当归饮子加减方对慢性荨麻疹抗过敏作用及机理研究
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摘要
目的：运用当归饮子加减方干预荨麻疹慢性期动物气血两虚症模型,观察养血活血通络法预防荨麻疹复发的作用,并探讨其抗过敏机制,为养血活血通络法的应用以防治慢性皮肤病复发提供科学依据。
方法：采用腹侧皮下注射0.3m1·kg-1利血平生理盐水溶液和皮下注射2%乙酰苯肼制作动物气血两虚模型。气血两虚模型制作成功后,结合Ⅰ-Ⅳ型变态反应模拟慢性荨麻疹发病,以KM小鼠或SD大鼠为受试对象,按体重随机分为空白组、模型组、氯雷他定组、玉屏风组、祛风止痒口服液组、当归饮子高、中、低剂量组分别研究：(1)尾静脉注射1%卵清蛋白合卡介菌疫苗稀释剂致大鼠被动皮肤过敏反应,测定每组动物蓝斑直径、蓝斑光密度(OD)及蓝斑抑制率,分析当归饮子加减方对大鼠被动皮肤过敏的影响。(2)小鼠背部皮下注射组胺,尾静脉注射1%伊文思兰生理盐水溶液,通过测定蓝斑光密度及蓝斑抑制率,分析当归饮子加减方对小鼠毛细血管通透性的影响。(3)尾静脉注射1%卵蛋白生理盐水溶液致大鼠肥大细胞脱颗粒反应,测定各组动物颅顶皮下组织肥大细胞脱颗粒及其抑制率,并对肥大细胞形态进行显微分析,分析当归饮子加减方对大鼠肥大细胞脱颗粒的影响。(4)尾静脉注射低分子右旋糖酐致小鼠全身瘙瘁,记录注射后30min内小鼠搔抓潜伏期、搔抓次数和瘙痒持续总时间,计算瘙痒抑制率；搔抓反应结束后,采用Elisa法测定每只动物血清组胺水平,分析当归饮子加减方对低分子右旋糖酐致小鼠全身瘙瘁的影响。(5)采用DNCB致小鼠耳廓超敏反应,测定每组动物耳廓肿胀度及其抑制率,分析当归饮子加减方对小鼠迟发型变态反应的影响。(6)采用卵蛋白+氢氧化铝合百日咳疫苗致小鼠变态反应,光镜计数外周血嗜酸性粒细胞(EOS)变化；采用Elisa法测试血清IgE水平；免疫组织化学检测血管壁组织中IL-4、IFN-γ表达量；RT-PCR检测血管壁组织IL-4mRNA、IFN-γ mRNA表达情况；血管壁组织的病理切片显微镜检测,研究当归饮子对慢性荨麻疹治疗作用机理。实验数据用SPSS18.0统计软件进行统计分析。计量数据均用(x±s)表示,组间比较用Dunnett t检验；计数资料用X2检验。
结果：当归饮子加减方可显著减小大鼠被动皮肤过敏蓝斑直径(P<0.01),降低蓝斑光密度吸收值(P<0.01)；显著抑制组胺致小鼠毛细血管通透性增高(P<0.01);显著抑制大鼠肥大细胞脱颗粒(P<0.01)；显著延长低分子右旋糖酐致小鼠瘙痒搔抓潜伏期(P<0.01),减少小鼠搔抓次数(P<0.01),缩短搔抓持续时间(P<0.01),降低动物血清组胺水平(P<0.01)；显著对抗DNCB致小鼠迟发型耳廓变态炎症反应(P<0.01)；显著降低致敏动物外周血嗜酸性粒细胞数量(P<0.01),降低动物血清IgE水平(P<0.01),降低致敏动物血管壁组织IL-4水平(P<0.01),升高IFN-γ(P<0.01)水平,下调致敏动物血管壁组织IL-4mRNA(P<0.01)表达,升高IFN-γ mRNA表达(P<0.01),显著降低荨麻疹动物血管壁组织水肿、淋巴滤泡增生及炎细胞浸润。且效果显著优于同等剂量的玉屏风口服液(P<0.05)和祛风止痒口服液(P<0.05)；
结论：当归饮子加减方可显著干预荨麻疹慢性期动物气血两虚症模型,主要表现在：当归饮子加减方可显著对抗气血两虚证动物变态反应,能有效抵抗大鼠被动皮肤过敏、肥大细胞脱颗粒;有效抑制小鼠毛细血管通透性、耳廓肿胀及低分子右旋糖酐致全身瘙痒反应；显著减少致敏动物外周血嗜酸性粒细胞数量,降低动物血清IgE水平,下调致敏动物血管壁组织IL-4水平,升高IFN-γ水平,下调致敏动物血管壁组织IL-4mRNA表达,升高IFN-γ mRNA表达,对气血两虚型慢性荨麻疹具有显著对抗作用。
Objective:Using the Angelica Yin Zi intervented in chronic urticaria of animal of deficiency both blood and qi model, to observe the nourishing blood and collaterals to prevent the recurrence of the role of urticaria, and research the mechanism of its anti-allergy, so as to provide scientific basis for prevention and treatment of chronic skin disease recurrence
Method:The reserpine saline solution0.3ml-kg"'and subcutaneous2%acetyl phenylhydrazine were injected from ventral subcutaneous to induce animal blood and qi deficiency model. After this, Ⅰ-Ⅳ type allergys were combinated to simulate chronic urticaria,then following experiments were studied:(1)1%egg white protein and BCG vaccine diluent were injected into rats intravenous to induce passive cutaneous anaphylaxis, through determinating animals' locus coeruleus, the locus coeruleus optical density (OD) and the locus coeruleus inhibition rate, analyse the effect of Angelica Yinzi on the rat passive cutaneous anaphylaxis.(2) Histamine was injected into mouse subcutaneous.then1%Evans blue saline solution were injected into intravenous, by measuring the optical density of the locus coeruleus and locus coeruleus inhibition rate to analyse Angelica Yinzi effecting on capillary permeability in mice.(3)1%saline solution of egg was injected into rats intravenous of albumin to induce rats" mast cell degranulation reaction, through determinating animals parietal subcutaneous tissue mast cell degranulation and its inhibition rate and the mast cell morphology of the microscopic analysis, to analyse Angelica Yinzi effecting on rats mast cell degranulation.(4) Dextran was injected into mices' intravenous to induce systemic itching, then mices scratching incubation period, scratching frequency and itching continuing time were recorded within30min after the injection, calculation itching inhibition rate; Elisa method was used to determinate histamine level to analyse Angelica Yinzi effecting on mice itching caused by the low molecular weight dextran.(5) DNCB was used to induce hypersensitivity of mouse ear, through determinating animals'ear swelling and the inhibition rate to analyse Angelica Yinzi effecting on mouse delayed type allergy.(6) The egg protein, aluminum hydroxide and pertussis vaccine were injected into mice to induce allergic reaction.then peripheral blood eosinophils (EOS) were counted; Elisa method was used to test serum IgE level; immunohistochemical was used to detect IL-4and IFN-γ in vessel wall tissue;RT-PCR was used to detect IL-4mRNA and IFN-γ mRNA expression of vessel wall tissue:and pathology of microscopic examination of the vessel wall to analyse the mechanism of Angelica Yinzi treating the chronic urticaria.
Result:Angelica Yinzi can significantly reduce blue spot diameter of passive cutaneous allergy rat (P<0.01). reduce the density of blue spots of light absorption values (P<0.01); significantly inhibite capillary permeability in mice (P<0.01);significant inhibit rats'mast cell degranulation (P<0.01); significantly extend the itching scratching incubation period in mice (P<0.01), reduce mice scratching the number (P<0.01).shorten the scratching duration (P<0.01), lower animal serum histamine levels (P<0.01);significantly inhibit mice delayed type auricle caused by DNCB (P<0.01); significant reduce the sensitized animal peripheral blood addicted acidic particles cell number (P<0.01), reduce animal serum IgE levels(P<0.01), reduce the vessel wall tissue IL-4levels (P<0.01), rise of IFN-y levels(P<0.01),reduce the sensitized animal vascular wall tissue of IL-4mRNA expression(P<0.01), increased]FN-y mRNA expression (P<0.01), significantly reduce the the hives animal vascular wall tissue edema, lymphoid follicular hyperplasia and inflammatory cell infiltration. And the results are significantly better than the same dose of Yupingfeng oral solution (P<0.05) and allergy oral solution (P<0.05).
Conclusion:Angelica Yinzi can significantly cure allergic reaction of animals of both blood and qi deficiency, resist rats passive cutaneous anaphylaxis, inhibit mast cell degranulation; inhibit capillary permeability in mice ear edema.itching reaction of the body:significantly reduce the sensitized animal peripheral blood eosinophil number, reduce animal serum IgE levels, and lower the vessel wall tissue of the sensitized animals IL-4levels and increase IFN-γlevels, reduce the sensitized animal vascular wall tissue IL-4mRNA expression and increase IFN-γ mRNA expression, blood deficiency, it has significant treat effect on chronic urticaria.
方法：采用腹侧皮下注射0.3m1·kg-1利血平生理盐水溶液和皮下注射2%乙酰苯肼制作动物气血两虚模型。气血两虚模型制作成功后,结合Ⅰ-Ⅳ型变态反应模拟慢性荨麻疹发病,以KM小鼠或SD大鼠为受试对象,按体重随机分为空白组、模型组、氯雷他定组、玉屏风组、祛风止痒口服液组、当归饮子高、中、低剂量组分别研究：(1)尾静脉注射1%卵清蛋白合卡介菌疫苗稀释剂致大鼠被动皮肤过敏反应,测定每组动物蓝斑直径、蓝斑光密度(OD)及蓝斑抑制率,分析当归饮子加减方对大鼠被动皮肤过敏的影响。(2)小鼠背部皮下注射组胺,尾静脉注射1%伊文思兰生理盐水溶液,通过测定蓝斑光密度及蓝斑抑制率,分析当归饮子加减方对小鼠毛细血管通透性的影响。(3)尾静脉注射1%卵蛋白生理盐水溶液致大鼠肥大细胞脱颗粒反应,测定各组动物颅顶皮下组织肥大细胞脱颗粒及其抑制率,并对肥大细胞形态进行显微分析,分析当归饮子加减方对大鼠肥大细胞脱颗粒的影响。(4)尾静脉注射低分子右旋糖酐致小鼠全身瘙瘁,记录注射后30min内小鼠搔抓潜伏期、搔抓次数和瘙痒持续总时间,计算瘙痒抑制率；搔抓反应结束后,采用Elisa法测定每只动物血清组胺水平,分析当归饮子加减方对低分子右旋糖酐致小鼠全身瘙瘁的影响。(5)采用DNCB致小鼠耳廓超敏反应,测定每组动物耳廓肿胀度及其抑制率,分析当归饮子加减方对小鼠迟发型变态反应的影响。(6)采用卵蛋白+氢氧化铝合百日咳疫苗致小鼠变态反应,光镜计数外周血嗜酸性粒细胞(EOS)变化；采用Elisa法测试血清IgE水平；免疫组织化学检测血管壁组织中IL-4、IFN-γ表达量；RT-PCR检测血管壁组织IL-4mRNA、IFN-γ mRNA表达情况；血管壁组织的病理切片显微镜检测,研究当归饮子对慢性荨麻疹治疗作用机理。实验数据用SPSS18.0统计软件进行统计分析。计量数据均用(x±s)表示,组间比较用Dunnett t检验；计数资料用X2检验。
结果：当归饮子加减方可显著减小大鼠被动皮肤过敏蓝斑直径(P<0.01),降低蓝斑光密度吸收值(P<0.01)；显著抑制组胺致小鼠毛细血管通透性增高(P<0.01);显著抑制大鼠肥大细胞脱颗粒(P<0.01)；显著延长低分子右旋糖酐致小鼠瘙痒搔抓潜伏期(P<0.01),减少小鼠搔抓次数(P<0.01),缩短搔抓持续时间(P<0.01),降低动物血清组胺水平(P<0.01)；显著对抗DNCB致小鼠迟发型耳廓变态炎症反应(P<0.01)；显著降低致敏动物外周血嗜酸性粒细胞数量(P<0.01),降低动物血清IgE水平(P<0.01),降低致敏动物血管壁组织IL-4水平(P<0.01),升高IFN-γ(P<0.01)水平,下调致敏动物血管壁组织IL-4mRNA(P<0.01)表达,升高IFN-γ mRNA表达(P<0.01),显著降低荨麻疹动物血管壁组织水肿、淋巴滤泡增生及炎细胞浸润。且效果显著优于同等剂量的玉屏风口服液(P<0.05)和祛风止痒口服液(P<0.05)；
结论：当归饮子加减方可显著干预荨麻疹慢性期动物气血两虚症模型,主要表现在：当归饮子加减方可显著对抗气血两虚证动物变态反应,能有效抵抗大鼠被动皮肤过敏、肥大细胞脱颗粒;有效抑制小鼠毛细血管通透性、耳廓肿胀及低分子右旋糖酐致全身瘙痒反应；显著减少致敏动物外周血嗜酸性粒细胞数量,降低动物血清IgE水平,下调致敏动物血管壁组织IL-4水平,升高IFN-γ水平,下调致敏动物血管壁组织IL-4mRNA表达,升高IFN-γ mRNA表达,对气血两虚型慢性荨麻疹具有显著对抗作用。
Objective:Using the Angelica Yin Zi intervented in chronic urticaria of animal of deficiency both blood and qi model, to observe the nourishing blood and collaterals to prevent the recurrence of the role of urticaria, and research the mechanism of its anti-allergy, so as to provide scientific basis for prevention and treatment of chronic skin disease recurrence
Method:The reserpine saline solution0.3ml-kg"'and subcutaneous2%acetyl phenylhydrazine were injected from ventral subcutaneous to induce animal blood and qi deficiency model. After this, Ⅰ-Ⅳ type allergys were combinated to simulate chronic urticaria,then following experiments were studied:(1)1%egg white protein and BCG vaccine diluent were injected into rats intravenous to induce passive cutaneous anaphylaxis, through determinating animals' locus coeruleus, the locus coeruleus optical density (OD) and the locus coeruleus inhibition rate, analyse the effect of Angelica Yinzi on the rat passive cutaneous anaphylaxis.(2) Histamine was injected into mouse subcutaneous.then1%Evans blue saline solution were injected into intravenous, by measuring the optical density of the locus coeruleus and locus coeruleus inhibition rate to analyse Angelica Yinzi effecting on capillary permeability in mice.(3)1%saline solution of egg was injected into rats intravenous of albumin to induce rats" mast cell degranulation reaction, through determinating animals parietal subcutaneous tissue mast cell degranulation and its inhibition rate and the mast cell morphology of the microscopic analysis, to analyse Angelica Yinzi effecting on rats mast cell degranulation.(4) Dextran was injected into mices' intravenous to induce systemic itching, then mices scratching incubation period, scratching frequency and itching continuing time were recorded within30min after the injection, calculation itching inhibition rate; Elisa method was used to determinate histamine level to analyse Angelica Yinzi effecting on mice itching caused by the low molecular weight dextran.(5) DNCB was used to induce hypersensitivity of mouse ear, through determinating animals'ear swelling and the inhibition rate to analyse Angelica Yinzi effecting on mouse delayed type allergy.(6) The egg protein, aluminum hydroxide and pertussis vaccine were injected into mice to induce allergic reaction.then peripheral blood eosinophils (EOS) were counted; Elisa method was used to test serum IgE level; immunohistochemical was used to detect IL-4and IFN-γ in vessel wall tissue;RT-PCR was used to detect IL-4mRNA and IFN-γ mRNA expression of vessel wall tissue:and pathology of microscopic examination of the vessel wall to analyse the mechanism of Angelica Yinzi treating the chronic urticaria.
Result:Angelica Yinzi can significantly reduce blue spot diameter of passive cutaneous allergy rat (P<0.01). reduce the density of blue spots of light absorption values (P<0.01); significantly inhibite capillary permeability in mice (P<0.01);significant inhibit rats'mast cell degranulation (P<0.01); significantly extend the itching scratching incubation period in mice (P<0.01), reduce mice scratching the number (P<0.01).shorten the scratching duration (P<0.01), lower animal serum histamine levels (P<0.01);significantly inhibit mice delayed type auricle caused by DNCB (P<0.01); significant reduce the sensitized animal peripheral blood addicted acidic particles cell number (P<0.01), reduce animal serum IgE levels(P<0.01), reduce the vessel wall tissue IL-4levels (P<0.01), rise of IFN-y levels(P<0.01),reduce the sensitized animal vascular wall tissue of IL-4mRNA expression(P<0.01), increased]FN-y mRNA expression (P<0.01), significantly reduce the the hives animal vascular wall tissue edema, lymphoid follicular hyperplasia and inflammatory cell infiltration. And the results are significantly better than the same dose of Yupingfeng oral solution (P<0.05) and allergy oral solution (P<0.05).
Conclusion:Angelica Yinzi can significantly cure allergic reaction of animals of both blood and qi deficiency, resist rats passive cutaneous anaphylaxis, inhibit mast cell degranulation; inhibit capillary permeability in mice ear edema.itching reaction of the body:significantly reduce the sensitized animal peripheral blood eosinophil number, reduce animal serum IgE levels, and lower the vessel wall tissue of the sensitized animals IL-4levels and increase IFN-γlevels, reduce the sensitized animal vascular wall tissue IL-4mRNA expression and increase IFN-γ mRNA expression, blood deficiency, it has significant treat effect on chronic urticaria.
引文
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