P2x7受体基于NLRP3炎症小体调控酒精性脂肪肝和肝纤维化的作用机制研究

发布时间：2018-03-15 06:18

本文选题：酒精性脂肪肝　切入点：肝纤维化　出处：《延边大学》2017年博士论文　论文类型：学位论文

【摘要】：目的:肝纤维化是一种发生在肝脏、循序而渐进的病理损伤,随着肝纤维化研究的不断深入,发现肝纤维化过程中炎症反应始终贯穿始终。P2x7受体(purinergic receptor,P2x7R)属于嘌呤受体家族,是P2x家族的7个不同亚型(P2x1-7)中的一种。它在人体中分布广泛,特别是与炎症反应过程直接相关。因而是否可以通过影响P2x7R来实现对酒精性脂肪肝的调控,摸索其中的潜在联系与机制,是我们要探究的问题。方法:(1)脂多糖(lipopolysaccharide,LPS)预处理巨噬细胞的培养基间接刺激人系肝星状细胞株LX2细胞与直接LPS刺激LX2细胞的组别相比较,ELISA与 Western Blot 检测细胞因子白介素-1β(Interleukin-1β,IL-1β),白介素-1α(Interleukin-1α,IL-1α)和肿瘤坏死因子(tumor necrosis factor alpha,TNF-α)表达情况。Western Blot与RT-PCR检测蛋白与mRNA的表达水平;LX2细胞给予LPS刺激4h,加入三磷酸腺苷(adenosine-triphosphate,ATP)时间为30min,加入 ATP 前 1Omin 给予 P2x7R 拮抗剂 A438079。Western Blot 与 RT-PCR 验证LX2细胞激活过程中炎症因子P2x7R、NOD样受体家族成员(NLRs)NLRP3和半胱氨酸的天冬氨酸蛋白水解酶 1(cysteinyl aspartate specificproteinas,caspase-1)表达。同时检测肝纤维化标志蛋白Ⅰ型胶原(type Ⅰ collagen,collagen I)和平滑肌肌动蛋白(alpha-smooth muscle actin,α-SMA)蛋白mRNA表达情况;沉默P2x7R后检测相关炎症小体与肝纤维化相关基因的表达。(2)建立硫代乙酰胺(Thioacetamide,TAA)肝纤维化模型,采用C57BL/6小鼠随机分为3组:正常组(n=5),模型TAA组(n=14)与模型TAA给药A438079组(n=5)。除正常组,第一周腹腔注射给予TAA(100 mg/kg)给药,从第二周到第六周内TAA(200 mg/kg),从第5周起给药组每周给予A438079(30mg/kg)3次,持续两周。第6周给药结束后处死,收取血清,肝脏等样本。苏木精-伊红染色法(hematoxylin-eosin staining,HE)、免疫组化法(Immunohistochemistry,IHC)、麻松三色染色(Masson's Trichrome Staining)、免疫荧光染色(Immunofluorescence Staining,IF)检测炎症相关因子 NLRP3、核转录因子(Nuclear factor kB,NF-kB)、髓过氧化物酶(myeloperoxidase,MPO)和F4/80的表达情况。结合Western Blot与RT-PCR检测肝纤维化相关标志collagenI、α-SMA的表达。(3)对小鼠肝脏细胞(AML12)给予酒精(50mM)、二甲双胍与AMP依赖的蛋白激酶(AMP dependent kinase,AMPK)通路抑制剂 Compound C 刺激 24h,利用 Wertern Blot检测炎症因子与脂肪代谢相关沉默信息调节因子(Sirtuin1,SIRT1)、过氧化物增殖剂激活受体(PeroxisomeproliferatoractivatedReceptor-alpha,PPARα)蛋白表达结果,凋亡相关因子半胱天冬酶3,即caspase-3表达情况。(4)慢性酒精加急性酒精灌胃模型,采用C57BL/6小鼠随机分为4组:对照饲料组,慢性酒精加酒精急性灌胃组,酒精给药组以及单独给药组。给药组每天皮下注射给药A438079(30mg/kg)。实验阶段,分别给予对照饲料与酒精饲料10天喂养。第11天单次酒精灌胃的计量为5g/kg,9小时后处死并收集样本。利用Western Blot与RT-PCR检测炎症因子与肝纤维化标志因子的蛋白与mRNA表达情况,对肝脏病理切片进行染色分析肝纤维化与炎症因子浸润情况。结果:(1)LX2细胞经过LPS刺激后分泌IL-1β和IL-1α。LX2细胞经过LPS与ATP联合作用,肝纤维化相关各项指标collagen I、α-SMA以及P2x7R和caspase-1、NLRP3炎症小体的mRNA与蛋白表达升高;预处理P2x7R拮抗剂A438079可以有效降低上述因子的表达。(2)TAA所致的肝纤维化模型中,A438079可以在体内降低TAA引起的caspase-1、NLRP3炎症小体与纤维化指标标collagen I、α-SMA和TIMP 1的蛋白与mRNA表达,在病理学上改善模型组的病变情况。(3)在AML12细胞中,A438079可以有效降低酒精诱发的PPARα、caspase-3、P-AMPK、SIRT 1的蛋白表达。(4)慢性加急性酒精灌胃模型中,P2x7R拮抗剂A438079可以降低酒精诱发的ALT/AST指标升高,在病理学上改善模型组的病变情况以及collagen I、α-SMA、P-AMPK、SIRT1、LIPIN 1的蛋白表达。结论:P2x7R介导的NLRP3炎症小体激活可以促使IL-1β分泌,间接促进肝纤维化的发生发展,在肝脏中调节酒精性脂肪肝进程。说明P2x7R在肝纤维化和酒精性脂肪肝治疗过程中会是一个有效的研究靶点。
[Abstract]:Objective: hepatic fibrosis is a pathological injury occurring in the liver, gradual and progressive, with the study of hepatic fibrosis further revealed inflammatory reaction in the process of liver fibrosis always runs through the.P2x7 receptor (purinergic receptor P2x7R) belongs to the purine receptor family, is one of 7 different subtypes of the P2x family (P2x1-7) in a. It is widely distributed in the human body, especially directly associated with the inflammatory process. Therefore, whether the influence of P2x7R to realize the regulation of alcoholic fatty liver, and explore the potential link mechanism, we want to explore issues. Methods: (1) lipopolysaccharide (lipopolysaccharide, LPS) medium pretreatment macrophage indirect stimulation system of hepatic stellate cell line LX2 and LPS directly stimulate LX2 cells compared with Western group, ELISA Blot detection of interleukin -1 beta (Interleukin-1 beta, beta IL-1), interleukin -1 alpha (Interleukin-1 alpha, IL-1 alpha and tumor necrosis factor (tumor) necrosis factor alpha, TNF- alpha) expression of.Western Blot and RT-PCR protein and mRNA; LX2 cells stimulated with LPS 4h, adenosine triphosphate (adenosine-triphosphate, ATP) with time 30min, before joining ATP 1Omin P2x7R antagonist A438079.Western Blot and RT-PCR to verify the LX2 cell activation of inflammatory cytokines in P2x7R process, members of the family of NOD like receptors (NLRs) aspartic acid proteases NLRP3 and cysteine (1 cysteinyl aspartate specificproteinas, caspase-1). The expression of simultaneous detection of liver fibrosis marker protein collagen type I (type I collagen, collagen I) and smooth muscle actin (alpha-smooth muscle actin alpha, -SMA) protein mRNA expression; the expression of inflammatory corpuscle with hepatic fibrosis gene silence detection after P2x7R (2). The establishment of thioacetamide (Thioacetamide, TAA) model of liver fibrosis, C57BL/6 mice were randomly divided into 3 groups: normal group (n=5), TAA model group (n=14) and model TAA administration A438079 group (n=5). In addition to the normal group, the first week of intraperitoneal injection of TAA (100 mg/kg) administration, from second sixth weeks in TAA (200 mg/kg), the drug group received A438079 from the fifth week (30mg/kg) 3 times, for two weeks. Sixth weeks after the administration were collected serum, liver samples. Hematoxylin eosin staining (hematoxylin-eosin staining HE), immunohistochemistry (Immunohistochemistry IHC, MA), pine (Masson's Trichrome Staining) staining, immunofluorescence staining (Immunofluorescence, Staining, IF) detection of inflammatory cytokines NLRP3, nuclear transcription factor (NF-kB Nuclear factor kB), myeloperoxidase (myeloperoxidase, MPO) and the expression of F4/80 and RT-PC combined with Western Blot. Mark R detection of liver fibrosis in collagenI, the expression of alpha -SMA. (3) on mouse liver cells (AML12) with alcohol (50mM), metformin and AMP dependent protein kinase (AMP dependent kinase AMPK Compound C 24h) pathway inhibitor stimulation, measured inflammatory cytokines and lipid metabolism by Wertern Blot silent information regulator check (Sirtuin1, SIRT1), peroxisome proliferator activated receptors (PeroxisomeproliferatoractivatedReceptor-alpha, PPAR alpha) protein expression, apoptosis related factors of caspase 3, namely, the expression of Caspase-3. (4) chronic alcohol and acute alcohol gavage model, C57BL/6 mice were randomly divided into 4 groups: control group, chronic alcohol and feed. Acute alcohol gavage group, alcohol administration group and single drug group. The drug group daily subcutaneous injection of A438079 (30mg/kg). The experimental stage, were given control diet and alcohol feed 10 Day eleventh days feeding. Measurement of single alcohol gavage for 5g/kg, were sacrificed after 9 hours and collect samples. By using Western Blot and RT-PCR detection of inflammatory cytokines and hepatic fibrosis markers expression factor protein and mRNA staining analysis of liver fibrosis and inflammation by infiltration of liver biopsy. Results: (1) LX2 cells after LPS stimulated secretion of IL-1 beta and IL-1 alpha.LX2 cells after the combined effect of LPS and ATP, the indexes of hepatic fibrosis and P2x7R collagen I, alpha -SMA and caspase-1, increased mRNA and protein expression of NLRP3 inflammasome; pretreatment with the P2x7R antagonist A438079 can effectively reduce the expression of the above factors. (2) liver fibrosis model induced by TAA, A438079 can be reduced in vivo by TAA caspase-1, NLRP3 inflammasome and fibrosis indexes collagen, I, -SMA and TIMP 1 alpha and mRNA protein in pathological improvement mode The lesion type group. (3) in AML12 cells, A438079 can effectively reduce the alcohol induced PPAR alpha, Caspase-3, P-AMPK, the expression of SIRT 1 protein. (4) chronic acute alcohol administration model, the P2x7R antagonist A438079 can decrease the ALT/AST index of alcohol induced pathological changes in the. The lesion of the model group, collagen I, P-AMPK, SIRT1, alpha -SMA, the expression of LIPIN 1 protein. Conclusion: NLRP3 inflammasome mediated P2x7R activation can induce IL-1 beta secretion, indirectly promote the occurrence and development of liver fibrosis, regulation of alcoholic fatty liver in the liver. P2x7R process will be an effective the research target in liver fibrosis and alcoholic fatty liver in the treatment process.

【学位授予单位】：延边大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R575

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