高盐培养基对人脐静脉内皮细胞株eNOS表达的影响及其机制

The effects and mechanism of high-salt medium on eNOS expression in human umbilical vein endothelial cell line

目的探讨高盐培养基干预人脐静脉细胞融合细胞(EA.hy926)对eNOS表达的影响及相关机制。方法予以EA.hy926细胞不同浓度的高盐培养基干预48h,选择137mmol/L氯化钠为高盐培养基的浓度;分别予以DMEM高糖培养基组(氯化钠浓度为109mmol/L)、高盐培养基(氯化钠浓度为137mmol/L)、高盐培养基(氯化钠浓度为137mmol/L)+RNA干扰、高渗对照组(甘露醇浓度为56mmol/L)干预48h,高效液相色谱法检测细胞上清液非对称二甲基精氨酸(ADMA)浓度,实时定量PCR和Western blot检测蛋白质精氨酸甲基转移酶1(PRMT-1)、RhoA、Rho激酶(ROCK)、内皮型一氧化氮合酶(eNOS)的表达及活性。结果高盐组上清液中ADMA浓度、PRMT-1表达、RhoA mRNA表达及ROCK活性较对照组显著增高,eNOS蛋白表达显著降低;高盐培养基+RNA干扰组较高盐组ADMA浓度、PRMT-1、RhoA mRNA及ROCK活性明显下降,eNOS蛋白表达显著上调。结论高盐培养基通过刺激ADMA生成并激活下游RhoA-ROCK通路而抑制eNOS的生成。

Objective To investigate the effects of high-salt medium on the expression of endothelial nitric oxide synthase （eNOS） and related mechanisms in EA. Hy926 cell line. Methods EA. hy926 cells were treated 48 h with different concentrations of high-salt medium; 137 mmol/L was selected as the target concentration intervention in further experiment. The cells were divided into four groups which received intervention for 48 h： DMEM high-glucose medium group （sodium chloride concentration of 109 mmol/L） , high-salt medium （sodium chloride concentration of 137 mmol/L）, high-salt medium （sodium chloride concentration of 137 mmol/L）＋RNA interference, and hypertonic control group （mannitol concentration of 56 mmol/L）. High-performance liquid chromatography was used to assay ADMA concentration in cell supernatant ; real-time quantitative PCR and Western blot were used to assay the expressions and activities of PRMT-1, RhoA, ROCK and eNOS. Results ADMA concentration, PRMT-1 expression, the mRNA expression of RhoA and ROCK activity increased significantly whereas the protein expression of eNOS significantly reduced in the high-salt group compared with those in the two control groups. High-salt medium ~ RNA interference restored the above biochemical indicators and upregulated the protein expression of eNOS significantly. Conclusion High-salt medium induces ADMA-mediated RhoA/ Rock-1 pathway activation and suppresses eNOS generation.