METTL3 Promotes Macrophage Pyroptosis In Alcoholic Steatohepatitis Via MiR-34a-5p/SIRT1 Axis

Alcoholic liver disease(ALD)is one of the most prevalent liver diseases in Europe and the United States.This disease may be caused by long-term daily drinking of more than a certain amount(generally considered to be more than 40 g of pure alcohol).The symptoms are atypical,the range of disease is wide,and there is no effective targeted treatment.The pathological process of ALD includes simple steatosis,alcoholic steatohepatitis,liver fibrosis,liver cirrhosis and even the development of liver cancer.Among them,alcoholic steatohepatitis(ASH)is an important turning point in the progression of the disease,and intervention in the formation of ASH is the focus of prevention and treatment of ALD.Previous studies have found that the activation of liver inflammation in ALD is caused by LPS(lipopolysaccharide)in the intestine entering the liver through the portal vein or upregulation of systemic inflammatory factors.Recent studies have found that biological factors released by hepatocyte apoptosis can activate immune cells.This discovery has caused hepatocyte apoptosis to rise from the level of ALD pathological results to inducement.In addition,there are also reports in the literature that in addition to apoptosis,there are other ways of programmed cell death in the pathological process of ALD.Pyroptosis is also one of the ways of programmed cell death.It is induced by the first signal LPS and the second signal such as Nig(Nigericin)and ATP(adenosine triphosphate).Pyroptosis is mediated by Caspase family proteins and is characterized by the process of pyrolysis.Then cells release a large amount of IL-1β and IL-18 to promote the activation of inflammation.1.Liver macrophage pyrolysis is involved in the occurrence of alcoholic hepatitis This experiment explored the role of cell pyroptosis in Kupffer cells in the liver of ASH mice fed with alcoholic diets.The experimental results showed that Kupffer cells in the liver of ASH mice were activated to promote alcoholic liver injury,and cell pyroptosis was partly or completely involved in it.These results suggesting that not only liver cells,but also the unprogrammed death of immune cells can also promote the occurrence of immune overreaction.2.SIRT1 inhibited the pyrosis of mouse bone marrow macrophages in vitro SIRT1 is a histone-modified deacetylase.A large number of studies have shown that it has the functions of anti-inflammatory,anti-aging,immune regulation and metabolic regulation.For this reason,we investigated whether it can alleviate the excessive release of inflammatory factors mediated by pyroptosis.We established an in vitro model of pyrolysis using mouse bone marrow macrophages.The cells were transfected with SIRT1 overexpression plasmids and silencing Si RNA.The results were consistent with our expectations,SIRT1 could inhibit the production of pyrolysis.3.miR-34a-5p can target and inhibit the expression of SIRT1 to promote macrophage pyrolysis in vitroAnother interesting thing is that the protein expression of SIRT1 in mouse ASH liver Kupffer cells is significantly lower than that of m RNA.We suspect this phenomenon may be related to protein degradation or translation inhibition.For this reason,we screened out miRNAs predicted to be able to bind to the 3’end of SIRT1 m RNA through 5 databases,and verified their expression in liver Kupffer cells through experiments.Finally,we determined that miR-34a-5p/SIRT1 regulates cell pyroptosis.In addition,we used mimics and inhibitors of miR-34a-5p to transfect mouse bone marrow macrophages to verify its effect on pyroptosis.4.METTL3 mediates macrophage pyrolysis by promoting the maturation of miR-34a-5pOur research group has been committed to studying the apparent regulation mechanism in liver diseases.The previous research of our research group found that in liver fibrosis,the level of m6A(N-6 methyladenosine)is abnormally increased.That promotes the synthesis and maturation of miRNA,not the degradation of m RNA.We found that Kupffer cells in ASH liver also showed high levels of m6A.In order to verify whether m6A is involved in the maturation of miR-34a-5p in ASH,we screened the level of m6A-related methylases,then we transfected BMDM with METTL3 virus to verifies its function.All in all,our research breakthrough incorporates Kupffer cell pyroptosis into the development of ASH disease,and proposes that METTL3 can increase Kupffer cell pyroptosis by regulating the miR-34a-5p/SIRT1 axis.It provides a new direction for the study of the mechanism of ALD and the discovery of new targets.