肝细胞线粒体NDUFA13蛋白缺陷可诱导小鼠自发性慢性肝纤维化

目的 探究肝细胞线粒体NDUFA13蛋白表达缺失在诱导小鼠肝脏纤维化发生中的作用及可能机制。方法 以肝脏特异性NDUFA13杂合敲除小鼠品系（NDUFA13fl/-；Alb-Cre）作为研究对象，以同窝NDUFA13fl/fl小鼠作为对照，8只/组，进行长达两年的实验观察，分别在实验早期和晚期处死小鼠获取肝组织样本。采用HE染色、Masson染色观察不同时期小鼠的肝组织病理变化及纤维化表型。Western blot检测肝组织中NDUFA13蛋白表达水平。免疫荧光分析巨噬细胞标志物F4/80与转化生长因子TGF-β1、肿瘤坏死因子TNF-α与白介素IL-1β的表达。免疫组化分析肝星型细胞活化标志物α-SMA、基质金属蛋白酶MMP-9与基质金属蛋白酶组织抑制剂TIMP-1、胶原纤维Collagen-Ⅰ与Collagen-Ⅲ的表达情况。结果 HE染色、Masson染色发现4周 龄NDUFA13fl/-小鼠肝组织炎症浸润明显但未出现纤维化表型；2年龄NDUFA13fl/-小鼠肝组织严重损伤且伴大量胶原纤维产生。Western Blot结果显示2年龄NDUFA13fl/-小鼠肝组织中NDUFA13蛋白表达水平较对照小鼠明显降低（P<0.05）。免疫荧光与免疫组化结果显示，NDUFA13fl/-小鼠肝组织中巨噬细胞F4/80聚集增多并伴随大量TGF-β1产生（P<0.05），以及TNF-α、IL-1β等炎症因子大量分泌（P<0.05）；同时，α-SMA、Collagen-Ⅰ及Collagen-Ⅲ表达明显增多（P<0.05），基质金属蛋白酶MMP-9表达降低（P<0.05），而其抑制剂TIMP-1表达明显增加（P<0.05）。结论 肝细胞线粒体NDUFA13蛋白缺陷能够诱导小鼠自发的慢性肝纤维化病理表型，可能与巨噬细胞/炎症因子信号诱导的肝星状细胞异常活化有关。

Mechanism of hepatocyte mitochondrial NDUFA13 deficiency-induced liver fibrogenesis: the role of abnormal hepatic stellate cell activation

Objective To investigate the role of hepatocyte mitochondrial NDUFA13 loss in the liver fibrogenesis in mice and explore the possible mechanisms. Methods We used liver-specific NDUFA13 heterozygous knockout mouse models (NDUFA13fl/- ; Alb-Cre) established previously by intercrossing NDUFA13fl/fl and Alb-Cre mice, with their littermate control NDUFA13fl/fl mice as the control (n=8). The mice were euthanized at the age of 4 weeks and 2 years, and the liver tissues were collected for HE and Masson staining to observe the pathological changes and fibrosis phenotypes. Western blotting was performed to detect the expression of NDUFA13 protein in the liver tissues, and the infiltration of F4/80+ macrophages and the expressions of TGF-β1, TNF-α and IL-1β were analyzed by immunofluorescence assay. The expression levels of α-SMA, matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of matrix metalloproteases 1 (TIMP-1), collagen-I and collagen-III were assayed by immunohistochemistry. Results HE and Masson staining showed obvious inflammatory infiltration but no significant fibrosis in the liver tissues of 4-week-old NDUFA13fl/- mice, but severe liver damage with massive fibrosis was observed in 2-year-old NDUFA13fl/- mice. NDUFA13 expression in 2-year-old NDUFA13fl/- mice markedly decreased compared with that in the control NDUFA13fl/fl mice as shown by Western blotting (P<0.05). Immunohistochemistry showed obvious infiltration of F4/80+ macrophages in the liver tissue with a large amount of TGF-β1 production (P<0.05) and TNF-α and IL-1β secretions in NDUFA13fl/- mice (P<0.05). NDUFA13 knockout obviously promoted α-SMA expression (P<0.05) and collagen- I and collagen-III deposition (P<0.05) while significantly decreased MMP-9 and increased TIMP-1 expression in the liver (P< 0.05). Conclusion Hepatocytes-specific NDUFA13 deficiency can trigger spontaneous and chronic liver fibrosis phenotypes in mice probably in association with abnormal activation of hepatic stellate cells induced by macrophages and inflammatory factors.