扶正排毒片干预环磷酰胺致免疫抑制小鼠免疫功能的效应

Effect of Fuzheng Paidu tablet on the immune function of mice with immunosuppression induced by cyclophosphamide

目的:观察扶正排毒片对环磷酰胺所致免疫低下小鼠各项免疫功能的影响,探讨扶正排毒片的益气滋阴、清热解毒的功效。方法:实验于2005-02/10在河南中医学院动物实验中心完成。①扶正排毒片主要成分为西洋参、黄芪、白术、防风、女贞子、山茱萸、南沙参、紫草、连翘、白花蛇舌草、甘草等(由河南中医学院第三附属医院提供,批号050601)。②选取昆明种小鼠180只,分别用于以下3项各自独立实验。每项实验选用60只小鼠,随机数字表法分为6组:空白对照组、模型组、香菇多糖片组、扶正排毒0.15,0.10,0.05g/mL浓度组,10只/组。③除空白对照组外,其余各组均建立环磷酰胺致小鼠免疫抑制模型。于造模第1天,扶正排毒0.15,0.10,0.05g/mL浓度组分别灌服各自对应浓度的扶正排毒片混悬液0.2mL/10g;香菇多糖片组灌服5g/L的香菇多糖片混悬液0.2mL/10g;模型组、空白对照组灌服同体积生理盐水。每天给药1次,连续给药7d。④环磷酰胺致免疫抑制小鼠腹腔巨噬细胞吞噬实验:第7天给药后2h,每组鼠均腹腔注射50g/L的鸡红细胞生理盐水液0.5mL,4h后处死。腹腔注入汉氏液2.5mL,在腹部剪一小口,吸取腹腔液,混匀后滴于载玻片上,37℃孵育30min,冲去附着的细胞,瑞氏染色,观察小鼠腹腔巨噬细胞的吞噬情况。⑤环磷酰胺致免疫抑制小鼠溶血素及溶血空斑形成实验:于给药第1天,各组小鼠均腹腔注射50g/L的鸡红细胞生理盐水混悬液0.2mL,于最后一次给药后2h,小鼠眼眶取血,分离血清,取上清液于UV-2000型分光光度计540nm处比色。处死小鼠后,取脾细胞混悬液,取上清液于UV-2000型分光光度计413nm处比色,检测溶血空斑形成情况。⑥环磷酰胺致免疫抑制小鼠外周血淋巴细胞转化实验:于给药第1,2,3天每组鼠肌注80mg/kg的植物血凝素0.01mL/g。于最后一次给药后2h,小鼠剪尾取血,瑞氏染色,油镜观察并计算外周淋巴细胞转化百分率。结果:180只小鼠均进入结果分析。①与模型组比较,香菇多糖片组及扶正排毒各浓度组均可提高免疫抑制小鼠腹腔巨噬细胞对鸡红细胞的吞噬百分率和吞噬指数(t=4.22～7.66,P<0.01),且以扶正排毒0.15,0.10g/mL浓度组效果明显。②与模型组比较,香菇多糖片组及扶正排毒各浓度组均可促进免疫抑制小鼠溶血素的形成(t=5.26～9.12,P<0.05或0.01);且均可促进免疫抑制小鼠溶血空白斑的形成(t=2.86～6.39,P<0.05或0.01),以扶正排毒0.15,0.10g/mL浓度组效果明显。③与模型组比较,香菇多糖片组及扶正排毒0.15,0.10,0.05g/mL浓度组均可显著提高淋巴细胞转化率[(32.2±5.6)%,(49.6±6.9)%,(59.9±6.9)%,(57.4±7.3)%,(51.4±6.8)%,P<0.01],且以扶正排毒0.15,0.10g/mL浓度组效果明显。结论:扶正排毒片可显著提高环磷酰胺所致免疫抑制小鼠腹腔巨噬细胞的吞噬功能,同时促进溶血素、溶血空斑的形成与外周血淋巴细胞的转化。

AIM： To observe the effect of Fuzheng Paidu tablet on the immune function of mice with immunosuppression caused by cyclophosphamide, and probe into the effect of Fuzheng Paidu tablet on supplementing qi and nourishing yin as well as expelling toxin by cooling. METHODS： The experiment was accomplished at Experimental Animal Center of the Henan College of TCM between February and October 2005. （1） Fuzheng Paidu was maily composed of American ginseng, radix astragall, atractylodes macrocephala, divaricate saposhnikovia root, glossy privet fruit, Asiatic cornelian cherry fruit, adenophorae, radix, lithospermi, radix, forsythia suspense, hedyotic diffusa and licorice root etc. （Which were provided by Third Hospital Affiliated to Henan College of TCM with the batch number of 050601）. （2） 180 Kunming mice were selected for the following experiments with 6 mice for each experiment. The mice were randomly divided into 6 groups： The blank control group, model group, Xianggu Duotang group and 0.15, 0.10, 0.05 g/mE Fuzheng Paidu groups with 10 mice in each group. （3） Except those in the blank control group, mice in other groups were made into cyclophosphamide-induced immunosuppressional models. On the 1^st day of modeling, mice in the 0.15, 0.10, 0.05 g/mL Fuzheng Paidu groups were administrated with 0.2 mL/10g Fuzheng Paidu suspension at corresponding concentration. Mice in the Xianggu Duotang group were drenched with 5 g/L of Xianggu Duotang suspension （0.2 mL/10 g）, while mice in the model group and the blank control group were drenched with normal saline at the same dose. The administration was given once a day for 7 continuous days. （4） Peritoneal macrophage experiment in rats with-immunosnpression induced by cyclophosphamide： At 2 hours on the 7^th day after administration, 0.5 mE of chicken red blood cell liquid （50 g/L） was injected into rat abdominal cavity, and rats were executed at 4 hours later. 2.5 mL of Han＇s solution was injected into the abdomen of mice, and then an incision was made on the peritoneum to suck the celiac solution, which was dropped on the glass after being mixed symmetrically to incubate at 37℃ for 30 minutes. Adherent cells were gotten rid off and then Wright＇s staining was conducted to observe the phagocytosis of macrophage in abdominal cavity under the microscope. （5） Hemolysin and hemolytic antibody plaques formation test in rats with immunosuppresion induced by cyclophosphamide： On the first day, mice were abdominally injected with 0.2 mL of chicken red blood cell solution （50 g/L）, and the blood sample was obtained from the eyepit of mice at 2 hours after the last administration to separate the serum. The level of hemolysin in blood serum was determined by using the spectrophotometer UV-2000 （λ=540 nm）. Then the mice were executed to break out the spleen suspension. The level of hemolysin and plaque forming cell were observed by using the spectrophotometer UV-2000 （λ=413 nm）. （6）Peripheral blood lymphocyte （PBL） transformation experiment in mice with immunosuppressioin induced by cyclophophamide： On the 1^st, 2^nd and 3^rd days of administration, the mice were injected with 80 mg/kg PHY （0.1 mL/10 g） by intramuscular injection. Two hours after the last medicine, the blood sample was adopted by cutting the mice tails, and Wright＇s staining and immersion objective were used to count the percent of the transformation efficiency of lymphocyte in peripheral blood. RESULTS： A total of 180 mice were involved in the analysis of results. （1） Compared with the model group, the phagocytic rate and index of macrophage in abdominal cavity could be significantly promoted in the large, middle and smafl-dose Fuzheng Paiclu groups and Xianggu Duotang group （t=4.22-7.66,P 〈 0.01）, and the effects were remarkable in all doses of Fuzheng Paidu groups. （2） Compared with the model group, the level of hemolysin was remarkably improved in the large, middle and small-dose Fuzheng Paidu groups and Xianggu Duotang groups （t=5.26-9.12 ,P 〈 0.05 or 0.01）, and the plaque forming cells were obviously suppressed （t=2.86-6.39,P 〈 0.05 or 0.01）, especially at the doses of 0.15 and 0.10 g/mL. （3） Compared with the model group, the large, middle and small-dose Fuzheng Paidu tablet and Xianggu Duotang tablet remarkably increased the transformation efficiency of lymphocyte in immunosuppressional mice [（32.2±5.6）%, （49.6±6.9）%, （59.9±6.9）%, （57.4±7.3）%, （51.4±6.8）%, P 〈 0.01], and the effects of FuzhengPaidu tablets were much remarkable at the doses of 0.15 and 0.10 g/mL. CONCLUSION： Fuzheng Paidu tablet can remarkably accelerate the phagocytosis of macrophage in abdominal cavity of immunosuppressional mice caused by cyclophosphamide and the level of hemolysin and plaque forming cell in blood serum and the transformation of peripheral lymphocyte in immunosuppressional mice.