肝豆汤对Wilson病模型TX小鼠脾脏免疫功能调节的作用

Effect of Gandou Decoction on Immune Function of Spleen in TX Mice of Wilson's Disease Model

目的:探讨肝豆汤对Wilson病模型TX小鼠脾脏免疫功能的调节作用,并探讨相关的作用机制。方法:将小鼠分为正常组、模型组、四硫钼酸铵组、肝豆汤低、中、高剂量组,每组20只。正常组选取DL小鼠常规饲养30 d;模型组和肝豆汤低、中、高剂量组选取TX小鼠分别予生理盐水、肝豆汤(22,44,66 g·kg^(-1))各2 mL·kg^(-1)·d^(-1)灌胃,四硫钼酸铵组予四硫钼酸铵2 mg·kg-1·d-1灌胃,每日2次,连续30 d。上述4组采用电感耦合等离子体-质谱法(ICP-MS)测脾组织微量元素,流式细胞术检测小鼠脾组织T淋巴细胞亚群CD4^+,CD8^+,CD4^+/CD8^+;蛋白免疫印迹法(Western blot)检测白细胞介素-2(interleukin-2,IL-2),白细胞介素-8(interleukin-8,IL-8),白细胞介素-17(interleukin-17,IL-17),白细胞介素-18(interleukin-18,IL-18),γ-干扰素(interferon-γ,IFN-γ),肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)的蛋白表达量。结果:ICP-MS结果显示:与模型组比较,肝豆汤可以降低TX小鼠脾组织铜含量(P <0. 01),流式细胞术检测显示,与正常组比较,模型组脾组织内CD4^+,CD8^+增高(P <0. 01),CD4^+/CD8^+降低(P <0. 01),与模型组比较,肝豆汤中、高剂量组脾组织内CD4^+,CD8^+降低(P <0. 05),CD4^+/CD8^+增高,但差异无统计学意义,肝豆汤低剂量组与模型组比较CD4^+,CD8^+降低,CD4^+/CD8^+增高,但差异均无统计学意义; Western blot检测显示,与正常组比较,模型组脾组织内IL-2,IL-8,IL-17,IL-18,TNF-α,IFN-γ的蛋白表达量增高(P <0. 01),与模型组比较,肝豆汤中、高剂量组及四硫钼酸铵组IL-2,IL-8,IL-17,IL-18,TNF-α,IFN-γ蛋白表达量均降低(P <0. 05,P <0. 01);与模型组比较,低剂量肝豆汤组IL-2,IL-8,IL-17,TNF-α相对表达量降低(P <0. 05)。结论:TX小鼠脾脏存在免疫功能紊乱,且以负向免疫调节为主。肝豆汤对TX小鼠脾脏免疫功能紊乱具有一定的改善作用。

Objective:To discuss the effect of Gandou decoction(GDD)on the immune index of spleen in TX mice of Wilson’s disease model.Method:The mice were divided into normal group,model group and GDD or tetrathiomolybdate(TM)treatment group,with 20 mice in each group.Each group was fed in various ways for30 successive days.Normal group:10 normal DL mice were randomly selected and feed normally.Model group:20 TX mice were randomly selected and feed with 2 mL·kg^-1·d^-1 ig saline by gavage twice per day.GDD or TM treatment group:80 TX mice were randomly selected and feed with 2 mL·kg^-1·d^-1 ig Gandou decoction 22,44,66 g·kg^-1 or tetrathiomolybdate by gavage twice per day.ICP-MS was used to compare the expressions of trace elements inside the mice’s spleens,flow cytometry was applied to detect the mice T lymphocyte subsets of splenic tissue CD4^+,CD8^+,CD4^+/CD8^+,and Western blot was used to detect the expressions of interferon-γ(IFN-γ),tumor necrosis factor-α(TNF-α),interleukin-2(IL-2),interleukin-8(IL-8),interleukin-17(IL-17)and interleukin-18(IL-18).Result:Flow ICP-MS results showed that GDD can reduce Cu of mice’s spleen,flow cytometry results showed that CD4^+and CD8^+in model group were increased than those in normal group(P<0.01),and CD4^+/CD8^+was decreased(P<0.01);compared with model group,CD4^+and CD8^+in middle and high-dose GDD groups were decreased(P<0.01),and CD4^+/CD8^+was increased.According to Western blot detection,compared with normal group,the expressions of IL-2,IL-8,IL-17,IL-18,TNF-α and IFN-γ in the model group were increased(P<0.01);compared with model group,the expressions of TNF-α,IFN-γ,IL-2,IL-8,IL-17 and IL-18 in the GDD middle and high or TM group were decreased(P<0.05,P<0.01).Compared with model group,the expressions of IL-2,IL-8,IL-17 and TNF-α in the GDD low were decreased(P<0.05).Conclusion:Spleen of TX mice shows the cellular immunity hyperfunction,which is mainly dominated by the negative immunoloregulation.GDD has a certain effect in regulating cellular immunity hyperfunctional state of TX mice,but it’s difficult to thoroughly change the negative immune regulation.