Peptide AWRK6 Relieves Palmitic Acid-induced Hepatocyte Steatosis Via GLP-1R

Metabolic associated fatty liver disease（MAFLD）is a chronic liver disease characterized by hepatic lipid accumulation and steatosis,with a high incidence worldwide and a rapid upward trend.MAFLD is closely associated with metabolic diseases such as type 2 diabetes mellitus（T2DM）and obesity,which is also a risk factor for the eventual development of hepatocellular carcinoma.The pathogenesis of MAFLD is complex and no drugs are currently approved for treatment of MAFLD,and clinical interventions in lifestyles such as diet and exercise are the main treatment methods.Therefore,there is a pressing need to explore more effective MAFLD treatment strategies and drugs.AWRK6（SWVGKHGKKFGLKKHKKH）is a novel cationic peptide with broad-spectrum antibacterial activity and higher stability,which is isolated from the skin and secretions of Rana dybowskii and optimized by this laboratory.Previous studies have shown that AWRK6 could effectively improve insulin resistance in T2MD mice and relieve MAFLD in nonalcoholic fatty liver model mice,but the specific mechanism remains unclear.In order to explore the role and mechanism of AWRK6 in steatosis caused by lipid accumulation,Hep G2 cells were induced by the treatment of palmitic acid to establish the cell model of hepatic steatosis and simulate early fat deposition of MAFLD in vitro.The experimental results showed that the triglyceride（TG）content was extremely significantly increased in steatosis cell model induced by 2.5×10^-4mol/L palmitic acid,and hepatic steatosis model of liver cells could be successfully constructed.After steatosis cells were treated with 1×10^-4 mol/L,1×10^-5 mol/L and1×10^-6 mol/L AWRK6 for 24 h,the TG contents in the three groups of cells were significantly decreased（p<0.05）.These results suggest that AWRK6 can effectively reduce the lipid accumulation and alleviate hepatic steatosis in Hep G2 steatosis cells induced by palmitic acid.Glucagon-like peptide-1 receptor（GLP-1R）is a key target of glucolipid metabolism regulated by its receptor agonists,therefore,q PCR was further used to detect the transcription level of h GLP-1R gene in steatosis cells treated with AWRK6in this study.The result shows that there was no significant difference in the transcription level of h GLP-1R gene between AWRK6 intervention group and control group（p>0.05）,which illustrates that AWRK6 alleviates lipid accumulation in steatotic cells independent of the regulation of h GLP-1R gene expression.Therefore,in this study,molecular docking was further used to simulate the structural interaction between AWRK6 and GLP-1R,the results show that AWRK6could bind to the GLP-1R extracellular domain via hydrogen bonding.Then,in order to explore their functional specific interacton,HEK293T cells with low GLP-1R expression were used as receptor cells,furthermore,p EGFP-h GLP-1R and pc DNA3.1（+）-h GLP-1R overexpression recombinant plasmid were constructed and transfected.The expression and internalization of GLP-1R were respectively observed by fluorescence microscope and confocal laser microscope.Meanwhile,the intracellular c AMP content was analyzed by ELISA technique,and the intracellular calcium ion level was determined by flow cytometry to analyze the activation of GLP-1R.The results showed that in HEK293T cells transfected with empty vector p EGFP-N1,the green fluorescence of the expressed GFP protein was evenly distributed in the cytoplasm,while the green fluorescence of GLP-1R-GFP fusion protein was concentrated on the membrane of HEK293T cells transfected with p EGFP-h GLP-1R,and the expression of GLP-1R was validated by q PCR and western-blot analysis.The mean intracellular fluorescence intensity was analyzed by Image J software.Compared with the control group,the mean intracellular fluorescence intensity was significantly increased after AWRK6 treatment（p<0.05）.The results of ELISA and flow cytometry analysis showed the intracellular c AMP level significantly increased after AWRK6 treatment（p<0.01）,and intracellular calcium ion level increased,which indicate AWRK6 could activate GLP-1R.These results suggest that AWRK6 is a GLP-1R agonist,may interact with GLP-1R and promote the internalization of GLP-1R,which further activates GLP-1R and alleviates the lipid accumulation of palmitic acid-induced Hep G2 steatosis cells.