干扰素调节因子1调控线粒体自噬参与小鼠肝缺血再灌注损伤

Effect of LRF-1 on murine liver ischemia-reperfusion injury by regulating mitophagy

【摘要】目的探讨干扰素调节因子-1（IRF-1）调控线粒体自噬对小鼠肝缺血再灌注损伤的影响。方法选择健康雄性C57BL／6小鼠建立活体小鼠肝缺血再灌注损伤模型，用随机数字表法分为假手术组（sham）和缺血再灌注组（IR），每组6只。血生化检测两组小鼠血清ALT、AST变化，罗丹明123染色（Rhodamine123）法检测肝细胞线粒体损伤情况，HE染色观察肝组织病理学改变，TUNEL法检测肝细胞凋亡情况。Westernblot检测IRF-1、Nix、LC3和Caspase-3蛋白表达。AML12细胞系建立离体肝细胞缺血再灌注损伤模型，分为siRNA-NC组和siIRF-1组。PI染色法检测AMLl2细胞凋亡情况，免疫荧光观察AML12细胞内自噬体形成情况，Westernblot检测IRF-1、Nix、LC3和Bax蛋白表达。结果与假手术组比较，缺血再灌注组小鼠血清ALT、AST明显升高（P〈0．0001）；罗丹明123染色法见缺血再灌注组线粒体膜电位明显下降，线粒体损伤严重；病理学检查见缺血再灌注组肝细胞肿胀、脂肪变性，肝窦狭窄，中性粒细胞浸润和片状坏死，肝组织结构破坏严重；TUNEL法检测见IR组肝细胞凋亡率明显增高（P〈0．0001）；Westernblot检测显示，缺血再灌注组IRF-1、Caspase-3蛋白表达水平明显高于假手术组，Nix、LC3Ⅱ表达水平明显低于假手术组。PI染色法示，SiRNA-NC组细胞凋亡率明显高于siIRF-1组（P〈0．0001）。免疫荧光示SiRNA-NC组细胞内自噬体个数明显低于siIRF-1组（P〈0．0001）；Westernblot检测显示SiRNA-NC组IRF-1、Bax蛋白表达水平明显高于siIRF-1组，Nix、LC3Ⅱ表达水平明显低于siIRF-1组。结论IRF-1加重小鼠肝缺血再灌注损伤，其机制可能与IRF-1抑制线粒体自噬，促进肝细胞凋亡有关。抑制IRF-1表达能够提高线粒体自噬，对肝缺血再灌注起到保护作用。

Objective To explore the effect of interferon regulatory factor-1 （IRF-1） on murine liver ischemia-reperfusion injury （IRI） by regulating mitophagy. Methods The living routine hepatic IRI model was established in the healthy male Male C57BL/6 mice. The male C57BL/6 mice were randomly allocated into sham group and IR group （n = 6 each）. Serum ALT and AST levels were determined in two groups by blood biochemical tests. The mitochondrial damage of liver cells was detected by Rh123 staining. The pathological changes of liver tissue were observed by HE staining. The apoptosis of liver cells was detected by TUNEL assay. The expression levels of IRF-1, Nix, LC3 and Caspase-3 proteins were detected by Western blotting. AML12 cells were cultured and divided into siRNA-NC group and silRF-1 group. PI staining was used to detect the apoptosis of AML12 cells. Immunofluorescence was used to observe the formation of autophagy in AML12 cells during IRI. Western blotting was used to detect the expression of IRF-1, Nix, LC3 and Bax. Results IR group had significantly higher levels of serum ALT and AST than in sham group （P〈0. 001）. Rhodamine 123 staining showed the decreased mitochondria membrane potential and mitochondrial damage. The hepatic histological changes were aggravated in IR group, consisted of hepatocytes swelling, fatty degeneration, sinusoids narrowing, neutrophils infiltration and large patchy necrosis. TUNEL staining showed IR group exhibited an increased hepatocellular apoptosis rate （P〈0. 001）. Western blotting showed the expression of IRF-1 and caspase-3 proteins was significantly higher in IR group than that in sham group. The Nix and LC3 II expression levels were significantly lower in IR group than those in sham group. PI staining showed the apoptosis rate in siRNA-NC group was significantly higher than in siIRF-1 group. Immunofluorescence showed the number of autophagosomes was significantly less in siRNA-NC group than in siIRF-1 group. Western blotting showed the expression levels of IRF-1 and Bax in siRNA-NC group were significantly higher than those in siIRF-1 group, and those of Nix and LC3 II were significantly lower than those in siIRF-I group. Conclusion IRF-1 promotes murine liver IRI, which may be related to the inhibition of mitophagy and induction of hepatocellular apoptosis. Inhibition of IRF-1 expression can improve mitophagy, and play a protective role in liver IRI.