核受体VDR在高糖诱导人血管内皮细胞氧化应激中的作用及机制
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摘要
目的：流行病学资料显示维生素D缺乏能促进动脉粥样硬化的发生和发展，从而增加心血管事件的风险。维生素D介导一系列生活学效应在动脉粥样硬化过程中可能起着很重要的保护作用，但维生素D抗动脉粥样硬化的具体机制尚不清楚。维生素D受体（VDR）在调控基因表达中需与视黄醇X受体（RXR）形成异源二聚体，共同调节多种基因的表达。我们前期已经发现RXR可能是机体抗动脉粥样硬化的重要内源性保护因素。因此本研究对VDR激动剂1,25(OH)_2D_3在高糖诱导人脐静脉血管内皮细胞（HUVECs）产生的氧化应激和细胞凋亡过程中的作用和机制，以及与RXR信号通路之间的相互关系进行探讨。
方法：本研究中采用原代培养的HUVECs，通过流式细胞学方法和荧光显微镜检测HUVECs凋亡率和细胞内的活性氧离子（ROS）水平，分别应用实时荧光定量PCR和免疫印迹杂交的方法检测烟酰胺腺嘌呤二核苷酸磷酸（NADPH）氧化酶亚基p22~(phox)和gp91~(phox)的表达水平，用免疫印迹杂交方法检测活化的PKC、caspase-3的蛋白表达水平。利用干扰RNA（SiRNA）技术分别沉默VDR或RXRα基因，研究VDR信号通路与RXR信号通路之间的相互作用关系。
结果：（1）高糖处理后HUVEC凋亡百分率和caspase-3蛋白表达显著增加，细胞ROS水平显著上升。高糖处理显著上调NADPH氧化酶p22~(phox)和gp91~(phox)亚基的mRNA和蛋白表达水平。（2）1,25(OH)_2D_3和RXR激动剂9-cis-RA均能抑制高糖诱导的caspase-3蛋白表达、内皮细胞凋亡、ROS生成、PKC的活化，以及NADPH氧化酶p22~(phox)和gp91~(phox)亚基mRNA和蛋白水平的表达，并且两者联合干预具有协同抑制效应。（3）预先转染VDR siRNA，1,25(OH)_2D_3抑制高糖诱导下caspase-3蛋白表达、内皮细胞凋亡、ROS生成、PKC的活化、p22~(phox)和gp91~(phox)亚基表达的作用明显减弱，而预先转染RXRα siRNA，1,25(OH)_2D_3抑制作用无明显影响。（4）然而当预先转染VDR siRNA再用9-cis-RA干预，9-cis-RA抑制氧化应激和内皮细胞凋亡的作用减弱。
结论：（1）VDR激动剂1,25(OH)_2D_3和RXR激动剂9-cis-RA均能抑制高糖诱导人血管内皮细胞的凋亡和氧化应激反应，并且两者联合干预具有协同抑制效应。（2）1,25(OH)_2D_3可能通过VDR—PKC—NADPH氧化酶—ROS信号通路抑制高糖诱导的内皮细胞氧化应激和细胞凋亡。（3）9-cis-RA抑制高糖诱导人血管内皮细胞氧化应激反应和细胞凋亡部分可能依赖VDR信号通路所介导。
Objective: Low levels of vitamin D are associated with increased risk ofcardiovascular disease and mortality. Vitamin D may inhibit cytokine-mediatedendothelial cell activation and adhesion molecule expression. However,themechanism by which vitamin D receptor (VDR) ligand acts in endothelial cellremains unclear．In this study,the effects of VDR agonists(1,25(OH)_2D_3) and RXRαagonists (9-cis-RA) on high-glucose—induced oxidative stress in human umbilicalvein endothelial cells(HUVECs) have been investigated．Our study may help us toseek and screen beneficial drugs preventing diabetic complications and a novel agentsfor preventing cardiovascular diseases.
Methods: HUVECs were isolated from healthy umbilical cords and cultured．Reversetranscription real-time PCR and immunoblotting were performed to determine theexpression of the components of NADPH oxidase(gp91~(phox) and p22~(phox)) andcaspase-3．ROS production and HUVECs apoptosis rate were detected by flowcytometry and confocal microscopy．Activatioan and expression of PKC weredetected by immunoblotting．The interrelation between VDR signaling pathway andRXR signaling pathway was demonstrated with transfection of small interferingRNA(siRNA)．
Results: Compared to control, the ROS production of HUVECs and HUVECsapoptosis rate were significantly higher in high glucose group. Treatment ofendothelial cells with1,25(OH)_2D_3（10-8M）or9-cis-RA（10-7M） resulted insignificant inhibition of high glucose induced oxidative stress, PKCactivation,expression of gp91~(phox), p22~(phox)and caspase-3, and HUVECs apoptosis.Furthermore,1,25(OH)_2D_3and9-cis-RA had a synergistic inhibitory effect by joint intervention. Our results also showed that transfection of VDR siRNA largelyabrogated the effects of VDR agonists，suggesting the inhibition of high glucoseinduced oxidative stress by VDR mediation. But we found that9-cis-RA on highglucose-induced HUVECs apoptosis, ROS generation, activation of PKC, mRNA andprotein expression of gp91~(phox)and p22~(phox)was weakened after VDR siRNAtransfection. While the pre-transfected RXRα siRNA,1,25(OH)_2D_3inhibition had nosignificant change.
Conclusions:(1)VDR agonist1,25(OH)_2D_3inhibited HUVECs apoptosis, theexpression of NADPH oxdase subunit gp91~(phox),p22~(phox)and ROS production ofHUVECs in induced with high glucose by VDR-PKC-NADPH oxidase-ROSsignaling pathway.(2)1,25(OH)_2D_3or9-cis-RA inhibited high glucose-inducedHUVECs apoptosis and oxidative stress, and both had a synergistic inhibitory effectby joint intervention.(3)9-cis-RA inhibited high glucose-induced oxidative stress ofvascular endothelial cells which may partly depend on VDR signaling pathwaymediation.
方法：本研究中采用原代培养的HUVECs，通过流式细胞学方法和荧光显微镜检测HUVECs凋亡率和细胞内的活性氧离子（ROS）水平，分别应用实时荧光定量PCR和免疫印迹杂交的方法检测烟酰胺腺嘌呤二核苷酸磷酸（NADPH）氧化酶亚基p22~(phox)和gp91~(phox)的表达水平，用免疫印迹杂交方法检测活化的PKC、caspase-3的蛋白表达水平。利用干扰RNA（SiRNA）技术分别沉默VDR或RXRα基因，研究VDR信号通路与RXR信号通路之间的相互作用关系。
结果：（1）高糖处理后HUVEC凋亡百分率和caspase-3蛋白表达显著增加，细胞ROS水平显著上升。高糖处理显著上调NADPH氧化酶p22~(phox)和gp91~(phox)亚基的mRNA和蛋白表达水平。（2）1,25(OH)_2D_3和RXR激动剂9-cis-RA均能抑制高糖诱导的caspase-3蛋白表达、内皮细胞凋亡、ROS生成、PKC的活化，以及NADPH氧化酶p22~(phox)和gp91~(phox)亚基mRNA和蛋白水平的表达，并且两者联合干预具有协同抑制效应。（3）预先转染VDR siRNA，1,25(OH)_2D_3抑制高糖诱导下caspase-3蛋白表达、内皮细胞凋亡、ROS生成、PKC的活化、p22~(phox)和gp91~(phox)亚基表达的作用明显减弱，而预先转染RXRα siRNA，1,25(OH)_2D_3抑制作用无明显影响。（4）然而当预先转染VDR siRNA再用9-cis-RA干预，9-cis-RA抑制氧化应激和内皮细胞凋亡的作用减弱。
结论：（1）VDR激动剂1,25(OH)_2D_3和RXR激动剂9-cis-RA均能抑制高糖诱导人血管内皮细胞的凋亡和氧化应激反应，并且两者联合干预具有协同抑制效应。（2）1,25(OH)_2D_3可能通过VDR—PKC—NADPH氧化酶—ROS信号通路抑制高糖诱导的内皮细胞氧化应激和细胞凋亡。（3）9-cis-RA抑制高糖诱导人血管内皮细胞氧化应激反应和细胞凋亡部分可能依赖VDR信号通路所介导。
Objective: Low levels of vitamin D are associated with increased risk ofcardiovascular disease and mortality. Vitamin D may inhibit cytokine-mediatedendothelial cell activation and adhesion molecule expression. However,themechanism by which vitamin D receptor (VDR) ligand acts in endothelial cellremains unclear．In this study,the effects of VDR agonists(1,25(OH)_2D_3) and RXRαagonists (9-cis-RA) on high-glucose—induced oxidative stress in human umbilicalvein endothelial cells(HUVECs) have been investigated．Our study may help us toseek and screen beneficial drugs preventing diabetic complications and a novel agentsfor preventing cardiovascular diseases.
Methods: HUVECs were isolated from healthy umbilical cords and cultured．Reversetranscription real-time PCR and immunoblotting were performed to determine theexpression of the components of NADPH oxidase(gp91~(phox) and p22~(phox)) andcaspase-3．ROS production and HUVECs apoptosis rate were detected by flowcytometry and confocal microscopy．Activatioan and expression of PKC weredetected by immunoblotting．The interrelation between VDR signaling pathway andRXR signaling pathway was demonstrated with transfection of small interferingRNA(siRNA)．
Results: Compared to control, the ROS production of HUVECs and HUVECsapoptosis rate were significantly higher in high glucose group. Treatment ofendothelial cells with1,25(OH)_2D_3（10-8M）or9-cis-RA（10-7M） resulted insignificant inhibition of high glucose induced oxidative stress, PKCactivation,expression of gp91~(phox), p22~(phox)and caspase-3, and HUVECs apoptosis.Furthermore,1,25(OH)_2D_3and9-cis-RA had a synergistic inhibitory effect by joint intervention. Our results also showed that transfection of VDR siRNA largelyabrogated the effects of VDR agonists，suggesting the inhibition of high glucoseinduced oxidative stress by VDR mediation. But we found that9-cis-RA on highglucose-induced HUVECs apoptosis, ROS generation, activation of PKC, mRNA andprotein expression of gp91~(phox)and p22~(phox)was weakened after VDR siRNAtransfection. While the pre-transfected RXRα siRNA,1,25(OH)_2D_3inhibition had nosignificant change.
Conclusions:(1)VDR agonist1,25(OH)_2D_3inhibited HUVECs apoptosis, theexpression of NADPH oxdase subunit gp91~(phox),p22~(phox)and ROS production ofHUVECs in induced with high glucose by VDR-PKC-NADPH oxidase-ROSsignaling pathway.(2)1,25(OH)_2D_3or9-cis-RA inhibited high glucose-inducedHUVECs apoptosis and oxidative stress, and both had a synergistic inhibitory effectby joint intervention.(3)9-cis-RA inhibited high glucose-induced oxidative stress ofvascular endothelial cells which may partly depend on VDR signaling pathwaymediation.
引文
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