Icariin protects vascular endothelial cells from oxidative stress through inhibiting endoplasmic reticulum stress
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摘要
Objective: To investigate the protective effect and underlying mechanism(s) of icariin(ICA) in preventing hydrogen peroxide(H_2O_2)-induced vascular endothelial cell injury via endoplasmic reticulum stress(ERS).Methods: To study the effects of ICA on H_2O_2-induced damage, we used the cell counting kit-8 assay to detect cell viability and the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay to determine cell adhesion and apoptosis, respectively. Spectrophotometry and enzyme-linked immunosorbent assay were used to measure the expression levels of superoxide dismutase(SOD) and glutathione peroxidase(GSH-Px). Subsequently, glucose-regulated protein 78(GRP78), activating transcription factor-4(ATF4) and eukaryotic initiation factor-2 a(eIF2 a) were detected using Western blotting.Results: In human umbilical vein endothelial cells, different concentrations of ICA exhibited multiple effects, including reduced H_2O_2 damage, improved cell viability and adhesion, reduced cell apoptosis and increased SOD and GSH-Px activity. Among the ICA concentrations used, only the H_2O_2+ 100 lmol/L ICA group had significant differences compared to the H_2O_2 group. ERS activators H_2O_2 and DL-dithiothreitol(DTT) significantly increased GRP78, ATF4 and eIF2 a expressions, decreased cell activity and reduced SOD and GSH-Px activity. In contrast, the H_2O_2+ 100 lmol/L ICA and H_2O_2+ 100 lmol/L ICA + DTT groups had significant inhibitory effects on the expressions of GRP78,ATF4 and eIF2 a proteins, showing enhanced cell viability and SOD and GSH-Px activity.Conclusion: The results showed the dose-dependent effects of ICA against H_2O_2-induced injury in vascular endothelial cells. The inhibition of GRP78, ATF4 and eIF2 a protein expressions in the ERS, and the subsequent alleviation of oxidative stress damage, might be the molecular mechanism.
Objective: To investigate the protective effect and underlying mechanism(s) of icariin(ICA) in preventing hydrogen peroxide(H_2O_2)-induced vascular endothelial cell injury via endoplasmic reticulum stress(ERS).Methods: To study the effects of ICA on H_2O_2-induced damage, we used the cell counting kit-8 assay to detect cell viability and the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay to determine cell adhesion and apoptosis, respectively. Spectrophotometry and enzyme-linked immunosorbent assay were used to measure the expression levels of superoxide dismutase(SOD) and glutathione peroxidase(GSH-Px). Subsequently, glucose-regulated protein 78(GRP78), activating transcription factor-4(ATF4) and eukaryotic initiation factor-2 a(eIF2 a) were detected using Western blotting.Results: In human umbilical vein endothelial cells, different concentrations of ICA exhibited multiple effects, including reduced H_2O_2 damage, improved cell viability and adhesion, reduced cell apoptosis and increased SOD and GSH-Px activity. Among the ICA concentrations used, only the H_2O_2+ 100 lmol/L ICA group had significant differences compared to the H_2O_2 group. ERS activators H_2O_2 and DL-dithiothreitol(DTT) significantly increased GRP78, ATF4 and eIF2 a expressions, decreased cell activity and reduced SOD and GSH-Px activity. In contrast, the H_2O_2+ 100 lmol/L ICA and H_2O_2+ 100 lmol/L ICA + DTT groups had significant inhibitory effects on the expressions of GRP78,ATF4 and eIF2 a proteins, showing enhanced cell viability and SOD and GSH-Px activity.Conclusion: The results showed the dose-dependent effects of ICA against H_2O_2-induced injury in vascular endothelial cells. The inhibition of GRP78, ATF4 and eIF2 a protein expressions in the ERS, and the subsequent alleviation of oxidative stress damage, might be the molecular mechanism.
Objective: To investigate the protective effect and underlying mechanism(s) of icariin(ICA) in preventing hydrogen peroxide(H_2O_2)-induced vascular endothelial cell injury via endoplasmic reticulum stress(ERS).Methods: To study the effects of ICA on H_2O_2-induced damage, we used the cell counting kit-8 assay to detect cell viability and the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay to determine cell adhesion and apoptosis, respectively. Spectrophotometry and enzyme-linked immunosorbent assay were used to measure the expression levels of superoxide dismutase(SOD) and glutathione peroxidase(GSH-Px). Subsequently, glucose-regulated protein 78(GRP78), activating transcription factor-4(ATF4) and eukaryotic initiation factor-2 a(eIF2 a) were detected using Western blotting.Results: In human umbilical vein endothelial cells, different concentrations of ICA exhibited multiple effects, including reduced H_2O_2 damage, improved cell viability and adhesion, reduced cell apoptosis and increased SOD and GSH-Px activity. Among the ICA concentrations used, only the H_2O_2+ 100 lmol/L ICA group had significant differences compared to the H_2O_2 group. ERS activators H_2O_2 and DL-dithiothreitol(DTT) significantly increased GRP78, ATF4 and eIF2 a expressions, decreased cell activity and reduced SOD and GSH-Px activity. In contrast, the H_2O_2+ 100 lmol/L ICA and H_2O_2+ 100 lmol/L ICA + DTT groups had significant inhibitory effects on the expressions of GRP78,ATF4 and eIF2 a proteins, showing enhanced cell viability and SOD and GSH-Px activity.Conclusion: The results showed the dose-dependent effects of ICA against H_2O_2-induced injury in vascular endothelial cells. The inhibition of GRP78, ATF4 and eIF2 a protein expressions in the ERS, and the subsequent alleviation of oxidative stress damage, might be the molecular mechanism.
引文
[1]Simplicio JA,Resstel LB,Tirapelli DP,D’Orléans-Juste P,Tirapelli CR.Contribution of oxidative stress and prostanoids in endothelial dysfunction induced by chronic fluoxetine treatment.Vascul Pharmacol 2015;73:124-37.
[2]Suchal K,Malik S,Khan SI,Malhotra RK,Goyal SN,Bhatia J,et al.Protective effect of mangiferin on myocardial ischemia-reperfusion injury in streptozotocin-induced diabetic rats:role of AGE-RAGE/MAPK pathways.Sci Rep 2017;7:42027.
[3]Cubillos-Ruiz JR,Bettigole SE,Glimcher LH.Tumorigenic and immunosuppressive effects of endoplasmic reticulum stress in cancer.Cell2017;168(4):692-706.
[4]Civelek M,Manduchi E,Riley RJ,Stoeckert Jr CJ,Davies PF.Chronic endoplasmic reticulum stress activates unfolded protein response in arterial endothelium in regions of susceptibility to atherosclerosis.Circ Res 2009;105(5):453-61.
[5]Boussabbeh M,Ben Salem I,Prola A,Guilbert A,Bacha H,Abid-Essefi S,et al.Patulin induces apoptosis through ROS-mediated endoplasmic reticulum stress pathway.Toxicol Sci 2015;144(2):328-37.
[6]Yang YN.Antagonistic effect of paeoniflorin on endoplasmic reticulum stress induced by H2O2in H9C2 myocardial cells.Mudanjiang Medical College;2017[Chinese].
[7]Namkoong S,Chung BH,Ha KS,Lee H,Kwon YG,Kim YM.Microscopic technique for the detection of nitric oxide-dependent angiogenesis in an animal model.Methods Enzymol 2008;441:393-402.
[8]Hu Y,Liu K,Yan M,Zhang Y,Wang Y,Ren L.Effects and mechanisms of Icariin on atherosclerosis.Int J Clin Exp Med 2015;8(3):3585-9.
[9]Zhang Q,Li H,Wang S,Liu M,Feng Y,Wang X.Icariin protects rat cardiac H9c2cells from apoptosis by inhibiting endoplasmic reticulum stress.Int J Mol Sci2013;14(9):17845-60.
[10]Wang Y,Yang X,Li XD,He XJ,Wang YJ.Estrogen reduces apoptosis induced by endoplasmic reticulum stress in vascular endothelial cells and the mechanism.Zhongguo Dong Mai Ying Hua Za Zhi 2016;24(3):217-23[Chinese with abstract in English].
[11]Zhang WP,Bai XJ,Zheng XP,Xie XL,Yuan ZY.Icariin attenuates the enhanced prothrombotic state in atherosclerotic rabbits independently of its lipidlowering effects.Planta Med 2013;79(9):731-6.
[12]Fu S,Yang L,Li P,Hofmann O,Dicker L,Hide W,et al.Aberrant lipid metabolism disrupts calcium homeostasis causing liver endoplasmic reticulum stress in obesity.Nature 2011;473(7348):528-31.
[13]Upton JP,Wang L,Han D,Wang ES,Huskey NE,Lim L,et al.IRE1a cleaves select microRNAs during ER stress to derepress translation of proapoptotic caspase-2.Science 2012;338(6108):818-22.
[14]Li F,Gao B,Dong H,Shi J,Fang D.Icariin induces synoviolin expression through NFE2L1 to protect neurons from ER stress-induced apoptosis.PLoS ONE2015;10(3):e0119955.
[15]Di SY,Fan CX,Yang Y,Jiang S,Liang MM,Wu GL,et al.Activation of endoplasmic reticulum stress is involved in the activity of icariin against human lung adenocarcinoma cells.Apoptosis 2015;20(9):1229-41.
[16]Wang SX,Zhang Q,Shang M,Wei S,Liu M,Wang YL,et al.Microvesicles derived from hypoxia/reoxygenation-treated human umbilical vein endothelial cells impair relaxation of rat thoracic aortic rings.Zhongguo Ying Yong Sheng Li Xue Za Zhi 2014;30(6):560-6[Chinese with abstract in English].
[17]Wang Y,Vera L,Fischer WH,Montminy M.The CREB coactivator CRTC2 links hepatic ER stress and fasting gluconeogenesis.Nature 2009;460(7254):534-7.
[18]Liu XH,Zhang ZY,Sun S,Wu XD.Ischemic postconditioning protects myocardium from ischemia/reperfusion injury through attenuating endoplasmic reticulum stress.Shock 2008;30(4):422-7.
[19]Schisano B,Harte AL,Lois K,Saravanan P,Al-Daghri N,Al-Attas O,et al.GLP-1analogue,Liraglutide protects human umbilical vein endothelial cells against high glucose induced endoplasmic reticulum stress.Regul Pept 2012;174(1-3):46-52.
[20]Chiang CK,Hsu SP,Wu CT,Huang JW,Cheng HT,Chang YW,et al.Endoplasmic reticulum stress implicated in the development of renal fibrosis.Mol Med2011;17(11-12):1295-305.
[21]Chang JR,Duan XH,Zhang BH,Teng X,Zhou YB,Liu Y,et al.Intermedin1-53attenuates vascular smooth muscle cell calcification by inhibiting endoplasmic reticulum stress via cyclic adenosine monophosphate/protein kinase Apathway.Exp Biol Med(Maywood)2013;238(10):1136-46.
[2]Suchal K,Malik S,Khan SI,Malhotra RK,Goyal SN,Bhatia J,et al.Protective effect of mangiferin on myocardial ischemia-reperfusion injury in streptozotocin-induced diabetic rats:role of AGE-RAGE/MAPK pathways.Sci Rep 2017;7:42027.
[3]Cubillos-Ruiz JR,Bettigole SE,Glimcher LH.Tumorigenic and immunosuppressive effects of endoplasmic reticulum stress in cancer.Cell2017;168(4):692-706.
[4]Civelek M,Manduchi E,Riley RJ,Stoeckert Jr CJ,Davies PF.Chronic endoplasmic reticulum stress activates unfolded protein response in arterial endothelium in regions of susceptibility to atherosclerosis.Circ Res 2009;105(5):453-61.
[5]Boussabbeh M,Ben Salem I,Prola A,Guilbert A,Bacha H,Abid-Essefi S,et al.Patulin induces apoptosis through ROS-mediated endoplasmic reticulum stress pathway.Toxicol Sci 2015;144(2):328-37.
[6]Yang YN.Antagonistic effect of paeoniflorin on endoplasmic reticulum stress induced by H2O2in H9C2 myocardial cells.Mudanjiang Medical College;2017[Chinese].
[7]Namkoong S,Chung BH,Ha KS,Lee H,Kwon YG,Kim YM.Microscopic technique for the detection of nitric oxide-dependent angiogenesis in an animal model.Methods Enzymol 2008;441:393-402.
[8]Hu Y,Liu K,Yan M,Zhang Y,Wang Y,Ren L.Effects and mechanisms of Icariin on atherosclerosis.Int J Clin Exp Med 2015;8(3):3585-9.
[9]Zhang Q,Li H,Wang S,Liu M,Feng Y,Wang X.Icariin protects rat cardiac H9c2cells from apoptosis by inhibiting endoplasmic reticulum stress.Int J Mol Sci2013;14(9):17845-60.
[10]Wang Y,Yang X,Li XD,He XJ,Wang YJ.Estrogen reduces apoptosis induced by endoplasmic reticulum stress in vascular endothelial cells and the mechanism.Zhongguo Dong Mai Ying Hua Za Zhi 2016;24(3):217-23[Chinese with abstract in English].
[11]Zhang WP,Bai XJ,Zheng XP,Xie XL,Yuan ZY.Icariin attenuates the enhanced prothrombotic state in atherosclerotic rabbits independently of its lipidlowering effects.Planta Med 2013;79(9):731-6.
[12]Fu S,Yang L,Li P,Hofmann O,Dicker L,Hide W,et al.Aberrant lipid metabolism disrupts calcium homeostasis causing liver endoplasmic reticulum stress in obesity.Nature 2011;473(7348):528-31.
[13]Upton JP,Wang L,Han D,Wang ES,Huskey NE,Lim L,et al.IRE1a cleaves select microRNAs during ER stress to derepress translation of proapoptotic caspase-2.Science 2012;338(6108):818-22.
[14]Li F,Gao B,Dong H,Shi J,Fang D.Icariin induces synoviolin expression through NFE2L1 to protect neurons from ER stress-induced apoptosis.PLoS ONE2015;10(3):e0119955.
[15]Di SY,Fan CX,Yang Y,Jiang S,Liang MM,Wu GL,et al.Activation of endoplasmic reticulum stress is involved in the activity of icariin against human lung adenocarcinoma cells.Apoptosis 2015;20(9):1229-41.
[16]Wang SX,Zhang Q,Shang M,Wei S,Liu M,Wang YL,et al.Microvesicles derived from hypoxia/reoxygenation-treated human umbilical vein endothelial cells impair relaxation of rat thoracic aortic rings.Zhongguo Ying Yong Sheng Li Xue Za Zhi 2014;30(6):560-6[Chinese with abstract in English].
[17]Wang Y,Vera L,Fischer WH,Montminy M.The CREB coactivator CRTC2 links hepatic ER stress and fasting gluconeogenesis.Nature 2009;460(7254):534-7.
[18]Liu XH,Zhang ZY,Sun S,Wu XD.Ischemic postconditioning protects myocardium from ischemia/reperfusion injury through attenuating endoplasmic reticulum stress.Shock 2008;30(4):422-7.
[19]Schisano B,Harte AL,Lois K,Saravanan P,Al-Daghri N,Al-Attas O,et al.GLP-1analogue,Liraglutide protects human umbilical vein endothelial cells against high glucose induced endoplasmic reticulum stress.Regul Pept 2012;174(1-3):46-52.
[20]Chiang CK,Hsu SP,Wu CT,Huang JW,Cheng HT,Chang YW,et al.Endoplasmic reticulum stress implicated in the development of renal fibrosis.Mol Med2011;17(11-12):1295-305.
[21]Chang JR,Duan XH,Zhang BH,Teng X,Zhou YB,Liu Y,et al.Intermedin1-53attenuates vascular smooth muscle cell calcification by inhibiting endoplasmic reticulum stress via cyclic adenosine monophosphate/protein kinase Apathway.Exp Biol Med(Maywood)2013;238(10):1136-46.