天津医药

天津医药 ›› 2018, Vol. 46 ›› Issue (10): 1045-1050.doi: 10.11958/20180682

• 实验研究 • 上一篇    下一篇

微小RNA-182-5p调控焦亡参与肝缺血再灌注损伤

杜晨阳 1，宋虎 1，王星星 1，王振 2，张建军 2△   

  1. 基金项目：天津市器官移植临床医学研究中心（项目编号：15ZXLCSY00070） 作者单位：1天津医科大学一中心临床学院（邮编300192）；2天津市第一中心医院器官移植中心 作者简介：杜晨阳（1992），男，硕士研究生，主要从事细胞焦亡、自噬在肝缺血再灌注损伤和肝癌中的作用及机制的研究 △通讯作者 E-mail：yzxzhangjianjun@163.com
  • 收稿日期:2018-04-28 修回日期:2018-07-14 出版日期:2018-10-15 发布日期:2018-11-09
  • 通讯作者: 杜晨阳 E-mail:duchenyang668@163.com
  • 基金资助:
    天津市器官移植临床医学研究中心

miR-182-5p regulates pyroptosis involving liver ischemia reperfusion injury

DU Chen-yang1, SONG Hu1, WANG Xing-xing1, WANG Zhen2, ZHANG Jian-jun2△   

  1. 1 The First Central Clinical College of Tianjin Medical University, Tianjin 300192, China; 2 Organ Transplant Center, Tianjin First Central Hospital △Corresponding Author E-mail: yzxzhangjianjun@163.com
  • Received:2018-04-28 Revised:2018-07-14 Published:2018-10-15 Online:2018-11-09
  • Supported by:
    Tianjin Clinical Research Center for Organ Transplantation Project

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摘要： 摘要：目的 探讨微小RNA-182-5p（miR-182-5p）靶向调控叉形头转录因子O亚型3a（FoxO3a）调节焦亡对肝 缺血再灌注损伤的影响。方法 建立小鼠肝脏缺血再灌注模型。按随机数字表法将40只小鼠分为5组，每组8只， 分别为假手术（sham）组，缺血再灌注（IR）各组（缺血1.5 h，按再灌注时间分为IR 2 h组、IR 6 h组、IR 12 h组和IR 24 h 组）。细胞实验分组分为两部分，（1）缺氧模型建立，分为 control 组和 IR 组。（2）缺氧/复氧模型建立，分为对照组、 mimic组，inhibitor组和inhibitor+siRNA组。HE染色观察肝组织病理变化；免疫细胞化学染色检测FoxO3a表达分布 情况；荧光实时定量PCR（qRT-PCR）和Western blot 分别从mRNA和蛋白水平检测miR-182-5p、FoxO3a、半胱天冬 酶-1（Caspase1）、白细胞介素（IL）-1β、IL-18 表达情况；分析 miR-182-5p 与 FoxO3a 基因相关性；免疫荧光检测 Caspase1表达情况；ELISA检测IL-1β和IL-18表达情况；CCK-8试剂盒检测细胞活性变化情况。结果 IR处理后小 鼠肝组织损伤增加，再灌注12 h时损伤最重，同时FoxO3a、Caspase1、IL-1β、IL-18表达增加（P＜0.05），诱导焦亡产 生；IR处理后小鼠肝组织内miR-182-5p表达水平较sham组升高（P＜0.05）。体外培养小鼠肝细胞AML12，IR处理 后miR-182-5p表达上调，FoxO3a表达下调，同时Caspase1表达升高（P＜0.05）。过表达miR-182-5p能降低FoxO3a 表达水平；反之能增加FoxO3a表达量，进而降低Caspase1、IL-1β、IL-18的表达，增加肝细胞活性（P＜0.05）。结论 激活miR-182-5p能加重肝缺血再灌注损伤，而抑制miR-182-5p能减轻肝缺血再灌注损伤，其机制可能与miR-182- 5p通过靶向调控FoxO3a激活肝细胞焦亡、影响Caspase1、IL-1β、IL-18表达有关。

关键词: 肝, 再灌注损伤, 微RNAs, 叉头转录因子类, 微小RNA-182-5p, 焦亡, 叉形头转录因子O亚型3a

Abstract: Abstract：Objective To investigate the effect of miR-182-5p targeting FoxO3a-mediated pyroptosis on hepatic ischemia-reperfusion (IR) injury. Methods Liver ischemia-reperfusion models of mice were established. According to the random number table method, 40 mice were divided into 5 groups with 8 rats in each group respectively, sham group, IR groups (2 h group, 6 h group, 12 h group and 24 h group). The cell experiments were divided into two parts: (1) the hypoxia models were established and divided into control group and IR group. (2) Hypoxia/reoxygenation models were established and divided into control group, mimic group, inhibitor group and inhibitor+siRNA group. HE staining was used to observe the pathological changes of liver tissues after IR. Immunocytochemistry staining was used to detect the expression of FoxO3a in liver cells. qRT-PCR and Western blot assay were carried out to detect the expressions of miR-182-5p, FoxO3a, Caspase1, IL-1β and IL-18. The gene correlation between miR-182-5p and FoxO3a was analyzed. Immunofluorescent staining was used to detect the Caspase1 expression. ELISA kit was used to detect the expression of IL-1β and IL-18. CCK- 8 kit was used to detect changes of cell viability. Results The liver injury after IR treatment was increased in mice, and the injury was the most severe at 12 h-reperfusion. At the same time, the expression levels of FoxO3a, Caspase1, IL-1β and IL- 18 were increased (P＜0.05), and pyroptosis was induced. MiR-182-5p level in liver tissue was higher after IR treatment compared with that in sham group (P＜0.05). After IR treatment for AML12 cells in vitro, miR-182-5p expression was up regulated, FoxO3a expression was down-regulated, and caspase1 expression was increased (P＜0.05). Overexpression of miR-182-5p decreased the expression of FoxO3a. On the contrary, it increased the expression of FoxO3a, which in turn decreased the expression levels of Caspase1, IL-1β and IL-18. In the end, the viabilities of hepatocytes were increased (P＜ 0.05). Conclusion The activation of miR-182-5p can aggravate hepatic ischemia-reperfusion injury, while the inhibition of miR-182-5p can reduce hepatic ischemia-reperfusion injury. The mechanism may be related to miR-182-5p activating FoxO3a to activate hepatocellular pyroptosis, and further influence the expression of Caspase1, IL-1β and IL-18.

Key words: reperfusion injury, liver, microRNAs, forkhead transcription factors, miR-182-5p, pyroptosis, FoxO3a