原花青素抑制脂多糖诱导的RAW264.7细胞PGE_2生成的机制

The mechanisms of proanthocyanidins Inhibits PGE_2 synthesis in lipopolysaccharide-Induced RAW264.7 cells

目的:观察原花青素对脂多糖诱导RAW264.7细胞PGE2生成的影响及其作用机制。方法:放射免疫法(RIA)检测原花青素对脂多糖诱导的RAW264.7细胞PGE2生成的影响;LPS诱导RAW264.7细胞9h,再加不同浓度原花青素作用30min,放射免疫(RIA)法检测原花青素对COx-2酶活性的影响;半定量逆转录-聚合酶联反应(RT-PCR)法检测原花青素对COx-2 mRNA表达的影响;提取核蛋白,蛋白免疫印迹(western blot)法检测原花青素对NF-κB/p65蛋白表达的影响;电泳迁移率变动分析(EMSA)法检测原花青素对NF-κB与DNA结合活性的影响。结果:0.8,4和20mg·L-1原花青素抑制LPS诱导RAW264.7细胞PGE2生成;0.8,4和20mg·L-1原花青素不影响LPS诱导RAW264.7细胞COx-2酶活性;0.8,4和20mg·L-1原花青素下调LPS诱导RAW264.7细胞COx-2 mRNA表达;4,20mg·L-1原花青素下调LPS诱导RAW264.7细胞NF-κB/p65蛋白表达;0.8,4和20mg·L-1的原花青素可明显降低LPS诱导下RAW264.7细胞的NF-κB活化。结论:原花青素显著抑制LPS诱导RAW264.7细胞PGE2生成的作用与原花青素抑制COx-2 mRNA表达有关,此作用可能是通过抑制NF-κB/p65蛋白表达和抑制NF-κB的DNA结合活性来实现。

Objective：To study the effect of proanthocyanidins on PGE2 synthesis in lipopolysaccharide-induced RAW264.7 cells and its mechanisms.Methods： The effect of proanthocyanidins on PGE2 synthesis in lipopolysaccharide-induced RAW264.7 cells was measured by radioimmunoassay（RIA）;After being pretreated with different concentrations of proanthocyanidins for 30 min,and then LPS 1 mg·L-1 for 9 h,the effect of proanthocyanidins on the activity of COx-2 enzyme in RAW264.7 cells was analysed by RIA;the expressions of COx-2 mRNA by RT-PCR; the nuclear protein was isolated and the expressions of NF-κB protein by Western blot; and the DNA-binding activity of NF-κB was measured by electrophoretic mobility shift assay（EMSA）. Results： PGE2 synthesis was inhibited by proanthocyanidins 0. 8 mg·L^-1,4 mg·L^-1 and 20 mg·L^-1 ; the activity of COx - 2 enzyme was not inhibited by proanthocyanidins 0.8 mg·L^-1 ,4mg·L^-1 and 20 mg·L^-1（P 〉 0, 05, vs LPS group） ; the expression of COx -2 mRNA was inhibited by proanthocyanidins 0. 8 mg·L^-1 ,4 mg·L^-1 and 20 mg·L^-1; the expression of NF-κB/p65 protein was inhibited by proanthocyanidins 4 mg·L^-1 and 20 mg·L^-1. The DNA-binding activity of NF-κB was reduced by proanthocyanidins 0.8 mg·L^-1 ,4 mg·L^-1 and 20 mg·L^-1. Conclusion： The mechanisms of proanthocyanidins inhibited PGE2 synthesis in lipopolysacc, haride-induced RAW264.7 cells is relat- ed to the inhibition of the expression of COx - 2 mRNA ,which is inhibited possibly by suppressing expression of NF-κB/p65 protein and DNA-binding activity of NF-κB.