| In this research,we observed the vascular effect induced by nobiletin,is the mainly active component deriving from orange peel's polymethoxyflavonoids extracts in rat vivo and vitro experiments.And we further explored the mechanism and the change of related with vasodilation and vasoconstriction induced by Nobiletin.These results will provide the solid theoretical basis for the use of orange peels,polymethoxyflavonoids extract and nobiletin as food additives or health food.Firstly,we selected 200-250 g SD male rats in rat vivo experiment.Then endothelim juried-hypertension rat model was prepared by peritoneal injection of 15mg/kg/d L-NNA with 28 d.The hypertension rats were divided into six groups,including control group,model group,drug group and positive control group.The control group is prepared by peritoneal injection of saline solution individually.The model group is prepared by peritoneal injection of L-NNA individually.The drug group included three groups.The low-dose group of nobiletin is prepared by peritoneal injection of L-NNA and gavage with low-dose nobiletin(1.8mg/kg/d).The middle-dose group of nobiletin is prepared by peritoneal injection of L-NNA and gavage with middle-dose nobiletin(3.6mg/kg/d).The high-dose group of nobiletin is prepared by peritoneal injection of L-NNA and gavage with high-dose nobiletin(7.2mg/kg/d).And the positive control group is prepared by peritoneal injection of L-NNA and gavage with telmisartan(80mg/kg/d).Each group included five rats.The rats were treated with gavege in 28 d.Then we detected the change of index.(1)The artery pressure of rat tail and heart rate was detected by the standard tail-cuff methods.And the weight of rat was detected with scale.These changes were detected at 14 d and 28 d.(2)After 28 d,we collected the artery plasma from experimental rats and tested the content of four active factors related with vasodilation and vasoconstriction,included PGI2,ANG2,EDHF and total NO.(3)After 28 d,we collected the thoracic aorta,heart and kidney sample from experimental rats,and observed the change of tissue structure after HE staining.The results included four aspects.(1)The artery pressure increased obviously with peritoneal injection of L-NNA after 28 d,compared with peritoneal injection of saline solution.The blood pressure was equal or greater than 140 mm Hg,and increased with 20 mm Hg compared with control group.This means the hypertension rat model was successfully established.(2)The blood pressure increased obviously after peritoneal injection of L-NNA,compared with control group.The data was 178±9.08 and showed statistic difference(P<0.05).The blood pressure decreased obviously after 14 d,compared with model group.The data was 123±1.56 and showed statistic difference(P<0.05).The blood pressure had no change after 28 d,compared with 14 d.The data was 135±13.23 and had no statistical difference.The blood pressure increased obviously after peritoneal injection of L-NNA,compared with control group in low-dose nobiletin group.The data is 184±29.46 and showed statistic difference(P<0.05).The blood pressure decreased obviously after gavege with 1.8mg/kg/d nobiletin,compared with peritoneal injection of L-NNA.The data was 127±11.59 and showed statistic difference(P<0.05).The blood pressure had no change after 28 d,compared with 14 d.And the data is 135±13.23 and had no statistic difference.The blood pressure increased obviously after peritoneal injection of L-NNA,compared with control group in middle-dose nobiletin group.The data is 196±34.02 and showed statistic difference(P<0.05).The blood pressure decreased obviously after gavege with 3.6mg/kg/d nobiletin,compared with peritoneal injection of L-NNA.The data was 124±9.00 and showed statistic difference(P<0.05).The blood pressure had no change after 28 d,compared with 14 d.And the data is 127±9.64 and had no statistic difference.The blood pressure increased obviously after peritoneal injection of L-NNA,compared with control group in high-dose nobiletin group.The data is 175±5.51 and showed statistic difference(P<0.05).The blood pressure decreased obviously after gavege with 7.2mg/kg/d nobiletin,compared with peritoneal injection of L-NNA.The data was 137±7.21 and showed statistic difference(P<0.05).The blood pressure had no change after 28 d,compared with 14 d.And the data is 142±10.50 and had no statistic difference.However,there was no statistic difference in low,middle and high-dose nobiletin groups.And there was no statistic difference compared with positive control group.The weight and heart rate had no obviously change and no statistic difference in experimental groups.These results showed nobiletin acted anti-hypertension effect in hypertension rats.(3)The results included three aspects with ELISA kit.(1)The change of PGI2 content were detected with ELISA kit.There was no statistic difference of the change of PGI2 content induced by 1.8mg/kg/d nobiletin,compared with peritoneal injection of L-NNA.The content of PGI2 increased obviously induced by 3.6mg/kg/d nobiletin,compared with low-dose nobiletin group,model group and control group.The data was 79.0±6.3 and showed statistic difference(P<0.05).There was no statistic difference of the change of PGI2 content induced by 7.2mg/kg/d nobiletin,compared with 3.6mg/kg/d nobiletin.The content of PGI2 increased obviously induced by 7.2mg/kg/d nobiletin,compared with low-dose nobiletin group,model group and control group.The data was 89.8±4.0 and showed statistic difference(P<0.05).(2)The change of ANG2 content were detected with ELISA kit.The content of ANG2 had no obviously change and statistic difference in experimental groups.(3)The change of EDHF content were detected with ELISA kit.The content of EDHF had no obviously change and statistic difference in experimental groups.(4)The change of total NO content were detected with ELISA kit.The total NO content increased obviously induced by after gavege with 1.8mg/kg/d nobiletin,compared with model group and control group.The data was 91.3±3.9 and had statistic difference(P<0.05).The total NO content increased obviously induced by after gavege with 3.6mg/kg/d nobiletin,compared with model group and control group.The data was 89.0±6.3 and had statistic difference(P<0.05).The total NO content increased obviously induced by after gavege with 7.2mg/kg/d nobiletin,compared with model group and control group.The data was 90.8±4.0 and had statistic difference(P<0.05).However,the total NO content had no obviously change and no statistic difference in low,middle and high-dose nobiletin groups.And the result was similar in nobiletin groups and positive control group.These results showed anti-hypertension effect induced by nobiletin was related with PGI2 and total NO content from plasma,but no related with ANG2 and EDHF content in hypertension rats.(4)After HE staining,the tissue structure of thoracic aorta,heart and kidney had no obviously changes with microscopic examination in experimental hypertension rats.Secondly,we selected 200-250 g SD male rats and isolated mesenteric arteries and pulmonary arteries in rat vitro experiment.(1)The rat isolated mesenteric and pulmonary arteries were given with baseline tension,and then precontracted after treated with 10-6M PE.The vasodilation induce by 10-9-10-5M Nobiletin was detected with vascular ring experiments.The results showed 10-5M nobiletin induced vasodilation in rat isolated mesenteric arteries.The relaxation rate was 36.87±3.57% and showed on a dose-dependent manner.The EC50 is 16.34±5.61?M.However,10-5M nobiletin had no vasodilation effect on rat isolated pulmonary arteries.(2)The change of vascular tension was detected after treated with 10-9-10-5M nobiletin on 10-6M PE-precontriction rat isolated endothelial-denuded mesenteric artery rings.The vasodilation effect induced by nobiletin almost disappeared.The results indicated that the vasodilation effect induced by nobiletin showed endothelim-dependent on rat isolated mesenteric arteries.(3)The vascular tension was detected after treated with e NOS inhibi?-NAME(10-4M)for 30 min and 10-9-10-5M nobiletin on 10-6M PE-precontriction rat isolated endothelial-denuded mesenteric artery rings.The rexalation rate induced by nobiletin decreased on rat isolated mesenteric arteries.The data is 21.90±2.06%.These results indicated the vasodialtion effect induced by nobiletin has related with endothelial e NOS partly on rat isolated mesenteric arteries.(4)We cultured rat aorta endothelial cells and prepared cell proteins after treated with 10-5M nobiletin.The expression of e NOS protein and activity of p-e NOS protein were detected with western blotting.We found that the activity of p-e NOS-1177 increased obviously in 10-5M nobiletin-treated rat aorta endothelial cells,but the expression of e NOS protein had no change.And the activity of p-e NOS-1177 was inhibited by e NOS inhibitor?-NAME.These results showed the vasodilation effect was induced by nobiletin through activating p-e NOS-1177.(5)NO production was detected using the fluorescence probe after treated with 10-5M nobiletin in cultured rat aorta endothelial cells.We found that NO production induced by 10-5M nobiletin increased obviously in rat aorta endothelial cells.And the increase effect of NO content was inhibited by e NOS inhibitor?-NAME.The results indicated that nobiletin induced vasodilation of rat isolated mesenteric arteries through activating p-e NOS-1177 and promoting NO production.(6)The vascular tension was detected after treated with PI3K/Akt inhibitor wortmannin,PKA inhibitor H-89,SIRT1 inhibitor nicotinamide or RORs inhibitor SR1001 respectively for 30 min and 10-9-10-5M nobiletin on 10-6M PE-precontriction rat isolated endothelial-integrallty mesenteric artery rings.We found that the vasodilation induced by nobiletin had no obviously change on rat isolated mesenteric arteries.These results indicated vasodilation induced by nobiletin had no related with PI3K/Akt,PKA,SIRT1 and RORs signaling pathway.(7)The vascular tension was detected after treated with Ca2+-free solution and 10-9-10-5M nobiletin on 10-6M PE-precontriction rat isolated endothelial-integrallty mesenteric artery rings.The vasodilation rate induced by nobiletin decreased obviously on rat isolated mesenteric arteries.The data was 12.83±2.22% and had statistic difference(P<0.05).These results indicated that Ca2+ in endothelial cells involved in vasodilation induced by nobiletin on rat isolated mesenteric arteries.(8)The change of Ca2+ content was detected after treated with 10-5M nobiletin in cultured rat aorta endothelial cells.We found that the Ca2+ content induced by nobiletin increased obviously in rat aorta endothelial cells.And the data is 1.24±0.13 and had statistic difference(P<0.05),compared with control groups.These results indicated that nobiletin increased the content of Ca2+in endothelial cells.And high concentration of Ca2+ in endothelial cells activated p-e NOS,promoted NO production and induced vasodilation effect.(9)The change of Ca2+ content in cultured endothelial cells was detected after treated with IP3 inhibitor 2-APB for 30 min and 10-5M nobiletin.We found that the increase effect of endothelial Ca2+ content was inhibited after inhibiting release of endothelial Ca2+.The data was 0.93±0.008 and had statistic difference(P<0.05),compared with control group.The content of endothelial Ca2+ induced by nobiletin decreased obviously after treated with Ca2+-free solution.The data was 0.98±0.01 and had statistic difference(P<0.05),compared with control group.These results indicated that the nobiletin-induced increase of endothelial Ca2+ concentration is related to both the release of intracellular Ca2+ and the entry of extracellular Ca2+. |
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