Circadian disruption aggravates non-alcoholic fatty liver disease by activating RIPK1-RIPK3-MLKL axis in mice

Circadian disruption represents a significant risk factor for non-alcoholic fatty liver disease (NAFLD); however, the underlying regulatory mechanisms remain poorly understood. This study aims to investigate the impact of circadian disruption on NAFLD development in mice and to elucidate the associated molecular pathways. First, a NAFLD mouse model was established by feeding mice a high-fat diet over a period of 12 weeks, during which an intervention to disrupt the circadian rhythm was also implemented. The experimental groups included: the control group, the NAFLD model group, the circadian disruption group (CCD), and the NAFLD combined with circadian disruption group (NAFLD + CCD). Lipid accumulation and liver function were assessed using biochemical assay kits from each group. Serum levels of inflammatory cytokines were measured via ELISA. Histopathological alterations in liver tissues were evaluated using HE staining, Masson staining, and Sirius Red staining. Cell apoptosis in liver tissues was detected using the TUNEL assay, while the expression levels of fibrosis-related (Collagen IV, Fibronectin, and α-SMA) proteins were determined through immunohistochemical analysis. Western blotting was employed to assess the expression of necroptosis-related (p-RIPK1/RIPK1, p-RIPK3/RIPK3, and p-MLKL/MLKL) proteins. Additionally, immunofluorescence triple staining was performed to detect the co-localization of RIPK3, IBA1, and Clec4F. The results showed that circadian disruption markedly enhanced lipid accumulation in both serum and liver tissue of NAFLD mice, thereby exacerbating hepatic functional impairment. Compared with the NAFLD group, the NAFLD + CCD group exhibited increased collagen fiber deposition and elevated expression levels of fibrosis-related and necroptosis-related proteins. Furthermore, circadian disruption significantly promotes necroptosis of kupffer cells in the liver tissue of NAFLD mice. In conclusion, this study reveals a novel mechanism by which circadian rhythm disruption promotes necroptosis in kupffer cells by activating the RIPK1/RIPK3/MLKL pathway, thereby exacerbating NAFLD, providing a potential new target for the prevention and treatment of this disease.