TXNIP-NLRP3炎症小体激活在镉暴露引起肝损伤中的作用与褪黑素拮抗效应研究

发布时间：2018-03-17 20:43

本文选题：NLRP3炎症小体　切入点：镉　出处：《第三军医大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究背景镉是一种广泛存在的极具毒性的职业污染物和环境污染物,它严重危害着人类健康。镉作为一种全身毒性重金属,它的环境暴露和健康危害在中国引起了公众的广泛关注。肝脏是镉暴露的主要靶器官,镉的肝毒性机制日趋成为研究热点。流行病学数据显示镉暴露与脂肪肝和肝纤维化等疾病密切相关。越来越多的证据表明炎症和氧化应激在镉暴露引起的肝毒性中起着重要作用。研究发现NLRP3(NLR pyrin domain containing 3)炎症小体是一个炎症信号通路中的关键组成部分,它能感受病原体相关分子模式(pathogen-associated molecular patterns,PAMPs)和危险信号相关分子模式(damage-associated molecular patterns,DAMPs)的入侵。NLRP3炎症小体激活可诱导促炎因子如IL-1β和IL-18释放,进而促进天然免疫应答反应。然而,尚未有文献报道NLRP3炎症小体参与镉暴露引起的肝炎症反应。褪黑素(N-acetyl-5-methoxytryptamine,melatonin)是一种主要由松果体、皮肤和小肠等部位合成的吲哚胺,具有多种生物效应,包括抗氧化,抗炎和抗凋亡等。基于氧化应激和炎症反应在镉暴露引起肝损伤中的重要作用,在治疗镉暴露引起的肝毒性时褪黑素可能是一种潜在药物。然而,褪黑素是否能够减轻镉暴露引起的肝脏炎性损伤以及其中分子机制仍然不清楚。因此,本课题旨在研究镉暴露引起的肝脏炎症损伤是否是通过NLRP3炎症小体活化,探讨褪黑素能否通过抑制硫氧还蛋白互作蛋白(thioredoxin-interacting protein,TXNIP)与NLRP3炎症小体的相互作用来执行其肝保护作用。研究内容(1)雄性C57BL/6小鼠随机分为对照组和镉暴露组。镉处理组小鼠腹腔注射2 mg/kg Cd Cl2生理盐水溶液,对照组小鼠注射等体积生理盐水。镉暴露0、6、12和24 h后立刻收集小鼠血浆样本,通过检测血浆中ALT/AST、LDH、IL-1β、IL-6和TNF-α的分泌以及组织切片染色来反映镉暴露引起的肝损伤和炎症反应。检测NLRP3、caspase-1和IL-1β的表达,明确镉暴露致肝组织炎症小体激活的效应。进一步应用NLRP3敲除鼠来证实NLRP3炎症小体在镉暴露导致小鼠肝脏炎性损伤中的作用。(2)应用10 mg/kg褪黑素预处理小鼠3天,再给予Cd Cl2暴露,检测血浆中ALT/AST和LDH活性以及肝脏组织病理形态,证实褪黑素预处理对镉暴露引起肝脏毒性的保护作用。进一步检测小鼠肝组织氧化应激水平,TXNIP的表达以及NLRP3炎症小体激活,明确TXNIP-NLRP3炎症小体信号通路在褪黑素拮抗镉暴露引起肝损伤中的作用。研究结果(1)镉暴露可升高小鼠血浆中ALT/AST和LDH活性,增加IL-1β、IL-6和TNF-α的含量以及肝组织中MPO的表达。组织病理切片分析显示镉暴露引起了肝细胞肿胀和坏死,肝脏轻度炎性细胞浸润和出血,提示镉暴露引起了炎症反应和肝功能损伤。同时,镉暴露显著增加肝组织中NLRP3、caspase-1和IL-1β的表达以及增强caspase-1的活性。与野生小鼠比较,敲除NLRP3的表达可显著减轻小鼠肝功能损伤,炎性细胞浸润以及NLRP3炎症小体活化,证实NLRP3炎症小体在镉暴露引起小鼠肝脏炎性损伤中起重要作用。(2)褪黑素预处理能显著减轻镉暴露引起的肝功能损伤、肝细胞坏死和炎性细胞浸润。ELISA结果显示褪黑素预处理显著降低镉暴露引起血浆中IL-1β、IL-6和TNF-α的含量。同时,褪黑素预处理能有效拮抗镉暴露引起的MDA含量,SOD、CAT和GSH活性以及TXNIP的表达。进一步结果显示,褪黑素预处理能够显著拮抗镉暴露引起的NLRP3炎症小体活化以及TXNIP与NLRP3的相互作用,证实褪黑素通过抑制TXNIP-NLRP3信号通路来拮抗镉暴露引起的肝损伤。研究结论本研究提出了一个全新的镉毒理作用机制,即镉暴露通过激活NLRP3炎症小体引起小鼠肝脏炎症反应和肝损伤。本实验进一步证明褪黑素对镉暴露引起肝炎症损伤的保护作用是通过抑制TXNIP与NLRP3炎症小体的相互作用。总而言之,我们的研究表明TXNIP-NLRP3炎症小体激活是镉暴露所致肝损伤的分子基础和新的干预靶点,同时也为褪黑素作为拮抗镉毒性的药物提供了新的实验证据。
[Abstract]:The research background of cadmium is a kind of very toxic occupation pollutants and environmental pollutants, it seriously endangers human health. As a systemic toxicity of heavy metals, its environmental exposure and aroused wide public concern in Chinese health hazard. The liver is a major target for cadmium exposure, liver toxicity mechanism of cadmium increasing has become a research hotspot. Epidemiological data showed that cadmium exposure associated with fatty liver and liver fibrosis and other diseases. There is increasing evidence that inflammation and oxidative stress exposure plays an important role in liver toxicity caused by cadmium. The results show that NLRP3 (NLR pyrin domain containing 3) inflammasome is a key signaling pathway in inflammation part, it can feel the pathogen associated molecular patterns (pathogen-associated molecular, patterns, PAMPs) and the danger associated molecular patterns (damage-associated molecu Lar patterns, DAMPs) the invasion of.NLRP3 inflammasome activation induced proinflammatory cytokines such as IL-1 and IL-18 beta release, and then promote the natural immune response. However, NLRP3 has not been reported inflammasome involved in hepatitis inflammation induced by cadmium exposure. Melatonin (N-acetyl-5-methoxytryptamine, melatonin) is a mainly by the pineal gland, indoleamine the synthesis of skin and small intestines, with a variety of biological effects including antioxidant, anti-inflammatory and anti apoptosis. Oxidative stress and inflammation caused by exposure to the important role in liver damage in cadmium exposure based on liver toxicity induced by cadmium in the treatment of melatonin may be a potential drug. However, melatonin is able to reduce cadmium exposure induced liver damage and the molecular mechanism is still unclear. Therefore, this paper aims at the study of inflammation of the liver injury caused by cadmium exposure is through NLR P3 inflammasome activation, melatonin can inhibit thioredoxin interacting protein (thioredoxin-interacting, protein, TXNIP) interacting with NLRP3 inflammasome to perform its function of protecting liver. The research contents (1) male C57BL/6 mice were randomly divided into control group and cadmium exposure group. Cadmium treatment group of mice by intraperitoneal injection of 2 mg/kg Cd Cl2 saline solution, control group were injected with saline. Cadmium exposure of 0,6,12 and 24 h plasma samples were collected immediately after mice by ALT/AST, detection of plasma LDH, IL-1 beta, IL-6 and TNF- secretion and tissue sections were stained to reflect liver injury and inflammation caused by cadmium exposure. Detection of NLRP3 expression. Caspase-1 and IL-1 beta, a clear effect of cadmium exposure induced liver tissue inflammasome activation. Further application of NLRP3 knockout mice that NLRP3 inflammasome in cadmium exposure caused liver damage in mice Role. (2) using 10 mg/kg melatonin pretreatment mice for 3 days, then given Cd Cl2 exposure, detection of plasma ALT/AST and LDH activity and liver tissue morphology, confirmed that melatonin pretreatment exposure induced liver protective effect on toxicity of cadmium. Further detection of oxidative stress levels in liver tissue of mice, the expression of TXNIP and NLRP3 inflammation body activation, clear TXNIP-NLRP3 inflammasome signaling pathway in melatonin exposure to cadmium induced liver injury in rats. Results (1) exposure to cadmium can increase ALT/AST and LDH activity in plasma of mice, increase the expression of IL-6 and IL-1 beta, TNF- alpha and MPO content in liver tissue. Histological analysis showed that cadmium exposure the liver cell swelling and necrosis of the liver, mild inflammatory cell infiltration and hemorrhage, suggesting that cadmium exposure caused by the inflammatory response and liver injury. At the same time, cadmium exposure significantly increased NLRP3 in liver tissue The expression of Caspase-1, IL-1 and beta and enhance caspase-1 activity. Compared with wild-type mice, knockdown of NLRP3 expression can significantly reduce the liver injury in mice, inflammatory cell infiltration and NLRP3 inflammasome activation, confirmed that NLRP3 inflammasome exposure plays an important role in liver inflammatory injury in mice in CD (2) melatonin pre. Treatment can significantly reduce the liver damage caused by cadmium exposure, liver cell necrosis and infiltration of inflammatory cells in.ELISA showed that melatonin pretreatment significantly decreased the cadmium exposure caused by plasma beta IL-1, content of IL-6 and TNF- alpha. At the same time, melatonin pretreatment can effectively antagonize cadmium exposure caused by the content of MDA, SOD, and CAT the activity of GSH and the expression of TXNIP. Further results show that melatonin pretreatment can cause NLRP3 inflammasome activation and TXNIP interaction with NLRP3 significantly antagonized the cadmium exposure, confirmed that melatonin inhibits TX The NIP-NLRP3 signaling pathway to liver injury caused by cadmium exposure. Antagonistic conclusions this study proposed a new mechanism that cadmium toxicity, exposure to cadmium induced liver inflammatory response in mice and liver injury through activation of NLRP3 inflammasome. This experiment further proved that melatonin on cadmium exposure caused by the protective effect of liver inflammation injury through interaction the inhibition of TXNIP and NLRP3 inflammasome. In summary, our study shows that TXNIP-NLRP3 inflammasome activation is the molecular basis of liver injury caused by cadmium exposure and new intervention targets, but also for melatonin as drug antagonistic toxicity of cadmium provides new experimental evidence.

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R114

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