南方医科大学学报

南方医科大学学报 ›› 2021, Vol. 41 ›› Issue (1): 55-63.doi: 10.12122/j.issn.1673-4254.2021.01.07

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NDUFA13失活在诱导小鼠自发性肝炎病理进程中的作用及机制

徐小惠，李 锐，曾 欣，王 欣，薛炳乾，黄道超，黄 轶
  

  • 出版日期:2021-01-26 发布日期:2021-01-26

Pathogenic role of NDUFA13 inactivation in spontaneous hepatitis in mice and the mechanism

  • Online:2021-01-26 Published:2021-01-26

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摘要：

目的 构建线粒体蛋白（NDUFA13）基因肝特异性敲除小鼠，探讨NDUFA13表达失活在小鼠自发性肝炎病理进程中的作用及可能机制。方法 采用Cre/loxP转基因技术，将NDUFA13 flox基因编辑小鼠NDUFA13fl/fl与Alb-Cre转基因小鼠杂交，得到肝脏特异性NDUFA13杂合敲除（NDUFA13fl/-；Alb-Cre）小鼠。经PCR扩增及琼脂糖凝胶电泳，对鼠尾与肝脏DNA基因进行分型鉴定。以同窝NDUFA13fl/fl小鼠为对照，将肝脏特异性NDUFA13fl/-小鼠作为研究对象，10只/组，分别在4周龄、2年龄时各处死5只，获取小鼠肝组织样本，通过HE染色分析肝组织病理变化，免疫组化分析NDUFA13、NF-κB/p65、NF-κB/p-p65及炎症小体NLRP3的表达，ROS染色试剂盒分析细胞总ROS及线粒体ROS水平；免疫组化与免疫荧光分析免疫细胞标志物CD45、MPO、F4/80以及炎症因子IL1β、IL33的表达情况。结果 PCR鉴定结果显示成功建立肝特异性NDUFA13杂合敲除小鼠；HE染色显示4周龄与2年龄NDUFA13fl/-小鼠肝组织均损伤严重；免疫组化检测显示，
4周龄与2年龄NDUFA13fl/-小鼠NDUFA13蛋白表达显著下调（P<0.05），NF-κB信号分子p65、p-p65（Ser536）及炎症小体NLRP3蛋白表达显著增加（P<0.05）；ROS测定发现NDUFA13基因杂合敲除导致小鼠肝脏细胞总ROS、线粒体ROS水平均显著增加；免疫组化与免疫荧光结果显示，肝组织CD45、MPO、F4/80等炎症细胞标志物的聚集以及IL1β、IL33等炎症因子的分泌均明显增多（P<0.05）。结论 NDUFA13蛋白表达失活可诱导小鼠自发性肝炎病理表型，其机制可能与活化的ROS/NF-κB/ NLRP3信号通路相关。

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Abstract:

Objective To investigate the role of NDUFA13 inactivation in the pathogenesis of spontaneous hepatitis in mice and explore the possible mechanisms. Methods Hepatocyte-specific NDUFA13 knockout (NDUFA13fl/- ) mice were generated by intercrossing NDUFA13fl/fl and Alb-Cre mice based on Cre/loxP transgenic technology, and tail and liver DNA of the mice was genotyped by PCR analysis. Ten NDUFA13fl/- mice and 10 littermate control NDUFA13fl/fl mice were housed, and in each group, 5 mice were euthanized at the age of 4 weeks and the other 5 at two years for pathological examination of the liver tissues with HE staining. Immunohistochemistry was used to verify the expression levels of NDUFA13, NF-κB/p65, NF-κB/p-p65 and inflammasome NLRP3. The total intracellular ROS and mitochondrial ROS levels were measured with a ROS staining kit. The expressions of the inflammatory cell markers (CD45, MPO, and F4/80) and inflammatory cytokines (IL1β and IL33) in the liver were detected with immunohistochemistry and immunofluorescence assay. Results Liver-specific NDUFA13 heterozygous knockout mice were successfully constructed as verified by PCR results. HE staining revealed severe liver damage in both 4-week-old and 2-year-old NDUFA13fl/- mice as compared with their littermate controls. Immunohistochemistry showed a significant decrease of NDUFA13 expression in both 4-week-old and 2-year-old NDUFA13fl/- mice (P<0.05). The expression levels of NF-κB signals p65, p-p65 and NLRP3 inflammasomes were all significantly increased in NDUFA13fl/- mice (P<0.05). The total intracellular ROS and mitochondrial ROS levels in NDUFA13fl/- mice were also significantly increased. NDUFA13 knockout obviously promoted the expression of the inflammatory cell markers (CD45, MPO and F4/80) and the secretion of IL-1β and IL-33 in the liver tissue of the mice (P<0.05). Conclusion Hepatocytes-specific NDUFA13 ablation can trigger spontaneous hepatitis in mice possibly mediated by the activation of ROS/NF-κB/NLRP3 signaling.

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