Effect Of G Protein-coupled Receptor Kinase 2 On Diabetic Aortic Smooth Muscle Cells Through INR/IRS/PI3K/Akt/GLUT4 Signaling Pathway And The Role Of Paeoniflorin-6’-O-benzene Sulfonate(CP-25)

Diabetes(DM)is a seriously disease which threated to global public health.The situation in China is particularly severe.China is one the countries with the largest number of M patients,accounting for about one-third of global patients.The main signs of DM are hyperglycemia,hyperlipidemia,and hyperviscosity.There are two main types of DM,type 1 diabetes and type 2 diabetes.T2 DM is non-insulin-dependent DM,and its pathogenesis mainly includes insufficient insulin secretion or insulin resistance(IR).DM can lead to a variety of complications,the most serious of which is diabetic vascular diseases.As vascular smooth muscle cells(VSMCs)play an important role in the process of diabetic vascular disease.Therefore,the regulation of VSMC to reverse diabetic vascular disease has a wide application prospect.VSMC has two types of phenotypes,contraction/synthesis.The contractile cell is fusiform in shape and contains many filaments that can contract.Synthetic cells are cobblestone-like and have a morphology similar to epithelial cells and rhomboid cells.The cells contain a large number of organelles needed to synthesize proteins.Under normal physiologicalconditions,the main role of VSMCs are to constrict blood vessels and maintain the elasticity of blood vessels,which are contractile,with low proliferation and migration activity.However,when IR occurs,the PI3K/Akt pathway is blocked,VSMC changes from contractile to synthetic,with enhanced proliferation and migration capabilities,participating in remodeling and proliferation of blood vessels,etc.Eventually thickening the vessel wall,narrowing the lumen,and reducing compliance.Similarly,VSMC undergoes a phenotypic transition to the vascular intima and is involved in the formation of atherosclerosis.Insulin receptor(INR)is a tetrameric structure distributed in various tissues and cells of the body.In the insulin signaling pathway,insulin first functions by binding to INR,recruiting and phosphorylating insulin receptor substrate(IRS)to activate phosphatidylinositol-3-kinase(PI3K)/protein kinase B(Akt)pathway or activation of heterotrimeric G protein,promote the synthesis of glucose transporter 4(GLUT4).Eventually,GLUT4 is translocated to the cell membrane,causing various biological effects such as glucose transport and glycogen synthesis.G-protein-coupled receptor kinase 2(GRK2)belongs to the class of silk/threonine kinases.It has been reported that the expression of GRK2 was increased in the IR state,GRK2 mainly inhibits the transduction of the downstream insulin signaling pathway PI3 K / Akt by binding to INR and IRS.Eventually,the efficiency of insulin uptake and glucose utilization decreases and hyperglycemia occurs.It can be seen that in the insulin signal pathway,when any link or signal molecule fails,it will hinder the normal signal transduction of insulin,leading to a decline in the ability of cells in the body to take up and utilize glucose,causing DM.In the early stage,the research group esterified paeoniflorin(Pae)to synthesize paeoniflorin-6’-O-benzene sulfonate(CP-25).Studies have found that total glucosides of paeony(TGP)and CP-25 have pharmacological effects such as immunomodulation and anti-inflammatory.TGP has been used in the treatment of diabetic nephropathy.Previous studies by the research group showed that the target of CP-25 is GRK2,but whether CP-25 can improve the insulin signaling pathway and function of Human Aortic Smooth Muscle Cell(HASMC)by regulating GRK2 is unclear.Therefore,C57 mice were used to construct a T2 DM animal model in this study.CP-25 was administered at a high dose to observe the effects of CP-25 on glucose metabolism and related indicators of diabetic mice,as well as pathological changes of aorta and myocardial tissue.HASMCs were cultured in vitro,and IR-HASMC models were induced by high concentration of insulin.Transwell,co-immunoprecipitation and Western blot were used to observe whether CP-25 could affect the function of IR-HASMC by regulating GRK2 and downstream insulin signaling pathway,and its effect on the interaction of INR and GRK2,provide a basis for revealing the mechanism of CP-25 to improve aortic smooth muscle injury.Objective:1.To identify the effect of GRK2 on diabetic aortic smooth muscle cells through insulin signaling pathway.2.To clarify the effect of CP-25 on IR-HASMC and its possible mechanism.Methods:Male C57BL/6J mice were fed with high glucose and high fat diet for 6 weeks,and then intraperitoneally injected with 70mg/kg streptozotocin(STZ)for 4 days.After 1week,fasting blood glucose was measured,fasting blood glucose level greater than11.1mmol/l was considered successful.The model mice were divided into normal group,model group,metformin group,Gliclazide group,CP-25 group,CP-25 combined with Gliclazide group,which were administered orally once daily for 5 weeks.The mice were sacrificed by dislocation after eyeball blood extraction,and the general indicators(blood glucose,body weight)and IPGTT(Intraperitoneal glucose tolerance test)of the mice were measured.Serum was separated and the levels of INS(Insulin),VEGF(Vascular endothelial growth factor),TNF-α(Tumor necrosis factor α),NO(Nitric oxide),and NOS(Nitric oxide synthase)in serum were detected by ELISA.The aorta and myocardial tissue of each group of mice were taken,and their pathological conditions were observed by HE staining.Taking HASMC as the research object,the IR-HASMC model was first established by the glucose oxidase method;the optimal insulin concentration was screened.The experiment was divided into control group,insulin(1μM)group,insulin(1μM)+ CP-25(1n M,10 n M,100 n M,1μM,10μM)group and insulin(1μM)+ Metformin(3m M)group,cell viability were detected by CCK-8 method;migration were detected by scratch and transwell chamber method;cell apoptosis were detected by Hoechst/PI double staining;protein expressions of GRK2,INR,IRS,p-IRS,PI3 K,GLUT4 in cells were detected by Western blot method;Co-immunoprecipitation and laser confocal methods were used to detect the interaction and co-localization of GRK2 and INR,GRK2 and p-IRS.Results:1.Effects of CP-25 on glucose metabolism,aortic pathology and other related indexes in diabetic mice The results of animal experiments showed that CP-25 combined with Gliclazide group reduced the blood glucose of mice and lowered the serum TNF-α level more obviously than gliclazide alone,improved the glucose tolerance of diabetic mice,increased the levels of NO and NOS and improved the pathological damage of aortic tissue to a certain extent.2.Effects of CP-25 on the proliferation,migration and apoptosis of IR-HASMC When HASMC develops IR,proliferation and migration are enhanced,and apoptosis is inhibited.CP-25 can significantly inhibit cell proliferation and migration,and promote cell apoptosis.3.Effects of CP-25 on the expression of GRK2,INR,IRS,p-IRS,PI3 K and GLUT4 in IR-HASMC When HASMC develops IR,the total expression and membrane expression of GRK2 are higher than those of normal cells.The expressions of INR,p-IRS,PI3 K and the membrane expression of GLUT4 are significantly lower than those of normal cells.CP-25 can down-regulate the membrane expression of GRK2,up-regulate the expression of INR,p-IRS,PI3 K and the membrane expression of GLUT4.4.Influences of CP-25 on the co-expressions of GRK2 and INR,GRK2 and p-IRS in IR-HASMC The co-expressions of GRK2 and INR,GRK2 and p-IRS are up-regulated by insulin.CP-25 can down-regulate the co-expression of GRK2 and INR,GRK2 and p-IRS.Conclusion:1.CP-25 can improve the glucose tolerance of T2 DM mice,down-regulate the expression of related inflammatory factors and improve the aortic tissue damage.2.Insulin up-regulates the co-expression of GRK2 and INR in HASMC,suggesting that GRK2 is over-transformed during IR,which may be related to INR desensitization;Insulin up-regulates the co-expression of GRK2 and p-IRS in HASMC,suggesting that elevated GRK2 can inhibit the tyrosine phosphorylation of IRS and inhibit the transmission of insulin signaling.It is suggested that the insulin signal is inhibited to promote the proliferation and migration of HASMC.3.CP-25 down-regulates the co-expressions of GRK2 and INR,GRK2 and p-IRS in HASMC,suggesting that CP-25 may down-regulate the inhibitory effect of GRK2 on insulin signaling by inhibiting GRK2’s excessive transmembrane and protein expression of GRK2,which may be one of the mechanisms that inhibits the proliferation and migration of HASMC.