白藜芦醇调节小鼠主动脉Orai1表达抑制动脉粥样硬化氧化应激损伤机制研究
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摘要
目的:探讨白藜芦醇(resveratrol,RES)是否可以通过调节血管组织中钙释放激活钙通道调节分子1(calcium release-activated calcium modulator1,Orai1)表达,激活血管组织中内皮型一氧化氮合酶(endothelial nitric oxide synthase,eNOS)表达发挥抗动脉粥样硬化(atherosclerosis,AS)效应。方法:雌性C57BL/6J小鼠卵巢切除后高脂喂养建立动脉粥样硬化模型,实验分为对照组、模型组、白藜芦醇0.05 g/kg、0.15 g/kg组及亚硝基左旋精氨酸甲酯(L-nitro-arginine methylester,L-NAME)0.007 g/kg干预组。16周后,全自动生化分析仪检测小鼠血脂4项及血清Ca~(2+)水平,HE染色及油红O染色观察血管壁动脉粥样硬化情况,免疫组化技术观察血管组织中eNOS表达,免疫印迹测定血管组织的eNOS、硝基络氨酸(nitrotyrosine,NT)及Orai1蛋白表达。结果:高脂喂养16周后,AS模型成功建立,模型组血管组织有明显的AS病理改变,0.15 g/kg白藜芦醇组AS病变明显减轻,内膜下脂质斑块减少;小鼠血清中CHOL、TG和LDL水平明显降低,HDL水平升高,同时Ca~(2+)水平降低;而0.007 g/Kg L-NAME干预组内膜增厚,内膜下脂质斑块增多;CHOL、TG和LDL-C增高,同时HDL含量下降,血清Ca~(2+)水平略有升高。Western blot结果显示补充白藜芦醇后血管组织中eNOS表达显著增加,NT表达降低,Orai1表达增强,而提前应用0.007 g/Kg L-NAME预处理后可逆转白藜芦醇诱导的eNOS表达增加及NT表达下降,但不能逆转Orai1表达的增加。结论:白藜芦醇可以通过上调Orai1表达,增加eNOS,降低NT蛋白表达,有效防止ONOO~-(过氧化亚硝基阴离子,peroxynitrite anion)介导的氧化应激损伤,改善内皮功能,防治动脉粥样硬化。
Objective: To investigate the anti-atherosclerotic effect of resveratrol and its mechanism on activating Orai1 and eN OS expressions.Methods: female C57 BL/6 J mice after ovariectomy were fed with high-fat diet to establish the atherosclerosis model,and then the mice were divided into the control group,resveratrol groups at the doses of 0. 05 g/kg and 0. 15 g/kg,the model group and 0. 007 g/kg L-NAME pretreated group. After 16 weeks of administration,serum was collected to detect the lipid level and calcium concentration. The changes of aorta structure were evaluated by HE staining and Oil Red O staining. The expression of eN OS was measured by DAB staining and western blot,and NT and Orai1 expressions were detected by western blot. Results: Atherosclerosis model was successfully established after 16 weeks' administration. Compared with the model group,total cholesterol( TC),triglyceride( TG),low density lipoprotein( LDL) and calcium concentration were decreased,while the high density lipoprotein( HDL) was increased. HE staining and Oil Red O staining showed that in LNAME group,aortic intima was thickened,and the cells had irregular arrangement. Compared with the model group,the eN OS and Orai1 expressions in the resveratrol group were increased,NT expression was increased,which was reversed in L-NAME pretreated group. Conclusion: Resveratrol can prevent and treat AS by increasing the Orai1 and eN OS expressions,decreasing NT expression and preventing ONOO-( peroxynitrite anion) mediated oxidative stress injury.
Objective: To investigate the anti-atherosclerotic effect of resveratrol and its mechanism on activating Orai1 and eN OS expressions.Methods: female C57 BL/6 J mice after ovariectomy were fed with high-fat diet to establish the atherosclerosis model,and then the mice were divided into the control group,resveratrol groups at the doses of 0. 05 g/kg and 0. 15 g/kg,the model group and 0. 007 g/kg L-NAME pretreated group. After 16 weeks of administration,serum was collected to detect the lipid level and calcium concentration. The changes of aorta structure were evaluated by HE staining and Oil Red O staining. The expression of eN OS was measured by DAB staining and western blot,and NT and Orai1 expressions were detected by western blot. Results: Atherosclerosis model was successfully established after 16 weeks' administration. Compared with the model group,total cholesterol( TC),triglyceride( TG),low density lipoprotein( LDL) and calcium concentration were decreased,while the high density lipoprotein( HDL) was increased. HE staining and Oil Red O staining showed that in LNAME group,aortic intima was thickened,and the cells had irregular arrangement. Compared with the model group,the eN OS and Orai1 expressions in the resveratrol group were increased,NT expression was increased,which was reversed in L-NAME pretreated group. Conclusion: Resveratrol can prevent and treat AS by increasing the Orai1 and eN OS expressions,decreasing NT expression and preventing ONOO-( peroxynitrite anion) mediated oxidative stress injury.
引文
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[11]Jin S W, Choi C Y, Hwang Y P. Betulinic Acid Increases eNOS Phosphorylation and NO Synthesis via the Calcium-Signaling Pathway. J Agric Food Chem, 2016; 64(4)∶785～791.
[12]Huang H, Koelle P, Fendler M, et al. Induction of inducible nitric oxide synthase (iNOS) expression by oxLDL inhibits macrophage derived foam cell migration. Atherosclerosis, 2014, 235(1)∶213～22.
[13]王腊梅, 唐娜, 钟华, 等. 钙离子信号蛋白TRPC1及Orai1参与了HUVEC经SOC和ROC介导的钙内流和NO生成. 中国病理生理杂志, 2017, 25(12)∶1189～1195.
[14]袁楚婷, 葛长江, 宋现涛, 等. 血小板钙库操纵性钙通道蛋白与冠状动脉斑块易损性的相关性研究. 心肺血管病杂志, 2018, 37(4)∶273～277.
[15]Rodríguez-Moyano M, Díaz I, Dionisio N, et al. Urotensin-II promotes vascular smooth muscle cell proliferation through store-operated calcium entry and EGFR transactivation. Cardiovasc Res, 2013, 100(2)∶297～306.
[16]He S, Yan X. From resveratrol to its derivatives:new sources of nature antioxidant. Curr Med Chem, 2013,20(8)∶ 1005～1017.
[17]阮景明, 朱鹏立, 蒋娜, 等. 白藜芦醇对兔动脉粥样硬化及PPARγ相关炎症因子表达的影响. 中国动脉硬化杂志, 2013,21(3)∶203～208.
[18]程静静, 陈莉, 李朝飞, 等. 白藜芦醇通过高脂减缓内质网应激及增加eNOS的表达改善高热量高胆固醇饮食引起的内皮损伤. 中国药理学通报, 2014, 30(12)∶1756～1762.
[19]Olas B, Nowak P, Ponczek M, et al. Resveratrol, a natural phenolic compound may reduce carbonylation proteins induced by peroxynitrite in blood platelets. Gen Physiol Biophys, 2006,25(2)∶215～222.
[20]Lu T, Zhou D, Gao P, et al. Resveratrol attenuates high glucose-induced endothelial cell apoptosis via mediation of store-operated calcium entry. Mol Cell Biochem, 2018, 442(1-2)∶73～80.
[21]Selvaraj S, Sun Y, Sukumaran P, et al. Resveratrol activates autophagic cell death in prostate cancer cells via downregulation of STIM1 and the mTOR pathway. Mol Carcinog, 2016, 55(5)∶818～831.
[22]Casas-Rua V, Alvarez I S, Pozo-Guisado E, et al. Inhibition of STIM1 phosphorylation underlies resveratrol-induced inhibition of store-operated calcium entry. Biochem Pharmacol, 2013, 86(11)∶1555～1563.
[2]Wang T H, Fu X D, Yang D, et al. Membrane estrogen receptor mediates the rapid nongenomic activation of endothelial nitric oxide synthase by estrogen. Sheng Li Xue Bao, 2003, 55(2)∶213～218.
[3]白藜芦醇通过调节iNOS抑制C57BL/6J小鼠动脉粥样硬化的机制探讨. 中国病理生理杂志,2015,31(9)∶1601～1605.
[4]王烨, 徐莲薇, 朱芝玲, 等. 调更汤对围绝经期雌性大鼠生殖内分泌影响的实验研究. 上海中医药杂志, 2014, 48(12)∶ 72～74.
[5]Cai X, Li X, Li L, et al. Adiponectin reduces carotid atherosclerotic plaque formation in ApoE-/- mice: roles of oxidative and nitrosative stress and inducible nitric oxide synthase. Mol Med Rep, 2015,11(3)∶ 1715～1721.
[6]O'Neal W T, Soliman E Z, Qureshi W, et al. Sustained pre-hypertensive blood pressure and incident atrial fibrillation: the Multi-Ethnic Study of Atherosclerosis. J Am Soc Hypertens, 2015,9(3)∶ 191～196.
[7]Castellon X, Bogdanova V. Chronic Inflammatory Diseases and Endothelial Dysfunction. Aging Dis, 2016, 7(1)∶81～89.
[8]Obuchowicz A, Kniazewska M, Zmudzińska-Kitczak J, et al. Concentrations of tumour necrosis factor-α and its soluble receptors in the serum of teenagers with atherosclerosis risk factors: obesity or obesity combined with hypertension. J Pediatr Endocrinol Metab, 2014, 27(11-12)∶ 1209～1212.
[9]Li T, Li D, Xu H, et al. Wen-Xin Decoction ameliorates vascular endothelium dysfunction via the PI3K/AKT/eNOS pathway in experimental atherosclerosis in rats. BMC Complement Altern Med, 2016, 16(1)∶27～36.
[10]Padmapriya Ponnuswamy, Angelika Schr?ttle. eNOS Protects from Atherosclerosis Despite Relevant Superoxide Production by the Enzyme in apoE-/- Mice. PLoS One, 2012, 7(1)∶ e30193.
[11]Jin S W, Choi C Y, Hwang Y P. Betulinic Acid Increases eNOS Phosphorylation and NO Synthesis via the Calcium-Signaling Pathway. J Agric Food Chem, 2016; 64(4)∶785～791.
[12]Huang H, Koelle P, Fendler M, et al. Induction of inducible nitric oxide synthase (iNOS) expression by oxLDL inhibits macrophage derived foam cell migration. Atherosclerosis, 2014, 235(1)∶213～22.
[13]王腊梅, 唐娜, 钟华, 等. 钙离子信号蛋白TRPC1及Orai1参与了HUVEC经SOC和ROC介导的钙内流和NO生成. 中国病理生理杂志, 2017, 25(12)∶1189～1195.
[14]袁楚婷, 葛长江, 宋现涛, 等. 血小板钙库操纵性钙通道蛋白与冠状动脉斑块易损性的相关性研究. 心肺血管病杂志, 2018, 37(4)∶273～277.
[15]Rodríguez-Moyano M, Díaz I, Dionisio N, et al. Urotensin-II promotes vascular smooth muscle cell proliferation through store-operated calcium entry and EGFR transactivation. Cardiovasc Res, 2013, 100(2)∶297～306.
[16]He S, Yan X. From resveratrol to its derivatives:new sources of nature antioxidant. Curr Med Chem, 2013,20(8)∶ 1005～1017.
[17]阮景明, 朱鹏立, 蒋娜, 等. 白藜芦醇对兔动脉粥样硬化及PPARγ相关炎症因子表达的影响. 中国动脉硬化杂志, 2013,21(3)∶203～208.
[18]程静静, 陈莉, 李朝飞, 等. 白藜芦醇通过高脂减缓内质网应激及增加eNOS的表达改善高热量高胆固醇饮食引起的内皮损伤. 中国药理学通报, 2014, 30(12)∶1756～1762.
[19]Olas B, Nowak P, Ponczek M, et al. Resveratrol, a natural phenolic compound may reduce carbonylation proteins induced by peroxynitrite in blood platelets. Gen Physiol Biophys, 2006,25(2)∶215～222.
[20]Lu T, Zhou D, Gao P, et al. Resveratrol attenuates high glucose-induced endothelial cell apoptosis via mediation of store-operated calcium entry. Mol Cell Biochem, 2018, 442(1-2)∶73～80.
[21]Selvaraj S, Sun Y, Sukumaran P, et al. Resveratrol activates autophagic cell death in prostate cancer cells via downregulation of STIM1 and the mTOR pathway. Mol Carcinog, 2016, 55(5)∶818～831.
[22]Casas-Rua V, Alvarez I S, Pozo-Guisado E, et al. Inhibition of STIM1 phosphorylation underlies resveratrol-induced inhibition of store-operated calcium entry. Biochem Pharmacol, 2013, 86(11)∶1555～1563.