The Effect And Underlying Mechanism Of MiR-29b On Alcoholic Liver Disease In Mice

Background Alcoholic liver disease?ALD?is a common chronic liver disease all over the world.Chronic excessive alcohol consumption may induce hepatocytes damage,inflammation,oxidative stress and also lead to microRNA?miRNA?dysfunction.Our previous study has demonstrated that miR-29b was expressed in a variety of liver parenchymal and non-parenchymal cells and was closely related to the development of hepatic fibrosis.Overexpression of miR-29b could significantly inhibit collagen deposition and alleviate hepatic fibrosis.However,the role of miR-29b in the early stage of alcoholic liver disease is not clear.Objective To investigate its effect on alcoholic liver disease and underlying mechanism.Methods 1.Wild-type?WT?and miR-29b knockout(miR-29b^-/-)mice were fed a Lieber-DeCarli liquid diet containing 5%alcohol for 10 days,then received gavage of a single dose of ethanol?5 g/kg body weight?.2.The serum levels of alanine aminotransferase?ALT?and aspartate aminotransferase?AST?were measured by automatic biochemical analyzer,which were used to represent degree of hepatic injury.3.The harvested liver tissue was fixed in 4%formaldehyde for 24 hours,embedded in paraffin,and cut into 4?m for hematoxylin and eosin?H&E?staining to observe hepatic pathological changes.Neutrophils infiltration and macrophages activation in liver tissue was observed by immunohistochemical staining of myeloperoxidase?MPO?and F4/80.The expression of 4-hydroxynonene aldehyde?4-HNE?was detected by immunohistochemistry to reflect oxidative stress.4.Hepatic lipid accumulation was observed by oil red O staining.5.Dual-luciferase reporter assay revealed that target relationship between miR-29b and STAT3.6.Real-time quantitative PCR?q-PCR?was used to detect mRNA expression of miR-29b,inflammatory mediators and lipogenic genes.7.Western blot?WB?was used to detect STAT3 protein level in liver tissues and cells in different groups.Results In Gao-Binge model,miR-29b expression was significantly decreased.miR-29b knockout mice were more sensitive to alcohol-induced liver injury with higher level of ALT and AST,presented increased infiltration of neutrophils and secretion of pro-inflammatory cytokines.In addition,compared with WT mice,miR-29b^-/-mice represent more severe oxidative stress induced by alcohol.We found that depletion of miR-29b upregulate STAT3 mRNA and protein level and promote inflammatory response by targeting 3'UTR of STAT3.Whereas overexpression of miR-29b could significantly reduce the level of STAT3,suppressing inflammation and alleviate alcohol-induced liver injury.Conclusion After alcohol consumption,expression of miR-29b was decreased,and the IL-6/STAT3 signaling pathway was activated by targeting STAT3,which promoted the inflammatory reaction and aggravated the alcoholic liver injury.