血管紧张素Ⅱ-血管紧张素Ⅱ1型受体途径在巨噬细胞生成促炎因子中的作用

Role of angiotensin Ⅱ -angiotensin Ⅱ type 1 receptor pathway in the production of proinflammatory cytokines in macrophage

目的 了解血管紧张素Ⅱ（AngⅡ）-血管紧张素Ⅱ1型受体（AT1R）途径在介导巨噬细胞促炎因子产生中的作用和初步机制.方法采用随机数字表法,将体外培养的小鼠单核巨噬细胞株RAW 264.7分为4组,每组3个培养皿,均采用含体积分数10%FBS的DMEM培养液培养.（1）空白对照组：常规培养6 h,不加任何刺激因素.（2）ZD7155组：预先用含38 μmol/L特异性AT1R拮抗剂ZD7155的培养液作用细胞1 h,换液后培养6 h.（3）AngⅡ组：用含0.01 μmol/LAngⅡ的培养液培养6 h.（4）ZD7155＋AngⅡ组：先用含38 μmol/L ZD7155的培养液处理1 h,再改用含0.01 μmol/L AngⅡ的培养液作用6 h.ELISA法检测各组细胞培养上清液中TNF-α和IL-1β含量,RT-PCR法检测细胞TNF-α和IL-1β mRNA表达,凝胶电泳迁移率变化分析法检测细胞NF-κB和激活蛋白1（AP-1）活性.对数据行单因素方差分析.结果 （1）AngⅡ组细胞培养上清液中TNF-α、IL-1β含量分别为（119±14）、（105±17）pg/mL,均显著高于空白对照组[（24±11）、（24±6）Pg/mL,F值分别为1.62、8.03,P值均小于0.01]、ZD7155组[（22±11）、（25±8）pg/mL,F值分别为1.62、4.52,P值均小于0.01]以及ZD7155＋AngⅡ组[（45±13）、（62±11）pg/mL,F值分别为1.16、2.29,P＜0.05或P＜0.01].（2）AngⅡ组细胞TNF-α和IL-1 β mRNA表达水平均显著高于空白对照组（F值分别为7.59、3.38,P＜0.05或P＜0.01）、ZD7155组（F值分别为10.66、2.24,P值均小于0.05）和ZD7155＋AngⅡ组（F值分别为5.10、5.09,P值均小于0.01）.（3）AngⅡ组细胞内转录因子NF-κB和AP-1活性分别为69 027±2502、36 752±2055,均显著高于空白对照组（45 709±1203、20 325±2695,F值分别为4.32、1.72,P值均小于0.01）、ZD7155组（46 303±1897、21 951±2517,F值分别为1.74、1.50,P值均小于0.01）和ZD7155＋AngⅡ组（38 271±690、22 365±3797,F值分别为13.13、3.41,P值均小于0.01）.结论 AngⅡ与AT1R结合可以介导巨噬细胞内转录因子NF-κB和AP-1活化,从而促进TNF-α和IL-1β的产生及释放.

Objective To investigate the role of angiotensin Ⅱ （Ang Ⅱ ） -angiotensin Ⅱ type 1 receptor （AT1 R） pathway in the production of proinflammatory cytokines in macrophage, and to analyze its mechanisms. Methods RAW264.7 macrophages were cultured in vitro in DMEM nutrient medium containing 10% FBS, and then they were divided into control group （ ordinary culture for 6 hours without any stimulation）, ZD7155 group （pretreated with 38 μmol/L AT1R-specific inhibitor ZD7155 for 1 hour, then cultured with fresh nutrient solution for 6 hours） , Ang Ⅱ group （cultured with 0.01 μ mol/L Ang Ⅱ for 6 hours）, and ZD7155 ＋ Ang Ⅱ group （pretreated with 38 μmol/L AT1R-specific inhibitor ZD7155 for 1 hour, then cultured with 0.01 μ mol/L Ang Ⅱ for 6 hours） according to the random number table. Contents of TNF-α and IL-1β in the supernatant were measured by ELISA. Expressions of TNF-α mRNA and IL-1β mRNA were determined by RT-PCR. Activity of NF-κB and AP-1 were examined by electrophoretic mobility shift assay. Data were processed with one-way analysis of variance. Results Compared with those in Ang Ⅱ group [（ 119 ± 14）, （ 105 ± 17） pg/mL, respectively], the levels of TNF-α and IL-1β in the supernat ant in control group [（24 ± 11 ）, （24 ± 6） pg/mL, with F value respectively 1.62, 8.03, P values all below 0.01], ZD7155 group [（ 22 ± 11 ）, （25 ± 8） pg/mL, with F value respectively 1.62, 4. 52, P values all below 0.01], and ZD7155 ＋ Ang Ⅱ group [（45 ± 13 ）, （62 ± 11 ） pg/mL, with F value respectively 1.16, 2.29, P 〈 0.05 or P 〈 0.01] were all obviously decreased. The expressions of TNF-α mRNA and IL-1β mRNA, and activity of NF-κB and AP-1 showed the similar changes as above： （ 1 ） The levels of TNF-α mRNA and IL-1 β mRNA in Ang Ⅱ group were all higher than those in control group （ with F value respectively 7.59, 3.38, P 〈 0.05 or P 〈 0. 01 ）, ZD7155 group （ with F value respectively 10.66, 2.24,P values all below 0.05） , and ZD7155 ＋ Ang Ⅱ group （with F value respectively 5.10, 5.09, P values all below 0.01 ）. （2） Activity of NF-κB and AP-1 was respectively 69 027 ±2502, 36 752 ±2055 in Ang Ⅱ group, all higher than those in control group （45 709 ± 1203, 20 325 ± 2695, with F value respectively 4. 32, 1.72, P values all below 0.01 ）, ZD7155 group （46 303 ± 1897, 21 951 ± 2517, with F value respectively 1.74, 1.50, P values all below 0.01）, and ZD7155 ＋ Ang Ⅱ group （38 271 ±690, 22 365 ± 3797, with F value respectively 13.13, 3.41, P values all below 0.01 ）. Conclusions Ang Ⅱ can mediate activation of transcription factor NF-κB and AP-1 via combination of AT1 R, thereby contributing to the production and release of proinflammatory cytokines TNF-α and IL-1β in macrophage.