A771726对体外培养胶原性关节炎大鼠腹腔巨噬细胞产生一氧化氮的影响

In vitro effect of A771726 on the secretion of nitric oxide of peritoneal macrophages from collagen-induced arthritis rats

目的:在体外培养环境中,研究来氟米特活性代谢产物A771726,对脂多糖(LPS)刺激的胶原性关节炎(collagen-induced arthritis,CIA)大鼠腹腔巨噬细胞(peritoneal macrophages,PMФ)产生一氧化氮(nitric oxide,NO)的影响及作用机制。方法:建立大鼠CIA模型,无菌制备CIA大鼠PMФ悬液,A771726体外给药,硝酸还原法测定A771726影响CIA大鼠PMФ分泌NO的量效和时效关系;MTT法检测A771726对CIA大鼠PMФ细胞活力的影响;RT-PCR法测定A771726对CIA大鼠PMФ中诱导型一氧化氮合酶(inducible NO synthase,iNOS)mRNA表达的影响;Western blot法检测A771726对CIA大鼠PMФ中iNOS蛋白表达的影响。结果:A771726体外用药,可有效降低CIA大鼠PMФ中NO的分泌,且抑制作用有明显的量效和时效关系;A771726在有效抑制NO分泌的浓度范围和作用时间内,对PMФ的细胞活力无明显作用;A771726(0.1、1、10μmol/L)能显著抑制CIA大鼠PMФiNOS mRNA和iNOS蛋白的表达。结论:A771726(0.1、1、10μmol/L)可能通过抑制CIA大鼠PMФiNOS mRNA的转录,进而影响i NOS蛋白的翻译,最终引起NO的分泌减少。该作用并非由A771726对PMФ的毒性引起。

AIM： To study the effect and mechanism of A771726,the active metabolite of leflunomide,on the production of nitric oxide（NO） of peritoneal macrophages（PMФ） from collagen-induced arthritis（CIA） rats.METHODS： Collagen-induced arthritis（CIA） model was induced in rats and the PMФ were collected.A771726 were used in vitro.The dose-dependent and time-dependent effects of A771726 on the secretion of NO of PMФ from CIA rats were checked by nitric acid reduction synthase method;MTT reagent was used to detect the effect of A771726 on the cell viability of PMФ from CIA rats;RT-PCR was used to detect the expression of inducible NO synthase（iNOS） mRNA.Western blot was used to detect the expression of iNOS protein.RESULTS： A771726 could inhibit the secretion of NO of PMФ from CIA rats in a dose-dependent and time-dependent manner.The cytotoxicity of A771726 was not detected during the time course and dose range.A771726（0.1,1,10 mol/L） significantly inhibited the expression of iNOS mRNA and iNOS protein in PMФ from CIA rats.CONCLUSION： A771726（0.1,1,10 mol/L） can inhibit the transcription of iNOS mRNA in PMФ from CIA rats,and reduce the translation of iNOS protein,which leads to suppressing the secretion of NO.And the suppression is not related to the cytotoxicity of A771726.