肝豆汤对Wilson病模型TX小鼠脾脏免疫功能调节的作用
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摘要
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.
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.
引文
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[14]常丽娜,王金萍,王佳佳,等.超声监测肝豆状核变性患者肝脾动脉血流动力学指标及其临床意义[J].中国中西医结合影像学杂志,2015,13(1):29-33.
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[23]汪美霞,陶庄,李祥,等.加味肝豆汤治疗Wilson病湿热内蕴证早期肾损伤的临床分析[J].贵州医药,2017,41(7):695-697.
[24] Hsu P Y, Yen H H, YANG T H, et al.Tetrathiomolybdate, a copper chelator inhibited imiquimod-induced skin inflammation in mice[J]. J Dermatol Sci,2018,92(1):30-37.
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[28]荆凡辉,吕玮,李太生. HIV感染者免疫功能重建新视角:CD4/CD8比值[J].中国艾滋病性病,2018,24(6):643-646.
[29]孙旺,高博,薛东波,等.肝硬化并发血细胞减少的机制和治疗进展[J].肝胆胰外科杂志,2014,26(1):83-85.
[30] LI Z F,ZHANG S,LV G B,et al. Changes in count and function of splenic lymphocytes from patients with portal hypertension[J]. World J Gastroenterol,2008,14(15):2377-2382.
[31] Cho C W,HAN C J,Young K R,et al. Cheonggukjang polysaccharides enhance immune activities and prevent cyclophosphamide-induced immunosuppression[J]. Int J Biol Macromol,2015,72(1):519-525.
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[34]翟志敏. IL-2对免疫激活和免疫耐受的双向调节作用[J].中国药理学通报,2013,29(3):319-322.
[35] Matthew W K,Timothy J M,Haruo U,et al. Outside-in acute fetal systemic inflammation in very preterm chronically catheterized sheep fetuses is not driven by cells in the fetal blood[J]. Am J Obstet Gynecol,2016,214(2):e1-e10.
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[2] Bandmann O,Weiss K H,Kaler S G. Wilson's disease and other neurological copper disorders[J]. Lancet Neurol,2015,14(1):103-113.
[3] Seessle J,Gotthardt D N,Schfer M,et al. Concomitant immune-related events in Wilson disease:implications for monitoring chelator therapy[J]. J Inherit Metab Dis,2016,39(1):125-130.
[4]董健健,韩咏竹,程楠.肝豆汤对Wilson病模型TX小鼠肝细胞内铜代谢通路的分子调控机制[J].中国实验方剂学杂志,2016,22(24):128-133.
[5]耿昊,李海,徐陈陈,等.肝豆汤对Wilson病模型铜负荷大鼠肝脏Wnt/β-catenin信号通路的调控作用[J].中国实验方剂学杂志,2019,25(7):75-81.
[6]徐陈陈,董健健,陈雪燕,等.肝豆汤改良方对TX小鼠脑组织ASM,Cer及p38 MAPK mRNA和蛋白表达的影响[J].中华中医药杂志,2017,32(11):5049-5055.
[7]张静,陈怀珍,艾文龙,等.肝豆汤联合DMPS驱铜治疗对Wilson病湿热内蕴型认知功能障碍的影响[J].中国实验方剂学杂志,2018,24(15):210-215.
[8]杨任民.肝豆状核变性[M].北京:人民卫生出版社,2015:371-372,404-406.
[9] Theophilos M B,Cox D W,Mercer J F,et al. The toxic milk mouse is a murine model of Wilson disease[J].Hum Mol Genet,1996,5(10):1619-1624.
[10]陈曦,王楚怀,丰岩清,等. TX小鼠铜代谢和肝损害的实验研究[J].中华肝脏病杂志,2009,17(9):688-690.
[11]谢文光,魏钰书,周良玉.儿童肝豆状核变性的体液免疫和急性期反应蛋白变化[J].四川医学,2000,21(2):15-16.
[12]许力,杨任民,洪铭范.肝豆状核变性患者的免疫功能监测[J].蚌埠医学院学报,2006,23(5):456-458.
[13]张春海,杨文明.肝豆状核变性合并脾功能亢进治疗进展[J].安徽医药,2010,14(4):380-382.
[14]常丽娜,王金萍,王佳佳,等.超声监测肝豆状核变性患者肝脾动脉血流动力学指标及其临床意义[J].中国中西医结合影像学杂志,2015,13(1):29-33.
[15] ZHANG L H,PAN J P,YAO H P,et al. Intrasplenic transplantation of IL-18 gene-modified hepatocytes:an effective approach to reverse hepatic fibrosis in schistosomiasis through induction of dominant Th1response[J]. Gene Ther,2001,8(17):1333-1342.
[16] Bahr M J,Manns M P. Function of the immune system in liver cirrhosis[J]. Z Gastroenterol,2001,39(8):601-607.
[17]张静,方媛,崔圣伟,等.肝豆汤联合二巯基丙磺酸钠对湿热内蕴型肝豆状核变性患者的影响[J].中国实验方剂学杂志,2017,23(17):190-194.
[18] CHEN D,ZHOU X,HOU H,et al. Clinical efficacy of combined sodium dimercaptopropanesulfonate and zinc treatment in neurological Wilson's disease with Dpenicillamine treatment failure[J]. Ther Adv Neurol Disord,2016,9(4):310-316.
[19] Maeda S,Matsubara H,Hiejima E,et al. Acute lymphoblastic leukemia in a girl with Wilson's disease[J]. Pediatr Int,2014,56(4):626-629.
[20] Farallo M,Amoruso C,Frattini C,et al. Nephrotic syndrome after treatment with d-penicillamine in a pediatric patient with Wilson's disease[J]. Pediatr Med Chir,2012,34(5):234-236.
[21] Dell'era L,Boati E,Nebbia G,et al. Wilson's disease treated with penicillamine and lupus erythematosus:related or distinct entities?[J]. Minerva Pediatr,2012,64(1):55-57.
[22]汪美霞,詹宇婷,杨文明.肝豆汤加减治疗湿热内蕴型肝豆状核变性的临床疗效研究[J].中国全科医学,2017,20(28):3573-3578.
[23]汪美霞,陶庄,李祥,等.加味肝豆汤治疗Wilson病湿热内蕴证早期肾损伤的临床分析[J].贵州医药,2017,41(7):695-697.
[24] Hsu P Y, Yen H H, YANG T H, et al.Tetrathiomolybdate, a copper chelator inhibited imiquimod-induced skin inflammation in mice[J]. J Dermatol Sci,2018,92(1):30-37.
[25] Przybylkowski A,Gromadzka G,Wawer A,et al.Neurochemical and behavioral characteristics of toxic milk mice:an animal model of Wilson's disease[J].Neurochem Res,2013,38(10):2037-2045.
[26]徐文,杨任民.肝豆状核变性的铁代谢研究[J].安徽中医学院学报,2009,28(1):62-64.
[27]更藏达杰,毛海明.分泌性中耳炎患者炎性因子、细胞免疫及体液免疫功能分析[J].海南医学院学报,2018,24(16):1529-1531.
[28]荆凡辉,吕玮,李太生. HIV感染者免疫功能重建新视角:CD4/CD8比值[J].中国艾滋病性病,2018,24(6):643-646.
[29]孙旺,高博,薛东波,等.肝硬化并发血细胞减少的机制和治疗进展[J].肝胆胰外科杂志,2014,26(1):83-85.
[30] LI Z F,ZHANG S,LV G B,et al. Changes in count and function of splenic lymphocytes from patients with portal hypertension[J]. World J Gastroenterol,2008,14(15):2377-2382.
[31] Cho C W,HAN C J,Young K R,et al. Cheonggukjang polysaccharides enhance immune activities and prevent cyclophosphamide-induced immunosuppression[J]. Int J Biol Macromol,2015,72(1):519-525.
[32] Nasibullin T R,Timasheva Y R,Tuktarova I A,et al.Combinations of cytokine gene network polymorphic markers as potential predictors of myocardial infarction[J]. Genetika,2014,50(9):987-993.
[33]岳文杰,刘懿,徐薇,等.溃疡性结肠炎肠黏膜中IL-2、IL-4、IL-17和IL-10的表达特点及其与疾病活动度的关系[J].复旦学报:医学版,2012,39(5):454-459.
[34]翟志敏. IL-2对免疫激活和免疫耐受的双向调节作用[J].中国药理学通报,2013,29(3):319-322.
[35] Matthew W K,Timothy J M,Haruo U,et al. Outside-in acute fetal systemic inflammation in very preterm chronically catheterized sheep fetuses is not driven by cells in the fetal blood[J]. Am J Obstet Gynecol,2016,214(2):e1-e10.
[36]张晓玲,刑荣格懿,韩国达,等.西黄胶囊辅助化疗治疗结肠癌术后肝转移疗效及对患者外周血IL-17和IL-6的影响[J].中草药,2015,46(6):871-874.
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