Study On The Effects And Mechanism Of α-Hederin From Stauntonia Chinensis DC. On Improving The Disorder Of Hepatic Glucose And Lipid Metabolism

Non-Alcoholic Fatty Liver Disease（NAFLD）is a chronic progressive liver disease with an increasing incidence,and its pathogenesis involves insulin resistance（IR）,lipid accumulation,and oxidative stress,inflammation,gut microbiota and other factors.At present,there is no specific targeted drug to treat NAFLD.Natural products are rich in sources and low in toxic and side effects,which are important sources of NAFLD drug development.Stauntonia chinensis DC.belongs to Stauntonia,containing a large amount of oleanane-triterpenoid saponins.Our previous study found that the total saponins of Stauntonia chinensis DC.had significant hypoglycemic and lipid-lowering activities in C57 db/db mice,but the effect and mechanism of the monomeric saponins needed to be further studied.Hederin（α-hederin）is a monomeric saponin isolated from Stauntonia chinensis DC..Preliminary studies had shown thatα-hederin could improve the IR status of human hepatoma cell Hep G2,but its effects on lowering lipid and the related mechanism had not been reported.This research will explore effects and mechanism ofα-hederin isolated from Stauntonia chinensis DC.on NAFLD based on the following three aspects.（1）Using nitrophenyl-α-D-glucopyranoside（p-Nitrophenyl-β-D-Galactopyranoside,PNPG）as the substrate and acarbose as the positive control to explore the inhibition effect and the mode of inhibiton ofα-hederin onα-glucose in vitro.The inhibitory effect ofα-hederin combined with acarbose was detected.The above is to explore the possibility ofα-hederin reducing postprandial blood glucose in vitro.The results showed that the half maximal inhibitory concentration(IC₅₀)ofα-hederin onα-glucosidase was 23.13μM,and the inhibitory effect was dose-dependent.The results of enzyme kinetics showed that the maximum reaction rate V_max decreased,while the Michaelis constant K_m value remained unchanged,which belonged to non-competitive inhibition effect.The combination ofα-hederin and acarbose showed antagonistic inhibition at low concentrations,and synergistic inhibition at high concentrations.（2）The IR model was established by co-inducing Hep G2 cells with 0.25 m M palmitic acid plus 30 m M glucose to explore the effect and mechanism ofα-hederin on glucose utilization in Hep G2-IR cells in vitro.The results showed thatα-hederin could increase the glucose consumption of Hep G2-IR cells and enhance the utilization of glucose by regulating the AKT/GSK3βsignaling pathway.In addition,it could reduce the intracellular lipid accumulation and improve cellular insulin status by down-regulating SREBP1c/FAS signaling pathway.（3）In order to explore the ameliorating effect and mechanism ofα-hederin on NAFLD mice in vivo,six-week-old C57BL/6 mice were fed with high-fat diet for 12weeks to establish the NAFLD model.Then C57 mice fed with normal diet were used as the normal control group,metformin（20 mg/kg）as the positive control group,whileα-hederin were divided into low-dose group（12.5 mg/kg）and high-dose group（25mg/kg）.Continuous intragastric administration for six weeks.The results showed thatα-hederin could significantly improve the disorder of glucose metabolism in NAFLD mice,which was manifested as reducing the body weight and fasting blood glucose level of NAFLD mice,improving oral glucose tolerance and insulin tolerance,and increasing insulin sensitivity.At the same time,α-hederin also regulated lipid metabolism disorder,reduced free fatty acid（FFA）content,serum triglyceride（TG）,total cholesterol（TC）,low-density lipoprotein（LDL-C）content,and increased serum high-density lipoprotein（HDL-C）levels.In addition,α-hederin could significantly ameliorate oxidative stress and inflammation in NAFLD mice by reducing malondialdehyde（MDA）content,increasing superoxide dismutase（SOD）and catalase（CAT）content,and reducing serum inflammation factors IL-6,IL-1β,TNF-αlevels.By 16s r RNA sequencing analysis of mice gut microbiota,we found thatα-hederin could modulate gut dysbiosis by increasing the relative abundance of Streptococcus and Lactobacillus,while decreasing the relative abundance of Vibrionaceae and Allobaculum in the gut of NAFLD mice.In addition,Western blot showed thatα-hederin improved insulin sensitivity,increased glucose absorption and utilization,and increased glycogen synthesis by activating the glucose metabolism pathway PI3K/AKT/GSK3β.It could also reduce adipogenesis by down-regulating the SREBP1c/FAS fat metabolism pathway and reduce fatty acid transport by reducing CD36 protein expression.In addition,it could reduce inflammation and oxidative stress in NAFLD mice by down-regulating the IKKβ/NF-κB signaling pathway.