Studies On The Antidiabetic Activities Of Sophora Davidii Flavonoids And Aloperine

Diabetes has become one of the most challenging health problems around the world and its prevalence has increased year by year.The number of people with diabetes in China has reached 114 million,ranking top in the world.Diabetes is usually divided into type 1 diabetes mellitus,type 2 diabetes mellitus?T2DM?,gestational diabetes and other special types.T2DM is the most common type of diabetes,accounting for more than 90%of all cases of diabetes.T2DM is mainly manifested as hyperglycemia,hyperlipidemia,hyperinsulinemia and so on.Long-term hyperglycemia can cause a range of diabetic complications,such as cardiovascular disease,blindness,kidney disease,diabetic foot,oral health,pregnancy-related complications,etc.Currently available diabetes therapies include insulin and various oral hypoglycemic agents such as sulfonylureas,biguanides,?-glucosidase inhibitors and glinides,which are usually used as monotherapy or combinations to achieve better effects of blood glucose regulation.However,many oral hypoglycemic agents also have side effects in some degree.Therefore,developing new targets for novel antidiabetic drugs with higher efficiency and lower side effects is becoming a research hotspot.Glucose transporter 4?GLUT4?is the most important protein for transporting glucose in insulin-sensitive target tissues.Disorders in GLUT4 regulation is associated with insulin resistance in muscle and adipose tissue,which plays a key role in the occurrence and development of T2DM.Increasing GLUT4 translocation and expression is beneficial for alleviating the symptoms of type 2 diabetes.In order to search for potential hypoglycemic agents from natural products,we established a drug screening system based on GLUT4 fluorescent labeling in rat skeletal muscle L6 cells.The effects of agents on GLUT4 translocation in L6 cells will be observed by laser confocal scanning microscopy.Through in vitro screening,we have found that a flavonoid-rich extract from Sophora davidi?Franch.?Skeels?SD-FRE?and aloperine have significant effects in promoting GLUT4 translocation.The present study investigates the antidiabetic activity of SD-FRE through in vitro and in vivo studies and the hypoglycemic activity of aloperine throug in vivo study.In vitro,SD-FRE significantly promoted GLUT4 translocation and expression in L6 cells.Specific inhibitors of signaling pathways associated with GLUT4 trafficking and expression were used.Compound C?AMPK inhibitor?significantly inhibited the effect of SD-FRE induced GLUT4 trafficking to the plasma membrane.However,Wortmannin?PI3K inhibitor?and G?6983?PKC inhibitor?had no influence,indicating that SD-FRE promoted GLUT4 expression and translocation to the plasma membrane in L6 cells through the AMPK signaling pathway,and ultimately leads to an increase in glucose uptake.In the in vivo experiments,spontaneously type 2 diabetic model KK-Ay mice were administered with SD-FRE for consecutive four weeks.The results showed that SD-FRE treatment significantly improved hyperglycemia,glucose intolerance,insulin resistance and hyperlipidemia in diabetic mice.Histopathological examination revealed that KK-Ay mice had significant hepatic steatosis,islet compensatory hypertrophy and adipocyte hypertrophy.After four weeks'treatment with SD-FRE,these pathological changes were significantly alleviated.Western blotting was used to detect the expression of related proteins in insulin target tissues?muscle,adipose tissue and liver?of KK-Ay mice.The results showed that SD-FRE treatment significantly promoted GLUT4 expression and AMPK phosphorylation in insulin target tissues of KK-Ay mice,indicating that SD-FRE regulated glucose metabolism and alleviated insulin resistance through the AMPK signaling pathway.SD-FRE treatment also regulated the expression of acetyl-CoA carboxylase?ACC?phosphorylation and peroxisome proliferator-activated receptor gamma?PPAR??in liver and adipose tissue of KK-Ay mice,playing the role of regulating glucose and lipid metabolism.In vitro and in vivo experiments showed that SD-FRE has promising antidiabetic activity.Aloperine is an alkaloid isolated from the traditional Chinese ethnic medicine Sophora alopecuroides L.Cooperators had found that aloperine promoted GLUT4expression and translocation through G protein-PLC-IP₃-IP₃R-Ca²⁺-PKC and PI3K/Akt pathways.In the present study,high-fat diet combined with low-dose streptozotocin?STZ?induced type 2 diabetic rats were involved and administered with aloperine for consecutive four weeks.After four weeks'high-fat diet feeding,SD rats received low-dose STZ intraperitoneal injection.After one week,rats with fasting blood glucose level over the standard of T2DM rats were selected for experiments.Long-term high-fat diet feeding leads to insulin resistance in SD rats.Low-dose STZ injection will selectively destroy?-cells at the time of hyperinsulinemia,causing symptoms like hyperglycemia.The physiological and pathological processes are similar to T2DM.Following four weeks treatment of aloperine,the fasting blood glucose level of rats in treatment groups were significantly lower than that of T2DM rats in the diabetic control group.Oral glucose tolerance results showed that aloperine treatment significantly improved glucose intolerance in diabetic rats.Serum biochemical analysis showed that aloperine treatment significantly reduced blood lipid levels in diabetic rats and improved lipid metabolism abnormalities.In addition,related protein expression in insulin target tissues were examined and the results showed that aloperine treatment promoted GLUT4 expression and activated PKC and Akt phosphorylation in insulin target tissues of diabetic rats.Histopathological observation showed that aloperine treatment significantly improved the symptoms of fatty liver in diabetic rats and alleviated pancreatic injury.The results of the present study indicated that the active fraction and compound found through in vitro screening via targeting GLUT4 have shown satisfactory antidiabetic activities in further in vitro and in vivo experiments.The established in vitro GLUT4 translocation screening assay can efficiently and accurately screen and discover natural products with potential hypoglycemic activities,providing new ideas for accelerating the search and development of antidiabetic drugs.