| Non-alcoholic fatty liver(NAFLD)is the most common chronic liver disease in the world.Its main characteristics are lipid metabolism disorders and increased oxidative stress.Finding natural products that can prevent NAFLD has became an important development trend in adjuvant treatment of NAFLD.In this paper,the main active substance in royal jelly samples——Major Royal Jelly Proteins(MRJPs)is the research object,the extraction process is optimized,and on this basis,oleic acid(OA)is used to construct lipid accumulation in liver cells model and HepG2 cell mitochondrial dysfunction model in vitro,to study the mechanism of MRJPs preventing NAFLD,aiming to provide a basis for the development of high value-added peptide drugs.The main research results are as follows:(1)Centrifugal dialysis was used to extract MRJPs from royal jelly.The optimal extraction conditions obtained are as follows:extract liquid to material ratio 8:1 mL/g,extract pH is 7,extraction time 4 h,dialysis time 24 h.Under the optimal process conditions,0.314±0.051 g of MRJPs can be extracted from 2.5 g of royal jelly,with an average yield of 12.56%.(2)The main components of MRJPs identified by SDS-PAGE are MRJP5(72kDa),MRJP4(68kDa),MRJP3(64kDa),MRJP1(57kDa)and MRJP2(49kDa).And the results obtained by ultra-high-speed centrifugation further confirmed that MRJP1 is the main component of MRJPs.(3)Incubating HepG2 cells with 1.00 mmol/L OA for 24 hours or 0.50 mmol/L OA for L02 cells for 24 hours can effectively construct an hepatocyte lipid accumulation model in vitro.A large number of lipid droplets appear in the cytoplasm and accumulate significantly.The content of intracellular triglyceride(TG)and total cholesterol(TC)increased,the activity of superoxide dismutase(SOD)decreased,and the content of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)in the medium increased.Compared with the model group,pre-incubate these two kinds of cells for 24 hours with 0.2,0.5,1.0 g/L MRJPs in advance showed that the accumulation of lipid droplets in the cytoplasm was reduced,the content of TG and TC in the cells was reduced,the activity of SOD was increased,and the content of ALT/AST in the medium was reduced.This shows that MRJPs can prevent lipid metabolism disorders and oxidative stress in liver cells.(4)Incubating HepG2 cells with 0.50 mmol/L OA for 48 hours can effectively construct an in vitro mitochondrial dysfunction model.The model cells have reduced mitochondrial membrane potential(MMP)and reduced adenosine triphosphate(ATP)content.Incubating HepG2 cells for 24 hours can maintain the MMP and ATP content of HepG2 cells.This shows that MRJPs can prevent damage to the integrity of mitochondrial function.(5)Incubating HepG2 cells with 0.50 mmol/L OA for 48 hours can effectively reduce the mRNA and protein levels of sirtuin 3(SIRT3),mitochondrial superoxide dismutase(SOD2),and cytochrome C oxidase Ⅳ(COX Ⅳ),and inhibit the phosphorylation of adenosine monophosphate dependent protein kinase(AMPK),and pre-incubating HepG2 cells with 0.2,0.5,1.0 g/L MRJPs for 24 hours can up-regulate the mRNA and protein expression of SIRT3,SOD2,and COXIV.At the same time,inhibiting the expression of SIRT3 will prevent MRJPs from exerting their preventive effects.This indicates that the up-regulation of SIRT3 expression mediated by MRJPs and the up-regulation of downstream SOD2 and COXIV expression may be the key link for MRJPs to prevent NAFLD,and AMPK signaling pathway may be an important pathway and molecular basis for MRJPs to up-regulate SIRT3 expression. |
