Foxo3a通过Bim转录促进NAFLD中Kupffer细胞自噬及抑制炎症小体激活的机制研究

发布时间：2018-04-20 20:54

  本文选题：叉头蛋白O3a + 自噬　； 参考：《重庆医科大学》2017年博士论文

【摘要】：目的:肝脏内过多的脂质沉积可阻断Kupffer细胞(Kupffer cells,KCs)的自噬流,导致炎症小体的激活,从而加重肝脏内脂质沉积导致的炎症反应。叉头蛋白O3a(forkhead box protein O3a,Foxo3a)是连接炎症反应和能量代谢的重要转录因子。但是,在高脂饮食(high fat diet,HFD)的条件下,Foxo3a调节KCs自噬并抑制炎症小体激活的机制尚不清楚。因此,本实验将研究非酒精性脂肪性肝病(nonalcoholic fatty liver disease,NAFLD)模型小鼠中,KCs中Foxo3a的变化以及调节KCs自噬流并抑制炎症小体活性的机制。方法:1.梯度浓度的棕榈酸(palmitic acid,PA)(0,0.16,0.32,0.64 m M)以及脂多糖(lipopolysaccharide,LPS)(100 ng/ml)联合刺激体外的KCs12 h:1)蛋白印记法(western blotting assay,WB)检测:(1)自噬相关蛋白如Beclin 1和LC3;(2)NLRP3炎症小体(nucleotide oligomerization domain(NOD)-like receptor family pyrin domain containing 3,NLRP3)相关蛋白如:NLRP3、caspase 1和凋亡相关斑点蛋白(apoptosis associated speck-like protein,ASC);(3)Foxo3a及上游相关蛋白如:腺苷酸活化蛋白激酶(adenosine monophosphate ativated protein kinase,AMPK)、磷脂酰肌醇-3-羟激酶(phosphatidyl inositol3-kinase,PI3K)和丝氨酸/苏氨酸蛋白激酶B(protein serine threonine kinase B,Akt)的表达变化。(2)酶联免疫吸附试验(enzyme-linked immuno sorbent assay,ELISA)检测细胞上清白介素1β(interleukin-1β,IL-1β)和IL-18的含量。(3)利用2′7′-二乙酰二氯荧光素(dichlorofluorescein diacetate,DCFH-DA)荧光探针并荧光显微镜观察KCs中活性氧产生的情况。2.(1)分别用Foxo3a过表达质粒(Foxo3a overexpression plasmid,Foxo3a-OE)和Foxo3a-短发夹RNA质粒(Foxo3a-short hairpin RNA plasmid,Foxo3a-sh RNA)转染KCs 48 h后,再利用PA和LPS联合刺激KCs 12 h:(1)WB检测自噬相关蛋白及NLRP3炎症小体相关蛋白的表达变化;(2)ELISA检测细胞上清IL-1β和IL-18的含量;(3)透射电镜观察KCs自噬小体的分布情况。(2)m RFP-EGFP-LC3缺陷腺病毒感染KCs 48 h后,先利用Foxo3a激动剂Iturin A预处理KCs 1 h,再利用PA和LPS联合刺激KCs 12 h,最后利用荧光显微镜观察LC3标记的自噬小体的分布情况。3.(1)Foxo3a-OE转染KCs 48 h后,利用PA和LPS联合刺激KCs 12 h,再利用荧光定量聚合酶链反应(real-time quantitative polymerase chain reaction,RT-PCR)检测Foxo3a转录下游靶分子的m RNA表达情况。(2)利用Bim-OE及Bim-sh RNA体外转染KCs 48 h后,先分别利用Foxo3a抑制剂SC97和激动剂Iturin A预处理KCs 1 h,再利用PA和LPS联合刺激KCs 12 h,检测:(1)WB检测自噬相关蛋白及NLRP3炎症小体相关蛋白的表达变化;(2)ELISA检测细胞上清IL-1β和IL-18的含量。4.24只清洁级小鼠随机分为3组,即正常饲料组(coarse food diet group,CFD),高脂饲料组(high fat diet,HFD)和高脂饲料联合Iturin A腹腔注射组(Iturin A),每组8只。3组小鼠均喂养16周后,分别取小鼠的肝脏及外周血:(1)HE染色检测肝脏脂肪变情况;(2)全自动生化分析仪检测小鼠肝功能及血脂水平;(3)透射电镜观察肝脏组织KCs中的自噬小体分布情况;(4)分离肝组织中KCs,并提取其总RNA和蛋白后:(1)WB检测自噬相关蛋白及NLRP3炎症小体相关蛋白的表达情况;(2)RT-PCR检测IL-1β和IL-18的m RNA水平。结果:1.PA联合LPS刺激体外KCs组与单独LPS处理KCs组比较:(1)自噬相关蛋白Beclin1表达下降,LC3Ⅱ/Ⅰ的表达比例升高;(2)NLRP3炎症小体相关分子NLRP3、ASC、caspase 1 p10、IL-1β和IL-18的表达水平升高;(3)Foxo3a表达下降,p-Foxo3a、p-Akt和p-PI3K的表达升高,而p-AMPK表达无变化;(4)ROS产生明显增多。2.(1)KCs过表达Foxo3a后,可明显促进KCs自噬流,并抑制由PA联合LPS诱导的NLRP3炎症小体的激活;(2)KCs沉默Foxo3a后,可进一步抑制KCs自噬流,以及进一步促进由PA联合LPS诱导的NLRP3炎症小体的激活。3.(1)KCs过表达Foxo3a后,PA联合LPS刺激组中只有Bim的m RNA表达明显低于单独过表达Foxo3a组;(2)(1)沉默KCs中的Bim,即使给予Foxo3a激动剂,KCs的自噬流仍然受阻,并促进由PA联合LPS诱导的NLRP3炎症小体的激活;(2)过表达KCs中的Bim,即使给予Foxo3a抑制剂,KCs的自噬仍然顺向进行,并抑制由PA联合LPS诱导的NLRP3炎症小体的激活。4.Iturin A组小鼠与HFD组小鼠比较:(1)肝组织脂肪变及炎症反应较轻;(2)肝功能及血脂水平较低;(3)肝组织KCs中的自噬小体分布较多;(4)KCs中Foxo3a与Bim的表达明显升高,自噬被激活,高脂饮食诱导的NLRP3炎症小体的活性较低。结论:1.游离脂肪酸通过影响Foxo3a的磷酸化,阻断KCs的自噬流,并促进NLRP3炎症小体的激活。2.Foxo3a通过恢复KCs的自噬流,抑制由游离脂肪酸诱导的NLRP3炎症小体的活性。3.Foxo3a通过促进Bim的转录活性,诱导KCs的自噬,并抑制由游离脂肪酸诱导的NLRP3炎症小体的活性。4.Foxo3a激动剂Iturin A通过促进Bim的转录活性,减轻小鼠肝脏由于脂质沉积导致的脂肪变和炎症反应。
[Abstract]:Objective: excessive lipid deposition in the liver blocks the autophagic flow of Kupffer cells (Kupffer cells, KCs), which leads to the activation of the inflammatory corpuscles and thus aggravates the inflammatory response in the liver. The forked head protein O3a (forkhead box protein O3a, Foxo3a) is an important transcription factor that connects the inflammatory response and energy metabolism. However, it is in high fat drink. Under the condition of high fat diet (HFD), the mechanism of Foxo3a to regulate autophagy and inhibit the activation of inflammatory corpuscles is not clear. Therefore, this study will study the changes in the non alcoholic fatty liver disease (nonalcoholic fatty liver disease, NAFLD) model mice and the mechanism of regulating the autophagic flow and inhibiting the activity of the inflammatory corpuscle in the KCs. Methods: palmitic acid, PA (PA) (0,0.16,0.32,0.64 m M) and LPS (lipopolysaccharide, LPS) (lipopolysaccharide, LPS) (100 ng/ml) combined stimulation of KCs12 h:1) assay: (1) autophagy related egg white such as 1 and 1; (2) inflammatory corpuscles (2) - Like receptor family pyrin domain containing 3, NLRP3) related proteins such as NLRP3, caspase 1 and apoptosis related speckle protein (apoptosis associated speck-like); (3) and upstream related proteins such as adenylate activation protein kinase, phosphatidylinositol Enzyme (phosphatidyl inositol3-kinase, PI3K) and serine / threonine protein kinase B (protein serine threonine kinase B, Akt). (2) enzyme linked immunosorbent assay (enzyme-linked immuno) detection of cell supernatant interleukin 1 beta and content. (3) use 2 '7' - two acetyl Two chlorofluorescein (dichlorofluorescein diacetate, DCFH-DA) fluorescence probe and fluorescence microscopy were used to observe the production of reactive oxygen species in KCs..2. (1) was transfected with Foxo3a overexpressed plasmids (Foxo3a overexpression plasmid, Foxo3a-OE) and Foxo3a- short hairpin RNA plasmids, respectively. PA and LPS were combined to stimulate KCs 12 h: (1) WB to detect the expression of autophagy related proteins and NLRP3 inflammatory corpuscle related proteins; (2) ELISA was used to detect the content of IL-1 beta and IL-18 in cell supernatant; (3) transmission electron microscopy was used to observe the distribution of autophagic corpuscles in KCs. (2) m RFP-EGFP-LC3 deficiency adenovirus infection 48 KCs 1 h, and then the combination of PA and LPS to stimulate KCs 12 h. Finally, the distribution of autophagic bodies marked by LC3 was observed by fluorescence microscopy.3. (1) Foxo3a-OE transfection KCs 48 h, using PA and joint stimulation 12, and then detected by fluorescence quantitative polymerase chain reaction. M RNA expression of downstream target molecules was transcribed. (2) after transfection of KCs 48 h with Bim-OE and Bim-sh RNA, Foxo3a inhibitor SC97 and activator Iturin A were pre treated with KCs 1 respectively, and then the combination stimulation was used to detect the changes in the expression of autophagy related protein and inflammatory corpuscle related protein; (2) The content of IL-1 beta and IL-18 in cell supernatant.4.24 mice was randomly divided into 3 groups, namely, normal diet group (coarse food diet group, CFD), high fat diet group (high fat diet, HFD) and high fat diet combined abdominal injection group, and each group of 8 rats were fed for 16 weeks, respectively, to take the liver and peripheral blood of mice, respectively: (1) dyeing Color detection of liver fatty change; (2) automatic biochemical analyzer to detect liver function and blood lipid level in mice; (3) transmission electron microscope to observe the distribution of autophagic corpuscles in liver tissue KCs; (4) separate KCs in liver tissue and extract its total RNA and protein: (1) WB detection of autophagy related protein and NLRP3 inflammatory corpuscle related protein expression; (2) R T-PCR detected the m RNA level of IL-1 beta and IL-18. Results: 1.PA combined with LPS stimulation in KCs group and single LPS treatment KCs group: (1) Beclin1 expression of autophagy related proteins decreased, LC3 II / I increased the expression ratio; (2) the expression level of inflammatory corpuscles, 1 The expression of oxo3a, p-Akt and p-PI3K increased, but the expression of p-AMPK was not changed. (4) ROS produced a significant increase in.2. (1) KCs over expression of Foxo3a, which could obviously promote the KCs autophagic flow and inhibit the activation of the NLRP3 inflammatory corpuscle induced by PA combined LPS; (2) after the silence, the autophagic flow can be suppressed and further promoted by the joint induction of PA. After the activation of.3. (1) KCs over expression of Foxo3a in the RP3 inflammatory body, the RNA expression of m in the PA combined LPS stimulus group was significantly lower than that of the single overexpressed Foxo3a group; (2) (1) the Bim in the silent KCs, even if given the agonist, was still blocked and promoted the activation of the inflammatory corpuscle induced by the combination. (2) Even if Foxo3a inhibitors were given, the autophagy of KCs was still in progress, and the activation of the NLRP3 inflammatory body induced by PA combined with LPS in the.4.Iturin A group was compared with that of the HFD mice: (1) the liver tissues were fat and the inflammatory response was lighter; (2) the liver function and blood lipid levels were low; (3) the autophagic bodies in the liver tissues were more distributed; (4) Foxo3a in KCs The expression of Bim was significantly elevated, autophagy was activated and the activity of NLRP3 inflammatory bodies induced by high fat diet was lower. Conclusion: 1. free fatty acids can inhibit the autophagic flow of KCs by affecting the phosphorylation of Foxo3a, and promote the activation of NLRP3 inflammatory corpuscle by restoring the autophagic flow of KCs and inhibiting the NLRP3 inflammatory body induced by free fatty acids. The active.3.Foxo3a induces autophagy by promoting the transcriptional activity of Bim and inhibits the autophagy of KCs, and inhibits the active.4.Foxo3a agonist, Iturin A, induced by free fatty acids, Iturin A, by promoting the transcriptional activity of Bim, and alleviating the fatty changes and inflammatory reactions in the liver of mice due to lipid deposition.

【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R575

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