RIP1在对乙酰氨基酚诱导AIF依赖性肝细胞程序性坏死中的作用

发布时间：2018-04-11 04:04

本文选题：对乙酰氨基酚 + 程序性坏死　；参考：《安徽医科大学》2014年硕士论文

【摘要】：目的过量对乙酰氨基酚（APAP）可诱导细胞死亡诱导因子（AIF）依赖性肝细胞死亡，但作用机制还不清楚。本研究探讨了在对乙酰氨基酚诱发小鼠急性肝损伤的过程中，程序性坏死关键调控因子受体相互作用蛋白激酶1（RIP1）在AIF介导肝细胞死亡中的作用。方法（1）为探讨GSH耗竭对APAP诱导肝脏RIP1和RIP3上调的影响，将小鼠统一禁食12h后进行腹腔注射APAP（300mg/kg），分别在不同的时点（0、1、2和4h）取材，检测肝脏RIP1与RIP3的水平、GSH含量和肿瘤坏死因子（TNF-α）的mRNA水平。（2）为探讨RIP1选择性抑制剂Nec-1预处理和后处理对APAP诱导急性肝损伤的影响，将小鼠随机分为对照组、Nec-1组、APAP组和APAP+Nec-1组，在给予APAP前或后30min腹腔注射Nec-1（0.125mg/mouse），分别在4h和24h取材，检测血清中ALT的水平和肝细胞死亡情况。（3）为探讨Nec-1对APAP在肝脏代谢中的效应，将小鼠随机分为对照组、Nec-1组、APAP组、APAP+Nec-1组，在给予APAP前30min进行Nec-1预处理，30min后取材，检测肝脏的GSH含量和CYP2E1水平。（4）为探讨Nec-1对APAP诱导肝脏JNK磷酸化、线粒体Bax和细胞核AIF的转位的影响，将小鼠随机分为对照组、Nec-1组、APAP组和APAP+Nec-1组，在给予APAP前30min进行Nec-1预处理，4h后进行取材，检测肝脏的JNK磷酸化水平、线粒体Bax水平和细胞核AIF的转位情况。（5）为探讨Nec-1是否通过其抗氧化活性来阻止APAP诱导的肝细胞死亡，将小鼠随机分为对照组、APAP组、APAP+Nec-1组和APAP+Nec-1+BSO组，在给予APAP前30min，，APAP+Nec-1组进行Nec-1预处理，在给予Nec-1前进行BSO（25mg/kg）预处理，4h后进行取材，检测血清中ALT的水平、肝脏的3-NT水平、GSH含量、细胞核AIF的转位和肝细胞死亡情况。结果RIP1选择性抑制剂Nec-1可阻止APAP诱导的肝细胞死亡，能够改善小鼠的生存率。肝细胞RIP1的水平在APAP处理1h后显著升高，且与肝细胞GSH耗竭相关。但是，Nec-1预处理并没有影响在给予APAP30min后肝脏的GSH水平，且Nec-1预处理并没有影响到早期肝脏CYP2E1的表达。此外，Nec-1明显抑制APAP诱导JNK激活，表明JNK是RIP1的下游靶点。Nec-1可明显抑制APAP诱导胞质Bax向线粒体转位。同时，Nec-1完全阻断APAP诱导的AIF从线粒体向细胞核转位。在Nec-1对细胞内GSH水平的作用完全被BSO所取消后，Nec-1仍然能够阻止APAP诱导的ALT水平的升高，仍明显地阻止APAP诱导的细胞核AIF的转位和肝细胞死亡。结论本研究结果表明肝脏RIP1的激活是APAP诱导小鼠急性肝衰竭的早期事件。RIP1选择性抑制剂Nec-1预处理和后处理均对APAP诱导小鼠急性肝衰竭具有显著地保护作用。而且，Nec-1抑制APAP诱导的肝脏的JNK的激活和Bax从胞质向线粒体的转位。在APAP诱导的急性肝衰竭的过程中，Nec-1抑制AIF调控的程序性坏死。因此，Nec-1是APAP诱导急性肝衰竭的有效解毒剂。
[Abstract]:Objective excessive acetaminophen (APAP) can induce the death of hepatocytes dependent on AIF-dependent cell death, but the mechanism is not clear.The purpose of this study was to investigate the role of protein kinase 1 (RIP1) in AIF mediated hepatocyte death in paracetamol induced acute liver injury in mice.Methods to investigate the effect of GSH depletion on the up-regulation of liver RIP1 and RIP3 induced by APAP, the mice were given intraperitoneal injection of APP 300 mg / kg at different time points after 12 h fasting, and the samples were collected at different time points (1h and 4h, respectively).In order to investigate the effect of Nec-1 pretreatment and post-treatment on APAP induced acute liver injury, mice were randomly divided into control group (n = 10) and APAP Nec-1 group (n = 10) to detect the level of RIP1 and RIP3 in liver and the mRNA level of TNF- 伪) in order to investigate the effects of Nec-1 pretreatment and post-treatment on APAP induced acute liver injury.Before and after APAP, 30min was injected intraperitoneally with Nec-1n 0.125mg / mouse.The serum ALT levels and hepatocyte death were measured at 4h and 24h, respectively. In order to investigate the effect of Nec-1 on APAP metabolism in liver, mice were randomly divided into control group (Nec-1 group) and APAP Nec-1 group.In order to investigate the effect of Nec-1 on APAP induced JNK phosphorylation, mitochondrial Bax and nuclear AIF translocation, 30min pretreated with Nec-1 for 30 minutes before APAP was used to detect the content of GSH and CYP2E1 level in liver.The mice were randomly divided into control group (Nec-1 group) and APAP Nec-1 group (control group). 30min was pretreated with Nec-1 for 4 hours before APAP. The phosphorylation level of JNK in liver was detected.The level of mitochondrial Bax and the translocation of nuclear AIF. 5) in order to investigate whether Nec-1 can prevent APAP induced hepatocyte death through its antioxidant activity, mice were randomly divided into two groups: control group, APAP Nec-1 group and APAP Nec-1 BSO group.Nec-1 pretreatment was performed 30 minutes before APAP and 25 mg / kg Nec-1 was pretreated for 4 h. The levels of ALT in serum, 3-NT level in liver, nuclear AIF translocation and hepatocyte death were measured.Results Nec-1, a selective inhibitor of RIP1, could prevent hepatocyte death induced by APAP and improve the survival rate of mice.The level of RIP1 in hepatocytes increased significantly after 1 h of APAP treatment, and was related to the depletion of GSH in hepatocytes.However, Nec-1 pretreatment did not affect the level of GSH in the liver after APAP30min, and Nec-1 pretreatment did not affect the expression of CYP2E1 in the early liver.In addition, Nec-1 inhibited the activation of JNK induced by APAP, suggesting that JNK was the downstream target of RIP1. Nec-1 could significantly inhibit the translocation of cytoplasmic Bax to mitochondria induced by APAP.At the same time, Nec-1 completely blocked APAP induced AIF translocation from mitochondria to nucleus.After the effect of Nec-1 on intracellular GSH level was completely cancelled by BSO, Nec-1 could still prevent the increase of ALT level induced by APAP, and still significantly prevent APAP induced nuclear AIF translocation and hepatocyte death.Conclusion the results suggest that the activation of RIP1 in liver is the early event of APAP induced acute liver failure in mice. The pretreatment and post-treatment of Nec-1, a selective inhibitor of RIP1, have a significant protective effect on APAP induced acute liver failure in mice.Moreover, Nec-1 inhibited the activation of JNK and the translocation of Bax from cytoplasm to mitochondria induced by APAP.In the course of acute hepatic failure induced by APAP, Nec-1 inhibits programmed necrosis regulated by AIF.Therefore, Nec-1 is an effective antidote for acute hepatic failure induced by APAP.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R99

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