Role And Mechanism Of Alfalfa Flavonoids Extracts And The Bioactive Apigenin Regulating Endoplasmic Reticulum Stress To Ameliorate Lipid Metabolism Disorder

Metabolic syndrome（Met S）is a clustered metabolic disturbance characterized by central obesity,hypertension,glucose metabolism disorder,and dyslipidemia,which is a risk factor for diabetes and cardiovascular disease.Among them,hyperlipidemia is a common symptom of Met S,and insulin resistance caused by abnormal lipid metabolism is the central link of metabolic syndrome.Endoplasmic reticulum stress（ERS）is the major intracellular pathophysiological insult for metabolic disorders,and chronic long-term ERS can induce cellular lipotoxicity.Studies have proved that excess saturated fatty acids and cholesterol can induce ERS and interfere with lipid metabolism in liver cells,macrophages,and adipocytes,leading to metabolic syndrome.Alfalfa is the most cultivated forage species in China and even in the world and has abundant natural secondary metabolites,it can not only be used for the treatment of gastric diseases,pernicious anemia,hypertension,liver function disorders,and eczema but also has the functions of regulating lipid metabolism,lowering cholesterol,anti-oxidation,and improving immunity.The active monomers of alfalfa flavonoids mainly included Luteolin,Chrysoeriol,Tricin,and Apigenin（AP）.Among them,as a natural antioxidant,AP has been reported to have a variety of biological activities,such as antiviral,antibacterial,anti-tumor,cardio-cerebrovascular protection,etc.However,the specific role and mechanism of AP in HFD mice and palmitate（PA）-induced lipid-rich cell models are still unclear.Therefore,the meaning of natural flavonoids with favorable bioactivities may have crucial academic value and translational significance for the improvement of alfalfa products.In this study,the Hep G2 cells induced by palmitic acid（PA）and HFD-fed mice were applied to construct the lipid dysregulation model.We further revealed the effects of AFE and AP on lipid metabolism disorders and the regulatory relationship between them in the process of lipid anabolism in vitro and in vivo,elucidating the profound mechanism of AP regulating endoplasmic reticulum stress to improve lipid metabolism disorders.The main results of this study are as follows:1.Alfalfa flavonoid extract（AFE）ameliorates dyslipidemia and Insulin Resistance（IR）in HFD-fed mice and PA-induced lipid-rich Hep G2 cellsIn this study,Oil red O staining was adopted to verify the lipid accumulation in Hep G2 cells and liver tissues of HFD-fed mice,and H&E staining was applied to identify the changes in the steatosis of the liver tissues of mice.Meanwhile,we utilized IPTT,OGTT,and biochemical analyzer distribution to determine the changes in blood glucose,fasting insulin,HOMA-IR,and blood lipid parameters in each group.In addition,RT-q PCR or Western blotting assay was applied to detect the expression of lipid biosynthesis-related genes and proteins（FAS,HMGCR,SCD1,SRBEP-1c,and SRBEP-2）and insulin-sensitive proteins(p-Akt^S473 and p-IRS1^Y612).The results uncovered that AFE significantly decreased lipid accumulation,down-regulated lipid biosynthesis-related genes and proteins（FAS,HMGCR,SCD1,SRBEP-1c,and SRBEP-2）,and up-regulated insulin resistance-related proteins(p-Akt^S473and p-IRS1^Y612)in PA-induced Hep G2 cells.Simultaneously,AFE can remarkably reduce the body weight and the wet weight of multiple organs in HFD-fed mice,reduce the blood glucose,fasting insulin,HOMA-IR,blood lipid,and hepatic lipid accumulation in HFD-fed mice,and ameliorate steatosis of the liver in HFD mice.Besides,AFE can also notably inhibit lipid synthesis pathways and improve insulin resistance in the liver of HFD-fed mice.Therefore,AFE can markedly improve dyslipidemia.However,for the complex constituents of AFE,the active genin of alfalfa flavonoids which has the greatest effect on the lipid metabolism of hepatocytes is still unclear.Therefore,it is necessary to further screen the role of active monomers of alfalfa flavonoids in the lipid metabolism of hepatocytes in later studies.2.Apigenin（AP）had the best lipid-lowering activity among the major four alfalfa-derived flavonoidsIn this study,Oil red O staining was adopted to identify the effects of four alfalfa flavonoid active monomers（Luteolin,Chrysoeriol,Tricin,Apigenin）on lipid accumulation in PA-induced Hep G2 cells,and RT-q PCR or Western blot assay was conducted to verify the effects of these monomers on lipid anabolism and insulin-sensitive proteins,to select the monomer with the best lipid-lowering effect.The results disclosed that Tricin and AP could reduce lipid accumulation in PA-induced Hep G2 cells,and AP had the strongest effect.However,only AP could signally inhibit lipid synthesis pathways and ameliorate insulin resistance.Therefore,AP is the potential flavonoid genin with the best lipid-lowering activity.3.Apigenin（AP）ameliorates dyslipidemia and Insulin Resistance（IR）in HFD-fed mice and PA-induced lipid-rich Hep G2 cells possibly by targeting Liver tissueSimilarly,in this study,Oil red O staining was performed to verify the effects of AP on lipid accumulation in PA-induced Hep G2 cells and liver tissues of HFD-fed mice,and a biochemical analyzer was utilized to confirm the effects of AP on blood glucose,fasting insulin,blood lipid and other related indicators of HFD mice.H&E staining was also applied to further identify the effect of AP on the steatosis of mouse liver tissue.Moreover,RT-q PCR or western blotting assay was applied to examine the effect of AP on lipid synthesis and insulin resistance-associated protein expression in insulin-sensitive organs including the liver,muscle,and inguinal fat.The results testified that AP can dramatically reduce lipid accumulation,inhibit lipid anabolism pathways and attenuate insulin resistance in lipid-rich model cells.Besides,AP can memorably improve lipid accumulation and liver steatosis,inhibit lipid synthesis and improve insulin resistance in HFD-fed mice.Therefore,AP can observably improve the dyslipidemia of HFD-fed mice and PA-induced Hep G2 cells in a dose-dependent manner.However,the precise mechanism of AP on the improvement of dyslipidemia is still unclear,so further studies on the mechanism of AP alleviating dyslipidemia are needed.4.AP inhibit ERS-PERK signaling pathway to improve SREBPs activating-induced dyslipidemiaIn this study,western blotting assays were conducted to verify the effects of AP on ERS and lipid synthesis pathways in lipid-rich cells and liver tissue of HFD-fed mice.The results proved that AP could inhibit the activation of ERS.And,the PERK pathway is the up-steam signaling mainly involved in the weakening effect of AP on lipogenesis in cells.Rescue assays also demonstrated that AP mediates the down-regulation of ERS and lipid anabolism by regulating the PERK pathway.Similarly,as a validation of experimental therapy for ERS target,H&E staining and Oil red O staining were applied to verify the effects of AP and 4-PBA（ERS alleviator）on the steatosis and lipid accumulation of liver tissue in HFD mice,and to explore the effects of AP and 4-PBA on blood glucose,fasting insulin,lipid and other related indicators in HFD mice.Furthermore,we further verified the effect of AP and 4-PBA on lipid synthesis and insulin sensitive-related protein expression by western blot assay.The results showed that both AP and 4-PBA could reduce body weight,viscera index,fasting blood glucose,fasting insulin,and HOMA-IR in HFD mice,and improve lipid accumulation and liver steatosis in HFD-fed mice.Meanwhile,AP is similar to 4-PBA in HFD-fed mice by inhibiting the lipid synthesis pathway by alleviating ERS.In conclusion,this study confirmed that AFE and AP can inhibit SREBPs-induced transcriptional activation of lipid synthesis downstream genes and improve lipid disorders in HFD-fed mice and PA-induced Hep G2 cells.Mechanically,the ERS pathway,especially the PERK-CHOP signaling,is the potential target of AP in curbing nutritional stress-induced SREBPs activation for hepatic de novo lipogenesis and improving hepatic insulin sensitivities and global metabolic disorders.