A Preliminary Study On The Mechanismof Insulin Resistance By 794ap (Salmeterol Xinafoate)

Research background Type 2 diabetes mellitus(T2DM)is a complex disorder of endocrine and metabolic diseases,which has become one of the third major non communicable diseases that threaten human health after cardiovascular disease and cancer.Insulin resistance is the basis of T2 DM,and runs through the whole process of the development of T2 DM.Peroxisome proliferator activated receptors(Peroxisome proliferators activated,PPARs)belong to the family of non steroidal nuclear receptor superfamily,which is an important target for the prevention and treatment of metabolic diseases in humans.PPAR? is one of the key targets of insulin resistance and PPAR? is in complex numerous signaling pathways and developing its biological effects,such as PPAR? and PI3K/AKT kinase signal pathway,PPAR? can promote the expression of AKT in the PI3 K signaling pathway,enhance insulin sensitivity,and promote the expression of Glu T4 and the uptake of glucose;PPAR? and ADIPOQ,ADIPOQ can increase insulin sensitivity,anti-inflammatory and protection of endothelial function,is a new target of PPAR?,can stimulate AMPK phosphorylation.Many effects are achieved through the AMPK pathway.A large number of literatures have shown that high inhibition of CDK5 mediated phosphorylation of PPAR? Ser273 is the best candidate for antidiabetic drugs.Recent studies have found that 794ap(Salmeterol xinafoate)can reduce blood glucose in T2DM-SD rats,and is highly associated with PPAR? receptor.This paper takes Hep G2 as the target cells,preliminary exploration of the mechanism to establish insulin resistance model combined with WB,IHC,ICC,q RT-PCR experiments on the hypoglycemic effct and improving insulin resistance.Objective The mechanism of 794 ap in improving insulin resistance and reducing blood sugar was preliminarily investigated.Method Using molecular docking to identify.the best binding conformation of 794 ap and PPAR?.Using the molecular dynamics simulation simulating the binding mode of PPAR? receptor agonist 794 ap and PPAR? and calculate the binding energy using MM-PBSA method with Ammber dynamics.To explore the optimal concentration and time of the optimal concentration of insulin,and to establish the model of IR-Hep G2.;MTT method was used to detect the effect of 794 ap on the IR-Hep G2 model.The survival rate of cells was calculated.And the appropriate drug concentration gradient was selected;794ap acts on the IR-Hep G2 model and makes the corresponding glucose test to reflect the effect of the drug;the content of blot protein was detected by western,and the effects of 794 ap on PPAR?,AMPK?,AKT2,JAK2,and ADIPOQ were observed with rosiglitazone as the positive drug;the expression of PPAR? and other target proteins in IR-rat liver and pancreatic tissue and IR-Hep G2 cell was studied by immunohistochemistry(IHC)and immunocytochemistry(ICC);q RT-PCR was used to test the m RNA expression of PPAR?,ADIPOQ and AMPK ?1.Result The complexes of 794 ap with 2XKW and 4EMA have reached a dynamic equilibrium after MD 30 ns,forming a stable complex structure.The binding effect of 794 ap and receptor 2XKW is best,and van Edward force plays a key role in the binding process.Insulin resistance in Hep G2 cells was successfully established and the effect of 24 h on insulin concentration induced by 10-7 mol/L was the best.According to the result of MTT,1 n M,10 n M,100 n M are the selection of high concentration in low dosing group.794 ap can inhibit the expression of p-PPAR? Ser273,up regulate the expression of p-PPAR? Ser112,AMPK ?1,p-AMPK?1,ADIPOQ,p-AKT2.794 ap did not up regulate the expression of JAK2,p-JAK2,ERK1/2 and p-ERK1/2.After adding inhibitor T0070907,the effect of 794 ap on the expression of PPAR? and other proteins remained unchanged;the IHC,ICC effect of 794 ap on PPAR? in SD rat liver,pancreas and IR-Hep G2 cells was consistent with WB;m RNA increased the expression of ADIPOQ and AMPK?1,but did not increase the level of PPAR? m RNA expression in 794 ap.Conclusion It is proved that 794ap(Salmeterol xinafoate)has the effect of TZDs on reducing blood glucose and improving insulin resistance in type 2 diabetic mice.Molecular dynamics simulations demonstrate that 794 ap is tightly bound to the receptor of PPAR? and is an agonist of PPAR?.WB,IHC,ICC and q RT-PCR showed that 794 ap can inhibit the expression of p-PPAR? Ser273,and activation of AMPK ?1,AKT2,and ADIPOQ to improve the insulin resistance of IR-SD rats and IR-Hep G2 cells,increased insulin sensitivity...