MiR-494 alleviates hepatic ischemia-reperfusion injury in rats by activating PI3K/AKT signaling pathway
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摘要:
目的 探讨微小核糖核酸(miRNA)-494对肝缺血-再灌注损伤(HIRI)的影响及相关作用机制。 方法 24只雄性SD大鼠随机均分为4组(每组6只):假手术组行腹部手术而未行肝脏缺血-再灌注;HIRI组大鼠行部分肝缺血60 min后,再灌注6 h;HIRI+agomir-miR-494组在术前7 d内每日腹腔注射agomir-miR-494(20 μL);HIRI+agomir-NC组在术前7 d内每日腹腔注射等量的agomir-NC。采用逆转录聚合酶链反应(RTPCR)法检测各组肝组织中miR-494的信使核糖核酸(mRNA)表达水平。采用相关试剂盒检测各组的肝损伤指标和氧化应激指标表达水平。观察各组肝组织病理学改变。采用试剂盒检测各组大鼠肝组织中凋亡细胞数和胞质组蛋白相关DNA片段。采用免疫印迹法检测各组凋亡相关蛋白以及磷脂酰肌醇-3-激酶(PI3K)/蛋白激酶(AKT)通路相关蛋白的表达量。 结果 HIRI+agomir-miR-494组大鼠肝组织miR-494的mRNA水平显著高于HIRI+agomir-NC组(P < 0.01)。HIRI+agomir-miR-494组的血清肝损伤指标以及血清氧化应激指标均显著低于HIRI+agomir-NC组(均为P < 0.01)。与HIRI+agomir-NC组相比,HIRI+agomir-miR-494组肝细胞坏死明显减少和细胞完整性提高(P < 0.05),TUNEL阳性细胞数量明显减少(P < 0.05)。HIRI+agomir-miR-494组的cleaved-多聚二磷腺苷核糖聚合酶(PARP)、cleaved-含半胱氨酸的天冬氨酸蛋白水解酶-3(Caspase-3)、Bax水平明显低于HIRI+agomir-NC组(均为P < 0.05)。HIRI+agomir-miR-494组大鼠DNA片段显著少于HIRI+agomir-NC组(P < 0.01)。HIRI+agomir-miR-494组大鼠肝脏p-AKT、p-哺乳动物雷帕霉素靶蛋白(mTOR)和p-p70S6K表达量明显高于HIRI+agomir-NC组(均为P < 0.05)。 结论 miR-494可以减轻大鼠HIRI,其作用机制与激活PI3K/ AKT信号通路有关。 Abstract:Objective To investigate the effect and related mechanism of microRNA (miR)-494 on the hepatic ischemia-reperfusion injury (HIRI). Methods Twenty-four male SD rats were randomly divided into four groups (n=6 in each group). In the sham operation group, abdominal surgery without hepatic ischemia-reperfusion was performed. In the HIRI group, partial liver ischemia was performed for 60 min, followed by 6 h perfusion. In the HIRI+agomir-miR-494 group, intraperitoneal injection of agomir-miR-494 (20 μL) was daily given within preoperative 7 d. In HIRI+agomir-NC group, an equivalent quantity of agomir-NC was daily injected intraperitoneally within preoperative 7 d. The expression level of miR-494 messenger RNA(mRNA) in the liver tissues in each group was detected by reverse transcription polymerase chain reaction (RT-PCR). The expression levels of liver injury and oxidative stress related indexes were measured by relevant kits. The histopathological changes of the liver in each group were observed. The quantity of apoptotic cells and cytoplasmic histone-related DNA fragments in the liver tissues of rats was detected by relevant kits. The expression levels of the proteins related to the phosphatidylinositol-3-kinase(PI3K)/protein kinase(AKT) signaling pathway were measured by Western blot. Results The expression level of miR-494 mRNA in the rat liver tissues in the HIRI+agomir-miR-494 group was significantly higher than that in the HIRI+agomir-NC group (P < 0.01). The levels of the serum liver injury and oxidative stress related indexes in the HIRI+agomir-miR-494 group were significantly lower than those in the HIRI+agomir-NC group (all P < 0.01). Compared with those in the HIRI+agomir-NC group, the quantity of cellular necrosis was significantly reduced, the cell integrity was considerably increased and the quantity of TUNELpositive cells was evidently decreased in the HIRI+agomir-miR-494 group (all P < 0.05). The expression levels of poly ADP-ribose polymerase(PARP), cysteinyl aspartate specific proteinase-3(Caspase-3) and Bax in the HIRI+agomirmiR-494 group were significantly lower than those in the HIRI+agomir-NC group (all P < 0.05). The quantity of DNA fragments in the HIRI+agomir-miR-494 group was significantly less than that in the HIRI+agomir-NC group (P < 0.01). The expression levels of p-AKT, p-mammalian target of rapamycin(mTOR) and p-p70S6K in the HIRI+agomir-miR-494 group were significantly higher than those in the HIRI+agomir-NC group (all P < 0.05). Conclusions miR-494 can alleviate the severity of HIRI in rats by activating the PI3K/AKT signaling pathway. -
图 1 各组大鼠miR-494水平、氧化应激指标、凋亡相关指标和DNA片段化分析的比较
A图为各组大鼠肝组织中的miR-494水平;B图为各组大鼠血清中氧化应激指标MDA、TOA和OSI的水平;C图为各组大鼠肝细胞凋亡相关蛋白cleaved-PARP、cleaved-Caspase-3、Bax的表达水平;D图为各组大鼠DNA片段化分析;与假手术组比较,aP < 0.05和bP < 0.01;与HIRI+agomir-NC组比较,cP < 0.01
Figure 1. Comparison of miR-494 levels, oxidative stress indicators, apoptosis related indicators and DNA fragmentation analysis of rats among each group
表 1 各组大鼠肝损伤指标的比较
Table 1. Comparison of liver injury indicators of rats among each group(x±s)
组别 n ALT(U/L) AST(U/L) LDH(IU/L) GDH(U/L) 假手术组 6 49±12 61±11 113±15 661±76 HIRI组 6 1 163±32a 1 325±25a 657±76a 1 985±89a HIRI+agomir-NC组 6 1 250±26 1 523±33 665±73 1 992±83 HIRI+agomir-miR-494组 6 421±21b 532±23b 258±51b 1 013±82b 与假手术组比较,aP < 0.01;与HIRI+agomir-NC组比较,bP < 0.01 -
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