【作者】 慕宁；

【导师】 傅志仁； 丁国善； 王全兴；

【作者基本信息】 第二军医大学 ， 外科学， 2008， 博士

【摘要】 研究背景肝移植已经成为治疗多数急、慢性终末期肝病的首选方法。但是存在于每一例肝移植过程中的缺血再灌注损伤(IRI)仍然是原发性移植物功能不良或无功能的主要原因。供体的短缺使得移植中心不断扩大使用边缘供体的尺度,包括老龄供体、无心跳供体、小体积供肝及脂肪肝。而边缘供体带来的基本问题仍然是IRI。因此,最大限度的减轻IRI不但可以增加适当的供体,也可以使更多的病人从肝移植手术中康复。而实现这一愿望的首要步骤就是更加充分的理解I/R损伤的机制。肝脏是对缺血再灌注损伤最敏感的器官之一。肝窦内皮细胞、Kupfer cells和中性粒细胞的活化以及氧自由基的产生在缺血再灌注损伤的病理过程中具有重要作用。氧自由基和炎症细胞因子、粘附分子进一步促进中性粒细胞的活化与聚集,介导脂质过氧化损伤等过程,最终导致肝组织损伤。1997年发现的Toll样受体系统(TLRs)在感染和炎症性疾病中扮演重要角色。其中的TLR-4信号通路被认为在心脏、肾脏和肝脏的IRI病理生理过程中居主导地位。TLR-4分子存在于所有肝实质、非实质细胞上,每种肝细胞群体都拥有完整的TLR-4信号通路。激活的肝脏非实质细胞(尤其Kupffer细胞)通过TLR-4信号通路在肝脏热缺血再灌注损伤中发挥重要作用。TLR-4的激活引出NF-κB激活,进一步触发TNF-a、IL-1、IL-6等炎症因子的大量产生。因为TLR-4信号通道在肝脏缺血再灌注损伤中的重要作用,通过抑制该信号通路达到减轻肝脏IRI的目的就显得前景诱人。HO-1是热休克蛋白32成员,肝脏HO-1最初由Kupffer细胞产生,作为TLR-4的配体之一,通过阻抑TLR-4信号通路产生较强的抗氧化、抗炎症功能从而起到保护肝脏IRI的作用。另一方面,细胞因子信号转导抑制分子1(Supressors of cytokine signalingl,SOCS1)是一种负性调节因子。能通过负性调控JAK-STAT信号途径而抑制ILs、IFN-γ、GH等多种细胞因子的信号转导途径。SOCS1还参与其它信号途径的调节,如TNF-a、LPS等。SOCS1的过表达在体外能明显减轻TNF-a引起的肝细胞凋亡,而TNF-a被认为是TLR-4活化的标志。提示SOCS1可能是负调节TLR-4及其信号转导系统的内源性因子之一。研究显示IL-10能够促进SOCS蛋白的产生,尤其是SOCS1和SOCS3。研究者在包括肝脏的许多缺血再灌注损伤模型中都能观察到IL-10的抑制炎症、保护组织的作用。因此如有药物能促进体内IL-10表达,则可能通过SOCS1负调节信号通道在肝脏IRI中发挥保护作用。落新妇甙(Astilbin)是从土茯苓的乙醇提取液中分离得到的3个二氢黄酮醇甙之一。黄酮类化合物有较强的抗氧化作用,可作为递氢体清除氧自由基,从而在抗炎、减轻缺血再灌注损伤等方面发挥作用。研究证实其具有利尿、镇痛、抗炎及减轻心肌细胞缺血再灌注损伤的作用。有研究报道落新妇甙能够通过干预氧自由基和脂质过氧化物生成,明显减轻四氯化碳(CC14)引起的肝损伤;在刀豆球蛋白A(Con A)诱导肝炎后,落新妇甙能够显著降低TNF-a的生成和肝脏Kupffer细胞的增殖,并且落新妇甙在接触性皮炎模型中能显著促进IL-10和SOCS1和SOCS3的表达,减轻炎症反应。基于上述研究背景,本课题进一步研究:1)落新妇甙对肝脏热缺血再灌注损伤的保护效果,包括肝功能、肝脏病理、SOD、MDA、MPO的检测;2)落新妇甙对肝脏缺血再灌注损伤模型中TLR-4、HO-1、NF-κB、TNF-a等分子蛋白和mRNA水平表达的影响,探讨其抑制炎症信号通路的分子机制;3)落新妇甙对IL-10、SOCS1蛋白和mRNA表达的影响,探讨其促进抑炎信号通路的分子机制。通过上述研究,为以后在同种异体小鼠肝移植模型中研究落新妇甙对缺血再灌注损伤保护,免疫抑制效果及机制进行基础准备。第一部分落新妇甙对肝脏热缺血再灌注损伤的干预作用目的:探讨落新妇甙在肝脏热缺血再灌注损伤中对肝功能和肝组织的保护效果。方法:C57BL/6小鼠随机分为4组(n=8):假手术组(Sham)、模型组(I/R)、落新妇甙小剂量(10mg/kg)干预组和落新妇甙大剂量(40mg/kg)干预组。缺血前24小时和1小时干预组小鼠腹腔注射分别给予10或40mg/kg的落新妇甙,建立肝左、中叶70%部分肝缺血再灌注模型,模型组和假手术组给予同样体积的生理盐水。小鼠肝脏左叶缺血90min、再灌注6h后各实验组采集血液和肝脏组织样本。检测血清中ALT活性作为肝功能损伤的指标,同时用ELISA检测肝组织中MPO、SOD和MDA含量。肝脏组织病理学检测。结果:与模型对照组相比,小、大剂量落新妇甙干预组血清ALT含量均明显下降(P＜0.01),大剂量干预组较小剂量组更明显(P＜0.01);落新妇甙大剂量干预后肝脏组织病理学表现明显改善;落新妇甙干预后肝组织MPO、MDA含量较模型对照组明显下降(P＜0.01),大剂量干预组较小剂量组更明显(P＜0.01);而SOD含量明显上升(P＜0.01),大剂量干预组较小剂量组明显(P＜0.05)。结论:落新妇甙干预能有效改善小鼠肝脏热缺血再灌注损伤引起的肝功能和肝脏病理损害;落新妇甙干预能够减轻小鼠肝脏热缺血再灌注损伤后中性粒细胞的聚集浸润和活化,从而减轻炎症反应;落新妇甙能减少缺血再灌注后氧自由基的产生,减轻脂质过氧化引发的组织损伤。第二部分落新妇甙对肝缺血再灌注损伤TLR-4信号通路的抑制作用目的:通过观察落新妇甙对肝脏热缺血再灌注损伤中炎症信号通路的影响,探讨其缺血再灌注保护作用的分子机制。方法:C57BL/6小鼠随机分为4组(n=5):假手术组(Sham)、模型组(I/R)、落新妇甙小剂量(10mg/kg)干预组和落新妇甙大剂量(40mg/kg)干预组。缺血前24小时和1小时干预组小鼠腹腔注射分别给予10或40mg/kg的落新妇甙,建立肝左、中叶70%部分肝缺血再灌注模型,模型组和假手术组给予同样体积的生理盐水。小鼠肝脏左叶缺血90min、再灌注6h后各实验组采集血液和肝脏组织样本。检测血清中TNF-a活性,Westernblot检测肝组织中TNF-a、TLR-4、NF-κB和HO-1蛋白含量,半定量RT-PCR检测上述分子mRNA表达情况。结果:落新妇甙小、大剂量干预后血清TNF-a含量较I/R对照组均明显下降(P＜0.01),且呈现剂量—效益关系(P＜0.01);落新妇甙小、大剂量干预组肝组织中TNF-a、TLR-4、NF-κB蛋白表达与I/R模型对照组比较均渐次降低,与半定量RT-PCR结果相符(小剂量干预组P＜0.05;大剂量干预组P＜0.01);而落新妇甙小、大剂量干预组肝组织中HO-1蛋白表达与I/R模型对照组比较均渐次升高,亦与半定量RT-PCR结果相符(小剂量干预组P＜0.05;大剂量干预组P＜0.01)。结论:落新妇甙干预能降低缺血再灌注损伤引起的血清TNF-a水平升高以及TNF-a蛋白及mRNA在肝组织中的高表达,从而减轻由TNF-a介导的进一步损伤;落新妇甙干预能降低IRI肝组织中TLR-4蛋白、mRNA和NF-κB蛋白的高表达,显示其对TLR-4炎症信号通路具有抑制作用。落新妇甙干预能促进IRI肝组织HO-1蛋白和mRNA的表达,显示其对TLR-4通路的抑制与促进HO-1的表达密切相关。第三部分落新妇甙对肝缺血再灌注损伤SOCS1、IL-10表达的影响目的:通过观察落新妇甙对肝脏热缺血再灌注损伤中抑炎症信号通路的影响,进一步探讨其缺血再灌注保护作用的分子机制。方法:C57BL/6小鼠随机分为4组:假手术组(Sham)、模型组(I/R)、落新妇甙小剂量(10mg/kg)干预组和落新妇甙大剂量(40mg/kg)干预组。缺血前24小时和1小时干预组小鼠腹腔注射分别给予10或40mg/kg的落新妇甙,建立肝左、中叶70%部分肝缺血再灌注模型,模型组和假手术组给予同样体积的生理盐水。小鼠肝脏左叶缺血90min、再灌注6h后各实验组采集血液和肝脏组织样本。Westernblot检测肝组织中SOCS-1、IL-10蛋白含量,半定量RT-PCR检测上述分子mRNA表达情况。结果:落新妇甙小、大剂量干预组肝组织中SOCS-1、IL-10蛋白表达与I/R模型对照组比较均渐次升高,与半定量RT-PCR结果相符(小剂量干预组P＜0.05;大剂量干预组P＜0.01)。结论:落新妇甙干预能促进缺血再灌注损伤肝组织中IL-10蛋白及mRNA在的高表达,从而起到抑制炎症的作用;落新妇甙干预能促进缺血再灌注损伤肝组织SOCS1蛋白和mRNA的表达,显示其可能通过上调SOCS1负调节通路产生抑制炎症的作用。更多还原

【Abstract】 Experimental Study on the molecular mechanism of the effect of astilbin on liver warm ischemia-reperfusion injuryLiver transplantation has become the method of choice for treatment of the majority of acute and chronic end-stage liver disease.However,ischemia-reperfusion injury(IRI) existing in every case of liver transplantation is still the main reason of primary graft dysfunction or non-function.The shortage of donor leads to the growing use of the marginal donor in most transplant centres,including the aging donor,non-heart-beating donors,small size and fatty liver donor,while the basic problem of the marginal donor is still IRI.So,reducing IRI maximatily would not only increase the appropriate donors,but also allow more patients recovery from liver transplant surgery.And to achieve this desire, firstly,we should understand the mechanisms of I/R injury more fully.Liver is one of the most sensitive organs to ischemia-reperfusion injury.The activation of sinusoidal endothelial cells,Kupfer cells and neutrophil and the formation of oxygen free radicals plays an important role in the pathological process of ischemia-reperfusion injury.Oxygen free radicals and inflammatory cytokines,adhesion molecules further promote neutrophil activation and aggregation,mediate lipid peroxidation injury process, which eventually lead to liver damage.The Toll-like receptors(TLRs) found in 1997 play an important role in the disease of infection and inflammation.The TLR-4 signaling pathway was considered to be dominated in the pathophysiological process of heart,kidney and liver IRI.TLR-4 molecule exists in all of hepatic parenchyma,non-parenchymal cells,each group of liver cells has a complete TLR-4 signaling pathway.Activated hepatic non-parenchymal cells(especially Kupffer cells) play an important role in the liver warm ischemia-reperfusion injury through TLR-4 signaling pathway.Activated TLR-4 leads to the activation of NF-κB,which further triggers great increase of inflammatory cytokines such as TNF-a,IL-1,IL-6,et al.Because of the important role of TLR-4 signaling pathway in liver ischemia-reperfusion injury,the purpose of reducing liver IRI by inhibiting this signaling pathway is of enticing prospects. HO-1 is a heat shock protein 32 members.Liver HO-1 was first produced by Kupffer cells. As one of the TLR-4 ligands,it has strong antioxidant,anti-inflammatory function thus protecting liver IRI by inhibiting TLR-4 signaling pathway.On the other hand,SOCS1 is a negative regulator molecule,which inhibit ILs,IFN-γ, GH,and other cytokine signal transduction pathway through negative regulation of JAK-STAT signaling pathway.SOCS1 also involves in the regulation of other signaling pathways,such as TNF-a,LPS.The over-expression of SOCS1 in vitro can significantly reduce the hepatocytes apoptosis induced by TNF-a,while TNF-a is considered TLR-4 activation marker,implying that SOCS1 may be one of the endogenous negative regulators of TLR-4 signal transduction systems.Studies show that IL-10 can promote the formation of SOCS protein,in particular the SOCS1 and SOCS3.The role of inhibiting inflammation and protecting organizations of IL-10 can be observed in many models of ischemia -reperfusion injury,including liver.So if drugs can promote IL-10 expression in vivo,it could play a protective role in the liver IRI through regulating SOCS1 negative signal pathway.Astilbin is one of the three flavanonols isolated from the ethanol extract of rhizoma. Flavonoids have strong function of antioxidant and can be used as delivery of hydrogen to scavenging oxygen free radicals,resulting in anti-inflammatory and reducing ischemia-reperfusion injury.Researches have confirmed its role of diuretic,analgesic, anti-inflammatory and reduce myocardial ischemia-reperfusion injury.As reported that astilbin could significantly reduce the liver damage induced by carbon tetrachloride(CC14) through the intervention of oxygen free radicals and lipid peroxide formation;In concanavalin A(Con A)-induced hepatitis,astilbin could significantly reduce TNF-a production and the proliferation of liver Kupffer cells.And in a contact dermatitis model, astilbin could significantly promote the expression of IL-10 and SOCS1 and SOCS3, reducing inflammatory response.Based on these research backgrounds,further study will be involved in this topic: 1)The protective effects of astilbin on liver warm ischemia-reperfusion injury,including liver function,liver pathology,SOD,MDA,the MPO detection;2) The effects of astilbin on the expression of TLR-4,HO-1,NF-κB,TNF-a protein and mRNA in the liver ischemia-reperfusion injury model,to investigate the molecular mechanisms of inhibiting inflammation signal pathway;3) The effects of astilbin on the expression of IL-10,SOCS1 protein and mRNA,to explore the molecular mechanism of promoting anti-inflammation signaling pathway.Based on these researches,to prepare for the further study on the mechanisms of protective and immunosuppressive effects of astilbin on liver ischemia and reperfusion injury,in the liver transplantation model of allogeneic mice.PARTⅠ.Effect of astilbin on liver warm ischemia-reperfusion injuryObjective:To investigate the protective effect of astilbin on liver function and hepatic tissues in the liver warm ischemia-reperfusion injury.Methods:C57BL/6 mice were randomly divided into four groups(n=8):sham-operated group(Sham),model control group(I/R),low dosage of astilbin treatment group(10mg/kg) and large dosage of astilbin(40mg/kg) treatment group.24 hours and one hour before Ischemia,treatment group mice were intraperitoneally injected 10 or 40 mg/kg astilbin.Then the hepatic ischemia-reperfusion model of 70 percent of liver,including the left and middle hepatic lobe,were established.The I/R model control group and the sham operation group were administered with the same volume of normal saline.After 90 min ischemia and 6h reperfusion of the partial hepatic lobe,blood and liver tissue samples were collected from the experimental groups.Serum ALT activity was detected as an indicator of liver function damage,and the content of MDA,MPO,SOD in liver tissues were detected by ELISA. Histopathology detection was performed.Results:Compared with the I/R model control group,serum ALT in both treatment group of the low and large dosage astilbin were significantly decreased(P＜0.01),even lower in the large dosage group than in the low dosage group(P＜0.01).Hepatic pathology performance improved significantly in the large dosage group;The content of MDA,MPO in liver tissues were significantly decreased in both treatment groups when compared with the I/R model control group(P＜0.01),also lower in the large dosage group than in the low dosage group(P＜0.01).And SOD levels significantly increased in treatment groups(P＜0.01),higher in the large dosage group than in the low dosage group(P＜0.05).Couclusiou:Treatment with astilbin can effectively improve the mouse liver function and liver pathology damage induced by liver warm ischemia-reperfusion injury;Intervention with astilbin can reduce the aggregation, infiltration and activation of neutrophil in the mouse liver induced by warm ischemia-reperfusion injury,thereby reducing inflammatory response;Intervention with astilbin can inhibit the formation of oxygen free radicals,and reduce the damage of lipid peroxidation induced by liver ischemia-reperfusion injury. PARTⅡ.Inhibiting Effect of Astilbin on TLR-4 Signaling Pathway in Liver warm ischemia-reperfusion injuryObjective:To study the molecular mechanism of the effect of astilbin on inhibiting the inflammation signaling pathway in liver warm ischemia-reperfusion injury.Methods: C57BL/6 mice were randomly divided into four groups(n=5):sham-operated group (Sham),model control group(I/R),low dosage of astilbin treatment group(10mg/kg) and large dosage of astilbin(40mg/kg) treatment group.24 hours and one hour before Ischemia, treatment group mice were intraperitoneally injected 10 or 40 mg/kg astilbin.Then the hepatic ischemia-reperfusion model of 70 percent of liver,including the left and middle hepatic lobe,were established.The I/R model control group and the sham operation group were administered with the same volume of normal saline.After 90 min ischemia and 6h reperfusion of the partial hepatic lobe,blood and liver tissue samples were collected from the experimental groups.Serum TNF-a activity was detected,and the content of TNF-a、TLR-4、NF-κB and HO-1 in liver tissues were detected by western blot.mRNA expression of these proteins was detected by semiquantitative RT-PCR.Results:Compared with the I/R model control group,serum TNF-a in both treatment group of the low and large dosage astilbin were significantly decreased(P＜0.01),even lower in the large dosage group than in the low dosage group(P＜0.01).The protein content of TNF-a、TLR-4、NF-κB in liver tissues were gradually decreased in both treatment groups when compared with the I/R model control group,also lower in the large dosage group than in the low dosage group. Same trends were observed in the mRNA expression of these proteins showed by semiquantitative RT-PCR(low dosage group P＜0.05;large dosage group P＜0.01).HO-1 levels gradually increased in treatment groups,higher in the large dosage group than in the low dosage group,the result of mRNA showed a same trend(low dosage group P＜0.05; large dosage group P＜0.01).Conclusion:Intervention with astilbin can reduce the high levels of serum TNF-a caused by ischemia-reperfusion injury,as well as increased expression of TNF-a protein and mRNA in the liver tissues,thereby reducing TNF-a-mediated further injury;Intervention with astilbin can reduce the high expression of TLR-4 protein and mRNA,and NF-κB protein in IRI liver tissues,demonstrating its effect of inhibiting TLR-4 inflammation signaling pathway.Treatment with astilbin can promote the expression of HO-1 protein and mRNA in IRI liver,demonstrating its effect of TLR-4 pathway inhibition and promotion of the expression of HO-1 is closely related.PARTⅢ.Effect of Astilbin on Expression of SOCS1、IL-10 in Liver warm ischemia-reperfusion injuryObjective:To study the molecular mechanism of the effect of astilbin on the anti-inflammation signaling pathway in liver warm ischemia-repeffusion injury.Methods: C57BL/6 mice were randomly divided into four groups(n=5):sham-operated group (Sham),model control group(I/R),low dosage of astilbin treatment group(10mg/kg) and large dosage of astilbin(40mg/kg) treatment group.24 hours and one hour before Ischemia, treatment group mice were intraperitoneally injected 10 or 40 mg/kg astilbin.Then the hepatic ischemia-reperfusion model of 70 percent of liver,including the left and middle hepatic lobe,were established.The I/R model control group and the sham operation group were administered with the same volume of normal saline.After 90 min ischemia and 6h reperfusion of the partial hepatic lobe,liver tissue samples were collected from the experimental groups.The content of SOCS-1 and IL-10 in liver tissues were detected by western blot.mRNA expression of the same proteins was detected by semiquantitative RT-PCR.Results:Compared with the I/R model control group,SOCS-1 and IL-10 protein levels gradually increased in treatment groups,higher in the large dosage group than in the low dosage group,the result of mRNA showed a same trend(low dosage group P＜0.05; large dosage group P＜0.01).Conclusion:Intervention with astilbin can promote the expression of IL-10 protein and mRNA in IRI liver,thus inhibiting the inflammation; Intervention with astilbin can promote the expression of SOCS-1 protein and mRNA in IRI liver,showing its role of negative regulation by upregulating SOCS1 pathway.更多还原