扶正固表方对免疫低下小鼠免疫功能的实验研究

Experimental study of Fuzheng Gubiao Decoction on immune function of immunocompromised mice

关键词:  扶正固表方  免疫低下  环磷酰胺  免疫调节

Key words:  Fuzheng Gubiao Decoction  immunodeficiency  cyclophosphamide  immunoregulation

摘要:

[目的] 研究扶正固表方对环磷酰胺诱导免疫低下小鼠的免疫调节作用。[方法] 48 只 Balb/c 小鼠随机分为对照组、模型组、扶正固表方组（7.15 g/kg）和西药组（盐酸左旋咪唑，30 mg/kg），除对照组外，其他各组小鼠腹腔注射环磷酰胺制备免疫低下小鼠模型。造模成功后，扶正固表方组和西药组分别给予灌胃干预 7 d。观察各组小鼠的一般状态和体质量变化，检测免疫器官指数；酶联免疫吸附法测定血清肿瘤坏死因子-α（TNF-α）、白介素-4（IL-4）和干扰素-γ（IFN-γ）的水平；苏木素-伊红染色法观察脾脏病理变化；流式细胞术检测脾淋巴细胞亚群 CD4⁺ T 淋巴细胞、CD8⁺ T 淋巴细胞及 CD4⁺/CD8⁺的分布与比例；实时荧光定量聚合酶链反应法与蛋白免疫印迹法检测脾脏组织中 NF-κB p65 和 IκBα mRNA 与蛋白的表达水平。[结果] 与对照组相比，模型组小鼠出现精神萎靡，毛发黯淡等体征，伴随体质量、脾脏指数和胸腺指数降低（P<0.01）；血清 TNF-α 水平升高（P<0.01），而 IL-4 和 IFN-γ 水平降低（P<0.01）；脾组织红、白髓界限不清，伴有红细胞和炎性细胞浸润；脾 CD4⁺淋巴细胞数下降（P<0.01），CD8⁺淋巴细胞数升高（P<0.01），CD4⁺/CD8⁺比例降低（P<0.01）；脾组织中 NF-κB p65 mRNA 与蛋白表达降低（P<0.01），IκBα mRNA和蛋白表达升高（P<0.01）。与模型组相比，扶正固表方组一般状态有所改善，体质量升高，脾脏指数和胸腺指数提升（P<0.01），血清 TNF-α 水平降低（P<0.01），IL-4 和 IFN-γ 水平升高（P<0.01），脾组织红、白髓界线逐渐清晰，脾CD4⁺淋巴细胞数增多，CD8⁺淋巴细胞数降低，CD4⁺/CD8⁺比例上升； 脾组织中 NF-κB p65 mRNA 与蛋白的表达水平上升（P<0.05），IκBα mRNA 和蛋白表达降低（P<0.05）。[结论] 扶正固表方可以调节免疫低下小鼠的免疫状态，增强机体的免疫功能。

Abstract:

[Objective] To study the immunoregulatory effect of Fuzheng Gubiao Decoction on immunosuppressive mice induced by cyclophosphamide. [Methods] 48 Balb/c mice were randomly divided into the control group and the model group，and the Fuzheng Gubiao Decoction group （7.15 mg/kg），and the western medicine group（levamisole hydrochloride，30 mg/kg）. In addition to the control group, the other groups were injected with cyclophosphamide intraperitoneally to prepare immunosuppression model. After the successful modeling，the Fuzheng Gubiao Decoction group and the Western medicine group were given intragastric intervention for 7 days. The general state and body weight of mice were observed，and the immune organ index was detected；the serum levels of TNF-α，IL-4 and IFN-γ were determined by ELISA；the pathological changes of spleen were observed by HE staining；the distribution and proportion of splenic lymphocyte subsets CD4⁺ T lymphocytes，CD8⁺ T lymphocytes and CD4⁺/CD8⁺ were detected by flow cytometry；the mRNA and protein expression levels of NF-κB p65 and IκBα in spleen tissues were detected by real-time quantitative PCR and Western Blot. [Results] Compared with the control group，the mice in the model group showed signs of listlessness and dull hair，accompanied by a significant decrease in body weight，spleen index and thymus index were significantly increased（P<0.01）；Serum TNF-α level was significantly increased（P<0.01）， The levels of IL-4 and IFN-γ were significantly decreased（P<0.01）；the red and white pulp of that spleen were not clearly demarcate，with the infiltration of red blood cells and inflammatory cell；and the number of CD4⁺ lymphocytes decreased significantly （P <0.01），the number of CD8⁺ lymphocytes increased significantly （P <0.01），and the proportion of CD4⁺/CD8⁺ decreased significantly（P<0.01）；the expressions of NF-κB p65 mRNA and protein in spleen were significantly decreased（P<0.01），and the expressions of IκBα mRNA and protein were significantly increased（P<0.01）.Compared with the model group，the general state of Fuzheng Gubiao Decoction group was improved，body mass was increased，spleen index and thymus index were significantly increased（P<0.01），serum TNF-α level was significantly decreased（P<0.01），IL-4 and IFN-γ levels were significantly increased（P<0.01）；and the red and white pulp lines in spleen tissue were gradually clear；The number of CD4⁺ lymphocytes increased，the number of CD8⁺ lymphocytes decreased，and the proportion of CD4⁺/CD8⁺ increased；the mRNA and protein expressions of NF-κB p65 in spleen were significantly increased（P<0.05），and the mRNA and protein expressions of IκBα were significantly decreased（P<0.05）. [Conclusion] Fuzheng Gubiao Decoction can regulate the immune state of immunocompromised mice and enhance the immune function of the body.