Bmal1 Regulates Macrophage Polarize Through Glycolytic Pathway In Alcoholic Liver Disease

Liver is the main metabolic organ of the body.Ethanol and its derivative acetaldehyde can cause steatosis,necrosis and regeneration of liver cells after long-term excessive drinking,thus leading to alcoholic liver disease(ALD).ALD covers a wide range of clinical and tissue.Most patients who drink alcohol develop alcoholic fatty liver disease(AFLD),and only a small number of people develop advanced alcoholic steatohepatitis(ASH)and alcoholic cirrhosis(AC).ALD,as a potential pathological state,is characterized by liver cell injury,fat deposition and tissue inflammatory infiltration.Notably,if the inflammatory response continues to worsen,it can lead to liver cell necrosis and further damage to liver function.In recent years,it has been found that the polarization of hepatic macrophages is closely related to the inflammation of ALD.Furthermore,Brain and Muscle ARNT-like protein-1(Bmal1)is involved in macrophage polarization during a variety of disease processes.However,whether Bmal1 is involved in macrophage polarization during the progression of ALD has not been reported.This study aims to investigate the potential mechanism of Bmal1 regulating macrophages polarization in ALD.Therefore,this study is mainly carried out from the following aspects:1.Changes of macrophage phenotype in RAW264.7 cells stimulated by ethanol and liver tissue of EtOH-FED miceIn vivo experiments,Gao-Binge model was used to replicate alcoholic liver disease model.H&E and oil red O staining results showed that large area of fat vacuoles,hepatic cord disorder and obvious lipid droplet deposition were observed in the liver tissue of EtOH-fed mice.In addition,the serum levels of total cholesterol(T-CHO),triglyceride(TG),alanine aminotransferase(ALT)and aspartate aminotransferases(AST)in mice fed with alcohol were significantly increased.Western blot and q RT-PCR results showed that M1 and M2 macrophage markers(TNF-α,IL-1β,IL-10,Arg-1)were significantly elevated.These results displayed that the mice model of alcoholic liver disease was successfully constructed and the polarization state of macrophages was changed.In vitro experiment,RAW264.7 cells were stimulated with 25 m M ethanol for 24 h.Western blot and q RT-PCR results showed that the polarization state of macrophages was changed after ethanol stimulation.In ethanol stimulated RAW264.7 cells,the surface markers of M1 and M2 macrophages(TNF-α,IL-1β,IL-10,Arg-1)were significantly increased,and the increase rate was further enhanced after the addition of LPS.The results of flow cytometry indicated that the M1 macrophage surface markers(CD86)and M2 macrophage surface markers(CD206)had a significant upward trend,but the proportion of M1 and M2-type macrophages was different.2.Effect of Bmal1 on the polarization of macrophagesImmunohistochemical analysis indicated that Bmal1 positive region in EtOH-fed mice liver tissue was significantly less than that of CD-fed group.Moreover,the results of immunofluorescence double staining also displayed that Bmal1 co-expressed with macrophage marker CD68 in the EtOH-fed mice liver tissue,and the expression was significantly decreased.After analyzing the expression of Bmal1 in EtOH-fed mice liver tissue and ethanol-induced RAW264.7 cells,we found that the protein and m RNA levels of Bmal1 were significantly reduced.After the mice injected with adenovirus 21 days to overexpress Bmal1 in vivo,we fed the mice with alcoholic liquid diet.The results of H&E and oil red O staining showed that EtOH-fed mice injected with AAV8 adenovirus significantly reduced lipid vacuoles and improved lipid droplet deposition.After AAV8 injection,the protein and m RNA expressions of M1-type macrophage markers in liver tissues were significantly decreased,while the expression of M2-type macrophage markers was increased.In vitro,the overexpression model of RAW264.7 cells was established using Bmal1-OE plasmid.Then,RAW264.7 cells were stimulated with ethanol for 24 h.Western blot and q RT-PCR analysis showed that after Bmal1 overexpression,the surface markers of M1-type macrophages were significantly decreased in ethanol stimulated RAW264.7cells,while the surface markers of M2-type macrophages were obviously increased.3.Bmal1 regulates macrophage polarization through the glycolytic pathway3.1 The changes of glycolytic pathway in EtOH-fed mice liver tissue and ethanol-stimulated RAW264.7 cellsWestern blot and q RT-PCR results indicated that the expression of glycolytic key enzymes(HK2,PFKP)in EtOH-fed mice liver tissue was increased,and the secretion of lactic acid in serum which is the product of glycolysis was also augmented.The protein and m RNA levels of key glycolytic enzymes increased,and the content of lactic acid in cell supernatant also increased significantly.Importantly,glycolytic levels in LPS induced M1 macrophages were elevated,but not in IL-4 induced M2 macrophages.3.2 Effects of Bmal1 on glycolysis in EtOH-fed mice and ethanol-stimulated RAW264.7cellsIn order to further explore the effect of Bmal1 on glycolysis in EtOH-fed mice and ethanol induced RAW264.7,mice were injected with AAV8 adenovirus through tail vein to overexpress Bmal1.Western blot and lactic acid detection showed that overexpression of Bmal1 reduced the expression of glycolytic key enzymes and lactic acid secretion.In vitro,western blot and XF-24 Extracellular Flux Analyzer detection results displayed that after Bmal1 overexpression,the levels of glycolytic key enzymes and lactic acid decreased significantly.Moreover,the glycolytic rate also down-regulated.3.3 Bmal1 changes the polarization state of M1-type macrophages through glycolysis The glycolysis pathway was blocked by the chemical inhibitor 2-DG,and the expression of markers on the surface of M1 macrophages was significantly reduced by Western blot and q RT-PCR analysis.Overexpression of Bmal1 resulted in the reduction of M1-type macrophages surface markers was reversed by the addition of oligomycin,a glycolytic agonist.This directly proved that Bmal1 negatively regulates the polarization of M1 macrophages through glycolysis.3.4 Bmal1 binds with S100A9 protein and inhibits the effect of glycolysis on the polarization of M1-type macrophagesIn vitro,Co-IP results displayed that Bmal1 could bind to S100A9 protein in M1 macrophages to further regulate the glycolysis pathway.In vivo injection of AAV8 adenovirus overexpressed Bmal1,resulting in the restriction of S100A9 protein expression.The expression of S100A9 protein was significantly restrained after overexpressed Bmal1.Moreover,when S100A9 was silenced,the glycolytic rate of M1 macrophages decreased significantly,and the expression of glycolytic key enzymes also reduced.Notably,the overexpression of S100A9 in addition to the overexpression of Bmal1 resulted in the expression of the glycolytic key enzyme proteins down-regulated.The results suggested that Bmal1 may directly inhibit S100A9 to block the positive regulation effect of glycolysis on the polarization of M1 macrophages.ConclusionIn the course of alcoholic liver disease,the low expression of Bmal1 binds to S100A9 and inhibits the polarization of M1-type macrophages by blocking the glycolytic pathway,thus improving the inflammatory response of alcoholic liver disease.