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目的:研究芍药苷对体外小鼠脾细胞增殖及细胞因子的作用,探讨其对细胞免疫功能的影响及机制。方法:昆明种小鼠颈椎脱臼处死,体外培养小鼠脾细胞,不同质量浓度的芍药苷溶液(62.5,12.5,250,500,1 000 mg·L-1),另设空白组,分别于培养3,6,9 d时取出细胞培养板,采用血球计数板计数细胞数法测定不同浓度芍药苷对小鼠脾细胞增殖活性的影响,酶联免疫吸附法(ELISA)测定培养上清中白细胞介素-2(IL-2),干扰素-γ(IFN-γ)和肿瘤坏死因子-α(TNF-α)的含量;作用9 d后取细胞,Western blot方法测定IL-2,IFN-γ,TNF-α和核因子-κB(NF-κB)蛋白表达;500 mg·L-1芍药苷溶液作用5 d后,流式细胞仪(FCM)检测细胞表型变化。结果:在3,6,9 d,不同浓度芍药苷给药组的脾淋巴细胞增殖活性均高于空白组(P<0.01);各给药组IL-2,IFN-γ,TNF-α的含量较空白组明显升高(P<0.05,P<0.01),并随芍药苷浓度的增加而增加;淋巴细胞表型分析显示,芍药苷能够刺激T淋巴细胞和B淋巴细胞增殖;不同质量浓度芍药苷能够升高IL-2,IFN-γ,TNF-α和NF-κB蛋白表达(P<0.05,P<0.01)。结论:芍药苷可通过激活NF-κB表达,促进细胞因子IL-2,IFN-γ和TNF-α的分泌和表达,促进细胞增殖,提高机体免疫功能。
Objective: To study the effects of paeoniflorin on the proliferation and cytokines of splenocytes in vitro in mice and its effects on cellular immune function and its mechanism. Methods: Kunming mice were sacrificed by cervical dislocation, splenocytes of mice were cultured in vitro, and different concentrations of paeoniflorin (62.5,12.5,250,500,1 000 mg · L -1) At 6 and 9 d, the cell culture plates were removed and the number of cells treated with different concentrations of paeoniflorin on the proliferation of mouse spleen cells was determined by counting the number of cells in the blood cell counting plate. The levels of interleukin- IL-2, IFN-γ and TNF-α were measured. After 9 days of treatment, the cells were harvested for determination of IL-2, IFN-γ, TNF The expression of NF-κB protein and NF-κB protein were detected by flow cytometry (FCM) after treated with 500 mg · L -1 paeoniflorin for 5 d. Results: On 3, 6, and 9 days, the proliferation activity of spleen lymphocytes in paeoniflorin group was higher than that in blank group (P <0.01). The levels of IL-2, IFN-γ and TNF- (P <0.05, P <0.01), and with the increase of paeoniflorin concentration increased; phenotype of lymphocytes showed that paeoniflorin can stimulate the proliferation of T lymphocytes and B lymphocytes; different concentrations of Paeoniflorin increased the expressions of IL-2, IFN-|Ã, TNF-|Á and NF-|ÊB (P <0.05, P <0.01). CONCLUSION: Paeoniflorin can promote the secretion of IL-2, IFN-γ and TNF-α by stimulating the expression of NF-κB, promote cell proliferation and enhance immune function.