Effects And Mechanisms Of Breviscapine On The Function Of VSMCs Cultured Under Hyperglycemic Conditions

Diabetes mellitus is a state of chronic hyperglycemia due to an absolute or relative deficiency of insulin secretion that may or may not be associated with insulin resistance. The world wide prevalence of diabetes was estimated to be2.8%in2000and is projected to reach4.4%by2030. Atherosclerosis is one of the most prevalent cardiovascular complications of diabetes mellitus and has become a major cause of morbidity and mortality in diabetic patients, especially in patients with type2diabetes. Rupture of an atherosclerotic plaque may result in myocardial infarction, stroke or peripheral vascular disease. Atherosclerosis in diabetes begins earlier and progresses more rapidly, and is difficult to diagnose in the early stage. But there are very serious medical consequences. Thus, we tried to seek for an ideal treatment to inhibit the pathway towards diabetic atherosclerosis in the earlier period, such as primary prevention.Atheroma formation involves endothelial damage, oxidized lowdensity lipoprotein (LDL) infiltration, macrophage activation, collagen deposition, a change in the phenotype of vascular smooth muscle cells (VSMCs), as well as an increase in proliferative and migratory capability of VSMCs. Many epidemiological and clinical studies have shown that chronic hyperglycemia is a major initiator of diabetic atherosclerosis, as high glucose regulates structural and functional changes of vessel involved in diabetic atherosclerosis via the activation of several signal transduction pathways. Studies have indicated that VSMCs cultured under hyperglycemic conditions displayed enhanced proliferative and migratory responses compared to cells cultured in euglycemic conditions and PKC signal transduction pathways might play an important role in this effect. Other studies have shown that activation of the MAPK pathway might contribute to increased proliferation and migration of VSMCs in response to hyperglycemia. Therefore, PKC and MAPK pathways might play an important role in diabetic atherosclerosis. So inhibiting the activity of PKC or MAPK may have preventative and therapeutical effect on the diabetic atherosclerosis.Breviscapine (C21H18O12) is a flavonoid extracted from Erigeron breviscapus, the essential active ingredient of which is flavone. Its pharmacologic action is dilating blood vessel, reducing blood viscosity and improving microcirculation. It also possesses an anti-platelet action and can decrease plasma fibrin content and promote fibrinolytic activity. Recent studies have shown that breviscapine could inhibit the expression of PKC, and decrease the protein expression of c-jun, c-fos and synthesis of type IV collagen induced by hyperglycaemia. For example, breviscapine has been demonstrated to be a PKC inhibitor and has a protective effect on diabetic nephropathy in a previous report. Previous studies have also demonstrated that breviscapine ameliorated cardiac hypertrophy induced by high glucose or angiotensin Ⅱ via PKC pathway. Along with the research going deep into, the effects of breviscapine on other signaling molecules were determined. A recent study has demonstrated that breviscapine inhibited the proliferation and collagen production of cardiac fibroblasts induced by angiotensin Ⅱ via suppression of p38and ERK1/2MAPK signaling pathways. Although a recent study has demonstrated that breviscapine had potential protective effect on atherosclerosis and caused a significant reduction in the atherogenic index in rat atherosclerotic models, little is known about whether breviscapine has protective role in diabetic atherosclerosis and its underlying mechanisms.On the basis of above considerations that hyperglycaemia increases proliferation and migration of VSMCs via PKC and MAPK signaling pathways, and breviscapine could inhibit the activations of PKC and MAPK, we hypothesized that breviscapine might have protective effect on the function injury of VSMCs exposed to high glucose, and coulid ameliorate diabetic atherosclerosis. The inhibition of high-glucose-activated PKC and MAPK might contribute to its effects. In this study, we use primary cultured rat VSMCs to investigate the influence and mechanism of breviscapine on high-glucose-enhanced proliferation and migration of VSMCs.Effects and mechanisms of breviscapine on the function of VSMCs cultured under hyperglycemic conditionsAim:To investigate the influence and mechanism of breviscapine on the proliferation and migration of VSMCs cultured in a high-glucose medium.Methods:Male Sprague-Dawley rats were killed by cervical dislocation and their thoracic aortas were isolated. The connective tissue of outer layer was removed and the outer membrane was stripped by the microforceps. The endomembrane was scraped off by scrapping the inner surface of the vessel gently with forceps. Then the tunica media was cut into chips and VSMCs were cultured in vitro using tissue explant method. Differential attachment purification was used to obtain more pure VSMCs. Only VSMCs from passages4-6were used for the experiments. The cultured VSMCs were divided into eleven groups:(1) normal glucose group (5mmol/L);(2) high glucose group (25mmol/L);(3) normal glucose plus mannitol (25mmol/L);(4) high glucose plus DMSO;(5) high glucose plus low dose of breviscapine (30mg/L,65μmol/L);(6) high glucose plus high dose of breviscapine (50mg/L,108μmol/L)(7) high glucose plus PKC inhibitor Ro-31-8220group (3μmol/L);(8) high glucose plus MEK1/2inhibitor PD98059group (MEK1/2:the upstream activator of ERK1/2;25μmol/L);(9) normal glucose plus breviscapine (50mg/L,108μmol/L);(10) normal glucose plus PKC inhibitor Ro-31-8220group (3μmol/L);(11) normal glucose plus MEK1/2inhibitor PD98059group (25μmol/L). After treatment for some time, Cell Counting Kit-8cell viability assay was used to evaluate cell proliferation. Wound healing assay and transwell migration assay were used to evaluate cell migration. The expression and activity of PKC-β2, ERK1/2, p38, and JNK MAPK were measured by western blot.Results:In our study, we demonstrated that VSMCs cultured in a high-glucose medium showed an increased capability of proliferation and migration as well as a higher activity of PKC-β2and ERK1/2MAPK compared with the control group. This was not simply the result of a change in osmolarity and incubation with25mmol/L mannitol had no effect on VSMC proliferation. We also noticed that breviscapine attenuated high-glucose-enhanced proliferation and migration of VSMCs, while the solvent dimethyl sulphoxide (DMSO) showed no effect. In addition, high-glucose-activated ERK1/2, but not PKC-β2, was inhibited by breviscapine.Conclusion:Our study demonstrated that high glucose increased proliferation and migration of VSMCs via PKC-β2and ERK1/2MAPK signaling pathways. Breviscapine ameliorated high-glucose-induced proliferation and migration of VSMCs via ERK1/2MAPK, rather than via PKC-β2signaling pathway.