The Effect Of Insulin On Rat Vascular Smooth Muscle Proliferation And Insulin-induced Inhibition Of SM-α By Down-regulating AP-1

Background and objectiveCurrently, the number of diabetes patients in C hina is about 4.085 million and it has become the second largest country with diabetes in the world, which is no more than India. With the improvement of living standards, the incidence of diabetes also increase. And it has become a common serious disease which is harmful to human’s health. When a patient is diagnosed with diabetes, he would often be found to have vascular complications, particularly the cardiovascular and cerebrovascular diseases with atherosclerosis. Recently, large research and clinical studies of diabetic vascular complication indicate that diabetes patients have more possibility to be diagnosed with atherosclerosis than non-diabetes patients, and insulin resistance appears in half of diabetes with atherosclerosis.Insulin resistance is the sensitivity of insulin targeted cells respond to insulin decrease, and thus the biological effect of insulin decreases, eventually leading to the pathophysiological conditions of increased levels of early compensatory insulin. Therefore, hyperinsulinemia is one of the signs of insulin resistance. N umerous clinical studies suggest that the body of diabetic patients with atherosclerosis is usually in a state of hyperinsulinemia. This suggests that the important risk factor of vascula r complications of diabetes is insulin resistance and subsequent init iation of compensatory hyperinsulinemia.Today’s study suggests that a common pathophysiology of diabetic vascular complications(including vessel atherosclerosis, hypertension and restenosis after angioplasty) is phenotype conversion of vascular smooth muscle cell(VSMC). The phenotypic conversion of VSMC occurs: static contractile phenotype of differentiation converts into synthesis and secretion phenotype of dedifferentiation, and then lead to the enhanced ability of VSMC’s migration as well as its proliferation and the dysfunction of VSMC ’s contraction and diastole. At present, among the molecular mechanisms of the development of vascular proliferative diseases, the molecular mechanisms of VSMC’ phenotype conversion and effect of hyperinsulinemia on VSMC have become a hot research.As VSMC’s marker proteins: α-actin(smooth muscle alpha-actin, SM- α)is abundantly expressed in static contractile phenotype of VSMC, however, when insulin stimulates VSMC to migrate and proliferate abnormally, its expression is significantly decreased, then resulting in VSMC ’s conversion from static contractile phenotype into synthesis and secretion phenotype, and ultimately lead to the increased ability of VSMC ’s migration and proliferation. Thus, insulin down- regulates the expression of VSMC ’s key molecules—SM-α,resulting in VSMC’s phenotypic conversion, eventually leading to the dysfunction of VSMC ’s biological effects. Thus, insulin plays a key role in the development of atherosclerotic vascular.As a transcription factor, activator protein 1(AP-1) is capable of binding a plurality of intracellular gene promoter to regulate the expression of genes to respond to a variety of stimuli, controlling of many cellular processes, including proliferation, differentiation, apoptosis and so on. AP-1 can also bind to specific promoter sequences of upstream of SM-α gene and thus play a series of biological effects. However, the specific mechanism of AP-1 gene expression in a condition that insulin induces VSMC ’s phenotype conversion by down-regulating the expression of SM-α gene is not very clear. Therefore, know more about the mechanism of the effect of insulin inducing VSMC ’s phenotypic conversion and its migration and proliferation has important clinical implications for studies of diabetic vascular complications in diabetic patients.Research Methods1、 Culture vascular smooth muscle cells of SD rat.2、 After culturing insulin-stimulated rat VSMC with different concentration(0 、25、50、100、125nmol/L)insulin, measure the cell cycle of VSMC using flow cytometry.3、 Design and synthesize plasmid vector of AP-1, and then transfect it into VSMC to culture 24 h, in the meantime, divide it into four groups of VSMC : control group, pure insulin stimulation, AP-1 vector transfection group, insulin stimulation +AP-1 vector transfection group.4、 Detect the expression levels of mRN A of SM-α gene and AP-1 gene in SD rat VSMC by RT-PCR.5、 Detect the expression levels of protein of SM-α gene and AP-1 gene in SD rat VSMC by Western blotting.Results1、 After being cultured 24 hours in the rest, VSMC is stimulated by different concentration of insulin with 24 hours, and then use flow cytometry to test the cycle of VSMC. The results show that with the increasing concentrations of insulin, the percentage of S phase of VSMC begins to decline after it gradually increases to a certain peak after being cultured 24h[(23.49±1.17)%、(28.08±0.68)%、(30.44±1.77)%、(47.47±0.88)%、(46.23±1.65)%,P<0.05], the percentage of S phase of VSMC reachs the peak after it was stimulated by 100nmol/L concentration insulin(47.47 ±0.88)%.2、 Culture VSMC in the quiescent condition with 24 hours, and add 100 nM insulin to stimulate VSMC 24 hours, then use RT-PCR and Western blot to test the protein and m RN A expression of AP-1 and SM-α. The results show that in insulin-stimulated group, the m RN A and protein levels of AP-1 [(0.47 ± 0.09),(0.46 ± 0.14)] and SM-α [(0.41 ± 0.03),(0.32 ± 0.03)] are lower than the control group(No insulin stimulation)(P<0.05).3、 AP-1 vector can upregulate the m RN A expression of SM-α gene(2.25±0.28)after it is transfected into VSMC(P<0.05), and then under the stimulation of insulin, m RN A expression of SM-α gene(1.32±0.15)and AP-1 gene(0.99±0.20)are both inhibited(P<0.05).4、 After the transfection into VSMC of AP-1 vector, the protein expression of SM-αincreases(1.61±0.23)(P<0.05). In addition, the protein expression of AP-1 gene(0.97±0.06)and SM-α gene(1.02±0.80) in VSMC decreases after being stimulating by insulin(P<0.05).Conclusion1、 Insulin can promote VSMC of SD rat to proliferate by increasing the percentage of S phase of the cell cycle in VSMC.2、 After the transfection into SD rat VSMC, transcription factor AP-1 can upregulatethe expression of SM-α gene.3、 Insulin can inhibit the expression of SM-α gene in SD rat VSMC, eventually resulting in the phenotypic transition of VSMC to promote its proliferation by down-regulating the expression of AP-1.