微小RNA-182-5p调控焦亡参与肝缺血再灌注损伤

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

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。

Objective To investigate the effect of miR-182-5p targeting FoxO3a-mediated pyroptosis on hepatic ischemia-repeffusion （IR） injury. Methods Liver ischemia-repeffusion 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 can＇ied out to detect the expressions of miR-182-Sp, FoxO3a, Caspasel, IL-1β and IL-18. The gene con＇elation between miR-182-Sp and FoxO3a was analyzed. Immunofluorescent staining was used to detect the Caspasel 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, Caspasel, 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 ceils in vitro, miR-182-5p expression was up- regulated, FoxO3a expression was down-regulated, and caspasel 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 Caspasel, IL-1β and IL-18. In the end, the viabilities of hepatocytes were increased （P 〈 0.05）. Conclusion The activation of miR-182-Sp 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 furlher influence the expression of Caspase 1, IL-1β and IL-18.