?-opioid Receptor Stimulation Improves Endothelial Function Via Akt-stimulated NO Production In Hyperlipidemic Condition

Backgroud Hyperlipidemia is an independent risk factor for many cardiovascular diseases including atherosclerosis.Hyperlipidemia alters endothelial function and triggers molecular events that disturb balances between vasodilation and vasoconstriction,thrombogenesis and fibrinolysis,inhibition and stimulation of smooth muscle cellproliferation.Thus endothelial dysfunction occurs,which is considered as an early event for atherosclerosis before angiographic or ultrasonic evidence.Elevation in serum lipids increases the production of reactive oxygen species(ROS),which reacts with NO to produce ONOO-and causes e NOS uncoupling.An approach that possesses functions of stimulating NO production may provide the best protection against vascular endothelial dysfunction.Our previous work demonstrated that ?-opioid receptor(?-OR)stimulation with U50,488 H directly dilates vessels in a NO-dependent manner.It also attenuates pulmonary arterial pressure in rats with hypoxic pulmonary hypertension and effectively protects pulmonary artery endothelium through preservation of e NOS activity and anti-apoptotic effect.The present study was designed to determine whether ?-OR stimulation with U50,488 H protects endothelial function in hyperlipidemia and its mechanism.Our study demonstrates that ?-opioid receptor stimulation improved endothelial ultrastructure and function through activation of the PI3K/Akt signaling pathway and increased production of NO under hyperlipidemic condition,suggesting that?-OR may be used as a protective drug target in the treatment of hyperlipidemia-induced endothelial dysfunction.Aims1.To explore the effects of ?-OR agonist U50,488 H on endothelial structure and function in hyperlipidemia;2.If ?-OR agonist has the abillity to improve endothelial structure and function,we then explore the underlying mechanisms.Methods1.In vivo study Sixty male,8-week-old Sprague Dawley rats were randomly divided into six groups:1)normal diet group(ND),2)high-fat diet group(HFD),3)high-fat diet+saline group(HFD+V)(0.3m L saline was intraperitoneally(i.p.)injected every 2 days),4)high-fat diet+U50,488 H group(HFD+U)(1.25mg/kg U50,488 H was i.p.injected every other day),5)high-fat diet+nor-BNI group(HFD+N)(2.0mg/kg nor-BNI,a selective ?-OR antagonist,was i.p.injected every other day),6)high-fat diet+U50,488 H+nor-BNI group(HFD+U+N)(2.0mg/kg nor-BNI was i.p.injected and 1.25mg/kg U50,488 H was i.p.injected 10min later every other day).ND group received a regular chow diet and all other groups received a high-fat(5% cholesterol supplemented)diet.After a high-fat diet feeding for 14 weeks,blood was collected for determination of total cholesterol(TC),triglyceride(TG),low-density lipoprotein cholesterol(LDL-C),high-density lipoprotein cholesterol(HDL-C)and fasting blood glucose by a biochemistry analyzer.Ultrastructural analysis of aorta was made under transmission electron microscope.Endothelial function of the aortic rings was determined.NO production,e NOS and i NOS activity were determined.Western blot was used to analyze Akt,e NOS and i NOS expression/phosphorylation.2.In vitro study Human umbilical vein endothelial cell lines(HUVECs)were incubated with sodium palmitate(450?mol/L)for 48h to mimic hyperlipidemic condition and then were divided into several groups as follows: 1)U50,488 H treated group;2)U50,488H+ nor-BNI(a?-OR antagonist)treated group;3)U50,488H+ LY294002(a PI3 K inhibitor)treated group;4)U50,488H+ MK-0026(an AKT inhibitor)treated group;5)U50,488H+ L-NAME(an e NOS inhibitor)treated group,Western blot was used to analyze endothelial ?-OR,Akt,e NOS expression/phosphorylation and the expression of caspase-3.NO production was determined by by the Griess reaction.HUVECs were incubated with sodium palmitate to mimic hyperlipidemic condition and then were divided into several groups as follows: 1)U50,488 H treated group;2)U50,488H+ ?-OR si RNA treated group;3)U50,488H+Akt si RNA treated group.Western blot was used to analyze endothelial ?-OR,Akt,e NOS expression/phosphorylation and the expression of caspase-3,Bax,Bcl-2.Results1.Effect of U50,488 H on serum glucose and serum lipid profiles After 14 weeks,serum total cholesterol(TC)and LDL-C concentrations dramatically increased in groups fed with a high-fat diet.However,fasting blood glucose,triglyceride(TG)and high-density lipoprotein cholesterol(HDL-C)concentrations did not change in these groups.Pretreatment with U50,488 H and nor-BNI elicited no significant effect on these parameters.2.Effect of U50,488 H on hepatic fatty degeneration Compared with the animals in normal diet group,apparent fatty degeneration in rat liver tissue after high-fat diet feeding was observed after 14 weeks.The hepatic fatty degeneration was little affected by saline,U50,488 H,nor-BNI or U50,488H+nor-BNI.3.U50,488 H alleviated hyperlipidemia-induced ultrastructural lesion of the aorta Abnormal ultrastructural changes of the aorta segments from the HFD and HFD+V groups were observed,which showed discontinuous endothelial basement membrane and swollen endothelial cells,sometimes with adhered platelets.U50,488 H alleviated the lesion,turning the intima towards normal morphological pattern.The effect of U50,488 H was blocked by nor-BNI.4.U50,488 H preserved ACh-induced vasorelaxation in aorta segment from hyperlipidemic rats To clarify whether U50,488 H protects endothelial function,isolated aorta rings(from aorta abdominalis)in different treatment groups were collected to conduct endothelium-dependent and-independent vasodilation as described.Compared with ND group,concentration-dependent vasorelaxation in response to acetylcholine(ACh)was impaired in all groups receiving a high-fat diet.However,vasorelaxation in response to the endothelium-independent vasodilator(S-nitroso-N-acetylpenicillamine,SNAP)was similar in all groups.These results indicated that hyperlipidemia caused significant endothelial dysfunction.Intriguingly,treatment with U50,488 H exerted a protective effect on endothelial function,which was demonstrated by a significant improvement of vasorelaxation in response to ACh in HFD+U group compared with other groups receiving a high-fat diet.This effect was blocked by nor-BNI.5.U50,488 H increased the production of NO in hyperlipidemic rats and palmitate-treated HUVECs To further investigate the mechanisms involved in the improvement of endothelial function,serum NO content was measured in all groups.Feeding with a high-fat diet for14 weeks did not significantly alter serum NO content.Administration of U50,488 H significantly elevated the serum NO level.The effect of U50,488 H was abolished by nor-BNI.Palmitate treatment greatly decreased NO production in HUVECs.Treatment with U50,488 H partly compensated this decrease.The effect of U50,488 H was blocked not only by nor-BNI but also by LY294002,a PI3 K inhibitor,MK-2206-2HCl,an Akt inhibitor,and L-NAME,an e NOS inhibitor,respectively in HUVECs.6.U50,488 H enhanced Akt/e NOS phosphorylation but attenuated i NOS expression in aorta of hyperlipidemic rats We next investigated the change of Akt/e NOS pathway and i NOS in all groups.Hyperlipidemia significantly impaired Akt/e NOS phosphorylation,whereas U50,488 H significantly restored Akt/e NOS phosphorylation.Interestingly,high-fat diet caused elevation of i NOS expression which was significantly attenuated by administration of U50,488 H.The effects of U50,488 H were abolished by nor-BNI.7.U50,488 H enhanced e NOS activity but attenuated i NOS activity in aorta of hyperlipidemic rats Hyperlipidemia significantly decreased e NOS activity and elevated i NOS activity.Administration of U50,488 H significantly restored e NOS activity and suppressed i NOS activity.The effects of U50,488 H were also abolished by nor-BNI.8.Effect of U50,488 H was mediated by the PI3K/Akt/e NOS pathway To confirm the role of the PI3K/Akt/e NOS pathway in the endothelial protective effect of U50,488 H,we treated palmitate-stimulated HUVECs with chemical inhibitors of?-OR(nor-BNI),PI3K(LY294002),Akt(MK-2206-2HCl)and e NOS(L-NAME).The effects of chemical inhibitors were tested by Western blot.Although excessive palmitate inmedium did not affect Akt/e NOS expression in HUVECs,it significantly reduced Akt/e NOS phosphorylation and NO production and increased the expression of total caspase-3 and cleaved caspase-3.Preincubation of HUVECs with U50,488 H restored the phosphorylation of Akt/e NOS and NO production and reduced the expression of total caspase-3 and cleaved caspase-3,Nor-BNI abolished the beneficial effects above of U50,488 H.LY294002 and MK-0026 exerted no significant effects on the expression of?-OR while blocked the effects of U50,488 H on Akt/e NOS phosphorylation and NO production and caspase-3 expression.L-NAME blocked the effect of U50,488 H on e NOS phosphorylation and NO production and caspase-3 expression.9.Effect of ?-OR si RNA and Akt si RNA on ?-OR-Akt-e NOS anti-apoptotic signaling pathway Palmitate-stimulated HUVECs were treated with si RNAs targeting ?-OR and Akt.The effects of si RNAs were tested by Western blot.Excessive palmitate in medium significantly reduced ?-OR expression,Akt/e NOS phosphorylation and Bcl-2 expression and increased the expression of total caspase-3,cleaved caspase-3 and Bax.Preincubation of HUVECs with U50,488 H increased ?-OR expression,Akt/e NOS phosphorylation and Bcl-2 expression and reduced the expression of total caspase-3,cleaved caspase-3 and Bax,which were abolished by ?-OR si RNA.Akt si RNA exerted no significant effects on the expression of ?-OR while reduced the expression of Akt phosphorylation/expression and blunted the effect of U50,488 H on e NOS phosphorylation and Bax and Bcl-2 expression.Conclusion1.?-OR stimulation by U50,488 H attenuates hyperlipidemia-induced endothelial dysfunction and ultrastructural lesion without affecting lipid level.2.?-OR stimulation by U50,488 H reduces endothelial cell apoptosis and alleviates vascular endothelial injury through activation of the ?-OR-Akt-e NOS signaling pathway and increase of NO production.