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目的:探讨血管紧张素Ⅱ(AngⅡ)对RAW264.7细胞Toll样受体4(TLR4)mRNA、蛋白表达及髓过氧化物酶(MPO)活性的影响及其致炎致动脉粥样硬化(AS)机制。方法:体外培养RAW264.7细胞,用不同浓度的AngⅡ(1、10、100、1000nmol/L)及100μg/L脂多糖(LPS)刺激RAW264.7细胞24h后,RT-PCR法测RAW264.7细胞TLR4 mRNA水平,Western blot检测RAW264.7细胞TLR4蛋白表达,比色法测细胞培养上清中MPO活性。同时,预先加入不同剂量的TLR4阻断剂(1mg/L、5mg/L)后,再用AngⅡ(100nmol/L)刺激RAW264.7细胞24h,检测细胞培养上清中MPO活性。结果:AngⅡ浓度依赖性地增加RAW264.7细胞TLR4 mRNA及蛋白表达(P<0.01),LPS也可诱导RAW264.7细胞TLR4 mRNA及蛋白表达(P<0.01);AngⅡ浓度依赖性地升高RAW264.7细胞MPO活性(P<0.01),LPS也可升高RAW264.7细胞MPO活性(P<0.01),而TLR4阻断剂明显抑制AngⅡ这一效应(P<0.01)。结论:AngⅡ上调RAW264.7细胞TLR4表达,通过跨膜受体TLR4诱导MPO分泌,加重炎症反应,促进AS的形成和发展。
Objective: To investigate the effects of Ang Ⅱ on Toll-like receptor 4 (TLR4) mRNA, protein expression and myeloperoxidase (MPO) activity in RAW264.7 cells and the effect of angiotensin Ⅱ-induced atherosclerosis (AS )mechanism. Methods: RAW264.7 cells were cultured in vitro and RAW264.7 cells were stimulated with different concentrations of AngⅡ (1, 10, 100, 1000 nmol / L) and 100 μg / L lipopolysaccharide (LPS) The level of TLR4 mRNA was detected by Western blot. The expression of TLR4 protein in RAW264.7 cells was detected by Western blot. The activity of MPO in cell culture supernatant was measured by colorimetric assay. At the same time, RAW264.7 cells were stimulated with AngⅡ (100nmol / L) for 24h before pretreatment with different doses of TLR4 blocker (1mg / L, 5mg / L). Results: AngⅡinduced TLR4 mRNA and protein expression in RAW264.7 cells in a concentration-dependent manner (P <0.01), while LPS induced TLR4 mRNA and protein expression in RAW264.7 cells (P <0.01). AngⅡin a concentration-dependent manner increased RAW264 (P <0.01). LPS also increased the MPO activity of RAW264.7 cells (P <0.01), while TLR4 inhibitor significantly inhibited the effect of AngⅡ (P <0.01). CONCLUSION: AngⅡ up-regulates TLR4 expression in RAW264.7 cells, induces MPO secretion through transmembrane receptor TLR4, aggravates the inflammatory response and promotes the formation and development of AS.