木瓜苷和儿茶素对小鼠细胞免疫功能的调节作用
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摘要
目的:
T细胞介导的细胞免疫应答在移植物排斥、移植物抗宿主反应、自身免疫和肿瘤免疫等方面起重要作用。小鼠迟发型超敏反应(delayed-type hypersensitivity,DTH)是由特异性致敏效应T细胞介导的细胞免疫反应。本实验采用2,4-二硝基氯苯(2,4-dinitrochlorobenzene,DNCB)诱导的小鼠DTH反应以及环磷酰胺(cyclophosphamide,Cy)增强或抑制DNCB诱导的小鼠DTH反应作为细胞免疫功能异常的模型,从整体、胸腺T细胞增殖以及胸腺T细胞产生的细胞因子等方面观察木瓜苷(glucosides of Chaenomeles speciosa,GCS)和儿茶素(catechin,Cat)对上述各模型的影响,研究GCS和Cat对细胞免疫的调节作用及其部分机制,并探讨Cat是否是GCS发挥作用的主要有效成份之一。
方法:
以DNCB诱导小鼠DTH反应,于DNCB致敏前3天腹腔注射(intragperitoneal injection,ip)Cy(250 mg·kg~(-1))和致敏后立即ip Cy(125 mg·kg~(-1))分别诱导小鼠增强和抑制的DTH反应;于致敏前3天GCS(60,120,240 mg·kg~(-1)·d~(-1))和Cat(30,60,120 mg·kg~(-1)·d~(-1))灌胃给药(intragastric administration,ig)(d_(-3)～d_4),连续7d。用耳肿胀度、胸腺和脾指数作为整体检测指标;以ConA诱导胸腺T淋巴细胞,观察不同剂量或浓度的GCS和Cat体内外对胸腺T淋巴细胞增殖及其产生的细胞因子IL-2、IL-4的影响。MTT法检测胸腺T淋巴细胞增殖,ConA诱导的胸腺T淋巴细胞培养上清中IL-2活性检测采用活化的小鼠胸腺细胞MTT比色法;ELISA法测定小鼠胸腺T淋巴细胞培养上清中IL-4水平。
结果:
1.GCS和Cat对正常DTH小鼠细胞免疫功能的影响GCS和Cat对正常小鼠DTH有明显的抑制作用。GCS(60,120,240 mg·kg~(-1)),Cat(120 mg·kg~(-1))能显著下调DTH小鼠的耳肿胀;GCS(120,240 mg·kg~(-1))和Cat(120 mg·kg~(-1))可显著下调DTH小鼠的胸腺及脾脏指数;GCS(60,120,240 mg·kg~(-1)),Cat(60,120 mg·kg~(-1))可显著下调DTH小鼠胸腺T淋巴细胞增殖反应以及产生IL-2的水平,使之接近正常水平。GCS(120,240 mg·kg~(-1))和Ca(t120 mg·kg~(-1))可显著上调DTH小鼠降低的胸腺T淋巴细胞产生IL-4的水平。
体外实验进一步证实,GCS(12.5～200μg·ml~(-1))及Cat(25～200μg·ml~(-1))能显著下调DTH小鼠胸腺T淋巴细胞增殖反应和产生IL-2的水平,GCS(25～200μg·ml~(-1))及Cat(50～200μg·ml~(-1))能显著上调DTH小鼠胸腺T淋巴细胞产生IL-4的水平。
2. GCS和Cat对Cy增强的DTH小鼠细胞免疫功能的影响
GCS和Cat对Cy增强的小鼠DTH有明显的抑制作用。GCS(60,120,240 mg·kg~(-1))和Cat(120 mg·kg~(-1))能显著下调Cy增强的DTH小鼠耳肿胀度;GCS(120,240 mg·kg~(-1))和Cat(120 mg·kg~(-1))能显著下调Cy增强的DTH小鼠胸腺指数;GCS(60,120,240 mg·kg~(-1))和Cat(60,120 mg·kg~(-1))可显著下调Cy增强的DTH小鼠脾指数;GCS(120,240 mg·kg~(-1))和Cat(60,120 mg·kg~(-1))可显著下调Cy增强的DTH小鼠胸腺T淋巴细胞增殖反应;GCS(60,120,240 mg·kg~(-1))及Cat(30,60,120 mg·kg~(-1))能显著下调Cy增强的DTH小鼠胸腺T淋巴细胞产生IL-2的水平;GCS(120,240 mg·kg~(-1))及Cat(120 mg·kg~(-1))能显著上调Cy增强的DTH小鼠胸腺T淋巴细胞产生IL-4的水平。
体外实验进一步证实,GCS(12.5～200μg·ml~(-1))及Cat(25～200μg·ml~(-1))能显著下调Cy增强的DTH小鼠胸腺T淋巴细胞增殖反应和产生IL-2的水平,GCS(25～200μg·ml~(-1))及Cat(50～200μg·ml~(-1))能显著上调Cy增强的DTH小鼠胸腺T淋巴细胞产生IL-4的水平。
3. GCS和Cat对Cy抑制的DTH小鼠细胞免疫功能的影响
GCS和Cat对受抑制的小鼠细胞免疫功能有增强作用。GCS(120,240 mg·kg~(-1))及Cat(60,120 mg·kg~(-1))能显著上调Cy抑制小鼠的耳肿胀度;GCS(60,120,240 mg·kg~(-1))及Cat(60,120 mg·kg~(-1))能显著上调Cy抑制小鼠胸腺指数和脾指数;GCS(60,120,240 mg·kg~(-1))及Cat(60,120 mg·kg~(-1))能显著上调Cy抑制小鼠胸腺T淋巴细胞增殖以及产生IL-2和IL-4的水平。
体外实验进一步证实,GCS(12.5～200μg·ml~(-1))及Cat(25～200μg·ml~(-1))能显著上调Cy抑制DTH小鼠胸腺T淋巴细胞增殖反应和产生的IL-4水平;GCS (12.5～200μg·ml~(-1))及Cat(12.5～200μg·ml~(-1))能显著上调Cy抑制的DTH小鼠胸腺T淋巴细胞产生的IL-2水平。.
结论:
GCS和Cat对免疫功能异常的小鼠的细胞免疫具有机能依赖性的双向免疫调节作用,其作用机制可能与调节小鼠胸腺T淋巴细胞增殖及其产生的细胞因子水平,恢复Th1(IL-2)与Th2(IL-4)之间以及Th与Ts之间的平衡有关。GCS是木瓜调节小鼠细胞免疫的有效部位,Cat可能是GCS调节细胞免疫的有效成分之一。
Aim: T-cells Play an important role in transplant rejection, graft-versus-host reaction, autoimmune and tumor immunity. Delayed-type hypersensitivity (DTH) in mice is a specific T cell-mediated immune response.To confirm whether glucosides of Chaenomeles speciosa (GCS) and catechins (Cat) can regulate cell immunity of mice, and to explore whether Cat is one of the main active ingredient of GCS, the study used DTH induced by 2, 4-dinitrochlorobenzene (DNCB) and cyclophosphamide (Cy) potentiated or suppressed DTH in mice as a model of immune dysfunction to observe the impact of GCS and Cat on the above-mentioned model from the level of the whole, the thymocytes proliferation and cytokines produced by thymocytes.
Methods: DTH of mice was induced by DNCB. The potentiated and suppressed DTH of mice was developed by Cy at a dose of 250 mg·kg~(-1) 3 days prior to the sensitization by DNCB and 125 mg·kg~(-1) simultaneously with the sensitization of the mice by DNCB respectively. GCS (60, 120, 240 mg·kg~(-1)) and Cat (30, 60, 120 mg·kg~(-1)) were given intragastrically (ig) 3 days prior to the sensitization once daily for 7 consecutive days. Ear swelling degree, thymus index and spleen index was used as evaluating indication. Thymus lymphocyte proliferation was induced by concanavalin A (ConA) to examine the influence of different doses or concentrations of GCS and Cat on the lymphocyte proliferation and secretion of interleukin-2 and interleukin-4 in vivo or vitro. ConA-induced thymocytes proliferation was measured by 3-(4,5-dimethylthiazol-2- -thiazolyl)-2,5-diphenyl-2H tetrazolium bromide (MTT) assay. IL-2 activity was measured by testing its ability to support ConA-induced mice thymocytes proliferation by the MTT assay. IL-4 were measured by enzyme-linked immunosorbent assay(ELISA).
Results:
1. the effects on cell immunity of DTH mice by GCS and Cat. GCS and Cat could significantly inhibite DTH response in Normal mice. GCS (60, 120, 240 mg·kg~(-1)) and Cat (120 mg·kg~(-1)) could significantly decrease the ear swelling; GCS (120, 240 mg·kg~(-1)) and Cat (120 mg·kg~(-1)) could significantly decrease the thymus index and the spleen index; GCS (60, 120, 240 mg·kg~(-1)) and Cat (60, 120 mg·kg~(-1)) could decrease thymocytes proliferation and IL-2 levels to close to the Normal levels; GCS (120, 240 mg·kg~(-1)) and Cat (120 mg·kg~(-1)) could significantly increase IL-4 levels in cultures of thymocytes from DTH mice.
In vitro, GCS (12.5～200μg·ml~(-1)) and Cat (25～200μg·ml~(-1)) could significantly decrease thymocytes proliferation and IL-2 levels. GCS (25～200μg·ml~(-1)) and Cat (50～200μg·ml~(-1)) could significantly increase IL-4 levels in cultures of thymocytes from DTH mice with a concentration-dependent dose-effect relationship.
2. the effects on cell immunity of Cy- potentiated DTH mice by GCS and Cat. GCS and Cat could significantly inhibite DTH response in Cy-potentiated DTH mice. GCS (60, 120, 240 mg·kg~(-1)) and Cat (120 mg·kg~(-1)) could significantly decrease the ear swelling of Cy-potentiated DTH mice; GCS (120, 240 mg·kg~(-1)) and Cat (120 mg·kg~(-1)) could significantly decrease the thymus index of Cy-potentiated DTH mice; GCS (60, 120, 240 mg·kg~(-1)) and Cat (60, 120 mg·kg~(-1)) could significantly decrease the spleen index of Cy-potentiated DTH mice; GCS (120, 240 mg·kg~(-1)) and Cat(60, 120 mg·kg~(-1)) could significantly decrease thymocytes proliferation; GCS (60, 120, 240 mg·kg~(-1)) and Cat (30, 60, 120 mg·kg~(-1)) could significantly decrease the IL-2 levels in the culture supernatants of thymocytes; GCS (120, 240 mg·kg~(-1)) and Cat (120 mg·kg~(-1)) could significantly increase IL-4 levels in cultures of thymocytes from the Cy- potentiated DTH mice.
In vitro, GCS (12.5～200μg·ml~(-1)) and Cat (25～200μg·ml~(-1)) could significantly decrease thymocytes proliferation and IL-2 production of thymocytes; GCS (25～200μg·ml~(-1)) and Cat (50～200μg·ml~(-1)) could increase IL-4 levels in cultures of thymocytes from the Cy- potentiated DTH mice.
3. the effects on cell immunity of Cy-suppressed DTH mice by GCS and Cat.
GCS and Cat could enhance DTH response in Cy-suppressed DTH mice. GCS (120, 240 mg·kg~(-1)) and Cat(60,120 mg·kg~(-1)) could significantly increase the ear swelling of Cy-suppressed DTH mice; GCS (60, 120, 240 mg·kg~(-1)) and Cat (60, 120 mg·kg~(-1)) could significantly increase thymus index, spleen index of the Cy- suppressed DTH mice; GCS (60, 120, 240 mg·kg~(-1)) and Cat (60, 120 mg·kg~(-1)) could significantly increase thymocytes proliferation, IL-2 and IL-4 levels in cultures of thymocytes from the Cy-suppressed DTH mice.
In vitro, GCS (12.5～200μg·ml~(-1)) and Cat (25～200μg·ml~(-1)) could significantly increase thymocytes proliferation and IL-4 production of thymocytes; GCS (12.5～200μg·ml~(-1)) and Cat (12.5～200μg·ml~(-1)) could significantly increase IL-2 production of thymocytes.
Conclusion: GCS and Cat might have dual immunomodulatory effects in a function dependent manner in mice with immune dysfunction by regulating functions of immune organ of mice, thymocytes proliferation and the level of cytokine production by thymocytes, and balancing Th1 (IL-2) and Th2 (IL-4), Th and Ts. GCS are active compound in regulating the cell immunity of mice, Cat might regulate cell immunity of mice as one of the active ingredients of GCS.
T细胞介导的细胞免疫应答在移植物排斥、移植物抗宿主反应、自身免疫和肿瘤免疫等方面起重要作用。小鼠迟发型超敏反应(delayed-type hypersensitivity,DTH)是由特异性致敏效应T细胞介导的细胞免疫反应。本实验采用2,4-二硝基氯苯(2,4-dinitrochlorobenzene,DNCB)诱导的小鼠DTH反应以及环磷酰胺(cyclophosphamide,Cy)增强或抑制DNCB诱导的小鼠DTH反应作为细胞免疫功能异常的模型,从整体、胸腺T细胞增殖以及胸腺T细胞产生的细胞因子等方面观察木瓜苷(glucosides of Chaenomeles speciosa,GCS)和儿茶素(catechin,Cat)对上述各模型的影响,研究GCS和Cat对细胞免疫的调节作用及其部分机制,并探讨Cat是否是GCS发挥作用的主要有效成份之一。
方法:
以DNCB诱导小鼠DTH反应,于DNCB致敏前3天腹腔注射(intragperitoneal injection,ip)Cy(250 mg·kg~(-1))和致敏后立即ip Cy(125 mg·kg~(-1))分别诱导小鼠增强和抑制的DTH反应;于致敏前3天GCS(60,120,240 mg·kg~(-1)·d~(-1))和Cat(30,60,120 mg·kg~(-1)·d~(-1))灌胃给药(intragastric administration,ig)(d_(-3)～d_4),连续7d。用耳肿胀度、胸腺和脾指数作为整体检测指标;以ConA诱导胸腺T淋巴细胞,观察不同剂量或浓度的GCS和Cat体内外对胸腺T淋巴细胞增殖及其产生的细胞因子IL-2、IL-4的影响。MTT法检测胸腺T淋巴细胞增殖,ConA诱导的胸腺T淋巴细胞培养上清中IL-2活性检测采用活化的小鼠胸腺细胞MTT比色法;ELISA法测定小鼠胸腺T淋巴细胞培养上清中IL-4水平。
结果:
1.GCS和Cat对正常DTH小鼠细胞免疫功能的影响GCS和Cat对正常小鼠DTH有明显的抑制作用。GCS(60,120,240 mg·kg~(-1)),Cat(120 mg·kg~(-1))能显著下调DTH小鼠的耳肿胀;GCS(120,240 mg·kg~(-1))和Cat(120 mg·kg~(-1))可显著下调DTH小鼠的胸腺及脾脏指数;GCS(60,120,240 mg·kg~(-1)),Cat(60,120 mg·kg~(-1))可显著下调DTH小鼠胸腺T淋巴细胞增殖反应以及产生IL-2的水平,使之接近正常水平。GCS(120,240 mg·kg~(-1))和Ca(t120 mg·kg~(-1))可显著上调DTH小鼠降低的胸腺T淋巴细胞产生IL-4的水平。
体外实验进一步证实,GCS(12.5～200μg·ml~(-1))及Cat(25～200μg·ml~(-1))能显著下调DTH小鼠胸腺T淋巴细胞增殖反应和产生IL-2的水平,GCS(25～200μg·ml~(-1))及Cat(50～200μg·ml~(-1))能显著上调DTH小鼠胸腺T淋巴细胞产生IL-4的水平。
2. GCS和Cat对Cy增强的DTH小鼠细胞免疫功能的影响
GCS和Cat对Cy增强的小鼠DTH有明显的抑制作用。GCS(60,120,240 mg·kg~(-1))和Cat(120 mg·kg~(-1))能显著下调Cy增强的DTH小鼠耳肿胀度;GCS(120,240 mg·kg~(-1))和Cat(120 mg·kg~(-1))能显著下调Cy增强的DTH小鼠胸腺指数;GCS(60,120,240 mg·kg~(-1))和Cat(60,120 mg·kg~(-1))可显著下调Cy增强的DTH小鼠脾指数;GCS(120,240 mg·kg~(-1))和Cat(60,120 mg·kg~(-1))可显著下调Cy增强的DTH小鼠胸腺T淋巴细胞增殖反应;GCS(60,120,240 mg·kg~(-1))及Cat(30,60,120 mg·kg~(-1))能显著下调Cy增强的DTH小鼠胸腺T淋巴细胞产生IL-2的水平;GCS(120,240 mg·kg~(-1))及Cat(120 mg·kg~(-1))能显著上调Cy增强的DTH小鼠胸腺T淋巴细胞产生IL-4的水平。
体外实验进一步证实,GCS(12.5～200μg·ml~(-1))及Cat(25～200μg·ml~(-1))能显著下调Cy增强的DTH小鼠胸腺T淋巴细胞增殖反应和产生IL-2的水平,GCS(25～200μg·ml~(-1))及Cat(50～200μg·ml~(-1))能显著上调Cy增强的DTH小鼠胸腺T淋巴细胞产生IL-4的水平。
3. GCS和Cat对Cy抑制的DTH小鼠细胞免疫功能的影响
GCS和Cat对受抑制的小鼠细胞免疫功能有增强作用。GCS(120,240 mg·kg~(-1))及Cat(60,120 mg·kg~(-1))能显著上调Cy抑制小鼠的耳肿胀度;GCS(60,120,240 mg·kg~(-1))及Cat(60,120 mg·kg~(-1))能显著上调Cy抑制小鼠胸腺指数和脾指数;GCS(60,120,240 mg·kg~(-1))及Cat(60,120 mg·kg~(-1))能显著上调Cy抑制小鼠胸腺T淋巴细胞增殖以及产生IL-2和IL-4的水平。
体外实验进一步证实,GCS(12.5～200μg·ml~(-1))及Cat(25～200μg·ml~(-1))能显著上调Cy抑制DTH小鼠胸腺T淋巴细胞增殖反应和产生的IL-4水平;GCS (12.5～200μg·ml~(-1))及Cat(12.5～200μg·ml~(-1))能显著上调Cy抑制的DTH小鼠胸腺T淋巴细胞产生的IL-2水平。.
结论:
GCS和Cat对免疫功能异常的小鼠的细胞免疫具有机能依赖性的双向免疫调节作用,其作用机制可能与调节小鼠胸腺T淋巴细胞增殖及其产生的细胞因子水平,恢复Th1(IL-2)与Th2(IL-4)之间以及Th与Ts之间的平衡有关。GCS是木瓜调节小鼠细胞免疫的有效部位,Cat可能是GCS调节细胞免疫的有效成分之一。
Aim: T-cells Play an important role in transplant rejection, graft-versus-host reaction, autoimmune and tumor immunity. Delayed-type hypersensitivity (DTH) in mice is a specific T cell-mediated immune response.To confirm whether glucosides of Chaenomeles speciosa (GCS) and catechins (Cat) can regulate cell immunity of mice, and to explore whether Cat is one of the main active ingredient of GCS, the study used DTH induced by 2, 4-dinitrochlorobenzene (DNCB) and cyclophosphamide (Cy) potentiated or suppressed DTH in mice as a model of immune dysfunction to observe the impact of GCS and Cat on the above-mentioned model from the level of the whole, the thymocytes proliferation and cytokines produced by thymocytes.
Methods: DTH of mice was induced by DNCB. The potentiated and suppressed DTH of mice was developed by Cy at a dose of 250 mg·kg~(-1) 3 days prior to the sensitization by DNCB and 125 mg·kg~(-1) simultaneously with the sensitization of the mice by DNCB respectively. GCS (60, 120, 240 mg·kg~(-1)) and Cat (30, 60, 120 mg·kg~(-1)) were given intragastrically (ig) 3 days prior to the sensitization once daily for 7 consecutive days. Ear swelling degree, thymus index and spleen index was used as evaluating indication. Thymus lymphocyte proliferation was induced by concanavalin A (ConA) to examine the influence of different doses or concentrations of GCS and Cat on the lymphocyte proliferation and secretion of interleukin-2 and interleukin-4 in vivo or vitro. ConA-induced thymocytes proliferation was measured by 3-(4,5-dimethylthiazol-2- -thiazolyl)-2,5-diphenyl-2H tetrazolium bromide (MTT) assay. IL-2 activity was measured by testing its ability to support ConA-induced mice thymocytes proliferation by the MTT assay. IL-4 were measured by enzyme-linked immunosorbent assay(ELISA).
Results:
1. the effects on cell immunity of DTH mice by GCS and Cat. GCS and Cat could significantly inhibite DTH response in Normal mice. GCS (60, 120, 240 mg·kg~(-1)) and Cat (120 mg·kg~(-1)) could significantly decrease the ear swelling; GCS (120, 240 mg·kg~(-1)) and Cat (120 mg·kg~(-1)) could significantly decrease the thymus index and the spleen index; GCS (60, 120, 240 mg·kg~(-1)) and Cat (60, 120 mg·kg~(-1)) could decrease thymocytes proliferation and IL-2 levels to close to the Normal levels; GCS (120, 240 mg·kg~(-1)) and Cat (120 mg·kg~(-1)) could significantly increase IL-4 levels in cultures of thymocytes from DTH mice.
In vitro, GCS (12.5～200μg·ml~(-1)) and Cat (25～200μg·ml~(-1)) could significantly decrease thymocytes proliferation and IL-2 levels. GCS (25～200μg·ml~(-1)) and Cat (50～200μg·ml~(-1)) could significantly increase IL-4 levels in cultures of thymocytes from DTH mice with a concentration-dependent dose-effect relationship.
2. the effects on cell immunity of Cy- potentiated DTH mice by GCS and Cat. GCS and Cat could significantly inhibite DTH response in Cy-potentiated DTH mice. GCS (60, 120, 240 mg·kg~(-1)) and Cat (120 mg·kg~(-1)) could significantly decrease the ear swelling of Cy-potentiated DTH mice; GCS (120, 240 mg·kg~(-1)) and Cat (120 mg·kg~(-1)) could significantly decrease the thymus index of Cy-potentiated DTH mice; GCS (60, 120, 240 mg·kg~(-1)) and Cat (60, 120 mg·kg~(-1)) could significantly decrease the spleen index of Cy-potentiated DTH mice; GCS (120, 240 mg·kg~(-1)) and Cat(60, 120 mg·kg~(-1)) could significantly decrease thymocytes proliferation; GCS (60, 120, 240 mg·kg~(-1)) and Cat (30, 60, 120 mg·kg~(-1)) could significantly decrease the IL-2 levels in the culture supernatants of thymocytes; GCS (120, 240 mg·kg~(-1)) and Cat (120 mg·kg~(-1)) could significantly increase IL-4 levels in cultures of thymocytes from the Cy- potentiated DTH mice.
In vitro, GCS (12.5～200μg·ml~(-1)) and Cat (25～200μg·ml~(-1)) could significantly decrease thymocytes proliferation and IL-2 production of thymocytes; GCS (25～200μg·ml~(-1)) and Cat (50～200μg·ml~(-1)) could increase IL-4 levels in cultures of thymocytes from the Cy- potentiated DTH mice.
3. the effects on cell immunity of Cy-suppressed DTH mice by GCS and Cat.
GCS and Cat could enhance DTH response in Cy-suppressed DTH mice. GCS (120, 240 mg·kg~(-1)) and Cat(60,120 mg·kg~(-1)) could significantly increase the ear swelling of Cy-suppressed DTH mice; GCS (60, 120, 240 mg·kg~(-1)) and Cat (60, 120 mg·kg~(-1)) could significantly increase thymus index, spleen index of the Cy- suppressed DTH mice; GCS (60, 120, 240 mg·kg~(-1)) and Cat (60, 120 mg·kg~(-1)) could significantly increase thymocytes proliferation, IL-2 and IL-4 levels in cultures of thymocytes from the Cy-suppressed DTH mice.
In vitro, GCS (12.5～200μg·ml~(-1)) and Cat (25～200μg·ml~(-1)) could significantly increase thymocytes proliferation and IL-4 production of thymocytes; GCS (12.5～200μg·ml~(-1)) and Cat (12.5～200μg·ml~(-1)) could significantly increase IL-2 production of thymocytes.
Conclusion: GCS and Cat might have dual immunomodulatory effects in a function dependent manner in mice with immune dysfunction by regulating functions of immune organ of mice, thymocytes proliferation and the level of cytokine production by thymocytes, and balancing Th1 (IL-2) and Th2 (IL-4), Th and Ts. GCS are active compound in regulating the cell immunity of mice, Cat might regulate cell immunity of mice as one of the active ingredients of GCS.
引文
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