外用甲氨蝶呤柔性纳米脂质体对类风湿性关节炎大鼠血清γ-干扰素和白细胞介素4的影响

Effect of the external use of flexible nanonipsomes methotrexate on the levels of serum interferon-gamma and interleukin-4 in rats with rheumatoid arthritis

目的:观察经控制释放材料纳米柔性纳米脂质体制成的外用甲氨蝶呤对类风湿性关节炎大鼠血清γ-干扰素和白细胞介素4的影响。方法:实验于2005-03/11在中南大学湘雅医院中西结合研究所完成。80只健康的SD大鼠随机分为5组:正常组10只,其余70只大鼠采用皮下注射牛Ⅱ型胶原和完全福氏佐剂制备类风湿性关节炎模型,再随机分为模型组10只,0.5%甲氨蝶呤组20只;0.25%甲氨蝶呤柔性纳米脂质体组20只,0.5%甲氨蝶呤柔性纳米脂质体组20只。正常对照组大鼠自由饮水,模型组每天灌服1.5mL蒸馏水,其余各组于初次免疫7d后给药,于大鼠右后足分别涂抹0.5%甲氨蝶呤外敷剂、0.25%和0.5%甲氨蝶呤柔性纳米脂质体,约2.0～4.0g/只(视关节大小而定),每周一次。采用关节炎指数积分法评定关节症状,关节指数积分越高,关节症状越严重。给药后第45天采用酶联免疫吸附法检测各组大鼠血清γ-干扰素和白细胞介素4水平。结果:纳入大鼠80只,均进入结果分析。①各组大鼠关节炎指数积分给药前14d无明显区别,模型组随着时间延长,关节炎指数积分增加;0.5%甲氨蝶呤组、0.25%甲氨蝶呤柔性纳米脂质体组和0.5%甲氨蝶呤柔性纳米脂质体组于给药后25d达高峰,但仍低于模型组(分别为1.51±0.99,0.98±0.57,0.88±0.49,1.68±1.24,t=2.956,5.461,5.244,P<0.01)。②给药后45d模型组、0.5%甲氨蝶呤组、0.25%甲氨蝶呤柔性纳米脂质体组和0.5%甲氨蝶呤柔性纳米脂质体组γ-干扰素水平明显高于正常对照组,白细胞介素4水平明显低于正常对照组[γ-干扰素分别为(120.75±1.43),(104.41±1.71),(88.75±1.58),(83.60±1.86),(56.15±2.07)ng/L,t=-64.016,-67.970,-36.928,-48.092,P<0.01;白细胞介素4分别为(23.28±0.13),(36.45±0.15),(46.46±0.16),(48.47±0.12),(61.23±0.27)ng/L,t=398.098,328.973,179.924,186.796,P<0.01]。0.5%甲氨蝶呤组、0.25%甲氨蝶呤柔性纳米脂质体组和0.5%甲氨蝶呤柔性纳米脂质体组γ-干扰素水平明显低于模型组(t=21.387,46.365,43.639,P<0.01),白细胞介素4水平明显高于模型组(t=-238.74,-514.575,-385.096,P<0.01)。0.25%甲氨蝶呤柔性纳米脂质体组和0.5%甲氨蝶呤柔性纳米脂质体组γ-干扰素水平明显低于0.5%甲氨蝶呤组(t=36.581,30.124,P<0.01),白细胞介素4水平明显高于0.5%甲氨蝶呤组(t=-282.406,-203.685,P<0.01)。0.25%甲氨蝶呤柔性纳米脂质体组γ-干扰素和白细胞介素4与0.5%甲氨蝶呤柔性纳米脂质体组相比,无显著差异(P>0.05)。结论:外用甲氨蝶呤柔性纳米脂质体通过其智能控制释放效应,下调血清γ-干扰素及上调血清白细胞介素4水平的作用明显优于普通外用甲氨蝶呤。

AIM： To study the changes in serum interferon-gamma （IFN-γ） and interleukin 4 （IL-4） in rats with rheumatoid arthritis （RA） after externally administrated with methotrexate （MTX） made by flexible nanonipsomes. METHODS： The experiment was conducted in the Institute of Combined Traditional Chinese Medicine and Western Medicine, Xiangya Hospital of Central South University between March and November 2005. Totally 80 healthy SD rats were randomly divided into 5 groups： Normal control group （n=10）, while other 70 rats were made into RA models by subcutaneous injection of type Ⅱ collagen and complete Freund Adjuvant, and then randomly divided into model group （n=10）, 0.5% MTX group （n=20）, 0.25% flexible nanonipsomes MTX group （n=20） and 0.5% flexible nanonipsomes MTX group （n=20）. Rats in the normal control group drank water freely; rats in the model group were perfused with 1.5 mL of distilled water once a day. Rats in other groups were administered at 7 days after primary immunization, and then were externally painted with 0.5% MTX, 0.25% flexible nanonipsomes MTX and 0.5% flexible nanonipsomes MTX on the right postpedes respectively with 2.0 g to 4.0 g for each rat （according to the size of the articles） once a week. Articular symptoms were evaluated with the integral of arthritis index, and the higher the index was, the severer the arthritis was. Enzyme linked immunosorbent assay （ELISA） was adopted to detect the level of IFN-γ and IL-4 in the serum of rats on the 45^th day after administration. RESULTS ： Totally 80 enrolled rats all entered the final analysis. ① There was no significant difference in the integral of arthritis index at 14 days before administration among all groups, while with the time prolonging, the integral of aithritis index in the model group increased and peaked in the 0.5% MTX group, 0.25% flexible nanonipsomes MTX group and 0.5% flexible nanonipsomes MTX group, but they were still lower than that in the model group （which were 1.51,-0.99, 0.98±0.57,0.88±0.49,1.68±1.24, t =2.956,5.461, 5.244, P 〈 0.01 respectively）.② Forty-five days after administration, the level of IFN-γ in the model group, 0.5% MTX group, 0.25% flexible nanonipsomes MTX group and 0.5% flexible nanonipsomes MTX group were obviously higher than that in the normal control group, whereas the level of IL-4 was obviously lower than that in the normal control group [IFN-γ：（120.75±1.43）, （104.41±1.71）, （88.75±1.58）, （83.60±1.86）, （56.15±2.07）ng/L,t =-64.016, -67,970, -36.928, -48.092, P 〈 0.01; IL-4： （23.28±0.13）, （36.45±0.15）, （46.46±0.16）, （48.47±0.12）, （61.23±0.27）ng/L, t = 398.098, 328.973, 179.924, 186.796, P 〈 0.01]. The level of IFN-γ in 0.5% MTX group, 0.25% flexible nanonipsomes MTX group and 0.5% flexible nanonipsomes MTX group were obviously lower than that in the model group （t =21.387,46.365,43.639,P 〈 0.01）, and the level of IL-4 was significantly higher than that in the model group （t =-238.74,-514.575,-385.096,P 〈 0.01）. The level of IFN-γ in 0.25% flexible nanonipsomes MTX group and the 0.5% flexible nanonipsomes MTX group were obviously lower than that in 0.5% MTX group （t =36.561, 30.124,P 〈 0.01）, while the level of IL-4 were remarkably higher （t =-282.406,-203.685,P 〈 0.01）. The levels of IFN-γ and IL-4 in 0.25% flexible nanonipsomes MTX group were not significantly different from those in 0.5% flexible nanonipsomes MTX group （P 〉 0.05）. CONCLUSION： External using of flexible nanonipsomes MTX can down-regulate IFN-γ and up-regulate the level of IL-4 through its intelligent controlled releasing, and the effect is obviously better than normal external application of MTX.