一氧化氮在巨噬细胞移动抑制因子抑制大鼠糖皮质激素受体活性中的作用

Role of nitric oxide in inhibition of glucocorticoid receptor binding activity by macrophage migration inhibition factor in rats

目的探讨一氧化氮（NO）在巨噬细胞移动抑制因子（MIF）抑制大鼠糖皮质激素受体活性中的作用。方法实验Ⅰ原代培养新生SD大鼠肝细胞，随机分为3组（n=48）：对照组（C组）、重组鼠MIF组（rMIF组）及rMIF＋一氧化氮合酶抑制剂（L-NAME）组，分别于1640培养液、含rMIF20ng／ml的1640培养液、含rMIF 20ng／ml及L-NAME100μmol／L的1640培养液中孵育3h，随后采用配体结合实验计算大鼠肝细胞糖皮质激素受体的平衡解离常数（Kd），糖皮质激素受体活性与Kd值呈反比。实验Ⅱ健康成年雄性SD大鼠32只，250—300g，随机分为4组（n=8），对照组（C组）经右侧股静脉注射生理盐水1ml；rMIF低剂量组（rMIF-L组）、rMIF中剂量组（rMIF-M组）及rMIF高剂量组（rMIF-H组）分别经右侧股静脉注射rMIF50、100、200ng（用生理盐水稀释至1ml）。于给药前即刻（T0）、给药后5min（T1）、3h（T2）、6h（T3）、12h（T4）及24h（T5）时测定血清NO2^-／NO3^-水平。结果实验Ⅰ与C组比较，rMIF组和rMIF＋L-NAME组Kd值增大（P〈0．05）；rMIF组和rMIF＋L-NAME组尉值比较差异无统计学意义（P〉0．05）。实验ⅡT0-5时4组大鼠血清NO2^-／NO3^-水平差异无统计学意义（P〉0．05）。结论MIF抑制大鼠肝细胞糖皮质激素受体活性的机制与NO无关。

Objective To investigate the role of nitric oxide （NO） in inhibition of glucocorticoid receptor （GR） binding activity by recombinant macrophage migration inhibition factor （rMIF） in rats. Methods The experiment was performed in 2 parts. Part Ⅰ ： The primarily cultured SD rat hepatocytes were randomly divided into 3 groups： control group （group C）, rMIF group and rMIF ＋ L-NAME （NO synthase inhibitor） group. The hepatocytes were incubated in 1640 liquid culture media alone or containing rMIF 20 ng/ml or rMIF 20 ng/ml ＋ L-NAME 100 μmol/L for 3 h. The GR binding activity was measured after addition of [^3 H] dexamethasone （the final concentrations were 5, 10, 15, 20, 25 and 45 nmol/L respectively）. Part Ⅱ ： Thirty-two male SD rats weighing 250-300 g were randomized to 4 groups （ n = 8 each） ： control group and 3 rMIF groups-low, medium and high dose rMIF （rMIF-L, M, H）. Right femoral artery was cannulated for drug administration and blood sampling. Control group received normal saline （NS） 1 ml iv, while the 3 rMIF groups received rMIF 50, 100 and 200 ng in NS 1 ml iv respectively. Blood samples were taken immediately before （TO , baseline） and at 5 min, 3, 6, 12 and 24 h （T1-5 ） after NS/rMIF administration for determination of serum NO2^-/NO3^- level. Results In part Ⅰ the GR specific binding sites were significantly decreased by 48% and 46%, and the Kd values were increased to 53% and 57% in group rMIF and rMIF ＋ L-NAME respectively as compared with group C. There was no significant difference between group rMIF and rMIF ＋ L-NAME. In Part Ⅱ there was no significant difference in the serum concentration of NO at T0-5 among the 4 groups. Conclusion NO is not involved in the inhibition of GR binding activity by MIF in rats.