The Effect And Mechanism Of Sang-Tong-Jian On The Insulin Resistance Of Db/db Mice With Type 2 Diabetes Mellitus And L6 Skeletal Muscle Cells

Objectives:Type 2 diabetes mellitus(T2DM)is a glucose and lipid metabolism disorders.Insulin resistance(IR)plays a crucial role in the pathogenesis of T2DM,while skeletal muscle is an important target of IR.At present,with the awareness of the side effects and inconvenience of oral hypoglycemic agents and insulin injection bring,people are increasingly interested in the role and mechanism of traditional Chinese herbal medicine hypoglycemic.Mulberry leaves as a antidiabetic Chinese medicine is widely used in clinic.In the previous workf this laboratory,the antidiabetic components of mulberry leaves were screened,and it was found that when the ratio of total flavonoids to total alkaloid was set at 6:1,they could significantly reduce the blood glucose of diabetic mice.The aim of the present study is to explore the effects and mechanism of STJ(Sang Tong Jian,STJ)on insulin resistance in db/db mice with type 2 diabetes mellitus and insulin resistance L6 skeletal muscle cells.It will provide experimental evidence for the treatment of diabetes mellitus with Chinese medicineMethods and results1 The effect and mechanism of STJ on blood glucose in db/db mice with type 2 diabetes mellitus1.1 The effect of STJ on glucose metabolism in db/db mice:Thirty db/db mice were randomly divided into model group,metformin(200mg/kg)group,and STJ(420mg/kg)group,and db/m mice were used as control group Vehicle or drugs were given by gavage for 12 weeks.The blood glucose ofmicewas determined in the first week to the seventh week.The levels of glycated hemoglobin,glycated serum protein,glycogen,muscle glycogen,TG,TC,LDL-C and HDL-C were measured at the 12th week.The results showed that STJ could significantly decrease the levels of blood glucose,glycosylated hemoglobin,glycated serum protein,TG,TC and LDL-C,and increased the levels of glycogen,glycogen and HDL-C in db/db mice.1.2 The effect of STJ on insulin resistance in db/db mice:The insulin-sensitive tissue(liver,thigh skeletal muscle,epididymis fat)were weighed at the 12th week;Histopathological changes of pancreas were evaluated by HE staining.The oral glucose tolerance,insulin tolerance and starch tolerance tests were performed at the 8th week,10th weeks and 11th weeks after drug treatment;Serum insulin was determined after treatments,and the insulin resistance index and insulin sensitivity index were calculated.The results showed that STJimproved the lesions of pancreas and insulin sensitive tissues in db/db mice,improved the glucose tolerance,insulin tolerance and starch tolerance,decrease the insulin resistance index and increase the insulin sensitivity index.1.3 The effect of STJ on the mitochondrial function and AMPK-PGC-la signaling pathway in skeletal muscle of db/db mice:After 12 weeks of drug treatment,the protein expressions of CPT-1,COXIV and NRF1 in skeletal muscle were detected by western blotting.The protein expressions of p-AMPK,PGC-1α and GLUT4 in skeletal muscle were detected by immunohistochemistry and western blotting.The results showed that STJ up-regulated the protein expressions of CPT-1,COXIV and NRF1 and improved the mitochondrial function of skeletal muscle in db/db mice,activated the AMPK-PGC-1α signal,increased protein expression of GLUT4 and promoted GLUT4 translocation to cell membrane.2 The effect of STJ,alkaloids and total flavonoids on palmitic acid-induced insulin resistance model of L6 or skeletal muscle cells?2.1 The effect of STJ,alkaloids and total flavonoids on glucose uptake in palmitic acid-induced insulin resistance model of L6 skeletal muscle cellsThe insulin resistance model of L6 cells was induced with palmitic acid at a concentration of 0.75 mmol/L.MTT assay was used to detect the proliferation of normal L6 myocytes.Glucose uptake was measured by the method of glucose oxidase-peroxidase.The results showed that STJ,alkaloids and total flavonoids increased the uptake of glucose,promoted translocation of GLUT4,and increased the expression of GLUT4 protein in L6 cells.2.2 The effect of STJ,alkaloids and total flavonoids on mitochondrial function of palmitic acid-induced insulin resistance model in L6 cells:The proten levels of CPT-1,COXIV and NRF1 were detected by Western blot.ROS and mitochondrial membrane potential were detected by what?probe,and ATP was detected by what?.The results showed that STJ,alkaloids and total flavonoids up-regulated the expressions of CPT-1,COXIV and NRFlreduced ROS production,increased mitochondrial membrane potential and ATP productionin L6 myocytes.2.3 STJ,Alkaloids and total Flavonoids increased glucose uptake in the L6 myoblast insulin resistance model via the AMPK-PGC-la signaling pathway:The protein expressions of GLUT4,p-AMPK and PGC-1α were detected by Western blot.The results showed that STJ,alkaloids and total flavonoids up-regulated the expression of p-AMPK and PGC-1α in L6 myocytes,promoted the translocation of GLUT4.In the presence of AMPK inhibitor Compound C,the beneficial effects STJ,alkaloid and total flavonoids were all inhibited in L6 myocytes.conclusion:1.STJ could significantly improve glucose and lipid metabolism disorders,increased skeletal muscle insulin sensitivity,and improved insulin resistance in db/db mice.2.STJ could significantly improve mitochondrial function in skeletal muscle and insulin resistance of db/db mice,which may be related to the activation of AMPK-PGC-1α signaling pathway.3.STJ,alkaloids and total flavonoids could significantly increase glucose uptake and improve mitochondrial function in insulin resistant L6 cells.4.STJ,alkaloids,total flavonoids improved insulin resistance and mitochondrial function,increased glucose uptake of insulin resistant L6 cells through the activation of AMPK signal pathway.