南方医科大学学报

南方医科大学学报 ›› 2024, Vol. 44 ›› Issue (2): 201-209.doi: 10.12122/j.issn.1673-4254.2024.02.01

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NDUFA13过表达可减轻CCl4诱导的小鼠肝纤维化：基于抑制NLRP3活化

徐小惠，冯金梅，罗 颖，何昕觎，臧金宝，黄道超   

  1. 重庆医科大学附属儿童医院儿科研究所//国家儿童健康与疾病临床医学研究中心//儿童发育疾病研究教育部重点实验室，重庆 400014；重庆医科大学附属儿童医院心内科//国家儿童健康与疾病临床医学研究中心//儿童发育疾病研究教育部重点实验室//国家临床心血管内科重点专科//重庆市卫生健康委儿童重要器官发育与疾病重点实验室，重庆 400014；重庆市九龙坡区第二人民医院检验科，重庆 400052
  • 发布日期:2024-03-14

Adeno-associated virus-mediated hepatocyte-specific NDUFA13 overexpression protects against CCl4-induced liver fibrosis in mice by inhibiting hepatic NLRP3 activation

XU Xiaohui, FENG Jinmei, LUO Ying, HE Xinyu, ZANG Jinbao, HUANG Daochao   

  1. Institute of Pediatrics, Children's Hospital of Chongqing Medical University; Department of Cardiology, Children's Hospital of Chongqing Medical University, Key Laboratory of Children's Important Organ Development and Diseases of Chongqing Municipal Health Commission; Department of Laboratory Medicine, Second People's Hospital of Jiulongpo District
  • Published:2024-03-14

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摘要： 目的 探究NDUFA13蛋白在小鼠急性肝损伤和肝纤维化中的保护作用及可能机制。方法 小鼠分为正常组（n=18）、CCl4组（n=18）、CCl4+AAV-NC组（n=18）、CCl4+AAV-NDU13组（n=18）。采用腹腔注射CCl4（2次/周）建立肝纤维化小鼠模型，连续给药3、5、7周后处死取材。病毒干预组小鼠则预先分别尾静脉注射AAV8-TBG-NC对照与AAV8-TBG-NDUFA13过表达病毒，7~10 d后再经CCl4进行肝纤维化诱导。通过HE染色、Masson染色观察CCl4致小鼠肝损伤及肝纤维化情况。Western blotting检测肝组织中NDUFA13、α-SMA蛋白表达水平。免疫荧光分析NDUFA13与炎症小体NLRP3、肿瘤坏死因子TNF-α与白介素IL-1β、肝星型细胞活化标志物α-SMA与胶原纤维Collagen Ⅲ的表达情况。结果 HE染色、Masson染色结果显示CCl4诱导小鼠肝组织肝小叶结构紊乱，肝细胞变性坏死，炎症细胞浸润且伴大量胶原纤维沉积（P<0.001）。Western blotting结果发现CCl4模型鼠肝脏NDUFA13蛋白表达水平相比正常对照组明显降低（P<0.001）；而NDUFA13蛋白过表达后，CCl4处理小鼠的肝脏炎症细胞聚集和纤维化均明显减少（P<0.001）。免疫荧光结果显示，NDUFA13蛋白过表达的CCl4诱导鼠肝组织中炎症小体NLRP3活化明显减弱（P<0.001），并伴随炎症因子TNF-α与IL-1β分泌显著减少（P<0.001），同时肝星状细胞活化（P<0.05）及其胶原形成均受到抑制（P<0.001）。结论 肝细胞线粒体NDUFA13蛋白过表达可通过抑制NLRP3炎症信号活化发挥抗纤维化作用。

关键词: NDUFA13过表达；NLRP3；炎症因子；肝星状细胞；肝纤维化

Abstract: Objective To investigate the protective effect of NDUFA13 protein against acute liver injury and liver fibrosis in mice and explore the possible mechanisms. Methods BALB/C mice (7 to 8 weeks old) were divided into normal group, CCl4 group, CCl4+AAV-NC group and CCl4+AAV-NDU13 group (n=18). Mouse models of liver fibrosis were established by intraperitoneal injection of CCl4 twice a week for 3, 5 or 7 weeks, and the recombinant virus AAV8-TBG-NC or AAV8-TBG-NDUFA13 was injected via the tail vein 7-10 days prior to CCl4 injection. After the treatments, pathological changes in the liver of the mice were observed using HE and Masson staining. Hepatic expression levels of NDUFA13 and α-SMA were detected with Western blotting, and the coexpression of NDUFA13 and NLRP3, TNF-α and IL-1β, and α-SMA and collagen Ⅲ was analyzed with immunofluorescence assay. Results HE and Masson staining showed deranged liver architecture, necrotic hepatocytes and obvious inflammatory infiltration and collagen fiber deposition in mice with CCl4 injection (P<0.001). NDUFA13 expression markedly decreased in CCl4-treated mice (P<0.001), while a significant reduction in inflammatory aggregation and fibrosis was observed in mice with AAV-mediated NDUFA13 overexpression (P<0.001). In CCl4+AAV-NDU13 group, immunofluorescence assay revealed markedly weakened activation of NLRP3 inflammasomes (P<0.001), significantly decreased TNF-α and IL-1β secretion (P<0.001), and inhibited hepatic stellate cell activation (P<0.05) and collagen formation in the liver (P<0.001). Conclusion Mitochondrial NDUFA13 overexpression in hepatocytes protects against CCl4- induced liver fibrosis in mice by inhibiting activation of NLRP3 signaling.

Key words: NDUFA13 overexpression; NLRP3; inflammatory cytokines; hepatic stellate cells; liver fibrosis