血管紧张素Ⅱ对肺泡巨噬细胞NF-kB信号通路的影响

Effects of angiotensin Ⅱ on NF-KB binding activity in alveolar macrophage

目的阐明血管紧张素Ⅱ（AngⅡ）对肺泡巨噬细胞核转录因子xB（NF-kB）信号通路的影响。方法收集正常或其他肺病患者正常肺叶组织肺泡灌洗液，分离、纯化、培养肺泡巨噬细胞。予10^-6MAngⅡ刺激15，30，60，120min。另外，予AT-1受体阻断剂irbesaitan（10^-6 M）预处理肺泡巨噬细胞60min后再予10^-6M AngⅡ刺激60min。设置对照组。凝胶电泳移动抑制实验（EMSA）检测NFxB的结合活性。免疫蛋白质印迹检测胞浆内IxBα的表达。RT-PCR检测TNF-a和ICAM-1的表达。应用SPSS13．0软件进行One-Way ANOVA方差分析，多重比较采用LSD法。以P〈0．05为差异具有统计学意义。结果AngⅡ刺激肺泡巨噬细胞15minNF-KB结合活性增强，60min达到峰值。AT-1受体阻断剂irbesartan可阻断NF-kB结合活性增强。与对照组（1．0）相比，AngⅡ处理组（0．29±0．11）IkBa表达减弱，且差异具有统计学意义（P=0．013）。与AngⅡ处理组相比，AngⅡ＋irbesartan处理组IkBa表达（0．83±0．12）则增强，且差异具有统计学意义（P=0．001）。与对照组（0．42±0．099）相比，AngⅡ处理组TNF-α（1．13±0．17）表达增强，且差异具有统计学意义（P=0．001）。与AngⅡ处理组相比，AngⅡ＋irbesartan处理组（0．77±0．15）减弱，且差异具有统计学意义（P=0．02）。与对照组ICAM-1表达（0．16±0．050）相比，AngⅡ处理组（0．55±0．08）增强且差异具有统计学意义（P=0．003）。与AngⅡ处理组相比，AngⅡ＋irbesartan处理组（0．32±0．07）减弱，且差异具有统计学意义（P=0．001）。结论AngⅡ对肺洵巨噬细朐NF-KB通路有诈向调节作用。

Objective To determine the effects of angiotensin Ⅱ （Ang Ⅱ ） on NF-kB DNA binding activity in alveolar macrophage. Method Human alveolar macrophages were isolated and made homogeneous from alveo- lar lavage fluid, and cultured in DMEM. Alveolar macrophages were treated with AngⅡ （10-6M） for 15 min, 30 min, 60 min and 120 min, respectively. Moreover, alveolar macrophages were pretreated with irbesartan （AngⅡ type 1 receptor blocker） for 1 hour before stimulated with Angiotensin Ⅱ for 1 hour. Electrophoretic gel mobility shift assay （EMSA） was used to detect NF-kB DNA binding activity. The protein expression of IKBα was examined by Western blot. Expressions of TNF-α and ICAM-1 mRNA were detected by using RT-PCR. Results EMSA re- vealed that there was a increase in up-regtdation of NF-KB DNA binding activity after alveolar macrophages were treated with AngⅡ for 15 min and peaked at 60 min. Irbesartan treatment reduced DNA binding activity. Compared with control group, the protein expression of IkBα decreased in Ang Ⅱ treatment group（0.29 ± 0.11, P = 0. 013 ）, and Irbesartan treatment significantly increased protein expression of IkBα（0.83±0.12, P = 0. 001 ）. The expressions of TNF-α and ICAM-1 mRNA were up-regulated by Angn in comparison with the control group （TNF- α：1.13±0.17 vs. 0.42±0.099; ICAM-1 0.55±0.08 vs. 0.16±0.050, P =0.003）. Irbesartan inhibited the expressions of TNF-α ,（0.77 ± 0.15 vs 1.13 ± 0.17, P = 0.02; ICAM-1 （0.32± 0.07 vs 0.55±0.08, P = 0.001）. Conclusions AngⅡ is capable to stimulate NF-kB signal pathway in alveolar macrophages.