Effects Of Epimedium Chaohoding C On Type 2 Diabetic Mice And Its Proteomic Study

At present,diabetes has become the third major chronic disease threatening human health in succession to tumors and cardiovascular diseases.Although the long-term use of chemically synthesized hypoglycemic drugs have a certain therapeutic effect on diabetes,it will produce large side effects.Screening safe and effective hypoglycemic factors from traditional Chinese medicine is an important research direction in the prevention and treatment of diabetes.Epimedii Folium,as a traditional medicinal and edible plant in China,has been confirmed to have a certain hypoglycemic effect both in ancient records and modern research.However,the pharmacological substance basis of Epimedii Folium has not been determined,there are few pharmacological studies on its monomer components.Screening and studying the monomer of Epimedii Folium has positive significance for the prevention and treatment of diabetes and the development and utilization of Epimedii Folium.In this study,we used the main flavonoid monomers of Epimedii Folium,epimedin A,epimedin B,epimedin C,epimedin F,and icariin as raw materials to perform computer simulation experiments and hypoglycemic enzyme experiments to find hypoglycemic monomer,insulin-resistant HepG2(IR-HepG2)cell model was used to verify the hypoglycemic activity of the monomers obtained from the initial screening.The mice model of type 2 diabetes mellitus(T2DM)was established,and the therapeutic effect of epimedin C on T2DM mice was investigated.Finally,its hypoglycemic effect was explored at the molecular level by Label-free proteomics technology.The main methods and results are as follows:(1)Molecular docking experiments and in vitro hypoglycemic experiments were used to initially screen monomers with hypoglycemic activity.Cells were used to further verify the hypoglycemic activity of the monomers obtained from the initial screening.First,the computer simulation method was used to analyze the effects of epimedin A,epimedin B,epimedin C,epimedin F,icariin and the target moleculeα-glucosidase,α-glucosidase inhibitor acarbose was used as control to verify the reliability of the experiment.The results of molecular docking experiments showed that the different monomers had certain affinity activities with α-glucosidase and the strength of the docking activities was as follows:epimedin C>epimedin A>epimedin F>epimedin B>icariin.The results of enzymology experiments found that the IC50 values of epimedin A inhibited α-amylase and α-glucosidase were:1.88mg/mL and 0.94mg/mL;The IC50 values of epimedin B inhibited α-amylase and α-glucosidase were:1.92mg/mL and 1.29mg/mL;The IC50 values of epimedin C inhibits α-amylase andα-glucosidase were:1.31mg/mL,0.57mg/mL;The IC50 values of epimedin F inhibitedα-amylase and α-glucosidase were:1.94mg/mL and 1.16mg/mL;The IC50 values of icariin inhibited α-amylase and α-glucosidase were:0.99mg/mL,1.31mg/mL.Comparing with other monomers,epimedin C had the best hypoglycemic activity.Further cell experiments were conducted to verify the hypoglycemic activity of epimedin C,through MTT experiments,it was found that concentration of 0.1-100μg/mL epimedin C and 1-100μg/mL insulin had no significant effect on the survival of HepG2 cells,when the insulin concentration was 60μg/mL,relatively stable IR-HepG2 cell model could be established,the experiments found that with the increase of epimedin C concentration,glucose consumption of IR-HepG2 cells gradually increased by glucose oxidase method,so it is further verified that epimedin C has a certain hypoglycemic effect.(2)The effect of epimedin C on T2DM was studied by using a high-fat diet plus streptozotocin(STZ)injection-induced mice model of type 2 diabetes mellitus(T2DM).The results showed that epimedin C could effectively alleviate the diabetic symptoms of T2DM mice,reduce the fasting blood glucose(FBG)value、GSP content and insulin resistance of T2DM mice and improve the glucose tolerance of mice and insulin(INS)content.It was found that epimedin C could improve the oxidative stress level of T2DM mice by measuring the activity of antioxidant-related enzymes and effectively alleviate the disorder of lipid metabolism of T2DM mice by the measurement of high and low density lipoprotein.The results found that epimedin C could improve the ability of liver to synthesize glycogen by measuring liver glycogen content,epimedin C had a certain repair effect on liver damage caused by T2DM and could protect the insulin tissue of mice by the determination of liver-related enzyme activity and the observation of liver and pancreas tissue sections.In summary,epimedin C can achieve the therapeutic effect on T2DM by regulating the FBG,fasting INS content,insulin resistance,oxidative stress,lipid level and liver damage in T2DM mice.(3)Label-free proteomics technology was used to study the mechanism of T2DM in the treatment of epimedin C.The livers of T2DM mice intervened by high-dose epimedin C(EC30),normal group mice(NC group),and model group mice(MC group)were used as materials to extract total protein from the liver,isolate proteins,identification,and related bioinformatics analysis.The research found that 310,324,and 331 differentially expressed proteins(DEPs)were identified in the comparison of NC vs EC30,NC vs MC,and EC30 vs MC,and these proteins participated in multiple metabolic pathways.Through GO analysis,these proteins were involved in multiple biological processes.It was found that "glycolysis and gluconeogenesis","PPAR signaling pathway",and "bile acid signaling pathway" may be the most important signaling pathways involved in these DEPs through the KEGG pathway.Therefore,it is speculated that these identified DEPs and metabolic pathways(differential proteins PFK,G6Pase,PEPCK in the glycolysis and gluconeogenesis pathway,ABCG2 in the bile secretion pathway,and CYP4a14,CYP4a12 in the pathway of lipid metabolism mechanisms)may play an important role in the hypoglycemic mechanism of epimedin C.