Study The Preventive And Therapeutic Effects Of Eleutheroside B On Diabetes And Diabetes Complicated With Myocardial Injury And Its Liposome Preparation

Objective:The mechanism of Eleutheroside B（EB）in the treatment of diabetes and the prevention of myocardial damage caused by diabetes was studied through in vivo and in vitro experiments.At the same time,in order to improve the solubility and bioavailability of EB,the preparation and process optimization of Eleutheroside B liposomes（EB-LP）were completed,which is conducive to the clear play of the efficacy of EB and provides a theoretical basis and new drug research and development programs for the treatment of diabetes with traditional Chinese medicine.Methods:（1）A diabetic zebrafish model was constructed based on 1.75%glucose immersion combined with high-fat diet,and the effect of EB on glucose and lipid metabolism in diabetic zebrafish was evaluated using this model.（2）By means of liquid chromatography-mass spectrometry（LC-MS）,each group of zebrafish samples was examined and the metabolic pattern of EB in zebrafish was investigated using principal component analysis and orthogonal partial least squares discriminant analysis.Through metabonomics technology,the endogenous markers and metabolic pathways of EB regulating the occurrence and development of diabetes in zebrafish were searched.（3）Cultured H9c2 rat cardiomyocytes were randomly divided into control group,model group,model+solvent group,and model+eleutheroside B low-dose,medium-dose and high-dose groups.The optimal concentration of eleutheroside B was screened by CCK-8 method.GLUT4 expression was detected by immunofluorescence（IF）.GLUT4 and HBP were detected by qRT-PCR and Western-blot.（4）The EB content detection method was established by high performance liquid chromatography and the methodology was investigated.The formulation of EB-LP was optimized by orthogonal experiment,and the appearance,particle size,ζpotential and stability of EB-LP were evaluated.Results:（1）Three doses of EB treatment group can significantly reduce the body mass index（BMI）of diabetic zebrafish and improve blood glucose and lipid indexes.（2）OPLS-DA results showed that EB treatment group and model group could be significantly distinguished,and samples within the group were relatively concentrated,indicating that there were differences in the metabolic status of zebrafish in the two groups,indicating that eleutheroside B could cause changes in the metabolism of diabetic zebrafish.The changes may be related to the regulation of m TOR signaling pathway,purine metabolism,caffeine metabolism,arginine and proline metabolism,fatty acid degradation,glycerol phospholipid metabolism and other pathways.（3）CCK-8 results showed that 5～100μg·m L^-1eleutheroside B had no cytotoxicity to H9c2 cells.Compared with control group,GLUT4protein expression and m RNA expression in model group were significantly decreased,while O-GLc NAc,OGT and GFAT protein expressions were significantly increased（P<0.01）.Compared with model group,GLUT4 protein expression and m RNA expression were significantly increased（P<0.05 or P<0.01）,while O-GLc NAc,OGT and GFAT protein expression were significantly decreased（P<0.05 or P<0.01）.（4）The experimental results of methodology are all good.The optimum conditions were the ratio of soybean phospholipid to EB 24:5,the ratio of soybean phospholipid to cholesterol 24:6 and the ratio of organic phase to water phase 1:11.EB-LP prepared under the optimal process has light blue light and no obvious odor.The encapsulation rate was（85.9±1.05）%,the particle size was（74.88±0.45）nm,the polydispersion index was（0.14±0.01）,theζpotential was（-3.1±0.58）m V,and the mass was stable at 4℃.Conclusion:This study shows that EB has hypoglycemic effect and can significantly improve the disorder of glucose and lipid metabolism in the body.At the same time,EB can effectively enhance the transport capacity of GLUT4 in cardiomyocytes,reduce lipid deposition in heart tissue,and improve the damage of H9c2 cardiomyocytes induced by high glucose.The mechanism may be to regulate the HBP flow rate by influencing the expression of the rate-limiting enzyme GFAT,and reduce the abnormally elevated O-Glc NAc level to achieve its control effect.The physical and chemical properties and stability of EB-LP prepared by the optimal process were good.The above studies can provide theoretical basis and basis for further research on EB treatment of diabetes mellitus and its concomitant myocardial injury.