Study On Theeffect And Mechanism Of Relaxation To Resveratrol In Isolated Human Intrapulmonary Arteries

Resveratrol belongs to non flavonoids polyphenolic compounds,it is a rich content of polyphenols in red Wine.The study found that resveratrol could antioxidant, anti-inflammatory, antitumor, anti proliferation and cardiovascular protection.Recent studies show that resveratrol has effects of vasodilation,But the specific mechanism is not clear,Vasodilation effects of resveratrol have been demonstrated in several studies,including porcine coronary artery, rat aorta, pulmonary, coronary and renal artery,but human intrapulmonary arteries of the effects have not been reported.In our present study, we use isolated human intrapulmonary artery rings to observe effects of resveratrol.To observe the effect of resveratrol on contraction of isolated human intrapulmonary arterirs and inferring its vasodilating mechanism.The main of vessels used for vascular tension experiment include resistance and elastic vessel,This study selected the diameter of 1～1.5 mm human intrapulmonary arteries belonging to the vascular vessel.We hope our study could provide laboratory data for the antihypertensive effect of resveratrol to guide clinical drug-use.Materials and Methods1.The source of the specimenSpecimen were collected from patients undergoing pulmonary lobectomy due to lung cancer in Guangdong general hospital from May 4,2014 to July 28,2014,ASA Ⅰ～Ⅱ,age from 20 to 73,men 22,women 13,pulmonary function examination is normal,exclude hypertension, diabetes, pulmonary hypertension.Patients were informed the collection process and purpose of specimen, and signed the informed consent. This study was approved by the Guangdong Provincial Medical Ethics Committee(No.2010079H).Normal lung tissue away no less than 5cm from the cancer tissue edge was selected.2.Preparation of artery ringsThe specimen was placed into a container fulfilled with ice-cold Kreb’s solution.The careful separation of intrapulmonary arteries Under a stereomicroscope and cut into vessel rings about 1.5-2mm in length,Each vessel rings were mounted in the chamber of a Multi Myograph System.After an equilibration period of 60 min,Kreb’s solution was replaced in the chamber every 15 Minutes,each ring was stretched to an optimal tension of 1.8-2mN.Each chamber contained 5ml Kreb’s solution that bubbled with 95% 02 plus 5% CO2 constantly. The room temperature maintained at 37℃.the endothelial layer was mechanically disrupted by gently rubbing the luminal surface with a tiny wire back and forth 30 times.3.Vascular function testAfter a 60-minute balance,The Kreb’s solution was replaced with high K+solution,high K+solution induced contractile response of the human intrapulmonary arteries,after the changing was stable,Kreb’s solution was used to wash for 4 times to resume the vascular tension to baseline, repeat twice of the steps.There is no more than 10% of vascular response to the next steps.4.The fuction test of endotheliumAfter a 60-minute of high K+solution,2 umol/L 5-HT was to induce,after the changing was stable,1 umol/L Ach was to induce to relax vascular tension,at least 60% relaxation,we considered endothelium fuction intact and less than 10% one is not intact.Kreb’s solution was used to wash for 4 times to resume the vascular tension to baseline.5.The experiment content5.1 Effect of resveratrol on the vascular tension of resting state 10、30、100、300、1000 umol/L concentration-accumulated of resveratrol was added,The control group added DMSO as control at the corresponding time and data was recorded at the same time.the concentration of DMSO at 1:500 have proved that there were not affected.5.2 Effect of resveratrol on precontracted arterial rings by U46619、ET-1、KClArterial rings were precontracted with 100 nmol/L U46619、30 nmol/L ET-1、 60 mmol/L KCl,after the changing was stable,10、30、100、300、1000 umol/L concentration-accumulated of resveratrol was added,data was recorded at the same time,The same volume of DMSO as control.5.3 The vasodilation effect of resveratrol between endothelium-intact and Endothelium-denudedEndothelium-intact and Endothelium-denuded(n=4)was precontracted with 100nmol/L U46619,after the changing was stable,10、30、100、300、1000 umol/L concentration-accumulated of resveratrol was added,data was recorded at the same time,The same volume of DMSO as control.5.4 Vasodilatory effect of resveratrol presence of L-NAMEIn the presence and absence of 100 umol/L L-NAME,Endothelium-intact rings were added into 100 nmol/L U46619,after the changing was stable,10、30、100、300、 1000 umol/L concentration-accumulated of resveratrol was added,data was recorded at the same time.5.5 Vasodilatory effect of resveratrol presence of indomethacinIn the presence and absence of 10 umol/L indomethacin, Endothelium-intact rings were added into 100 nmol/L U46619,10、30、100、300.1000 umol/L concentration-accumulated of resveratrol was added,data was recorded at the same time.5.6 The role of K+on vasodilation effects of resveratrolEndothelium-intact rings were added into 100 nmol/L U46619 under the presence and absence of glibenclamide、BaCl2. TEA+,resveratrol was added,data was recorded at the same time.5.7 The role of Ca2+ on vasodilation effects of resveratrol5.7.1 Endothelium-denuded rings were added into 100 nmol/L U46619 under the presence and absence of nifedipine,10、30、100、300、1000 umol/L concentration-accumulated of resveratrol was added,data was recorded at the same time.5.7.2 Endothelium-denuded rings were added into nifedipine after 30 min、10、30、 100 umol/L resveratrol was added,100 nmol/L U46619 were added into contracting arterial rings,data was recorded at the same time.5.7.3 Some rings incubated in a Ca2+-free,then added into nifedipine,without and with resveratrol(10、30、100 umol/L),100 nM/L U46619 contract arterial rings,data was recorded at the same time.6 Measurements of data and data analysisThe Emax was represented the maximal response percentage.the concentrations of drugs increased the maximal contraction by 50% refereed to EC50,The negative logarithm of the dilator (or contractor) concentration that resulted in half of the maximal relaxation or contraction (pD2) was calculated. pD2=-1g(EC50).Data were expressed as the mean±S.E.M,SPSS 13.0 was used to analyzing,Graphpad was used to analyze curves.One-way ANOVA followed by LSD test was used when more than two groups were compared. P<0.05 was considered significant.Results1.Effect of resveratrol on the vascular tension of resting stateConcentration-accumulated of resveratrol in the resting state in endothelium-intact human intrapulmonary arteries tension without affecting.2.Effect of re sveratrol on precontracted arterial rings by U46619、ET-1、KClResveratrol induced relaxation on precontracted arterial rings by U46619、ET-1、 KCl in a concentration-dependent manner.pD2 is respectively:3.82±0.20, 3.84±0.57,3.68±0.27, Emaxis respectively:(99.58±0.83)%,100%, (99.65±0.98)%,there is no difference between control group.3.The vasodilation effect of resveratrol between endothelium-intact and Endothelium-denudedResveratrol induced relaxation on Endothelium-denuded arterial rings in a concentration-dependent manner.pD2 is 4.04±0.05, Emax is 100%.4.Vasodilatory effect of resveratrol presence of L-NAME Resveratrol induced relaxation on arterial rings in the presence of L-NAME,pD2 is 3.64±0.10, Emax is (98.97±2.30)%,L-NAME may cause the resveratrol diastolic function right,resveratrol may promote endothelial release of NO.5.Vasodilatory effect of resveratrol presence of indomethacinIn the presence of indomethacin,Resveratrol induced relaxation on arterial rings in the presence of indomethacin,pD2:3.91±0.31, Emax:100%,there is no ralation to cyclooxygenase.6.The role of K+on vasodilation effects of resveratrol Resveratrol induced relaxation on arterial rings in the presence of glibenclamide、 BaCl2. TEA+,pD2 is respectively:4.04±0.04、3.77±0.07、3.92±0.1, Emax is respectively:(100±6.81)%、(100±5.98)%、(100±7.18)%,relaxation of resveratrol does not rely on the K+channel.7.The role of Ca2+on vasodilation effects of resveratrol7.1 Resveratrol induced relaxation on arterial rings in the presence of nifedipine,pD2:3.92±0.06, Emax:(99.25±2.6)%,the two curves have difference, relaxation of resveratrol rely on the L-type VGCC.7.2 Endothelium-denuded rings were added into nifedipine and resveratrol,U46619 could contract arterial rings,but resveratrol Can partially inhibited the U46619 induced contract response,relaxation of resveratrol rely on the TXA2-related ROCC.7.3 Under the condition of Ca2+-free Kreb’s solution,resveratrol Can inhibited the U46619 induced contract response,relaxation of resveratrol rely on the extracellular Ca2+.Conclusion1.Resveratrol produces relaxation in human in intrapulmonary arteries;2.The possible mechanism of the relaxation of resveratrol may involve in promote endothelial release of NO,extracellular Ca2+via VOCC and ROCC.