Vascular Relaxation Induced By Physcion And Its Mechanism Of Action

ObjectiveRheum palmatum L.is one of the four traditional Chinese medicine that has a bitter cold taste.It has diarrhea,detoxification,anti carbuncle,removing blood stasis,dehumidification and cooling blood for hemostasis functions.It also has a wide range of pharmacological effects,such as lowering blood glucose and lipid,antihypertension,diuresis,antioxidant and so on.It has widely been used for the treatment of cardiovascular disease and inflammation.The main active components of Rheum palmatum L.include physcion,emodin,chrysophanol,aloe-emodin and rhein,and all of these five active components are anthraquinones.It has been known that physcion has anti-inflammatory,antioxidant and antiapoptotic effects.However,vascular relaxation induced by physcion and its mechanism of action are unknown.This study was designed to investigate the vascular relaxation induced by physcion and mechanism at the tissue,cellular and molecular levels in vitro.The purpose was to clarify the antihypertensive mechanism and provide scientific basis for the development and utilization of rhubarb and its active components in china.Methods1.Effect of physcion on vascular relaxation in the normal rat thoracic aortaPhyscion was examined for its vascular relaxant effects in isolated phenylephrine?PE,1?M?-precontracted rat thoracic aorta using an isolated vascular ring perfusion model.To record the changes in isometric tension,a biological laboratory system was used.The thoracic aorta was isolated from rats and the tension of aortic rings was measured with or without enthothelium.To define the mechanisms by which physcion induced vascular relaxation,another series of experiments were done in rat aortic rings.The rings were incubated to various modulating agents,and then vascular relaxation was carried out by cumulative addition of physcion.2.Effect of physcion on the expression of Akt and eNOS protein in human umbilical vein endothelial cellsHuman umbilical vein endothelial cells?HUVECs?were cultured and treated with physcion in vitro.Cell viavility was determined by using MTT assay.Nitrate reductase assay was used to detect the release of NO.The levels of Ca²⁺were detected by confocal microscopy.Protein expression of Akt and endothelial nitric oxide synthase?eNOS?were detected by Western blot.Results:1.In endothelium-intact and denuded rings,physcion relaxed PE-precontracted isolated aortic rings in a dose-dependent manner.Physcion-induced vascular relaxation was significantly attenuated by pretreatment with L-NAME?30?M?,a non-selective NOS inhibitor,or ODQ?10?M?,an inhibitor of soluble guanylyl cyclase?sGC?for 20 min in endothelium-intact aortic rings,whereas there had no significant effect in endothelium-denuded rings.Physcion-induced vascular relaxation was unaffected by indomethacin?10?M?.Extracellular Ca²⁺depletion and treatments with 2-aminoethyl diphenylborinate?2-APB,75?M?,modulator of the store-operated Ca²⁺entry?SOCE?significantly attenuated the physcion-induced vasorelaxation.Wortmannin?0.1?M?,inhibitor of PI₃K/Akt,significantly attenuated the physcion-induced vasorelaxation.Atropine?1?M?and propranolol?1?M?had no effect on the physcion-induced vasorelaxation.The pretreatment of endothelium-denuded aortic rings with TEA?1 mM?,iberiotoxin?0.1?M?,and glibenclamide?10?M?,but not apamin?0.3?M?,charybdotoxin?0.1?M?,BaCl₂ and 4-aminopyridine,attenuated the physcion-induced vasorelaxation.2.Physcion increased the survival rate of HUVECs in a dose-dependent manner.Physcion promoted the release of NO in HUVECs.Pretreatment of HUVECs with 2-APB,an inhibitor of store-operated calcium channel?SOCE?,and wortmannin,an inhibitor of phosphatidylinositol 3-kinase?PI₃K?/Akt,inhibited the physcion-induced NO release.Confocal microscopy and Western blot showed that physcion elevated intracellular Ca²⁺levels and increased phosphorylation of Akt and eNOS.Conclusions:1.This study suggested that physcion induced both endothelium-dependent and endothelium-independent relaxation.The mechanism was associated with the following aspects:?1?PI₃K/Akt-and Ca²⁺-eNOS-NO signaling pathway were likely involved in the endothelium-dependent relaxation;?2?Activation of large conductance calcium activated potassium channel(BK_Ca)and ATP sensitive potassium channel?KATP?contributed in part to the endothelium-independent relaxation.2.Physcion promoted the release of NO in vascular endothelial cells,and the underlying mechanism may be related with the increased protein expression of Akt and eNOS.