Effects Of Apelin On Inflammatory Response In Alcoholic Liver Disease

BackgroundAlcoholic liver disease is a liver disease caused by long-term and excessive drinking.In the early stage,it usually manifests as fatty liver,then fatty liver may envolve into alcoholic hepatitis,liver fibrosis and cirrhosis,and eventually form hepatocellular carcinoma.Alcoholic liver disease is a global public health problem,there are millions of ALD patients worldwide each year,and it has a high rate of morbidity and mortality.Apelin is a significant bioactive peptide,and it is an endogenous ligand of the specific receptor APJ that belongs to the G protein-coupled receptor family,which activates and binds its receptor APJ through endocrine,paracrine and autocrine ways,and hence produce important effects on physiology and pathophysiology of function.At present,the role of apelin/APJ in liver diseases is still being explored,the study of apelin in alcoholic liver disease has scarcely been reported up to now.We observed that the expression of apelin increased significantly after inflammatory injury in the liver so far,consequently,the exploration of apelin expression in alcoholic liver and its relationship with the development of alcoholic liver disease has gradually become a new research hotspot.ObjectiveTo investigate the interaction and effects of apelin and inflammatory factors in alcoholic liver disease.To verify whether apelin promotes the expression of inflammatory factors in alcoholic liver disease and explore the mechanism of apelin promoting liver inflammation.Methods1.RAW264.7 cells were inoculated into the cell culture plates and divided into groups according to the experimental group: control group;0.05 μmol/L;0.1 μmol/L;0.2 μmol/L;24 h;alcohol aging relationship group: 0 h;6 h;12 h;24 h;treatment concentration was 0.1μmol/L,and qRT-PCR was used to explore the changes of apelin and inflammatory factors.We observe intracellular lipid accumulation by oil red staining.2.Observe the changes of inflammatory factors after apelin-13 at various concentrations and at different time points was added to RAW264.7 cells for 24 h by Western blot and qRT-PCR.3.After incubated in RAW264.7 cells with alcohol(0.1μmol/L)for 24 h,apelin-13 at various concentrations and at different time points was added to stimulate the changes of inflammatory factors by Western blot and qRT-PCR.4.The plasmids of pCMV/pCMV-apelin(1.6 μg)were transfected into RAW264.7 cells,divided into groups according to the experimental group: control group(pCMV-flag);con(pCMV-flag)+alcohol(0.1μmol/L);pCMV-apelin(1.6 μg);pCMV-apelin(1.6 μg)+ alcohol(0.1μmol/L);and transfected cells were cultivated for 24 hours,after 24 hours of alcohol treatment,the changes of inflammatory factors were detected by Western blot and qRT-PCR,and the changes of cell fluorescence expression were observed by phalloidin staining.5.RAW264.7 cells were divided into si-con group,si-con+ alcohol(0.1μmol/L)group,si-apelin group,si-apelin+ alcohol(0.1μmol/L)group,and treated with alcohol for 24 h after transfection for 24 h.The changes of inflammatory factors were detected by Western blot and qRT-PCR,and the changes of cell fluorescence expression were observed by phalloidin staining.6.Construction of alcoholic liver disease model: C57BL/6 male mice,19~23g,were selected for 6~8 weeks.After one week of adaptive feeding,the wild male mice were randomly divided into three groups: normal control group,alcohol-treated group,apelin combined with alcohol.After 10 weeks,the mice were sacrificed and liver tissues were stored in 4% neutral formaldehyde solution.The remaining liver tissue was frozen in liquid nitrogen and then transferred to a-80 °C refrigerator for long-term storage.7.Mouse liver protein was extracted,Western blot was used to detect the expression of inflammatory factors in mice;immunohistochemical technique was used to detect the expression of inflammatory cytokines in mice;and the liver tissue of mice was homogenized by ultrasonication to extract total RNA,the transcription level of inflammation-related factors in liver tissue was detected by PCR.The liver tissue of mice with alcoholic liver was stained with HE after a series of treatments.The degree of liver damage was observed from the morphology of mice,the accumulation of lipids in cells was observed by oil red staining.Results1.Alcohol stimulation at various concentrations and at different time points was given in RAW264.7 cells,the results of qRT-PCR showed that the transcription levels of apelin,APJ and inflammatory factors IL-6、IL-1β and chemotactic protein MCP-1 increased with alcohol time and dose-dependent.2.Oil red staining detected lipid accumulation in RAW264.7 cells with increasing alcohol time point and concentration point gradient.。3.Apelin-13 at various concentrations and at different time points can increase the expressi on of inflammatory cytokines IL-6、IL-1β and chemotactic proteins MCP-1 and PCNA in a concen tration-dependent and time-dependent manner in RAW264.7 cells.4.Alcohol was added for 24 hours and then stimulated with apelin-13 at various concentrations and at different time points,resulting in an intensify in the expression of inflammatory factors and chemotactic proteins MCP-1 and PCNA in RAW264.7 cells.5.In RAW264.7 cells,Western blot and qRT-PCR results showed that the expression levels of inflammatory factors IL-6、IL-1β、chemotactic protein MCP-1 and PCNA were increased in the overexpressed apelin group compared with the control group.Apelin combined with alcohol((0.1μmol/L)group of inflammatory factors IL-6、 IL-1β and chemotactic protein MCP-1、PCNA expression was most up-regulated.6.The results of phalloidin staining showed that the over-expressed apelin group and apelin combined with alcohol((0.1μmol/L)group had significantly enhanced cell fluorescence expression than the control group and apelin group.7.In RAW264.7 cells,the results of Western blot and qRT-PCR showed that the expression levels of PCNA were significantly lower in the knockdown apelin group than in the control group,such as IL-6、IL-1β and chemotactic protein MCP-1.8.Phalloidin staining showed that knocking down apelin in RAW264.7 cells significantly attenuated the cell’s fluorescence expression.9.Apelin enhances liver inflammation through ERK signaling in RAW264.7 cells.10.In animal experiments,the results of HE staining showed that hepatocytes degeneration and necrosis were observed by alcohol stimulation.Apelin and ethanol combined treatment showed extensive necrosis of hepatocytes and obvious inflammatory cell infiltration.Oil red staining showed that alcohol stimulation caused fatty degeneration of hepatocytes,and apelin treatment significantly increased lipid accumulation in hepatocytes.11.The results of immunohistochemistry showed that the brown particles of apelin,MCP-1 and PCNA were significantly increased in the alcohol group compared with the control group.The apelin and alcohol combined treatment group had the most brown particles,and the results were consistent with the results of Western blot.12.In animal experiments,qRT-PCR results showed that the expression levels of inflammatory factors and chemokine MCP-1 were significantly increased in the apelin and alcohol combination group compared with the alcohol group.13.In animal experiments,Western blot results showed that the expression levels of MCP-1,PCNA and apelin proteins in the liver tissue of mice with alcoholic liver disease were significantly increased.Conclusions1.Apelin promotes liver inflammation by activating ERK signaling pathway in RAW264.7 cells.2.In the model of alcoholic liver disease in mice,apelin can aggravate hepatic steatosis and promote inflammation.