β-arrestin2介導(dǎo)TLR4通路參與阿片激動(dòng)劑肝保護(hù)作用的機(jī)制研究
發(fā)布時(shí)間：2018-08-15 17:47
【摘要】：研究背景和目的:肝缺血再灌注損傷(Hepatic Ischemia Reperfusion Injury,HIRI)是臨床上常見(jiàn)的病理生理過(guò)程,多見(jiàn)于肝葉切除、肝移植等肝臟外科相關(guān)手術(shù),是導(dǎo)致圍術(shù)期嚴(yán)重并發(fā)癥的重要因素。目前,肝臟IR損傷后發(fā)生的肝功能障礙仍然是臨床上急需解決的一個(gè)問(wèn)題,如何防治這類損傷是圍手術(shù)期肝保護(hù)相關(guān)領(lǐng)域的熱點(diǎn)。前期研究證實(shí)阿片激動(dòng)劑通過(guò)誘導(dǎo)i NOS的生成發(fā)揮肝保護(hù)效應(yīng),表明TLR4炎癥通路與阿片激動(dòng)劑的肝保護(hù)作用存在內(nèi)在的聯(lián)系。β-arrestin在激活阿片受體的信號(hào)轉(zhuǎn)導(dǎo)及調(diào)控中起著重要的作用。β-arrestin作為GPCR的負(fù)調(diào)控因子,當(dāng)G蛋白偶聯(lián)受體激活時(shí)可易位至胞膜與受體結(jié)合,促進(jìn)受體內(nèi)化脫敏。β-arrestin與細(xì)胞內(nèi)蛋白結(jié)合參與調(diào)節(jié)胞內(nèi)信號(hào)通路傳導(dǎo),在細(xì)胞生長(zhǎng)、凋亡、免疫調(diào)控等過(guò)程中發(fā)揮重要作用。已有文獻(xiàn)已證實(shí)β-arrestin參與調(diào)節(jié)機(jī)體的炎癥和免疫反應(yīng)過(guò)程,通過(guò)TLR下游關(guān)鍵分子腫瘤壞死因子受體相關(guān)因子6(TRAF6)調(diào)節(jié)TLR/IL-1R/NF-κB的活化,并與TRAF6結(jié)合成為負(fù)調(diào)控因子,參與多種炎癥性疾病的發(fā)生發(fā)展。此外,有研究證實(shí)μ阿片受體與β-arrestin2的親和力較其它亞型更強(qiáng)。本實(shí)驗(yàn)擬探討β-arrestin2與TLR4受體在阿片激動(dòng)劑肝保護(hù)作用中的具體機(jī)制。研究方法:1、采用小鼠70%肝臟缺血再灌注損傷模型,比較假手術(shù)組、肝臟缺血再灌注損傷組、阿片受體激動(dòng)劑預(yù)處理組、阿片受體激動(dòng)劑對(duì)照組之間血清轉(zhuǎn)氨酶、肝臟病理學(xué)變化的差異。為觀察TLR4受體在阿片藥物預(yù)處理肝保護(hù)中的作用,比較TLR4敲除小鼠和野生型小鼠的肝臟缺血再灌注組和阿片激動(dòng)劑預(yù)處理組的肝臟組織的TNF-α、IL-6 m RNA表達(dá)水平。并通過(guò)體外的LPS刺激RAW264.7模型模擬體內(nèi)肝臟缺血再灌注損傷,比較阿片激動(dòng)劑預(yù)處理組與LPS處理組的TLR4蛋白表達(dá)差異。2、采用70%的小鼠肝缺血再灌注模型,將小鼠隨機(jī)分為sham組,阿片激動(dòng)劑預(yù)處理組、肝缺血再灌注組、阿片激動(dòng)劑對(duì)照組,檢測(cè)各組小鼠肝組織的β-arrestin2的表達(dá)。體外采用RAW264.7細(xì)胞模擬肝臟Kupffer細(xì)胞,觀察阿片激動(dòng)劑預(yù)處理RAW264.7細(xì)胞后的β-arrestin2蛋白表達(dá)。通過(guò)采用si RNA干擾RAW264.7細(xì)胞的β-arrestin2表達(dá),觀察阿片激動(dòng)劑對(duì)LPS引起的細(xì)胞的活力和凋亡的影響。3、通過(guò)采用si RNA干擾RAW264.7細(xì)胞的β-arrestin2表達(dá),觀察阿片激動(dòng)劑預(yù)處理對(duì)LPS處理后的細(xì)胞的p-ERK、p-JNK蛋白表達(dá)以及β-arrestin2和TRAF6免疫共沉淀結(jié)合的影響。結(jié)果:1、阿片激動(dòng)劑預(yù)處理組的小鼠血清轉(zhuǎn)氨酶和肝臟組織損傷均較缺血再灌注對(duì)照組明顯減低,野生型小鼠組阿片激動(dòng)劑預(yù)處理減輕肝缺血再灌注小鼠的肝臟炎性因子釋放,TLR4敲除小鼠組的阿片激動(dòng)劑的肝保護(hù)作用消失。體外實(shí)驗(yàn)中,阿片激動(dòng)劑減輕LPS引起的細(xì)胞中TLR4蛋白表達(dá)。2、阿片激動(dòng)劑預(yù)處理引起缺血再灌注小鼠肝臟的β-arrestin2表達(dá)升高,體外的細(xì)胞實(shí)驗(yàn)中,阿片激動(dòng)劑的預(yù)處理促進(jìn)細(xì)胞中β-arrestin2的表達(dá),并有向胞膜募集的趨勢(shì)。采用si RNA干擾RAW264.7細(xì)胞的β-arrestin2后,阿片激動(dòng)劑預(yù)處理對(duì)LPS導(dǎo)致的細(xì)胞損傷的保護(hù)作用消失。3、阿片激動(dòng)劑預(yù)處理減低LPS刺激后升高的p-ERK、p-JNK蛋白表達(dá),干擾β-arrestin2后,該作用消失。而且阿片激動(dòng)劑促進(jìn)β-arrestin2和TRAF6免疫復(fù)合物的形成。結(jié)論:1、阿片激動(dòng)劑的預(yù)處理減輕小鼠的肝臟缺血再灌注損傷,并通過(guò)TLR4途徑實(shí)現(xiàn)其肝保護(hù)效應(yīng)。2、阿片激動(dòng)劑通過(guò)β-arrestin2發(fā)揮肝保護(hù)作用,干擾β-arrestin2后阿片激動(dòng)劑的肝保護(hù)作用消失。3、阿片激動(dòng)劑通過(guò)β-arrestin2影響ERK1/2、JNK蛋白活化水平,并通過(guò)增加β-arrestin2與TRAF6免疫復(fù)合物形成抑制LPS引起的炎癥反應(yīng)。
[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.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R614
本文編號(hào)：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.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R614
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
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
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