β-arrestin2介导TLR4通路参与阿片激动剂肝保护作用的机制研究
发布时间：2018-08-15 17:47
【摘要】：研究背景和目的:肝缺血再灌注损伤(Hepatic Ischemia Reperfusion Injury,HIRI)是临床上常见的病理生理过程,多见于肝叶切除、肝移植等肝脏外科相关手术,是导致围术期严重并发症的重要因素。目前,肝脏IR损伤后发生的肝功能障碍仍然是临床上急需解决的一个问题,如何防治这类损伤是围手术期肝保护相关领域的热点。前期研究证实阿片激动剂通过诱导i NOS的生成发挥肝保护效应,表明TLR4炎症通路与阿片激动剂的肝保护作用存在内在的联系。β-arrestin在激活阿片受体的信号转导及调控中起着重要的作用。β-arrestin作为GPCR的负调控因子,当G蛋白偶联受体激活时可易位至胞膜与受体结合,促进受体内化脱敏。β-arrestin与细胞内蛋白结合参与调节胞内信号通路传导,在细胞生长、凋亡、免疫调控等过程中发挥重要作用。已有文献已证实β-arrestin参与调节机体的炎症和免疫反应过程,通过TLR下游关键分子肿瘤坏死因子受体相关因子6(TRAF6)调节TLR/IL-1R/NF-κB的活化,并与TRAF6结合成为负调控因子,参与多种炎症性疾病的发生发展。此外,有研究证实μ阿片受体与β-arrestin2的亲和力较其它亚型更强。本实验拟探讨β-arrestin2与TLR4受体在阿片激动剂肝保护作用中的具体机制。研究方法:1、采用小鼠70%肝脏缺血再灌注损伤模型,比较假手术组、肝脏缺血再灌注损伤组、阿片受体激动剂预处理组、阿片受体激动剂对照组之间血清转氨酶、肝脏病理学变化的差异。为观察TLR4受体在阿片药物预处理肝保护中的作用,比较TLR4敲除小鼠和野生型小鼠的肝脏缺血再灌注组和阿片激动剂预处理组的肝脏组织的TNF-α、IL-6 m RNA表达水平。并通过体外的LPS刺激RAW264.7模型模拟体内肝脏缺血再灌注损伤,比较阿片激动剂预处理组与LPS处理组的TLR4蛋白表达差异。2、采用70%的小鼠肝缺血再灌注模型,将小鼠随机分为sham组,阿片激动剂预处理组、肝缺血再灌注组、阿片激动剂对照组,检测各组小鼠肝组织的β-arrestin2的表达。体外采用RAW264.7细胞模拟肝脏Kupffer细胞,观察阿片激动剂预处理RAW264.7细胞后的β-arrestin2蛋白表达。通过采用si RNA干扰RAW264.7细胞的β-arrestin2表达,观察阿片激动剂对LPS引起的细胞的活力和凋亡的影响。3、通过采用si RNA干扰RAW264.7细胞的β-arrestin2表达,观察阿片激动剂预处理对LPS处理后的细胞的p-ERK、p-JNK蛋白表达以及β-arrestin2和TRAF6免疫共沉淀结合的影响。结果:1、阿片激动剂预处理组的小鼠血清转氨酶和肝脏组织损伤均较缺血再灌注对照组明显减低,野生型小鼠组阿片激动剂预处理减轻肝缺血再灌注小鼠的肝脏炎性因子释放,TLR4敲除小鼠组的阿片激动剂的肝保护作用消失。体外实验中,阿片激动剂减轻LPS引起的细胞中TLR4蛋白表达。2、阿片激动剂预处理引起缺血再灌注小鼠肝脏的β-arrestin2表达升高,体外的细胞实验中,阿片激动剂的预处理促进细胞中β-arrestin2的表达,并有向胞膜募集的趋势。采用si RNA干扰RAW264.7细胞的β-arrestin2后,阿片激动剂预处理对LPS导致的细胞损伤的保护作用消失。3、阿片激动剂预处理减低LPS刺激后升高的p-ERK、p-JNK蛋白表达,干扰β-arrestin2后,该作用消失。而且阿片激动剂促进β-arrestin2和TRAF6免疫复合物的形成。结论:1、阿片激动剂的预处理减轻小鼠的肝脏缺血再灌注损伤,并通过TLR4途径实现其肝保护效应。2、阿片激动剂通过β-arrestin2发挥肝保护作用,干扰β-arrestin2后阿片激动剂的肝保护作用消失。3、阿片激动剂通过β-arrestin2影响ERK1/2、JNK蛋白活化水平,并通过增加β-arrestin2与TRAF6免疫复合物形成抑制LPS引起的炎症反应。
[Abstract]:BACKGROUND AND OBJECTIVE: Hepatic ischemia-reperfusion injury (HIRI) is a common pathophysiological process in clinic. It is common in liver surgery such as lobectomy and liver transplantation. It is an important factor leading to severe complications during perioperative period. At present, liver dysfunction after liver IR injury is still a clinical problem. Previous studies have shown that opioid agonists exert hepatoprotective effects by inducing the production of iNOS, suggesting that there is an intrinsic relationship between the inflammatory pathway of TLR4 and the hepatoprotective effect of opioid agonists. As a negative regulator of GPCR, beta-arrestin translocates to the membrane when G protein-coupled receptor is activated and promotes receptor desensitization. The binding of beta-arrestin with intracellular proteins participates in the regulation of intracellular signal transduction, cell growth, apoptosis and immune regulation. It has been proved that beta-arrestin participates in the regulation of inflammation and immune response, regulates the activation of TLR/IL-1R/NF-kappa B by tumor necrosis factor receptor-related factor 6 (TRAF6), a key downstream molecule of TLR, and binds with TRAF6 to become a negative regulator, and participates in the occurrence and development of many inflammatory diseases. This study was designed to explore the specific mechanism of the protective effects of beta-arrestin 2 and TLR4 receptors on the liver of opioid agonists. Methods: 1. 70% hepatic ischemia-reperfusion injury model was used to compare the sham operation group, hepatic ischemia-reperfusion injury group, opioid receptor agonists. To observe the role of TLR4 receptor in liver protection after opioid preconditioning, the expression of TNF-alpha and IL-6m RNA in liver tissues of TLR4 knockout mice and wild-type mice after hepatic ischemia-reperfusion and opioid agonist preconditioning were compared. Levels of TLR4 protein were compared between LPS group and OPA preconditioning group. Seventy percent of the mice were randomly divided into sham group, OPA preconditioning group, liver ischemia-reperfusion group and OPA preconditioning group. In vitro, RAW264.7 cells were used to simulate Kupffer cells. The expression of beta-arrestin2 protein in RAW264.7 cells pretreated with opioid agonists was observed. The expression of beta-arrestin2 in RAW264.7 cells was interfered by Si RNA, and the effect of opioid agonists on LPS-induced cells was observed. 3. By interfering with the expression of beta-arrestin2 in RAW264.7 cells with Si RNA, the effects of opioid agonist pretreatment on the expression of p-ERK, p-JNK protein and the binding of beta-arrestin2 and TRAF6 in LPS-treated cells were observed. In vitro, opioid agonists attenuated the expression of TLR4 protein in LPS-induced cells. In vitro, pretreatment with opioid agonists increased the expression of beta-arrestin2 in the liver of mice with ischemia-reperfusion injury. In vitro, pretreatment with opioid agonists promoted the expression of beta-arrestin2 in the cells and tended to recruit into the cell membrane. The protective effect disappeared. 3. Opioid agonist preconditioning decreased the expression of p-ERK, p-JNK protein after LPS stimulation, and the effect disappeared after interfering with beta-arrestin2. Moreover, opioid agonists promoted the formation of immune complexes beta-arrestin2 and TRAF6. Conclusion: 1. Opioid agonist preconditioning alleviated liver ischemia-reperfusion injury in mice and through TL. R4 pathway realizes its hepatoprotective effect. 2. Opioid agonists exert hepatoprotective effect through beta-arrestin 2. After interfering with beta-arrestin 2, the hepatoprotective effect of opioid agonists disappears. 3. Opioid agonists affect ERK1/2 through beta-arrestin 2, JNK protein activation level, and inhibit LPS-induced inflammation by increasing beta-arrestin 2 and TRAF6 immune complex formation. The reaction of the disease.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R614
本文编号：2184950
[Abstract]:BACKGROUND AND OBJECTIVE: Hepatic ischemia-reperfusion injury (HIRI) is a common pathophysiological process in clinic. It is common in liver surgery such as lobectomy and liver transplantation. It is an important factor leading to severe complications during perioperative period. At present, liver dysfunction after liver IR injury is still a clinical problem. Previous studies have shown that opioid agonists exert hepatoprotective effects by inducing the production of iNOS, suggesting that there is an intrinsic relationship between the inflammatory pathway of TLR4 and the hepatoprotective effect of opioid agonists. As a negative regulator of GPCR, beta-arrestin translocates to the membrane when G protein-coupled receptor is activated and promotes receptor desensitization. The binding of beta-arrestin with intracellular proteins participates in the regulation of intracellular signal transduction, cell growth, apoptosis and immune regulation. It has been proved that beta-arrestin participates in the regulation of inflammation and immune response, regulates the activation of TLR/IL-1R/NF-kappa B by tumor necrosis factor receptor-related factor 6 (TRAF6), a key downstream molecule of TLR, and binds with TRAF6 to become a negative regulator, and participates in the occurrence and development of many inflammatory diseases. This study was designed to explore the specific mechanism of the protective effects of beta-arrestin 2 and TLR4 receptors on the liver of opioid agonists. Methods: 1. 70% hepatic ischemia-reperfusion injury model was used to compare the sham operation group, hepatic ischemia-reperfusion injury group, opioid receptor agonists. To observe the role of TLR4 receptor in liver protection after opioid preconditioning, the expression of TNF-alpha and IL-6m RNA in liver tissues of TLR4 knockout mice and wild-type mice after hepatic ischemia-reperfusion and opioid agonist preconditioning were compared. Levels of TLR4 protein were compared between LPS group and OPA preconditioning group. Seventy percent of the mice were randomly divided into sham group, OPA preconditioning group, liver ischemia-reperfusion group and OPA preconditioning group. In vitro, RAW264.7 cells were used to simulate Kupffer cells. The expression of beta-arrestin2 protein in RAW264.7 cells pretreated with opioid agonists was observed. The expression of beta-arrestin2 in RAW264.7 cells was interfered by Si RNA, and the effect of opioid agonists on LPS-induced cells was observed. 3. By interfering with the expression of beta-arrestin2 in RAW264.7 cells with Si RNA, the effects of opioid agonist pretreatment on the expression of p-ERK, p-JNK protein and the binding of beta-arrestin2 and TRAF6 in LPS-treated cells were observed. In vitro, opioid agonists attenuated the expression of TLR4 protein in LPS-induced cells. In vitro, pretreatment with opioid agonists increased the expression of beta-arrestin2 in the liver of mice with ischemia-reperfusion injury. In vitro, pretreatment with opioid agonists promoted the expression of beta-arrestin2 in the cells and tended to recruit into the cell membrane. The protective effect disappeared. 3. Opioid agonist preconditioning decreased the expression of p-ERK, p-JNK protein after LPS stimulation, and the effect disappeared after interfering with beta-arrestin2. Moreover, opioid agonists promoted the formation of immune complexes beta-arrestin2 and TRAF6. Conclusion: 1. Opioid agonist preconditioning alleviated liver ischemia-reperfusion injury in mice and through TL. R4 pathway realizes its hepatoprotective effect. 2. Opioid agonists exert hepatoprotective effect through beta-arrestin 2. After interfering with beta-arrestin 2, the hepatoprotective effect of opioid agonists disappears. 3. Opioid agonists affect ERK1/2 through beta-arrestin 2, JNK protein activation level, and inhibit LPS-induced inflammation by increasing beta-arrestin 2 and TRAF6 immune complex formation. The reaction of the disease.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R614
【参考文献】
相关期刊论文 前1条
1 ;Expression of toll-like receptor 4 and MD-2 gene and protein in Kupffer cells after ischemia-reperfusion in rat liver graft[J];World Journal of Gastroenterology;2004年19期
，本文编号：2184950
本文链接：https://www.wllwen.com/yixuelunwen/waikelunwen/2184950.html
最近更新
教材专著
