Studies On The Anti-diabetic Activity And Mechanism Of The Aerial Part Of Sophora Alopecuroides L

Diabetes mellitus（DM）is a disorder of glucose metabolism that affects human health.In recent years,the prevalence of diabetes has increased year by year,and the age of the disease has also become younger,which has become a serious public health problem.Type 2 diabetes mellitus（T2DM）is a common type of diabetes,and its main pathological characteristics are insulin resistance and isletβcell function deficits.The essence of insulin resistance（IR）is the decrease of insulin-mediated glucose metabolism,which is mainly manifested in the decrease of insulin sensitivity and reactivity of target tissues and target organs.Glucose transporter 4（GLUT4）is the main glucose transporter in skeletal muscle and adipocytes,which plays an important role in regulating glucose and lipid metabolism.This study aims to investigate the mechanism of GLUT4 in glucose transport and glucose metabolism.At present,traditional drugs for T2DM treatment are mainly insulin,metformin and other hypoglycemic drugs.These drugs have some side effects,and also have the disadvantages of single mechanism and drug resistance.As a natural product,natural medicinal plants have the advantages of long-lasting curative effect,less side effects,extensive resources and low cost,which has become a new trend of medicine.Therefore,in this subject,we selected the aerial part of the medicinal plant Sophora alopecuroides to obtain the extracts,and studied their mechanism of action on GLUT4 transport and expression through experiments.First,we extracted the phenol-rich extract from Sophora alopecuroides.L（SA-PRE）,and detected the main monomer compounds by UPLC-Q-TOF/MS hyphenated technique.Then the effect of SA-PRE on GLUT4 activity in L6 cells in vitro and glycolipid metabolism in diabetic mice in vivo were studied.The results showed that SA-PRE mainly contained 14monomer compounds,which not only promoted the uptake of glucose and GLUT4translocation in L6 cells,but also promoted GLUT4 protein expression through AMPK/PKC pathways.Further validation of pathway inhibitors showed that GLUT4 protein expression mediated by SA-PRE was significantly weakened under the action of AMPK inhibitor compound C and PKC Inhibitor G（?）6983,which confirmed that SA-PRE promoted GLUT4transport and expression through AMPK/PKC signaling pathways in vitro,thus promoting glucose uptake.In addition,we also found that cytoplasmic Ca²⁺signal was also involved in the glucose uptake of L6 cells induced by SA-PRE.In vivo experiments,type 2 diabetes model KK-Ay mice were established.After 4 weeks of gavage,the SA-PRE treatment significantly improved hyperglycemia,glucose intolerance,insulin resistance and hyperlipidemia in diabetic mice.Histopathological sections results showed that after 4 weeks of SA-PRE treatment,hepatic steatosis,adipocyte hypertrophy and compensatory hypertrophy of islets in KK-Ay mice were significantly alleviated.Chronic low-grade inflammation is involved in the occurrence and development of insulin resistance.Therefore,RT-PCR was used to study the relationship between inflammation and IR,and the results showed that SA-PRE can effectively suppress the expression of proinflammatory factors IL-6and IL-1β、CRP and tumor necrosis factor TNF-αto alleviate IR.Finally,we detected the expression of related proteins in insulin target tissue muscle and white adipose tissue of KK-Ay mice by Western blotting.The results showed that SA-PRE treatment significantly promoted GLUT4 expression and AMPK,AKT and PKC protein kinases phosphorylation in muscle and white adipose tissue of KK-Ay mice,indicating that SA-PRE activated AKT/AMPK/PKC/GLUT4 signaling pathway in vivo to improve glucose and lipid metabolism disorder in diabetic mice.Next,we detected the glucose uptake on L6 cells by total alkaloids extracted from aerial part of Sophora alopecuroides（TASA）with glucose detection kit,and then dynamically tracked the GLUT4 expression and transport in L6 cells with laser confocal microscope.Finally,Western blot was used to investigate GLUT4 protein expression and phosphorylation levels of AMPK,AKT and PKC protein kinases.The results showed that TASA could effectively increase glucose uptake and GLUT4 translocation and expression in L6 cells.The results of protein phosphorylation and signal pathway inhibitor showed that the expression of GLUT4 protein mediated by TASA was weakened only under the action of G（?）6983.These results indicated that TASA activate the intracellular activity of GLUT4 through up-regulating PKC signaling pathway,and then promote glucose uptake in L6 cells.At the same time,TASA also promoted the uptake of glucose in L6 cells by inducing the release of Ca²⁺in L6cells.The above results showed that TASA had a certain hypoglycemic effect.To sum up,two natural products SA-PRE and TASA were screened in vitro activity experiment targeting GLUT4 in this project,and studied the related hypoglycemic signal mechanism in vitro and vivo.These studies not only provided new ideas for the screening antidiabetic drugs,but also provided scientific support for the comprehensive development and utilization of Sophora alopecuroides resources.