AngⅡ/AT_1R通路下调内皮型一氧化氮合酶磷酸化的机制研究  

AngⅡ/AT_1R pathway leads to down-regulation of endothelial nitric oxide synthase phosphorylation

作　　者：姜君财 丁菁[1] 张倩[1] 骆妍蓓 于敏 王胜男[1] 杨飞[1] 方沛钰 陆德琴[1] JIANG Jun-cai;DING Jing;ZHANG Qian;LUO Yan-bei;YU Min;WANG Sheng-nan;YANG Fei;FANG Pei-yu;LU De-qin(Department of Pathophysiology, Guizhou Medical University, Guiyang 550025, Chin)

机构地区：[1]贵州医科大学病理生理教研室,贵州贵阳550025

出　　处：《中国病理生理杂志》2018年第5期839-844,共6页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.31460267);贵州省科学基金资助项目(黔科合LH字[2014]7088);贵州省科技厅国际合作项目(黔科合外G字[2010]7019号)

摘　　要：目的:探讨血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)/血管紧张素Ⅱ1型受体(angiotensinⅡtype 1 receptor,AT_1R)通路通过激活蛋白磷酸酶2 A(protein phosphatase 2 A,PP2 A)导致大鼠肠系膜动脉中内皮型一氧化氮合酶(endothelial nitric oxide synthase,e NOS)磷酸化水平下调的机制。方法:采用体重160~180 g成年雄性SD大鼠90只,在无菌条件下分离大鼠肠系膜动脉。首先明确AngⅡ下调大鼠肠系膜动脉中e NOS(Ser1177)磷酸化的效应,将肠系膜动脉随机分为正常对照(control)组和AngⅡ组,AngⅡ组用浓度为1×10^(-7)mol/L、1×10^(-6)mol/L和1×10^(-5)mol/L AngⅡ分别孵育离体大鼠肠系膜动脉血管6 h、12 h和24 h;然后进一步探讨AngⅡ使eNOS(Ser1177)发生磷酸化下调的分子机制,将肠系膜动脉随机分为正常对照(control)组、AngⅡ组和坎地沙坦(candesartan,CAN;AT_1R特异性抑制剂)+AngⅡ组(用1×10^(-5)mol/L CAN预处理大鼠肠系膜动脉血管1 h后,再用1×10^(-7)mol/L AngⅡ继续孵育12 h)。采用Western blot法检测肠系膜动脉中eNOS蛋白表达和eNOS(Ser1177)磷酸化水平,以及PP2Ac的蛋白表达、PP2Ac(Tyr307)磷酸化水平和PP2A内源性抑制蛋白I^2^(PP2A)的表达水平,并用PP2A活性检测试剂盒测定大鼠肠系膜动脉PP2A活性变化。结果:(1)与control组比较,AngⅡ孵育离体大鼠肠系膜动脉血管6 h、12 h和24 h后,eNOS(Ser1177)磷酸化水平均明显降低(P<0.05),且12 h组和24 h组eNOS(Ser1177)磷酸化水平下降均出现明显浓度依赖性,但不同浓度组间eNOS蛋白表达水平差异均无统计学显著性;(2)与control组比较,1×10^(-7)mol/L AngⅡ孵育肠系膜动脉血管12 h后,eNOS(Ser1177)磷酸化水平降低(P<0.05);CAN预处理可明显上调eNOS(Ser1177)磷酸化水平(P<0.05),且各组间eNOS蛋白表达水平差异无统计学显著性;(3)与control组比较,1×10^(-7)mol/L AngⅡ孵育肠系膜动脉血管12 h后,PP2Ac(Tyr307)磷酸化水平和I_2^(PP2A)蛋白表达均降低(P<0.05);CAN预处理可使PP2Ac(AIM： To investigate the mechanism of angiotensin Ⅱ（ Ang Ⅱ）/angiotensin Ⅱ type 1 receptor（ AT1R） pathway activating protein phosphatase 2 A（ PP2A） which leads to down-regulation endothelial nitric oxide synthase（ eNOS） phosphorylation level in mesenteric arteries of rats. METHODS： METHODS： The mesenteric arteries of adult male SD rats（ weighing 160 ^ 180 g; n = 90） were isolated under aseptic conditions. Firstly,to determine the effect of angiotensinⅡ down-regulated eNOS（ Ser1177） phosphorylation level,the mesenteric arteries were randomly divided into normal control（ control） group and AngⅡ group. The mesenteric arteries in AngⅡ group were incubated with AngⅡ at 1× 10^（-7） mol/L,1 × 10^（-6） mol/L and 1 × 10^（-5） mol/L for 6 h,12 h and 24 h,respectively. Secondly,to investigate the molecular mechanism by which angiotensin Ⅱ activated PP2A leading to down-regulation eNOS（ Ser1177） phosphorylation level,the mesenteric arteries were randomly divided into control group,Ang Ⅱ group and candesartan（ CAN; a specific AT_1R blocker） ＋ AngⅡ group. The mesenteric arteries were pretreated with 1 × 10^（-5） mol/L CAN for 1 h,then incubated with 1 × 10^（-7） mol/L AngⅡ for 12 h in CAN ＋ AngⅡ group. The protein levels of eNOS,p-eNOS（ Ser1177）,PP2Ac,pPP2Ac（ Tyr307） and protein phosphatase 2 A inhibitor 2（ I_2^（PP2A）） in the arteries were determined by Western blot. The activity of PP2A in the arteries was detected by PP2A activity kit. RESULTS： Compared with the control group,the protein level of p-eNOS（ Ser1177） in the mesenteric arteries was decreased after incubated with AngⅡ for 6 h,12 h and 24 h（ P〈0. 05）. The decreasing tendency of p-eNOS（ Ser1177） showed concentration-dependently,especially in 12 h and 24 h groups. The expression of eNOS protein showed no significant difference in each group. Compared with the control group,the mesenteric arteries of the rats were incubated with Ang Ⅱ at 1 × 10^

关 键 词：血管紧张素Ⅱ 血管紧张素Ⅱ1型受体 蛋白磷酸酶2A 内皮型一氧化氮合酶 

分 类 号：R363[医药卫生—病理学] R543[医药卫生—基础医学]