内皮素-1对大鼠血管平滑肌细胞一氧化氮及硫化氢体系的影响

Influence of endothelin- 1 on nitric oxide and hydrogen sulfide pathways in vascular smooth muscle cells in rats

目的探讨不同浓度内皮素-1（ET-1）对大鼠血管平滑肌细胞（A7r5细胞株）内源性一氧化氮（NO）及硫化氢（H2S）体系的影响。方法将A7r5细胞分为对照组和实验组，在实验组中分别加入浓度为10－8～10－6 mol/L的ET-1，对照组加入等体积的无菌磷酸盐缓冲液（PBS），48 h后分别应用NO和H2S探针原位检测A7r5细胞中NO及H2S水平，分别采用NO测定试剂盒和敏感硫电极法检测细胞上清中NO及H2S水平，应用Western blot法检测A7r5细胞中诱导型一氧化氮合酶（NOS2）、内皮型一氧化氮合酶（NOS3）、胱硫醚-γ-裂解酶（CSE）、胱硫醚-β-合成酶（CBS）及细胞增殖核抗原（PCNA）的表达。结果10－8、10－7及10－6 mol/L ET-1处理的A7r5细胞内NO水平的相对荧光强度（0.078±0.080、0.075±0.002、0.056±0.009）均低于对照组（0.094±0.061），差异有统计学意义（F＝15.248，P〈0.05）；细胞上清中NO水平[（1.391±0.134） μmol/L、（1.219±0.280） μmol/L、（1.116±0.181） μmol/L]均明显低于对照组[（2.131±0.484） μmol/L]，差异有统计学意义（F＝20.833，P〈0.01）；NOS2 （0.457±0.097、0.462±0.116、0.438±0.180）明显低于对照组（0.721±0.222），差异有统计学意义（F＝6.196，P〈0.05）；NOS3蛋白表达差异无统计学意义（F＝2.669，P〉0.05）。10－8、10－7、10－6 mol/L ET-1组A7r5细胞内H2S水平的相对荧光强度（0.063±0.002、0.056±0.008、0.042±0.009）低于对照组（0.082±0.006），差异有统计学意义（F＝16.297，P〈0.01）；细胞上清中H2S水平[（19.961±3.428） μmol/L、（17.516±5.144） μmol/L、（14.481±4.885） μmol/L]均明显低于对照组[（29.439±4.236） μmol/L]，差异有统计学意义（F＝12.518，P〈0.01）；CBS蛋白表达（0.359±0.096、0.270±0.038、0.174±0.051）均低于对照组（0.707±0.107），差异有统计学意义（F＝20.833，P〈0.01），CSE蛋白表达差异无统计学意义（F＝0.708，P〉0.05）。10－7 mol/L ET-1组细胞PCNA表达高于对照组（0.686±0.180比0.437±0.191），差异有统计学意义（t＝－2.840，P〈0.01）。结论ET-1刺激可导致血管平滑肌细胞增殖，并使其内源性NO和H2S体系下调。

ObjectiveTo explore the effect of different concentrations of endothelin-1 （ET-1） on the endogenous nitric oxide （NO） and hydrogen sulfide （H2S） pathways of vascular smooth muscle cells （A7r5 cell lines）in rats.MethodsA7r5 cell lines were divided into the control group and the experimental group.ET-1 at a concentration of 10-8 -10-6 mol/L was added into the experimental group, and as for the control group, the same volume of sterile phosphate buffered saline （PBS） buffer solution was added.The content of NO and H2S in A7r5 cell lines was detected by fluorescent NO probe and H2S probe after ET-1 stimulation for 48 h, respectively.The content of NO in the supernatant was measured by NO assay kit at 48 h of the incubation.The content of H2S in the supernatant was measured by polarographic H2S sensor at 48 h of the incubation.The expressions of inducible nitric oxide synthase （NOS2）, endothelial nitric oxide synthase （NOS3）, cystathionine-γ-lyase （CSE）, cystathionine-β-synthase （CBS） and proliferating cell nuclear antigen （PCNA） were detected by the Western blot method.ResultsThe relative fluorescence intensity of the content of NO in the A7r5 cell lines of ET-1 10-8, 10-7 and 10-6 mol/L groups （0.078±0.080, 0.075±0.002, 0.056±0.009） was markedly lower than that in the control group（0.094±0.061）, and the differences were statistically significant（F=15.248, P〈0.05）; Compared with the control group[（2.131±0.484） μmol/L], the content of NO in the supernatant of the experimental groups [（1.391±0.134） μmol/L, （1.219±0.280） μmol/L, （1.116±0.181） μmol/L]was significantly decreased, and the differences were statistically significant（F=20.833, P〈0.01）; NOS2 protein expression（0.457±0.097, 0.462±0.116, 0.438±0.180） was decreased markedly compared with that of the control group（0.721±0.222）, and the differences were statistically significant（F=6.196, P〈0.01）, but the expression of NOS3 showed no significant differences（F=2.669, P〉0.05）. The relative fluorescence intensity of the content of H2S in the A7r5 cell lines of ET-1 10-8, 10-7 and 10-6 mol/L groups （0.063±0.002, 0.056±0.008, 0.042±0.009） was markedly lower than that in the control group （0.082±0.006）, and the differences were statistically significant（F=16.297, P〈0.01）; Compared with the control group[（29.439±4.236） μmol/L], the content of H2S in the supernatant of the experimental groups [（17.516±5.144） μmol/L, （14.481±4.885） μmol/L] was significantly decreased, and the differences were statistically significant （F=12.518, P〈0.01）. CBS protein expression（0.359±0.096, 0.270±0.038, 0.174±0.051）was decreased markedly compared with that of the control group（0.707±0.107）, and the differences were statistically significant （F=20.833, P〈0.01）, and the expression of CSE showed no significant differences（F=0.708, P〉0.05）. The data showed that PCNA protein expression in the 10-7 mol/L ET-1 group（0.686±0.180）significantly increased compared with that of the control group（0.437±0.191）, and the difference was statistically significant （t=-2.840, P〈0.01）.ConclusionET-1 stimulation can lead to the proliferation of vascular smooth muscle cells and down-regulate its endogenous NO and H2S pathways.