The Preventive Effect Of Total Saponins From Panax Japonicas On Insulin Resistance In Adipocytes Induced By A High-fat And Its Molecular Mechanism

Objective In this study,we aimed to investigate the effects of total saponins from Panax japonicas（TSPJ）on insulin resistance and mitochondrial damage induced by high-fat.We successfully established insulin resistance models in C57BL/6J mice and 3T3-L1 by high-fat diet（HFD）and palmitic acid（PA）.We observed the effects of TSPJ on insulin resistance and mitochondrial damage of adipocytes.In vitro,we overexpressed and knocked out mitochondrial fusion protein 2（MFN2）in 3T3-L1 adipocytes to explore the role of mitochondria in improving insulin resistance of adipocytes by TSPJ.We provided experimental basis for the clinical application of Panax Japonicus in the prevention and treatment of metabolic diseases.Methods In vivo,C57BL/6J mice were fed with high-fat diet to establish insulin resistance model.Low dose（30 mg/kg·d）and high dose（90 mg/kg·d）of TSPJ were given by gavage for 16 weeks.Glucose tolerance and insulin resistance test were conducted during feeding.After administration,blood was taken from eyeball,and adipose tissue and other tissues were quickly taken out to detect the insulin resistance associated indicators.In vitro,3T3-L1 adipocytes were induced by palmitic acid to establish insulin resistance model.The drug treated group was given 25μg/m L and 50μg/m L TSPJ respectively and detected insulin resistance associated indicators.Detection methods of TSPJ affecting insulin resistance of adipocytes.⑴Glucose tolerance test（GTT）and insulin resistance test（ITT）were used to detect the glucose tolerance and insulin sensitivity.⑵Biochemical enzyme method was used to detect the levels of glucose（Glu）,triglyceride（TG）,total cholesterol（TC）,high density lipoprotein（HDL）and low density lipoprotein（LDL）in serum of mice and medium.⑶HE staining was used to observe the morphology of adipose tissue induced by high fat.⑷Oil red staining was used to observe the changes of lipid droplets in adipocytes.⑸2-Deoxy-D-Glucose kit was used to detect the uptake of 2-DG by adipocytes.⑹Real time PCR was used to detect the expression of glucose transporter 4（GLUT4）,hormone-sensitive lipase（HSL）and adipose triglyceride lipase（ATGL）in adipose tissue.⑺Western blot was used to detect the expression of insulin resistance related protein insulin receptor substrate 1,（IRS1）,phosphotidylinositol3-kinase（PI3K）,protein kinase B（AKT）,GLUT4 and lipolysis related protein.Detection methods of TSPJ affecting mitochondrial damage of adipocytes:⑴The morphological and structural changes of mitochondria in mouse adipocytes and 3T3-L1adipocytes were observed by transmission electron microscope.⑵The morphological changes of mitochondria in 3T3-L1 cells were observed by laser scanning confocal microscope after labeling with mitochondrial fluorescent tracer.⑶ATP detection kit and fluo-4 kit were used to detect the expression of ATP and Ca²⁺in mitochondria of adipose tissue and 3T3-L1 adipocytes.⑷Flow cytometry was used to detect the expression of ROS in mitochondria of 3T3-L1 adipocytes.⑸Real time PCR and Western blot were used to detect the m RNA and protein expression of dynamin-related protein 1（DRP1）,Mitochondrial fission protein 1（FIS1）,Mitofusin 2（MFN2）,Optic Atrophy 1（OPA1）,Nuclear respiratory factor1（NRF1）,Mitochondrial transcription factor A（TFAM）,peroxisome proliferators-activated receptors（PPARγ）and PPAR coactivator?1α（PGC?1α）in adipocytes.The effects of mitochondrial function on insulin resistance of adipocytes improved by TSPJ:⑴In vitro,We established the adipocyte model of overexpression and knockdown of MFN2.⑵Real time PCR were used to detect the m RNA expression of GLUT4 in adipocytes.⑶Western blot were used to detect the protein expression of PI3K and AKT in 3T3-L1 cells.Results The effects of TSPJ on insulin resistance of adipocytes:⑴Compared with the normal diet（ND）control group,the body weight and epididymal fat index of the mice on the high-fat diet increased significantly;after the intervention with TSPJ,the body weight and epididymal fat index of the mice were significantly reduced（p<0.05）.⑵Compared with the ND group,the fasting blood sugar（FBS）of the mice on the high fat diet increased significantly,and the mice developed glucose intolerance and insulin resistance;after TSPJ treatment,the FBS of the mice decreased,and the glucose tolerance and insulin resistance of the mice were significantly improved（p<0.01）.⑶Compared with the normal control group,the glucose uptake of adipocytes in the PA model group under insulin stimulation decreased;after TSPJ intervention,the glucose uptake of adipocytes increased,and the difference was statistically significant（p<0.001,p<0.05）).⑷Compared with the normal control group,the expression of GLUT4 m RNA in the adipocytes in model group decreased;after TSPJ intervention,the expression of GLUT4 m RNA in the adipocytes increased significantly（p<0.01）.⑸Compared with the normal control group,the expression of IRS1,PI3K,AKT and GLUT4 in adipocytes in model group decreased;after TSPJ intervention,the expression of IRS1,PI3K,AKT,GLUT4 in adipocytes increased significantly,and the difference was statistically significant（p<0.01）.⑹Compared with the ND group,the concentration of TC,TG and LDL in the serum of mice on a high-fat diet increased,while the concentration of HDL decreased;after TSPJ intervention,the expression of TC,TG and LDL in the mice decreased,and the expression of HDL increased highly.⑺Compared with the normal control group,both high fat diet and palmitic acid increased adipocytes volume and fat droplets;after TSPJ intervention,adipocytes volume and large fat droplets decreased significantly（p<0.01）.⑻Compared with the normal control group,the expression of lipolysis-related genes HSL,ATGL,and p-AMPK in the adipocytes in model group decreased;after TSPJ intervention,the expression of lipolysis-related genes in adipocytes increased（p<0.05,p<0.01）.The effects of TSPJ on mitochondria injury of adipocytes:⑴Compared with the normal control group,high fat induced adipocyte mitochondrial cristae breakage,showing"fragmentation";after TSPJ intervention,the adipocyte mitochondrial damage was significantly improved（p<0.01）.⑵Compared with the normal control group,the expression of reactive oxygen species（ROS）in adipocytes in the PA model increased;after TSPJ intervention,the relative levels of reactive oxygen species in 3T3-L1 adipocytes were significantly reduced（p<0.01）.（3）Compared with the normal control group,ATP and Ca²⁺in adipocytes in the model group decreased;after the intervention of TSPJ,the content of ATP and Ca²⁺in the mitochondria of adipocytes was significantly increased（p<0.01）.（4）Compared with the normal control group,the expression of mitochondrial division genes DRP1 and Fis1 in adipocytes of mice on high-fat diet increased,mitochondrial fusion genes MFN2,OPA-1 and biogenesis-related gene NRF1 and TFAM expression decreased;after TSPJ intervention,the expression of mitochondrial division genes in adipocytes decreased,the expression of mitochondrial fusion genes and mitochondrial biogenesis-related genes increased（p<0.05,p<0.01）.（5）Compared with the normal control group,the expression of PPARγand PGC-1αin adiposcytes in model group decreased;after TSPJ intervention,the expression genes related to regulating mitochondrial biogenesislevels in adiposcytes increased significantly.The difference was statistically significant（p<0.01）.The effects of mitochondrial function on TSPJ in improving insulin resistance of adipocytes:（1）Compared with the mock group,the expression of MFN2 in adipocytes of negative control（NC）group had no change;compared with the NC group,the expression of MFN2 in adipocytes of MFN2 knockdown model decreased（p<0.05）,and the expression of MFN2 in adipocytes of MFN2 overexpression model increased significantly（p<0.01）.（2）Compared with the mock group,the expressions of GLUT4,PI3K and p-AKT in PA model group were decreased;after intervention with TSPJ,the expressions of GLUT4,PI3K and p-AKT in adipocytes were significantly increased（p<0.05）;compared with the NC group,the expressions of GLUT4,PI3K and p-AKT in MFN2 knockdown group were decreased,and the expressions of GLUT4,PI3K and p-AKT in MFN2 overexpression group were significantly increased（p<0.01）.Conclusion⑴TSPJ reduced fasting blood glucose,improved glucose tolerance and increased insulin sensitivity of adipocytes in obese mice.TSPJ effectively maintained glucose homeostasis,promoted glucose uptake of adipocytes to improve insulin resistance of adipocytes.TSPJ intervened PI3K/AKT/GLUT4 insulin signaling pathway to improved the insulin resistance of adipocytes induced by high fat.（2）TSPJ maintained the normal morphology of mitochondria in adipocytes,promoted the fusion of mitochondria in adipocytes,interfered with the separation and expression of fusion genes,and improved the biogenesis of mitochondria,so as to improve the mitochondrial damage in adipocytes induced by high fat.⑶TSPJ could not improve insulin resistance of adipocytes after inhibiting the expression of MFN2 in adipocytes,but overexpression of MFN2 in adipocytes could increase the effects of TSPJ on insulin resistance of adipocytes.