Protective Effect Of κ-opioid Receptor Activation Against Palmitate-induced Vascular Endothelial Cells Injury

Background:In the past decades,lifestyle in China has changed rapidly with economic development.Western meat-rich diet and physical inactivity cause systemic lipid metabolic disorder,which eventually leads to hyperlipidemia.Meanwhile,the morbidity of metabolic syndrome(MS)characterized by insulin resistance,hypertension and obesity has been arising anually,with an increasing adult population and rising prevalence among young adults.Hyperlipidemia may cause macrovascular lesion,thus increase the risk of cardiovascular events,which is of great danger to human health[1].Vascular endothelial structural change and dysfunction are two major factors contributing to early macrovascular lesion development.Research data have demonstrated that under hyperlipidemia,the balance between various vasoconstrictor and vasodilator released by vascular endothelial cells are disturbed[2],characterized by reduction of vasodilators and increase of vasoconstrictors,which leads to vascular dysfunction.Thus,improving vascular endothelial function and restoring vasodilator level are of great significance in prevention and treatment of hyperlipidemia-induced vascular injury.In addition,the levels of TNF-α,IL-1β,IL-6 and cell adhesion molecules are significantly increased in patients with hyperlipidemia.These high levels of inflammatory factors will cause vascular dysfunction.Therefore,it is important to control the level of inflammatory factors and the inflammatory response in order to prevent and cure vascular injury caused by hyperlipidemia.κ-opioid receptor(κ-OR)is a major isoform of ORs widely expressed in cardiovascular system[3].Heart can synthesis and release endogenous κ-opioid peptide,which then bind to κ-OR and regulate cardiovascular function.Our previous study found that U50488 H,a selective κ-OR activator,protects vessels against hyperlipidemia-induced endothelial dysfunction and vascular diastolic dysregulation via activation of κ-OR/PI3K/Akt/e NOS/NO signaling pathway[4].Visibly,k-OR activation can promote the generation of NO and protect vascular endothelial function.It is well known that NO is one of the most important vascular relaxing factors.Endothelial cells produce NO by phosphorylation of e NOS,and the decrease of NO level is considered as a key marker of endothelial dysfunction.It is also reported that[5] caveolin-1 can directly interact with e NOS and inhibit e NOS activity,thus reduce NO production and finally lead to endothelial cells injury.However,it is largely unknown whether caveolin-1 expression is increased in hyperlidemic condition and leads to inhibition of NO production.Whether or not the role of κ-OR in the treatment of endothelial dysfunction induced by hyperlipidemia being related to the regulation of caveolin-1 function is still unknown.It is also revealed that κ-OR activation inhibits TNF-α production during myocardial ischemia/reperfusion(MI/R)injury by activating PI3K/Akt/e NOS signaling pathway[6].However,hyperlipidemia causes the inflammatory reaction by increasing the release of inflammatory factors such as TNF-α and IL-1β,the expression of adhesion molecules,the adhesion rate of monocytes and neutrophils to HUVEC.Whether this inflammatory reaction is inhibited by activation of κ-OR remains to be elucidated in our present study.Objectives: Based on these facts,the objectives of our study are:(1)to determine whether activation of κ-OR can exert vascular endothelium protection under hyperlipidemia;(2)if so,to determine if it is via regulating caveolin-1 expression and stimulating e NOS activity to restore NO production and improve vascular endothelium injury;In addition,to determine if it’s by activating PI3K/Akt/e NOS signaling to exert anti-inflammatory effect.Materials and methods: 1.Cultured human umbilical vein endothelial cells(HUVECs)were incubated with sodium palmitate(SP)for 48 h to mimic high fat-induced cell injury.Caspase 3 expression topped at 300μmol/L SP concentration,suggesting that 300μmol/L SP induced most HUVECs apoptosis.Thus 300μmol/L SP was used in subsequent study.2.HUVECs were divided into 6 groups when investigating whether the κ-OR exerts its functions in protecting endothelium via activating caveolin-1/e NOS signal pathway,the group are as following: control,U50488 H group,SP group,SP+U50488H group,SP+ U50488H+nor-BNI(a κ-OR blocker)group,and SP+U50,488H+L-NAME(an e NOS inhibitor)group.HUVECs were divided into 8 groups when investigating whether the κ-OR exerts its functions in anti-inflammation by activating PI3K/Akt/e NOS signal pathway under SP induction,the group are as following: control,U50488 H group,SP group,SP+U50488H group,SP+ U50488H+nor-BNI(a κ-OR blocker)group,SP+U50,488H+ LY294002(a PI3 K inhibitor)group,SP+U50,488H+MK2206-2HCL group(an Akt inhibitor),and SP+U50,488H+L-NAME(an e NOS inhibitor)group.3.CCK-8 kit was adopted to determine cell viability.4.Flow cytometry was used to analyze apoptosis rate.5.NO assay kit was used to determine NO content in cell culture media.6.Western blot was used to determine caspase-3,p-e NOS,total-e NOS,caveolin-1,caspase 1 and NLRP3 expression.7.Co-localization of caveolin-1 and e NOS in HUVECs was detected by laser scanning confocal microscopy.8.The changes of intracellular ROS levels were detected by ROS Kit.9.ELISA was performed to determine TNF-α、IL-1β、IL-6、IL-10、ICAM-1、VCAM-1、P-selectin、E-selectin levels in cell culture media.10.Cell counting was used to determine the adherence rate of neutrophils and monocytes.Results: I.Protective effect of κ-OR activation in endothelial cells 1.CCK-8 test showed that cell viability was impaired with 300μmol/L SP incubation,while U50488 H significantly improved cell viability.This effect of U50488 H was abrogated by nor-BNI,and L-NAME.2.Flow cytometry showed that SP induced HUVECs apoptosis,which was significantly inhibited by advanced U50488 H incubation.This effect of U50488 H on HUVECs apoptosis was blocked by nor-BNI,and L-NAME.3.SP incubation significantly decreased NO production.Advanced U50488 H treatment increased NO production,which was blocked by nor-BNI,and L-NAME.II.Regulatory effect of κ-OR activation on caveolin-1/e NOS signaling 1.Western blot showed that SP incubation significantly increased caveolin-1 expression and inhibited p-e NOS expression,which was attenuated by U50488 H administration.κ-OR blocker and e NOS inhibitor abolished e NOS activation stimulated by U50488 H.2.Co-localization experiments showed that the co-localization of caveolin-1 and e NOS was observed in HUVECs.SP incubation significantly increased caveolin-1 expression,which was reversed by U50488 H.This effect of U50488 H was abolished by nor-BNI,and L-NAME.III.Regulation of κ-OR activation on inflammatory factors and adhesion molecules 1.SP treatment upregulated caspase 1 and NLRP3 expression,which was abrogated by U50488 H.This effect of U50488 H was abolished by nor-BNI,LY294002,MK-2206-2HCL and L-NAME.2.Intracellular ROS examination showed that SP significantly increased ROS level in HUVECs.While U50488 H reduced ROS level incresed by SP.This effect of U50488 H was abolished by nor-BNI,LY294002,MK-2206-2HCL and L-NAME.3.ELISA showed that SP incubation upregulated TNF-α,IL-1β,IL-6,ICAM-1,VCAM-1,P-selectin,E-selectin expression and downregulated IL-10 expression.U50488 H administration significantly lowered TNF-α,IL-1β,IL-6,ICAM-1,VCAM-1,P-selectin,E-selectin expression and increased IL-10 expression.These effects of U50488 H were abolished by nor-BNI,LY294002,MK-2206-2HCL and L-NAME.4.Compared with the control group,the adhesion rate of monocytes and neutrophils to HUVECs in the SP group was significantly increased.U50488 H pre-treatment lowered the adhesion rate,which was abrogated by nor-BNI,LY294002,MK-2206-2HCL and L-NAME.Conclusion: κ-OR activation exerts protective effect against the endothelial cell injury induced by high fat.The possible mechanisms may be:(1)κ-OR activation inhibits SP-induced vascular endothelial cell injury via activating caveolin-1/e NOS signaling pathway.(2)κ-OR activation ameliorates SP-induced inflammation by activating PI3K/Akt/e NOS signaling pathway,thus improves endothelial function.The present study provides evidence suggesting that κ-OR activation exerts protective fuction against high fat-induced vascular endothelial injury and reveals the underlying molecular mechanism.κ-OR can be a promising target in prevention of high fat diet-induced vascular endothelial injury.