Study On The Underlying Mechanism By Which Nuciferine Improves Hepatic Steatosis Caused By Fructose-containing Diet

Fructose is widely used as a main component of sweeteners in the processing of foods.In Western countries,the intake of fructose among adolescents and adults exceeds the health range recommended by the World Health Organization.Experimental and clinical studies have shown that long-term intake of fructose can lead to non-alcoholic fatty liver disease(NAFLD).The body's intake of fructose not only provides a substrate for de novo lipogenesis(DNL),but also activates DNL in the liver to induce the initial symptoms of NAFLD,liver hepatic degeneration.In addition,fructose is capable of generating endogenous advanced glycation end-products(AGEs)by Maillard reaction with protein lysine and arginine residues.However,compared with the Maillard reaction occurring in the body,the ?-dicarbonyl compound(glyoxal)produced by fructose during metabolism can modify the protein lysine residue more quickly to produce AGEs(carboxymethyl lysine).Carboxymethyl-lysine(CML)).AGEs bind to advanced glycation end-products(RAGE)and activate downstream mitogen-activated protein kinase(MAPK)signaling pathways.From this we speculate that fructoseinduced AGEs may lead to liver steatosis.Studies have shown that lotus leaf extracted from Lotus Leaf has a significant lipid-lowering function,and lotus tablets containing lotus leaf can treat fatty liver and hyperlipidemia.Therefore,this article will explore whether lotus leaf has an improved effect on high-fructose diet-induced hepatic steatosis and its underlying molecular mechanisms.We used Sprague Dawley rats for 10 weeks with 10%by volume of fructose drinking water,and after 7 weeks,we applied nuciferine(7,14 and 28 mg/kg)and pioglitazone(4 mg/kg).Intervention.The combination of nuciferine and pioglitazone significantly reduced the serum triglyceride(TG),total cholesterol(TC),and low density lipoprotein cholesterol(LDL-c)in the serum of the fructose model group.The levels of aspartate aminotransferase(AST)and alanine aminotransferase(ALT)and the levels of TG and TC in liver tissue.In vitro,we stimulated rat primary hepatocytes and BRL-3A cells with 5 mM fructose for 72 h,and simultaneously administered nuciferine(5,10 and 20 uM)and pioglitazone(10 uM).Compared with the fructose model group,both nuciferine and pioglitazone reduced the intracellular TG and TC levels.Compared with the normal control group,the levels of AGEs in the liver and serum of the rats in the fructose model group were significantly increased.In BRL-3A cells,5 mM fructose significantly up-regulated intracellular and extracellular AGEs levels as well as intracellular TG and TC levels compared to normal controls;and increased extracellular AGEs levels and changes in intracellular TG and TC levels There is a significant correlation between them.Therefore,we speculate that fructose-induced AGEs production may be associated with steatosis in BRL-3A cells.The results of coimmunoprecipitation(Co-IP)showed that the level of RAGE protein increased with the prolongation of BRL-3 A cells treated with 5 mM fructose,and the protein level of CML bound to RAGE also increased.Compared with the fructose model group,RAGE small interfering RNA(siRNA)can significantly reduce the levels of TG and TC in BRL-3A cells,suggesting that fructose can induce fat degeneration of BRL-3A cells by promoting the binding of CML and RAGE.Based on the NAFLD and AGEs-RAGE signaling pathways to construct a proteinprotein interaction(PPI)network and analyze its topological parameters,we hypothesized that fructose may activate the liver through the ERK1/2-mTOR-SREBP1 signaling pathway downstream of AGEs and RAGE.DNL inside,which induces hepatic steatosis.The results showed that the high fructose diet significantly upregulated the levels of RAGE,sterol regulatory element binging protein-1(SREBP1)and promoted extracellular regulated kinase 1(ERK1/2)and phosphorylation of mammalian mammalian target of rapamycin(mTOR)protein in rat liver tissue compared with the normal control group.In the primary hepatocytes and BRL-3A cells,the levels of SREBP1 nuclear protein and the levels of RAGE,p-ERK1/2 and p-mTOR proteins in the fructose model group were significantly higher than those in the normal control group.In liver tissue,liver primary hepatocytes and BRL-3A cells,nuciferine can significantly reduce p-mTOR and SREBP1 protein levels in the fructose model group.In addition,the nuciferine can inhibit the entry of the transcription factor SREBP1 into the nucleus of the primary hepatocytes and BRL-3A cells stimulated by fructose.To confirm the above results,we observed that RAGE siRNA transfection inhibited phosphorylation of ERK1/2 and mTOR proteins using siRNA to reduce RAGE protein levels on BRL-3A cells.The mTOR protein phosphorylation inhibitor rapamycin was used again to verify that rapamycin can significantly reduce the levels of p-mTOR and SREBP1 protein in fructose-stimulated BRL-3A cells and primary hepatocytes,and reduce intracellular TG and TC levels.In conclusion,fructose can up-regulate AGEs levels and RAGE protein levels,promote the binding of CML and RAGE,activate the RAGE-ERK1/2-mTOR-SREBP1 signaling pathway,induce SREBP1 into the nucleus and eventually lead to hepatic steatosis.RAGE siRNA and mTOR protein phosphorylation inhibitor rapamycin inhibited the activation of RAGE-ERK1/2-mTOR-SREBP1 signaling pathway and decreased TG and TC levels in fructose-stimulated BRL-3A cells.Nuciferine can alleviate hepatic steatosis by down-regulating p-mTOR and SREBP1 protein levels and inhibiting SREBP1 entry into the nucleus.The above results suggest that nuciferine is one of the active constituents of Chinese herbal lotus leaf,which has significant improvement in hyperlipidemia,hepatic steatosis and liver damage in NAFLD rats caused by high fructose diet,and can inhibit fructose-activated RAGE.-The phosphorylation of mTOR protein and the transcription factor SREBP1 into the nucleus of the ERK1/2-mTOR-SREBP1 signaling pathway.The research in this paper provides an experimental basis for the treatment of NAFLD by nuciferine.