阻断HMGB1及TLR4相关信号途径对慢加急性肝功能衰竭大鼠的影响

发布时间：2018-01-31 15:16

本文关键词： 高迁移率族蛋白B1 慢加急性肝功能衰竭 HMGB1单克隆抗体 Toll样受体肿瘤坏死因子吡咯烷二硫基甲酸盐慢加急性肝功能衰竭高迁移率族蛋白B1　出处：《武汉大学》2014年博士论文　论文类型：学位论文

【摘要】：第一部分：阻断 HMGB1对慢加急性肝功能衰竭大鼠的影响目的：慢加急性肝功能衰竭是一种以肝功能损害,以及肝细胞大量坏死为核心症状严重疾病,具有很高的死亡率。HMGB1是近年来发现的一种强大的炎症调节因子。本研究应用HMGB1单克隆抗治疗慢加急性肝功能衰竭大鼠,观察HMGB1单克隆抗体对慢加急性肝功能衰竭大鼠的保护作用。方法：大鼠慢加急性肝功能衰竭模型由人血白蛋白,D—氨基半乳糖以及LPS联合诱导。大鼠被随机分为3组,正常组,模型组以及HMGB1单克隆抗体治疗组。HMGB1单克隆抗体治疗组大鼠在模型诱导后12小时及24小时时间点由尾静脉注射HMGB1单克隆抗体200μg/kg,正常组及模型组注射等剂量的生理盐水作为对照。采用鲎试剂检测各组大鼠血清内毒素水平,应用ELISA方法检测了血清HMGB1, TNF-α, IFN-γ水平,同时检测了各组大鼠肝脏组织学及肝细胞凋亡情况,并且应用Western b1ot方法检测了各组大鼠肝织中HMGB1, TLR4和P65的表达情况。结果：HMGB1单克隆抗体能够明显降低血清中内毒素,HMGB1, TNF-α, IFN-γ水平,改善肝组织的病理损害,减少肝细胞的凋亡,同时下调肝组织中HMGB1,TLR4, P65的表达量。结论：HMGB1单克隆抗体对慢加急性肝功能衰竭大鼠具有保护作用,即使是在模型诱导后24小时仍然有效。此效应可能与TLR4信号途径相关。第二部分：TLR4单克隆抗体对慢加急肝功能衰竭大鼠的影响目的：慢加急性肝功能衰竭是临床上常见危重症之一,TLR4信号途径及炎症因子HMGB1在其发病过程中发挥重要作用。本研究采用TLR4单克隆抗体对慢加急性肝功能衰竭大鼠进行治疗,观察TLR4信号途径被阻断后对慢加急性肝功能衰竭大鼠及炎症因子HMGB1的影响。方法：将32只雄性Wistar大鼠随机分为正常组、模型组、IgG对照组及TLR4单克隆抗体治疗组。除正常组大鼠外,其余每组大鼠采用人血白蛋白,D-氨基半乳糖以及LPS联合诱导的大鼠慢加急性肝功能衰竭模型。IgG对照组大鼠在急性攻击24小时后,予以尾静脉注射兔抗鼠IgG100μg/kg作为对照。TLR4单克隆抗体治疗组大鼠在急性攻击24小时后,予以尾静脉注射兔抗鼠TLR4单克隆抗体100μg/kg。模型组大鼠尾静脉注射等量的生理盐水作为对照。观察TLR4单克隆抗体对大鼠肝脏组织学、血清ALI、TNF-α、IFN-γ和HMGB1水平以及肝组织中HMGB1水平的影响。结果：与正常组相比,模型组大鼠肝组织明显破坏,炎症细胞浸润明显,血清中ALT、TNF-α、IFN-γ和HMGB1水平明显升高(P0.05)。TLR4单克隆抗体能够明显改善慢加急性肝功能衰竭大鼠肝脏组织病理学损害,降低血清中ALT、TNF-a、IFN-γ和HMGB1水平,并且降低肝组织中HMGB1水平(P0.05)。而与模型组相比,以上指标在IgG对照组中无明显差异(P0.05)。结论：TLR4单克隆抗体能够保护慢加急性肝功能衰竭大鼠并减少HMGB1胞外释放以及肝组织中HMGB1的产生。TLR4单克隆抗体,慢加急性肝功能衰竭,高迁移率族蛋白B1 第三部分：阻断NF-κB和TNF-α对慢加急性肝功能衰竭大鼠的影响目的：在慢加急性肝功能衰竭的发病机制中,炎症介质HMGB1发挥着重要作用。而HMGB1主要通过TLR4-NF-κB信号通路在发挥其作用。而在TLR4-NF-κB信号通路中,NF-κB和TNF-α又是两个处于中心地位的炎症因子。本研究的目的在于探索NF-κB和TNF-α被阻断后对于慢加急性肝功能衰竭大鼠的影响。方法：本研究应用人血白蛋白,D一氨基半乳糖及脂多糖联合诱导大鼠慢加急性肝功能衰竭模型,并采用TNF-α多克隆抗体(TNF-α抑制剂)及吡咯烷二硫基甲酸盐(PDTC, NF-κB抑制剂)治疗肝功能衰竭大鼠,观察TNF-α多克隆抗体和PDTC对慢加急性肝功能衰竭大鼠的影响。本研究进一步采用PMA诱导U937细胞成为巨噬细胞,并应用LPS刺激,观察了PDTC对巨噬细胞的影响。结果：TNF-α和NF-κB抑制剂都能够有效降低肝脏和外周血中HMGB1水平以及血清中炎症因子的水平,并能够抑制TLR4-NF-KB信号通路。TNF-α抑制剂还能够延长慢加急性肝功能衰竭大鼠的存活时间提高生存率,相比之下,NF-κB抑制剂PDTC却加速了慢加急性肝功能衰竭大鼠的死亡。为了进一步研究PDTC在炎症反应中的作用,我们进一步检测了PDTC在U937细胞中的作用,结果显示PDTC能够提高U937细胞的活性,并且降低HMGB1水平以及血清中炎症因子的水平,并能够抑制TLR4-NF-κB信号通路。结论：TNF-α抗体和PDTC都能够抑制TLR4-NF-κB信号通路的激活,减少炎症因子的释放,抑制炎症反应。TNF-α抗体对慢加急性肝功能衰竭大鼠具有保护作用,而PDTC不能保护肝功能衰竭大鼠。
[Abstract]:Part one: blocking
Effect of HMGB1 on rats with chronic acute liver failure
Objective: acute on chronic liver failure is a damage to the liver function, and massive necrosis of liver cells as the core symptoms of the serious disease, with high mortality of.HMGB1 is a powerful inflammatory regulatory factor found in recent years. In this study, using HMGB1 monoclonal antibody treatment of acute on chronic liver failure in rats, to observe the protection effect of HMGB1 monoclonal antibody in acute on chronic liver failure in rats.
Methods: the rat model of acute on chronic liver failure by Human Albumin, D and LPS induced by D-galactosamine. Rats were randomly divided into 3 groups, normal group, model group and HMGB1 treatment group.HMGB1 monoclonal antibody monoclonal antibody treated rats in the model induced after 12 hours and 24 hours from the time point intravenous injection of HMGB1 monoclonal antibody 200 g/kg, normal saline group and model group injected dose by limulus reagent as control. The serum levels of endotoxin, ELISA method was used to detect serum HMGB1, TNF- alpha, gamma IFN- level were detected and liver cell apoptosis in liver tissue was the rat, and used Western b1ot method to detect the liver of rats in the fabric HMGB1, expression of TLR4 and P65.
Results: HMGB1 monoclonal antibody could significantly reduce the levels of endotoxin, HMGB1, TNF-, IFN-, and improve the pathological damage of liver tissue, reduce the apoptosis of liver cells, and down regulate the expression of HMGB1, TLR4 and P65 in liver tissue.
Conclusion: HMGB1 monoclonal antibody has protective effect on rats with acute on chronic liver failure, even after 24 hours of induction. This effect may be related to TLR4 signaling pathway.
The second part: the effect of TLR4 monoclonal antibody on rats with chronic acute liver failure
Objective: acute on chronic liver failure is one of the common clinical critical disease, TLR4 signal pathway and inflammatory factor HMGB1 play an important role in its pathogenesis. This study used in acute on chronic liver failure rats were treated with TLR4 monoclonal antibody, observe the effect of acute on chronic liver failure rats and inflammatory factors HMGB1 is blocking TLR4 signaling.
Methods: 32 male Wistar rats were randomly divided into normal group, model group, IgG control group and TLR4 monoclonal antibody treatment group. In addition to the normal group rats, the rats in each group by Human Albumin, D- and LPS induced by D-galactosamine in rats with chronic model of acute liver failure.IgG control group rats in the acute attack after 24 hours of intravenous injection of Rabbit anti rat IgG100 monoclonal antibody g/kg as the control.TLR4 treated rats in the acute attack after 24 hours of saline to intravenous injection of Rabbit anti rat TLR4 monoclonal antibody 100 g/kg. model rats tail vein injected as control. Observe TLR4 monoclonal antibody to rat liver tissue, serum ALI, TNF- alpha, gamma and IFN- affect the level of HMGB1 and HMGB1 level in liver tissue.
Results: compared with normal group, the liver tissue of model rats obviously damage, inflammatory cell infiltration, serum ALT, TNF- alpha, IFN- gamma and HMGB1 levels were significantly higher (P0.05).TLR4 monoclonal antibody can significantly improve the pathological liver tissue of rats with acute on chronic liver failure damage, reduce the levels of ALT, TNF-a IFN-, gamma and HMGB1 levels, and reduce the level of HMGB1 in liver tissue (P0.05). Compared with the model group, the above indexes had no significant difference in the IgG control group (P0.05).
Conclusion: TLR4 McAb can protect rats from chronic acute liver failure and reduce HMGB1 extracellular release and the production of HMGB1 in liver tissue,.TLR4 monoclonal antibody, slow acute liver failure, high mobility group protein B1.
The third part: the effect of blocking NF- kappa B and TNF- alpha on rats with chronic acute liver failure
Objective: in the pathogenesis of acute on chronic liver failure, inflammatory mediators play an important role. HMGB1 and HMGB1 mainly through the TLR4-NF- B pathway to play its role in the TLR4-NF- B pathway, NF- kappa B and TNF- alpha is two at the center of the inflammatory factor. The purpose of this study is to explore the effect of acute on chronic liver failure rats NF- kappa B alpha and TNF- was blocked.
Methods: This study used Human Albumin D, a D-galactosamine and lipopolysaccharide induced rat acute on chronic liver failure model, and using TNF- alpha polyclonal antibody (TNF- alpha inhibitor) and pyrrolidine two Sbased formate (PDTC, NF- kappa B inhibitor) treatment of hepatic failure in rats, observe the effect alpha TNF- polyclonal antibody and PDTC in acute on chronic liver failure rats. This study further by PMA induced U937 cells into macrophages, and the application of LPS stimulation, the effect of PDTC on macrophages.
Results: TNF- and NF- alpha kappa B inhibitor can effectively reduce liver and peripheral blood HMGB1 levels and serum levels of inflammatory factors, and TLR4-NF-KB signal pathway of.TNF- alpha inhibitors could also prolong acute on chronic liver failure rats survival time and improve the survival rate, compared with NF- kappa B inhibitor PDTC it accelerates ACLF death in rats. In order to further study the role of PDTC in inflammation, we further examined the role of PDTC in U937 cells, the result shows that PDTC can improve the activity of U937 cells, and decrease the level of HMGB1 and serum levels of inflammatory factors, which can inhibit the expression of TLR4-NF- the B signaling pathway.
Conclusion: both TNF- alpha antibody and PDTC can inhibit the activation of TLR4-NF- kappa B signaling pathway, reduce the release of inflammatory factors and inhibit the inflammatory response..TNF- alpha antibody has protective effects on rats with chronic acute liver failure, while PDTC can not protect liver failure rats.

【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R575.3

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