Protective Effects Of Dopamine D₄ Receptors On Vascular Endothelial Injury Of Diabetes Mellitus And Its Mechanism

Objective:Diabetes mellitus is a chronic disease that will produce many complications in the course of its development.Diabetic vascular disease is one of the major complications.Many previous studies have shown that endothelial dysfunction is considered as the base of diabetic vascular complications,and hyperglycemia is the main culprit leading to endothelial dysfunction.Therefore,correcting endothelial dysfunction,improving endothelial function may have important implication for preventing these complications.Dopamine is an endogenous catecholamine neurotransmitters and play a protective role by its receptors.Dopamine receptors are a G protein-coupled receptors,which belong to the rhodopsin class family,including two subtypes depending on its structure and pharmacological properties,namely D1-like receptors(D1 and D5)and the D2-like receptors(D2,D3,D4).In recent years,there were literature reported that dopamine and its receptors more and more involved in the protection of diabetes and the cardiovascular system.Previous studies had mostly focused on dopamine D1 and D2 receptors,which can reduce hyperglycemia and hyperinsulinemia,activate endothelial function,and play a role to resist diabetes.Zarei et al's research found that dopamine D2-like receptors only existed in human aortic endothelial cells and human umbilical vein endothelial cells,but no dopamine D1-like receptors.This shows that D4 receptors exist in the endothelial cells.Our previous research found that D4 receptors are expressed in the SHR and WKY rats' vascular smooth muscle,and connected with insulin receptors and AT1 receptors.D4 receptors inhibited proliferation and migration of insulin or angiotensin ? mediated vascular smooth muscle cell,the role of D4 receptor inhibition of vascular smooth muscle cells benefited from lowering the role of insulin receptors and AT1 receptors.These explain that D4 receptors protect the diabetes and vascular function of hypertension through the regulation of vascular smooth muscle cell.However,so far,whether the intervention of dopamine D4 receptors for diabetic vascular endothelial dysfunction have a protective effect is unknown.Therefore,the purpose of this study is to verify dopamine D4 receptor on diabetic rat's endothelial function and high glucose-induced endothelial damage if there is the same protective effect.Methods:Part I: Animals experiment1.20 SPF healthy male SD Rats of 8-10 weeks,weight(220±20g),were randomly divided into two groups: control group 10 rats;diabetic group 10 rats,one-time intraperitoneal injection of STZ(35 mg/kg).The rats followed by a standard diet were feed food and water freely for 4 weeks.Fasting blood glucose of >10mmol/L is a successful symbol.Plasma glucose concentrations were determined by using Accu-Chek Advantage glucose monitoring system after fasting experimental animals in the 12 h.2.10 control group rats and 10 diabetic group were anesthetized by 30 mg/kg with pentobarbital sodium,opening the chest,quickly isolating and removing the thoracic aorta.After stripping of fat and connective tissue around,blood vessels were put in the fluid of the PBS,fixed at one end,cut along the longitudinal axis of the vessel,with a cell scraper to gently scrape the inner surface of blood vessels,endothelial tissue collected by centrifugation spare.Westen Blot measures the expression levels of dopamine D4 receptor in the thoracic aorta endothelium.Part II: Vitro study1.Cell culture and grouping: HUVECs were cultured with containing 5.5mmol/L glucose,10% FBS,DMEM complete medium containing double antibody at 37?,under 5% CO2.We will be in each group after the logarithmic growth phase cells were treated with different factors:(1)control group(glucose concentration of 5.5mmol/L,Control);(2)high glucose group(glucose concentration of 30mmol/L,HG);(3)high glucose+ PD168077 group(join 10-7mol/L PD168077,HG+PD);(4)high glucose+PD168077+ L745870 group(join 10-7mol/L PD168077 and 10-6mol/L L745870,HG+PD+L);(5)high glucose+PD168077+LY294002 group(HUVECs first by medium containing 5 ×10-5mol/L LY294002 for 30 min,other treatments with high glucose+PD168077 group,HG+PD+LY);(6)high glucose+PD168077+L-NAME group(HUVECs were treated firstly by 10-4mol/L L-NAME for 30 min,other treatments with high glucose+PD168077 group,HG+PD+e NOSI).2.Westen Blot detects the expression of D4 receptors on HUVECs which are treated by high glucose.The expression levels of Akt,P-Akt,eNOS,P-eNOS were measured.Cell viability was detected by MTT assay.Apoptosis of HUVECs was detected by TUNEL.Results:Part I: Animals experiment1.Compared with contol group,body weight was falling and fasting blood glucose level was increased in diabetic control group(P<0.05).Blood glucose was elevated over 20mmol/L.There show that diabetic rat model is successful.2.The expression of D4 receptor in diabetic vascular endothelium was decreased [diabetic group(5.50 ± 0.71),control group(26.20 ± 1.82),P<0.05].Part II: Vitro study1.Westen Blot:The expression of D4 receptors was decreased significantly in t he high glucose-induced HUVECs[high glucose group(0.71±0.04),control group(1.30±0.038),L-glucose group(1.22±0.17),mannitol group(1.13±0.35),P<0.05].Compared w ith contol group,the expression of P-Akt and P-e NOS sere decreased significantly i n high glucose group: P-Akt[HG(0.53±0.058),Control(1.64±0.06),P<0.05] and P-eN OS [HG(0.59±0.063),Control(1.62±0.098),P<0.05].In the case of PD168077,the do wnregulation effect of high glucose on P-Akt and P-e NOS was significantly inhibite d: P-Akt[HG(0.53±0.058),HG+PD(1.44±0.07),P<0.05] and P-e NOS[HG(0.59±0.063),HG+PD(1.44±0.084),P<0.05] The effect of PD168077 can be blocked by PI3 K inhi bitor LY294002 and e NOS inhibitor L-NAME: P-Akt[HG+PD+LY(0.59±0.092),HG+P D+e NOSI(0.62±0.071),HG+PD(1.44±0.072),P<0.05] and P-e NOS[HG+PD+LY(0.617±0.05),HG+PD+e NOSI(0.655±0.076),HG+PD(1.44±0.084),P<0.05].2.MTT:Compared with contol group,high glucose significantly reduced HUVECs' viability(P<0.05).In the case of PD168077,the downregulation effect of high glucose on HUVECs' viability was significantly inhibited(P<0.05).The effect of PD168077 can be blocked by D4 receptor inhibitor L745870,LY294002 and L-NAME(P<0.05).3.TUNEL staining: Compared with ccontrol group,the percent of apoptotic HUVECs in HG group was significantly increased[HG(28.3±0.8%),Control(6.1±0.6%),P<0.05].In the case of PD168077,the upregulation effect of high glucose on HUVECs' apoptosis was significantly inhibited[HG(28.3±0.8%),HG+PD(11.5±0.4%),P<0.05].The effect of PD168077 can be blocked by L745870[HG+PD+L(23.5±0.3%),HG+PD ?(11.5±0.4 %),P<0.05].Conclusions:This experiment finds that the expression of dopamine D4 receptors is falling in diabetic vascular endothelium and HUVECs.Dopamine D4 receptors can improve cell viability of high glucose-induced human umbilical vein endothelial cells in vitro and block apoptosis,but this protective effect can be blocked by PI3 K inhibitor LY294002 and e NOS inhibitor L-NAME.The experimental results show that PD168077 on the protection effects of high glucose-induced endothelial cell injury may be mediated via PI3K/Akt/e N0 S pathways.