PTP1B Regulates Macrophage Activation In Alcoholic Liver Injury Through The NF-?B Pathway

Alcoholic liver injury(ALI)is a liver related disease caused by long-term heavy drinking(alcoholism).These diseases include steatohepatitis,Alcoholic fibrosis,cirrhosis and hepatocellular carcinoma.In recent years,the incidence and mortality of liver diseases caused by alcohol consumption in China have been on the rise.There are several pathogenesis of ALI,mainly including kupffer cells(KCs)activation and hepatocyte apoptosis caused by alcohol and endotoxin.Therefore,inhibiting KCs activation and hepatocyte apoptosis can effectively slow down the progression of alcoholic liver injury.KCs are the intrinsic macrophages of the liver,which have functions such as phagocytosis,immune regulation and secretion of inflammatory factors,its activation plays an important role in alcoholic liver injury.Continuous inflammatory state can destroy the normal function and structure of the liver,and thus progress to more serious pathological state of the liver.Protein tyrosine phosphatases(PTPs)belong to a super family of proteins that dephosphorylate tyrosine on their protein substrates and play a key role in a number of biological processes.Protein tyrosine phosphatase 1B(PTP1B)is an intracellular PTPs,which is involved in the regulation of many cell signaling molecules.It has a variety ofregulatory roles in liver regeneration,hepatocyte apoptosis and liver cancer,but its role in ALI has not been fully elucidated.This paper will focus on the role of PTP1 B in macrophage activation in alcoholic liver injury and the mechanism of NF-?B signaling pathway in this process.In this study,in vivo and in vitro experiments were combined.In vivo experiments: 8-10 weeks C57 mice were randomly divided into two groups(normal group and model group)and kept in the animal experiment center.NIAAA experimental method was adopted for modeling.After 15 days of control and alcohol diet,blood and liver tissue were harvested after anesthesia.Serum levels of inflammatory factors and related indicators of liver damage were detected by ELISA and other kits.Liver tissues were processed for histological experiments such as HE,immunohistochemistry and fluorescence,and extracted for WB and QPCR.In vitro experiments: mouse macrophages RAW264.7 were stimulated with alcohol plus LPS,and then transfected with si RNA and p EX3-PTP1 B.Mouse hepatocytes AML-12 were stimulated with an adaptive concentration of alcohol.Protein and total RNA were extracted after 24 h appropriate culture.Protein expression of PTP1 B and inflammatory factors was detected by WB experiment,and gene expression of PTP1 B and inflammatory factors was detected by QPCR experiment.The expression levels of NF-?B related P65 and phosphorylated P65 were detected by using the corresponding kit.The corresponding levels of inflammatory factors in the cell culture supernatant were detected by ELISA.Results: the establishment of the animal model was confirmed by histological staining and serological experiments.In vivo experiments,we found that the expression levels of PTP1 B gene and protein were up-regulated,and the expressionlevels of inflammatory factors such as IL-1??IL-6 and TNF-? were increased in serum and liver tissues.In vitro studies showed no significant changes in the gene and protein expression of PTP1 B in AML-12 cells after alcohol stimulation.But the expression levels of PTP1 B gene and protein in mouse RAW264.7 cells were significantly increased when they were activated.In addition,the expression level of phosphorylated P65 protein in the nucleus increased after overexpression of PTP1 B and the expression level of inflammatory factors was up-regulated as well.While the expression level of phosphorylated P65 protein decreased after silencing PTP1 B and the expression level of inflammatory factors was down-regulated,indicating that silencing or overexpression of PTP1 B could regulate the activation or inhibition of NF-?B signaling pathway.Conclusion: in the alcoholic liver damage model,PTP1 B gene and protein expression level were both rised.Silence and overexpression of PTP1 B can regulate inflammation factors expression level such as IL-1??IL-6 and TNF-?,as well as the expression of the NF-?B pathway proteins,suggesting that PTP1 B may regulate macrophage activation in the alcoholic liver damage and the expression levels of inflammatory markers through regulating the NF-?B signaling pathways.