Study On The Role And Mechanism Of RIP3 In Macrophages In Liver Fibrosis

Background and Aims:Liver fibrosis is an important pathophysiological change in liver injury.A protein kinase,receptor interacting protein（RIP）3,plays a crucial role in mediating different diseases.RIP3 is a member of the serine-threonine kinase family,which is recognized as the central mediator in necrotizing apoptosis and plays an important role in the regulation of many human diseases.However,the role of RIP3 in macrophages in liver fibrosis has not yet been studied.In this study,RIP3 expression was found upregulated in liver tissues of patients and increased in liver tissues and macrophages in mice with liver fibrosis.Absence of RIP3 in macrophages could alleviate inflammation and macrophage or neutrophil accumulation in mice after carbon tetrachloride（CCl₄）or bile duct ligation（BDL）treatment.Importantly,RIP3 deficiency in macrophages could decrease CCl₄-induced and BDL-induced liver fibrosis in mice.Ras homologous gene family member A（Rho A）or Rho associated coil binding protein kinase（ROCK）is a"molecular switch"in LPS-mediated activation and synthesis of monocyte pro-inflammatory response.Moreover,RIP3 deficiency could inhibit the TLR4-NF-κB pathway through suppressing Rho-associated coiled-coil containing protein kinase（ROCK）1 in macrophages.NF-κB is a downstream effector molecule of TLR4 signaling pathway.It is a common nuclear transcription factor which mediate a lot of inflammatory processes.To explore the relationship between ROCK1 and RIP3 in macrophages of mice with liver fibrosis in vivo,RIP3-deficient macrophages with the overexpression of ROCK1 were infused to macrophage RIP3-deficient mice,which resulted in increased inflammation and liver fibrosis.In general,RIP3 plays a crucial proinflammatory role in promoting liver fibrosis through regulating the ROCK1-TLR4-NF-κB signaling pathway in macrophages and therefore may be a potential therapeutic target for immune-mediated liver fibrosis.Methods:32 samples were collected from patients with hepatic fibrosis who underwent hepatectomy.Intraperitoneal injection of CCl₄for 8 weeks or by BDL was used to establish a mouse model of hepatic fibrosis in WT mice.Then,we use q RT-PCR,Western blotting and immunofluorescence techniques to explore the relationship between RIP3and liver fibrosis.Subsequently,bone marrow transplantation was performed to produce the following chimeric mice:WT→WT（RIP3 expressed in both myeloid and non-myeloid cells）and RIP3^-/-→WT（RIP3 expressed only in non-myeloid cells）,to explore the effect of RIP3 deficiency in macrophages on inflammation and aggregation of macrophages or neutrophils in mice with liver fibrosis.To explore the role of RIP3 in macrophages in liver fibrosis,RIP3^-/-→WT and WT→WT mice were induced by BDL for two weeks or injected with CCl₄for eight weeks for inducing hepatic fibrosis.Western blotting and q RT-PCR were used to identify the molecular mechanism of RIP3 in macrophages in liver fibrosis.At the same time,lentivirus experiment was used to construct an effective functional recovery experiment,which further clarified the specific molecular dependent mechanism of RIP3 regulating macrophages on liver fibrosis.Results:The expression of RIP3 was increased in liver tissues of patients and was overexpressed in liver tissues and macrophages in mice of liver fibrosis.RIP3 deficiency in macrophages reduced inflammation and macrophage or neutrophil accumulation in mice with liver fibrosis.Meanwhile,in RIP3^-/-→WT mice,the serum levels of IL-1β,IL-6,TNF-α,and CXCL-10 were lower,while the serum levels of IL-4 and IL-10 were higher.In CCl₄treated RIP3^-/-→WT mice,the expression level of CD11b and Ly6G were decreased.In addition,RIP3 deficiency in macrophages alleviated CCl₄induced and BDL-induced liver fibrosis.Systematic molecular mechanism research showed ROCK1-dependent manner in macrophages.Furthermore,in vivo recovery experiments showed that the infusion of macrophages with overexpression of ROCK1 into macrophage RIP3 deficient mice increased inflammation and macrophage or neutrophils accumulation in RIP3-deficient mice,and also increased the degree of liver fibrosis in macrophage RIP3-deficient mice.Conclusion:In conclusion,macrophage RIP3 deficiency can reduce liver inflammation and fibrosis through ROCK1 via TLR4-NF-κB signaling pathway,which suggests that RIP3should be considered as a potential target for the treatment of liver fibrosis.