阿魏酸下调CYP 2E1和抑制TLR4介导的炎症减轻对乙酰氨基酚诱导的肝毒性

发布时间：2018-05-11 13:21

本文选题：阿魏酸 + 对乙酰氨基酚　；参考：《重庆医科大学》2017年硕士论文

【摘要】：目的:研究阿魏酸(Ferulic acid,FA)对对乙酰氨基酚(acetaminophen,APAP)诱导的肝毒性的保护作用并探讨其机制。方法:将Balb/c小鼠随机分成六组,分别为空白对照组、阿魏酸对照组、模型组,低、中、高剂量的阿魏酸干预组。禁食过夜的小鼠腹腔注射APAP(350 mg/kg)建立急性肝损伤模型;分别给予10、30、100 mg/kg的阿魏酸每隔8h一次,连续灌胃3次,然后腹腔注射APAP,作为低、中、高剂量的阿魏酸干预组;以100 mg/kg的阿魏酸每隔8h一次,连续灌胃3次作为阿魏酸对照组。给予APAP18 h后检测血清丙氨酸转氨酶(alanine aminotransferase,ALT)、天冬氨酸转氨酶(aspartate transaminase,AST)水平;肝脏组织石蜡切片苏木精-伊红(hematoxylin-eosin,HE)染色评价病理变化;末端脱氧核苷酸转移酶介导d UTP缺口末端标记测定法(Terminal deoxynucleoitidyl transferase-mediated d UTP nick end labeling,TUNEL)和Caspase-3活性测定评价凋亡情况;蛋白免疫印记法(western blot)和定量逆转录聚合酶链式反应(quantitative reverse transcription polymerase chain reaction,q RT-PCR)检测肝组织中细胞色P450 2E1(cytochrome P450 2E1,CYP 2E1)的表达;谷胱甘肽(glutathione)的含量测定和过氧化氢酶(catalase,CAT)、超氧化物歧化酶(superoxide dismutase,SOD)的活性检测评价肝脏的抗氧化能力;酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)检测血清中炎性介质TNF-?和IL-1?的表达;Western blot分析肝脏组织中Toll样受体4(Toll-like receptor 4,TLR4)、p-IRAK1、p-I?B、p-p38蛋白表达水平。结果:1.阿魏酸呈剂量依赖性地降低了APAP诱导的ALT、AST水平。2.阿魏酸减轻了APAP诱导的肝脏病理改变和凋亡。HE染色结果显示阿魏酸减少了APAP导致的坏死和出血程度。TUNEL染色和Caspase-3活性检测结果表明了阿魏酸预处理后的细胞凋亡程度降低。3.阿魏酸显著抑制了APAP诱导的CYP 2E1的m RNA和蛋白表达。4.阿魏酸明显抑制了APAP诱导的氧化应激反应。阿魏酸干预后,肝组织GSH的含量显著增多,SOD、CAT的活性显著增强。5.阿魏酸显著降低了APAP诱导的炎性因子TNF-?和IL-1?的表达。6.阿魏酸下调APAP诱导的TLR4、p-IRAK1、p-I?B、p-p38蛋白表达。结论:阿魏酸能减轻APAP诱导的肝毒性,这种保护作用可能与下调CYP 2E1表达,负性调控TLR4信号通路,抑制炎症反应有关。
[Abstract]:Aim: to study the protective effect of Ferulic acidfon (FFA) on hepatic toxicity induced by acetaminophenophenol (APAP) and its mechanism. Methods: Balb/c mice were randomly divided into six groups: blank control group, ferulic acid control group, model group, low, medium and high dose ferulic acid intervention group. The model of acute liver injury was established by intraperitoneal injection of APAP(350 mg / kg in overnight fasting mice, the rats were given 10 ~ 30100 mg/kg of ferulic acid every 8 hours, and then intraperitoneally injected with ferulic acid as low, medium and high doses of ferulic acid. Ferulic acid (100 mg/kg) was used as the control group for 3 times, once every 8 h. Serum alanine aminotransferase (Alanine aminotransferase) and aspartate transaminase (aspartate transaminase) were detected after APAP18, and the pathological changes were evaluated by paraffin section of liver tissue. Terminal deoxynucleoitidyl transferase-mediated d UTP nick end labeling and Caspase-3 activity were used to evaluate the apoptosis of terminal deoxynucleoitidyl transferase-mediated d UTP nick end labelingn by terminal deoxynucleotidyl transferase mediated d UTP Nick end labeling assay. The expression of P450 2E1(cytochrome P450 2E1C CYP2E1) was detected by Western blot and quantitative reverse transcription polymerase chain reactionQ RT-PCRs in liver tissue by Western blot and quantitative reverse transcriptase polymerase chain reaction (RT-PCR). The determination of glutathione content and the activity of catalase CATX, superoxide dismutase (SOD) were used to evaluate the antioxidant ability of liver. The enzyme linked immunosorbent assay (Elisa) was used to detect the inflammatory mediators TNF-TNF in serum. And IL-1? The expression of Toll like receptor 4(Toll-like receptor 4 and TLR4 was analyzed by Western blot. The result is 1: 1. Ferulic acid decreased the level of alt induced by APAP in a dose-dependent manner. Ferulic acid alleviated the pathological changes and apoptosis induced by APAP. The results of HE staining showed that ferulic acid reduced the degree of necrosis and bleeding induced by APAP. Tunel staining and the detection of Caspase-3 activity showed that the apoptosis degree of the cells pretreated with ferulic acid was decreased by .3. Ferulic acid significantly inhibited the expression of m RNA and protein in CYP 2E1 induced by APAP. Ferulic acid significantly inhibited the oxidative stress induced by APAP. After ferulic acid intervention, the content of GSH in liver tissue increased significantly and the activity of cat in liver tissue increased significantly. Ferulic acid significantly reduced the inflammatory factor TNF-induced by APAP. And IL-1? 6. Ferulic acid down-regulated APAP induced expression of TLR4, p-IRAK1, p38. Conclusion: ferulic acid can attenuate the hepatotoxicity induced by APAP, which may be related to the down-regulation of CYP 2E1 expression, negative regulation of TLR4 signaling pathway and inhibition of inflammation.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R965

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