissues. Thus, supplementing antiseptic hepatic injury treatment with GSH may be beneficial in the clinical setting.更多还原