Xiaohui Xu1, Rui Li1, Xin Zeng1, Xin Wang1, Bingqian Xue1, Daochao Huang1, Yi Huang1. 1. Institute of Pediatrics, Children's Hospital of Chongqing Medical University, Chongqing Key Laboratory of Child Infection and Immunity// Key Laboratory of Child Development and Disorders of Ministry of Education//National Clinical Research Center for Child Health and Disorders//China International Science and Technology Cooperation Base of Child Development and Critical Disorders, Chongqing 400014, China.
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). CONCLUSIONS: Hepatocytes-specific NDUFA13 ablation can trigger spontaneous hepatitis in mice possibly mediated by the activation of ROS/NF-κB/NLRP3 signaling.
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). CONCLUSIONS: Hepatocytes-specific NDUFA13 ablation can trigger spontaneous hepatitis in mice possibly mediated by the activation of ROS/NF-κB/NLRP3 signaling.