Effect And Improving Mechanism Of A Novel Peptide AWRK6 On Non-alcoholic Fatty Liver

Purpose:Non-alcoholic fatty liver disease(NAFLD)is a liver injury disease closely related to insulin resistance.It has become the most common chronic liver disease in the world and one of the world’s public health problems.Studies have shown that improving insulin resistance can effectively relieve NAFLD.AWRK6 is an 18-amino acid cationic peptide identified and transformed from the skin secretions of Rana chensinensis in the laboratory.It has various functions such as improving the body’s insulin sensitivity,alleviating insulin resistance,and improving type 2 diabetes in mice.This study intends to analyze the role of AWRK6in improving NAFLD and further explore its mechanism of action to provide a new strategy for the treatment of NAFLD.Methods:Oleic acid(OA)was used to induce the accumulation of triglyceride(TG)in HepG2 cells to construct an in vitro cell model with NAFLD symptoms.Used MTT method to detect cell survival rate and analyzed the change of TG content in the cells to optimize and determine the best conditions of AWRK6(5×10~-88 mol/L,4 h).We intervened the model cells induced by oleic acid with 5×10~-88 mol/L AWRK6 for 4 h.And then detected the intracellular TG content,and evaluated the effect of AWRK6 on improving NAFLD at the cellular level.At the same time,the mice were fed with high-fat feed for 12 weeks to construct the NAFLD mouse models.After modeling,the mice were randomly divided into NC group(common feed),NAFLD group(negative control),AWRK6 group(experimental group)and Exendin-4 group(positive control).The mice in NC group and NAFLD group were injected intraperitoneally with saline.AWRK6 and Exendin-4 mice were injected intraperitoneally with AWRK6(100 nmol/kg)and Exendin-4(10 nmol/kg)respectively.After administration for 4 weeks,measured body weight changes,serum biochemical indicators such as TC,TG and so on.Detected serum insulin content and calculated insulin resistance index.HE staining and oil red O staining were used to analyze liver pathological changes and accumulation of fat particles in NAFLD mice,and the anti-NAFLD effect of AWRK6 was analyzed at the animal level.And then used Western-blot technology to detect the amount of adenylate activated protein kinase(AMPK),p-AMPK,acetyl-CoA carboxylase(ACC)and p-ACC in HepG2 cells and mouse livers,and analyzed the mechanism of action of AWRK6.Results:Treated cells with 5×10~-44 mol/L of OA for 24 h,which changed the morphology of HepG2 cells,increased the content of AST and ALT in the cell culture fluid and added the lipid accumulation in the cells significantly.It is the best condition for the induction of NAFLD cell model.The intervention of AWRK6 can reduce the accumulation of TG in model cells(P<0.001).At the animal level,compared with the control group,the body weight(P<0.01),liver weight(P<0.001)and liver coefficient(P<0.001)of the AWRK6 group decreased significantly.HOMA-IR has been decreased effectively(P<0.001).The intervention of AWRK6can reduce the serum insulin,LDL-C,TC and TG content of the mice,and the difference was extremely significant(P<0.001)compared with the control group.And the serum HDL-C has increased(P<0.01).HE staining and oil red O staining showed that the liver pathological changes of mice in the AWRK6 group were significantly improved,the cell density was increased,the cell gap was reduced,the fat vacuoles in the liver were significantly reduced,and the lipid accumulation was improved.Western blot analysis showed that compared with the control group,the content of p-AMPK and p-ACC in HepG2 cells and mouse liver increased significantly after adding AWRK6,and the differences were P<0.05 and P<0.001respectively.While the differences in protein contents of AMPK and ACC were not significant(P>0.05).Conclusion:AWRK6 can reduce the accumulation of TG in HepG2 induced by OA.At the individual level,AWRK6 can improve insulin resistance,reduce lipid accumulation,reverse NAFLD induced by high-fat diet in mice,and has the effect of improving NAFLD.AWRK6 can promote the phosphorylation of AMPK and ACC proteins and up-regulate the content of p-AMPK and p-ACC,which indicates that AWRK6 can regulate lipid metabolism through AMPK/ACC signaling pathway and improve NAFLD.