Research About The Role And Mechanism Of NLRC4 Inflammasome Activation And Its Triggered Pyroptosis In Nonalcoholic Fatty Liver Disease Development

Lipid accumulation in the liver is an important cause of non-alcoholic fatty liver disease(NAFLD),which triggers a series of immune and inflammatory responses that promote the development of simple fatty liver(NAFL)to nonalcoholic steatohepatitis(NASH).Simple fatty liver does not require medication and can be alleviated by adjusting diet and lifestyle.Progression of simple fatty liver to hepatitis increases the prevalence of cardiovascular disease and malignancy,and the treatment of patients will be more complicated.Therefore,in-depth analysis of NAFL conversion to NASH helps control the disease progression of NAFLD.However,the specific mechanism is unclear,and this topic focuses on this issue.We used Hep G2 cells and THP-1 macrophages to establish an external coculture system.The in vitro NAFLD cell model was established by stimulating the cells with a mixture of FFAs,containing palmitic acid and oleic acid.Pre-experimental results show that the accumulation of FFAs in hepatocytes leads to a variety of organelle dysfunction and cell damage accompanied by the release of a series of inflammatory factors including TNF-?,IL-18,IL-1?.We focused on the activation of inflammasome since IL-18 and IL-1? can be induced and released by inflammasome activation and we explored this question with various experimental technologies including WB,qPCR,IF,Flow cytometry,Confocal microscopy and genome editing by siRNA and plasmid.The results suggested that NLRC4 inflammasome was activated in vitro NAFLD cell model and pyroptosis was triggered.Immunofluorescence results showed that NLRC4 was translocated to mitochondria after stimulation.The mRNA level of pro-IL-18 and pro-IL-1? as well as the proteins level of IL-18 and IL-1? were reduced in parallel with the inhibition of NLRC4 and increased with the overexpression of NLRC4,which indicated that the release of IL-18 and IL-1? was induced by NLRC4 increase.The inhibition of TNF-a with TNF-a-targeting siRNA after FFA treatment showed that NLRC4 and its downstream proteins,IL-18 and IL-1?,were reduced.So it could be asserted that NLRC4 expression was regulated by TNF-a.In conclusion,TNF-a expression is rapidly increased by FFA stimulation,and activates NLRC4.The activation of NLRC4 triggers pyroptosis,together with NLRC4 translocation.