| Objective:1.To establish Type 2 diabetes mellitus(T2DM)model in SD rats,and to explore the effect of oligosaccharides of Ophiopogon japonicus on Insulin resistance(IR),glucose and lipid metabolism in T2DM model SD rats.Based on PI3K/Akt insulin signaling pathway,the molecular mechanism of oligosaccharides of Ophiopogon japonicus improving IR and glucose and lipid metabolism disorder was elucidated.2.To establish an IR model of HepG2 cells and study the effect of oligosaccharides of Ophiopogon japonicus on IR,glucose and lipid metabolism in HepG2 cells.To explore the molecular mechanism of oligosaccharides of Ophiopogon japonicus in improving IR and the disorder of glucose and lipid metabolism,PI3K/Akt insulin signaling pathway was investigated.Methods:1.Regulation of oligosaccharides of Ophiopogon japonicus on PI3K/Akt insulin signaling pathway in T2DM ratsAfter 4 weeks of high fat diet,streptozocin(STZ)30 mg/kg was injected intraperitoneally to detect the fasting blood glucose(FBG)of SD rats.If FBG≥16.7 mmol/L,the model of T2DM was established successfully.SD rats were randomLy divided into model group,metformin group(250 mg/kg),oligosaccharides of Ophiopogon japonicus high,medium and low dose groups(450,225 and 112 mg/kg),with 8 rats in each group,Another normal control group was set up.The model group and normal group were given normal saline by gavage once day for 4 weeks.The liver,pancreas and kidney were taken out and the index of them were measured.The pathological changes of liver tissue were observed by HE staining.The contents of glycogen,triglyceride(TG)and total cholesterol(TCH)in liver tissue were determined.RT-PCR was used to detect the mRNA expression levels of IRS-1,INSR,PI3K,Akt,GLUT2 and GSK-3β in the liver tissue of all rats.Western blot was used to detect the protein expression levels of PI3K(p85),p-Akt(Ser473),Akt,p-GSK-3β(Ser9)and GSK-3β in the liver tissue of all rats.2.Regulation of oligosaccharides of Ophiopogon japonicus on PI3K/Akt insulin signaling pathway in HepG2 cellsThe cytotoxicity of oligosaccharides of Ophiopogon japonicus(1600,400,100,25,6.25,1.56,0.39 mg/L)on HepG2 cells was detected by MTT assay.HepG2 cells were induced by lmmol/L palmitic acid combined with 2 mmol/L oleic acid for 24 hours to establish IR model,and were divided into model group,metformin group(1 mmol/L),oligosaccharides of Ophiopogon japonicus high,medium and low dose groups(6.25,1.56 and 0.39 mg/L),and normal control group.After administration for 24 h,The content of glucose consumption,glycogen content,TG and TCH of all groups were measured.RT-PCR was used to detect the mRNA expression levels of IRS-1,INSR,PI3K,Akt,GSK-3β and GLUT2.Western blot was used to detect the protein expression levels of p-IRS-1(Ser307),IRS-1,PI3K(p85),p-Akt(Ser473),Akt,p-GSK-3β(Ser9),GSK-3βand GLUT2.Results:1.Regulation of oligosaccharides of Ophiopogon japonicus on PI3K/Akt insulin signaling pathway in T2DM ratsAfter 4 weeks of oligosaccharides of Ophiopogon japonicus administration,compared with the model group,the liver index of high and middle dose groups decreased significantly(P<0.01),while the liver index of low dose group decreased,but there was no statistical significance.Compared with the model group,the pancreatic index increased,while the renal index decreased,but there was no statistical significance.The results of HE staining showed that oligosaccharides of Ophiopogon japonicus could improve the liver tissue structure disorder,irregular arrangement,severe steatosis and other pathological phenomena in the model group.Oligosaccharides of Ophiopogon japonicus could increase the content of glycogen(P<0.01 or P<0.001),decrease the content of TG(P<0.05 or P<0.01)and TCH(P<0.01)in T2DM model SD rats.RT-PCR results showed that compared with the model group,oligosaccharides of Ophiopogon japonicus could up regulate the mRNA expression levels of IRS-1,INSR,PI3K,Akt and GLUT2,and down regulate the mRNA expression levels of GSK-3β(P<0.05 orP<0.01 or P<0.001).Western blot results showed that compared with the model group,the protein expression levels of PI3K,p-Akt/Akt and p-GSK-3β/GSK3β were increased in different dose groups(P<0.05 or P<0.01).2.Regulation of oligosaccharides of Ophiopogon japonicus on PI3K/Akt insulin signaling pathway in HepG2 cellsThe results of MTT assay showed that the survival rate of HepG2 cells was more than 90%when the concentration of oligosaccharides of Ophiopogon japonicus was 0.39～25 mg/L,indicating that the concentration range had no significant effect on cytotoxicity.After IR-HepG2 cells were treated with different concentrations of oligosaccharides of Ophiopogon japonicus for 24 hours,compared with the model group,the glucose consumption of high,medium and low dose groups was significantly increased(P<0.01).The content of glycogen of high and medium dose groups was significantly increased(P<0.05),while the content of glycogen of low dose group was increased,but there was no statistical significance.The content of TG and TCH of high,medium and low dose groups were significantly decreased(P<0.05 or P<0.01 or P<0.001).RT-PCR results showed that compared with the model group,oligosaccharides of Ophiopogon japonicus could up regulate the mRNA expression levels of IRS-1,INSR,PI3K,Akt and GLUT2,and down regulate the mRNA expression level of GSK-3β(P<0.05 orP<0.01 or P<0.001)in HepG2 cells of IR model in different degrees.Western blot results showed that compared with the model group,the protein expression levels of p-IRS-1(Ser307)/IRS-1 were decreased,PI3K(p85),p-Akt(Ser473)/Akt,GLUT2,and p-GSK-3β(Ser9)/GSK-3β were increased in different dose groups(P<0.05 or P<0.01).Conclusion:This study shows that oligosaccharides of Ophiopogon japonicus can improve IR,the disorder of glucose and lipid metabolism in T2DM model SD rats and HepG2 cells,and its mechanism is to up regulate the mRNA expression levels of IRS-1,INSR,PI3K,Akt and GLUT2,down regulate the mRNA expression levels of GSK-3β,and down regulate the protein expression levels of p-IRS-1(Ser307)/IRS-1,up regulate the protein expression levels of PI3K(p85),p-Akt(Ser473)/Akt,GLUT2,and p-GSK-3β(Ser9)/GSK-3β protein expression levels. |
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