Mechanisms Underlying Depression And Vascular Relaxation Induced By An Ethanol Extract Of Rumex Acetosa L.

ObjectiveRumex acetosa L.(RA)is an important traditional Chinese folk medicine plant.It has a wide range of pharmacological effects,such as clearing heat and detoxicating,kill worms,anti-bacterial,anti-viral,anti-oxidant properties.It contains anthraquinone,flavonoid,naphthalene derivatives,styrene,tannins and other biologicallyactive ingredients.Although many studies associated with the anti-hypertensive effect of RA have been carried out,there is no detailed study about its mechanism.The aim of the present study was to define the hypotensive and vasorelaxant effects and mechanisms of ethanol extract of Rumex acetosa L.(ERA)in rats.Methods1.Effect of ERA on blood pressure and heart rate in rats.Carotid artery was catheterized to measure the changes in blood pressure(BP)and hear rate(HR)in anesthetized rats.The changes of BP and HR were recorded using a biological laboratory system(Model BL-420S).ERA(0.2,0.5,1.0 mg/kg)is administered intravenously and the effects are observed in 60 min.2.Effect of ERA on vascular relaxation in rat thoracic aorta.The experimental apparatus is an isolated vascular ring model.ERA was examined for their vascular relaxant effects in isolated phenylephrine(PE,1 μmol/L)-precontracted rat thoracic aorta.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 ERA induced vascular relaxation,another series of experiments were done in rings.The rings were incubated to various modulating agents,and then vascular relaxation was carried out by cumulative addition of ERA.3.Effect of ERA on the expression of Akt and eNOS protein in vascular endothelial cellsThe change of ERA-induced human umbilical vein endothelial cells(HUVECs)viavility was detected using MTT method.Western blot method was used to detect the levels of Akt and eNOS protein.Optimal concentration and treatment time of ERA were screened out.Wortmannin(WT)and LY294002(LY),inhibitors of PI3K/Akt,were used to define the mechanisms by which ERA affect the Akt and eNOS protein levels.Results:1.ERA reduced systolic blood pressure(SBP),diastolic blood pressure(DBP),mean blood pressure(MBP)and heart rate(HR)in rats.2.ERA relaxed PE-precontracted isolated aortic rings in a dose-dependent manner.In contrast,denudation of the functional endothelium abolished ERA-induced vasorelaxation.ERA-induced vascular relaxation was significantly attenuated by pretreatment of endothelium-intact aortic rings with L-NAME(10 μmol/L),a non-selective NOS inhibitor,and ODQ(10 μmol/L),an inhibitor of soluble guanylyl cyclase(sGC),but not by indomethacin(Indo,10 μmol/L).The ERA-induced vasorelaxation was significantly attenuated by Ca2+-free Krebs buffer.Similarly,modulators of the store-operated Ca2+ entry(SOCE)such as 2-APB(75 μmol/L),Gd3+(10 μmol/L)and thapsigargin(TG,1 μmol/L),significantly attenuated the ERA-induced vasorelaxation.Pretreatment of endothelium-intact aortic rings with wortmannin(0.1 μmol/L)and LY294002(LY,30 μmol/L),inhibitors of PI3K/Akt,significantly attenuated the ERAinduced vasorelaxation.The pretreatment with TEA(1 mmol/L),a selective blocker of KCa channels,glibenclamide(Gli,10 μmol/L),an ATP-sensitive potassium channels(KATP)blocker,or 4-aminopyridine(4-AP,0.1 μmol/L),a voltage-dependent potassium channels(Kv)blocker,had no effects on the ERA-induced vasorelaxation.The large-conductance KCa channels(BKCa)blocker iberiotoxin(Iber,0.1 μmol/L),an intermediate-conductance KCa channels(IKCa)blocker charybdotoxin(Cha,0.1 μmol/L),a small-conductance KCa channels(SKCa)selective blocker apamin(Apa,0.3 μmol/L),had no significant affect the ERA-induced vascular relaxation.However,pretreatment of aortic rings with BaCl2(100 μmol/L),a selective KIR channels blocker significantly reduced ERA-induced vasorelaxation.Pretreatment of aortic rings with atropine(1 μmol/L),an inhibitor of muscarinic receptors,or propranolol(1 μmol/L),a non-selective blocker of β-adrenoreceptors,had no effect on ERA-induced vasorelaxation.3.The ERA-treated HUVECs showed increase in cell viability at the dose of 10-30 μg/ml.Western blot results revealed that,compared with the control group,there were significant up-regulation of the protein levels of p-Akt,Akt in HUVECs after treatment with ERA for 2-15 min,and the peak time was 5 min(P<0.01).there were significant up-regulation of the protein levels of p-eNOS,eNOS in HUVECs after treatment with ERA for 2-10 min,and the peak time was 5 min(P<0.01).The effect was also dose dependent,and peaked at 30 μg/ml ERA(P<0.01).Wortmannin(0.1 μM)and LY(30 μmol/L)blocked ERA-induced Akt and eNOS phosphorylation.Conclusions:The present study suggests that ERA induces depression and vasorelaxation via endothelium-dependent two-step signaling pathway: an activation of the PI3 K /Akt-and Ca2+-eNOS-NO signaling in the endothelial cells and then subsequent stimulation of the NO-sGC-cGMP-KIR channel signaling in the vascular smooth muscle cells.