氧化应激对Dahl盐敏感性高血压大鼠主动脉Rho激酶的直接作用

Direct Effect of Oxidative Stress on Rho Kinase in the Aorta of Hypertensive Salt-sensitive Rats

背景氧化应激与原发性高血压的发生密切相关。近年来研究表明氧化应激信号分子(ROS)可导致高血压动物血管功能紊乱,最近证明RhoA及其下游效应分子Rho激酶参与高血压发病,但参与机制还有许多不明之处。目的观察抗氧化剂N乙酰半胱氨酸(NAC)对Dahl盐敏感性(DSS)高血压大鼠主动脉Rho/Rho激酶(ROK)表达的影响,探讨盐敏感性高血压发病机制中ROS与Rho信号通路的关系。方法DSS高血压大鼠随机分为4组:正常饮食组;高盐饮食组(高盐组);高盐饮食辅以NAC治疗组(高盐+NAC组);正常饮食辅以NAC治疗组(正常饮食+NAC组),每组6只。每周测定鼠尾动脉血压,共4周;收集24 h尿,处死后取主动脉组织标本。DCF荧光法检测尿中氧化应激生物标志物过氧化氢(H2O2)水平;光泽精化学发光法检测主动脉超氧阴离子生成量;RT-PCR及Western Blot检测主动脉RhoA、ROK mRNA及蛋白表达。结果高盐饮食组与正常饮食组相比,血压明显升高[(219.7±5.3)比(152.8±3.8)mmHg,P<0.01],24 h尿中H2O2排出量显著增加[(663.8±67.7)比(18.9±4.5)nmol/d,P<0.01];高盐饮食大鼠主动脉超氧阴离子生成量较正常大鼠增多[(12.8±0.5)比(6.9±0.3)counts/(min.mg),P<0.01],主动脉ROK mRNA和蛋白表达亦明显增加(P<0.01)。NAC显著降低了高盐饮食大鼠血压[(161.8±2.9)mmHg,P<0.01]、尿H2O2排出量[(243.0±49.3)nmol/d,P<0.01]和主动脉超氧阴离子的生成[(7.7±0.6)counts/(min.mg),P<0.01],抑制了ROKmRNA和蛋白的过表达。但是,作为ROK上游的小G蛋白,RhoA表达在4组大鼠间并无差异(P>0.05)。结论DSS高血压大鼠主动脉ROK表达增加,表明Rho信号参与盐敏感性高血压的发生发展;RhoA可能不是此类高血压动物ROK激活的决定因素。NAC阻断后ROK表达显著下调,其机制可能与ROS的诱导作用降低有关,ROS可能作为上游信号直接参与DSS高血压大鼠ROK的激活。

Background Recent studies demonstrated the role of reactive oxygen species （ROS） in vascular dysfunction in hypertension; Studies also reveal RhoA and its downstream effector, Rho kinase, involved in the development of hypertension, however, the mechanism remained unclear. Objective To study the antioxidant effect of N acetylcysteine （NAC） on the expression of Rho/Rho kinase （ROK） in the aorta of hypertensive saltsensitive rats and investigate effect of ROS on Rho/ROK pathway in the development of salt-sensitive hypertension. Methods Dahl salt-sensitive （DSS） rats were randomly assigned to receive a normal （N） diet; a high-salt （HS） diet; a high-salt diet plus NAC supplement（HS-t-NAC）, or a normal diet plus NAC supplement（N＋ NAC） for 4 weeks. SBP was measured in awake rats with the tail-cuff method every week. 24 h urine was gathered and aorta tissues were obtained. Urinary H2O2 levels were measured by DCF fluorometry; Vascular superoxide production was determined using Luciginin chemiluminescence; Expression of RhoA, Rho kinase is accessed by the methods of RT - PCR and Western Blot. Results Compared with normal diet rats , high salt diet Dahl rats achieved markedly elevated systolic arterial pressure[（219.7±5.3） vs （152.8±3.8） mmHg, P〈0.01], urinary excretion of hydrogen peroxide （H2O2）[（663.8±67.7） vs （18.9±4.5） nmol/d, P〈0.01], and increased superoxide productionS（12.8±0.5）vs （6.9±0. 3） counts/（min · mg）, P〈0. 01] in aorta, as well as Rho kinase expression of aorta at the levels of mRNA and protein（P〈0.01）. NAC reduced the increases in SBP[（161.8± 2.9）mmHg, P〈0.01] and counteracted the elevation of urinary of H2O2 [（243.0 ± 49.3）nmol/d, P〈0.01], aortic superoxide production [（7. 7±0. 6） counts/（min · mg）, P〈0.01] in rats of HS＋NAC group. The overexpression of ROK mRNA and protein of aorta were also markedly downregulated in high-salt-loaded DSS rats by NAC supplement. But no obvious changes of expression of RhoA, the upstream samll G protein, was shown in four groups. Conclusion The overexpression of ROK in the aorta of hypertensive DSS rats revealed Rho signals in the development and progression of salt-sensitive hypertension. It seems that RhoA is not a determinant to activate ROK in these models of hypertensive animals. The downregulation of expression of ROK by NAC antioxidant may be attributed to the attenuation by ROS. ROS may serve as substrate that directly participates in ROK activation in hypertensive DSS rats.