Role And Mechanism Of RIP1 Kinase In Regulating Nonalcoholic Steatohepatitis Pathogenesis

Cell death and inflammation are intricately connected,mutually contributing to the progression of each other.Their crosstalk has been reported to play key roles in the pathogenesis of many diseases pathological.The kinase receptor-interacting protein 1(RIP1),as a crucial regulator determining the cell fate,is one of critical mediators regulating apoptotic and necrotic cell death as well as inflammation.RIP1 kinase has been reported to play a critical role in mediating the pathological processes of a variety of inflammatory diseases.However,it still remains elusive regarding the role of RIP1 in metabolic inflammatory disease,such as nonalcoholic steatohepatitis(NASH).NASH,as a metabolic inflammatory disease,is caused by multiple factors.It is widely reported that hepatic lipid accumulation such as increased free fatty acid(FFA)and free cholesterol causes hepatic steatosis and further leads to hepatocyte death and liver tissue damage through different mechanisms including endoplasmic reticulum stress,oxidative stress or mitochondrial damage,which is accompanied by chronic inflammation and liver fibrosis.During this process,however,the molecular basis for regulating hepatic cell death and inflammation through excessive lipids accumulation in liver tissue still remains unclear.Considering the critical role of RIP1 kinase in the regulation of cell death and inflammation,we hypothesized that it might also contribute to the pathological development of metabolic inflammatory diseases.In this study,we comprehensively investigated the role of RIP1 kinase in the pathogenesis of NASH and the underlying mechanisms using different animal models and cell models,and found that:1.RIP1 kinase activity was involved in NASH pathogenesis: Wild-type(WT)and RIP1 kinase-dead(Rip1K45A/K45A)mice were fed with high-fat diet(HFD)or methionine-choline deficient diet(MCD)to establish steatohepatitis models.Rip1K45A/K45 A mice exhibited significantly lower steatosis,liver injury and fibrosis than WT mice in both NASH models.2.RIP1 kinase activity contributed to the pathogenesis of NASH through prompting hepatic cell death and liver inflammation: We further explored the underlying molecular mechanism of RIP1 kinase promoting the pathogenesis of NASH.Our results indicated that hepatic apoptosis and necroptosis and liver inflammation were significantly reduced in Rip1K45A/K45 A mice than WT control in HFD-induced NASH model,suggesting that RIP1 kinase may promote the pathological progression of NASH by mediating cell death and inflammation.3.RIP1 kinase activity in macrophages was critical for the disease progression of steatohepatitis: Our results demonstrated that RIP1 kinase was mainly activated in hepatic macrophages in NASH models.Consistent with results from RIP1 kinase was also significantly activated in liver tissue of NASH patients,and immunofluorescence costaining results showed that RIP1 was mainly phosphorylated and activated in hepatic macrophages.Saturated fatty acid(palmitic acid,PA)treatment also induced kinase activation of RIP1 in liver macrophages in vitro.In bone marrow derived macrophages(BMDMs)and primary Kupffer cells,RIP1 kinase mediated palmitatic acids-induced inflammasome activation,apoptosis and necroptosis.Results from chimeric mice established through lethal irradiation and bone marrow transplantation further confirmed that the RIP1 kinase in hematopoietic-derived cells contributed mostly to the disease progression in NASH.4.RIP1 kinase is required for inflammasome activation induced by endoplasmic reticulum stress(ER stress): ER stress has been reported to play important roles in NASH pathogenesis.Therefore,we also investigated the role of RIP1 in ER stress-induced inflammasome activation in vitro.RIP1 kinase inhibitor Necrostatin 1(Nec-1)or si RNAmediated RIP1 knockdown significantly reduced ER stress-induced caspase-1 cleavage and IL-1? secretion in both BMDMs and J774 A.1 macrophages,suggesting that RIP1 kinase is involved in the regulation of ER stress-induced inflammasome activation,potentially through regulating ROS and Drp1In conclusion,our study shows that HFD feeding or saturated fatty acid stimulation can activate RIP1 kinase in macrophages,and then mediate cell death,inflammasome activation and pathological progression of NASH.RIP1 kinase in macrophages might be a potential therapeutic target for NASH or other metabolic inflammatory diseases.Majdi et al.at the same time also reported that RIP1 kinase inhibitors have shown positive therapeutic effects in experimental NASH models,and RIP1 kinase inhibitors have reached clinical trial stages.Our results provide evidence for broadening the potential clinical application of RIP1 kinase inhibitor.