多因素复合作用与维医异常黑胆质证候模型研究
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摘要
目的:研究多因素复合作用与维医异常黑胆质证侯模型及其免疫与下丘脑-垂体-肾上腺轴功能紊乱的关系。方法:选用健康雄性ICR小鼠45只,随机分成正常对照组、双因素组和三因素组。双因素组采用间断足底电击作为应激源,输出电压40±5V,不定时改变电压,间隔0.2～0.5s,每天连续刺激20min,并置于人工气候箱干寒(温度10±1℃,相对湿度36～40%)的饲养环境10小时/天,予以正常的普通饲料喂养,产生慢性应激共21天。三因素组采用间断足底电击作为应激源,置于人工气候箱干寒(温度10±1℃,相对湿度36～40%)的饲养环境10小时/天,并给予干寒属性饲料(按每公斤普通小鼠饲料中加入大麦150mg和芫荽籽150mg,然后制成颗粒状干饲料)喂养,共21天。观察各组小鼠生物表征的变化,第21天并取实验动物外周血应用流式细胞仪测定小鼠外周血CD4+、CD8+细胞百分比含量,ELISA法测定小鼠血清IL-2、IL-6、IFN-γ、TNF-α、IgM、IgG和IgA含量以及血清中CRH、ACTH、CORT的含量。结果:与正常组相比,三因素组实验动物第21天出现舌干暗紫,有瘀斑,少苔,皮毛黯淡无光泽,蜷缩少动,对刺激敏感度降低,反应迟缓,倦怠嗜睡,易惊,争食饮水,大便成形,小便色浅黄。外周血T淋巴细胞亚群CD4+细胞百分比与CD8+细胞百分比明显下降(P<0.01),CD4+/CD8+比值下降(P<0.05);血清中IL-2的含量与INF-γ含量明显降低(P<0.01),IL-6的含量与TNF-α的含量明显升高(P<0.01);血清中IgM的含量与IgG的含量明显升高(P<0.01);而血清中IgA含量明显降低(P<0.01);血清中CRH含量、ACTH含量、CORT含量明显升高(P<0.01)。双因素组实验动物第21天出现舌有瘀斑苔滑;皮毛暗淡无光泽、蜷缩少动,扎堆眯眼,倦怠淡漠,对刺激敏感度降低,反应迟缓,争食饮水,大便成形而湿,小便色清。外周血T淋巴细胞亚群CD4+细胞百分比下降(P<0.01),CD8+细胞百分比未见明显差异(P>0.05),CD4+/CD8+比值下降(P<0.01);血清中IL-2的含量明显低于正常对照组(P<0.01),血清中IL-6的含量与正常对照组相比未见明显差异(P>0.05),血清中INF-γ含量与正常对照组相比明显降低(P<0.01),血清中TNF-α的含量与正常对照组明显升高(P<0.01)。血清中IgM的含量与正常组相比明显升高(P<0.01),IgG的含量明显高于正常对照组(P<0.01),IgA含量明显低于正常对照组(P<0.01)。而血清中CRH含量、ACTH含量、CORT含量明显升高(P<0.01)。与三因素组相比,双因素组实验动物外周血T淋巴细胞亚群CD4+细胞百分比未见显著差异,CD8+细胞百分比上升(P<0.01),CD4+/CD8+比值下降(P<0.05)。血清中IL-2含量升高(P<0.05);IL-6含量明显降低(P<0.01);IFN-γ含量与TNF-α的含量未见显著性差异(P>0.05);血清中IgM的含量明显降低(P<0.01);IgG的含量与IgA含量未见明显差异(P>0.05);其血清中CRH含量、ACTH含量、CORT含量明显低于三因素组(P<0.01)。结论:⑴与正常对照组比较,三因素复合作用后模型动物表现出的生物表征变化更加符合人体异常黑胆质证侯临床表现,三因素复合作用组不仅有生物表征的改变,同时有免疫指标的改变,以及下丘脑—垂体—肾上腺轴功能的紊乱。⑵双因素复合作用模型动物在相同时间段表现出的生物表征与三因素复合作用组相比有程度上的差异,且尚不完全符合人体异常黑胆质证侯临床表现。其下丘脑—垂体—肾上腺轴功能亢进以及细胞免疫和体液免疫功能紊乱程度明显低于三因素组。⑶以往研究和有关文献可知干寒环境、不良精神刺激、干寒属性食物等多因素复合作用所建立的异常黑胆质证侯动物模型不仅符合人体异常黑胆质证侯临床表现,而且具有稳定性、可靠性和可重复性。
Objective: Study the relationship between co-effect of multi–factor and establishment of the model of abnormal savda syndrome in Uighur medicine, observe the immunological indicators and the changes of functional indicators of Hypothalamus-pituitary-adrenal axis. Methods: 45 healthy male ICR mice were randomly divided into normal control group, twi-factor group, and tri-factor group. Twi-factor group was stressed with intermittent foot shock, the output voltage 40±5V, changing the voltage from time to time, interval 0.2～0.5s, continuous stimulation 20mins everyday, and placed in cold Phytotron (temperature 10±1℃, relative humidity 36～40%) for 10 hours/day, and finally made it produce chronic stress. Tri-factor group was stressed with intermittent foot shock in cold Phytotron (temperature 10±1℃, relative humidity 36～40%) for 10 hours/day, and were fed dry and cool food, and the period lasted 21 days. We observed the change of biological characterization of each group, and the cell percentages of CD4+and CD8+in experimental mice’s peripheral blood were measured by flow cytometry, and the contents of IL-2, IL-6, IFN-γ, TNF-α, IgM, IgG, IgA, CRH, ACTH and CORT in the miceˊ s serum were examined by ELISA on the 21th day. Results: Compared to control group, tri-factor group showed petechiae, small moss, dark purple of tongue, fur lackluster, curled, reduced sensitivity to stimulation, slow response;lassitude somnolence, frequent arousals, more diet, more drink, forming stool, cervine urine on the 21th day. The percentage of CD4+ and the percentage of CD8+decreased (P < 0.01), the CD4+/CD8+ratio decreased (P<0.05);in peripheral blood:the contents of IFN-γand IL-2 decreased significantly in serum (P<0.01), TNF-αand IL-6 increased significantly (P<0.01), IgM and IgG increased significantly (P < 0.01), however, IgA decreased significantly (P<0.01), CRH?ACTH and CORT increased significantly (P<0.01). Twi-factor group showed lavender apex of tongue; fur lackluster, curled, tiredness indifferent, reduced sensitivity to stimulation, slow to react, more diet, more drink, forming and stool wet, pale colour of urine on the 21th day. The percentage of CD4+decreased (P<0.01), The percentage of CD8+ had no significant difference (P>0.05), CD4+/CD8+ratio decreased (P<0.01) in peripheral blood; compared to normal control group, the content of IL-2 in serum decreased significantly (P<0.01), IL-6 had no significant difference (P>0.05), INF-γdecreased significantly (P<0.01), TNF-αincreased significantly (P<0.01), IgM and IgG increased significantly (P<0.01), IgA decreased significantly (P<0.01), however, CRH?ACTH and CORT increased significantly (P<0.01). Compared to tri-factor group, the percentage of CD4+of twi-factor group in peripheral blood had no significant difference, the percentage of CD8+increased (P<0.01), CD4+/CD8+ratio decreased (P<0.05). The content of IL-2 increased in serum (P<0.05), IL-6 decreased significantly (P<0.01), IFN-γand TNF-αhad no significant difference (P>0.05), and CRH?ACTH and CORT decreased significantly (P<0.01). Conclusions:⑴Compared to normal control group, the changes in the biological characterization of tri-factor group were closer to the clinical manifestations of abnormal savda syndrome in humans, tri-factor group had not only the changes of biological characterization, but also the changes of immune parameters and Hypothalamus-pituitary- adrenal axis function disorder simultaneously.⑵The extent of the changes of biological characterization showed by twi-factor model group was lower than that of tri-factor model group during the same period, and the changes of biological characterization was not entirely consistent with the clinical manifestations of abnormal savda syndrome in humans. At the same time, the extent of Hypothalamus-pituitary-adrenal axis hyperfunction, as well as cellular and humoral immunity dysfunction was lower than that of tri-factor group.⑶The model established by co-effect of multi-factor of dry and cool environment, hostile psych-stimulation, dry and cool food and so on was not only consistent with the clinical manifestations of abnormal savda syndrome in humans, but also stable and reliable.
Objective: Study the relationship between co-effect of multi–factor and establishment of the model of abnormal savda syndrome in Uighur medicine, observe the immunological indicators and the changes of functional indicators of Hypothalamus-pituitary-adrenal axis. Methods: 45 healthy male ICR mice were randomly divided into normal control group, twi-factor group, and tri-factor group. Twi-factor group was stressed with intermittent foot shock, the output voltage 40±5V, changing the voltage from time to time, interval 0.2～0.5s, continuous stimulation 20mins everyday, and placed in cold Phytotron (temperature 10±1℃, relative humidity 36～40%) for 10 hours/day, and finally made it produce chronic stress. Tri-factor group was stressed with intermittent foot shock in cold Phytotron (temperature 10±1℃, relative humidity 36～40%) for 10 hours/day, and were fed dry and cool food, and the period lasted 21 days. We observed the change of biological characterization of each group, and the cell percentages of CD4+and CD8+in experimental mice’s peripheral blood were measured by flow cytometry, and the contents of IL-2, IL-6, IFN-γ, TNF-α, IgM, IgG, IgA, CRH, ACTH and CORT in the miceˊ s serum were examined by ELISA on the 21th day. Results: Compared to control group, tri-factor group showed petechiae, small moss, dark purple of tongue, fur lackluster, curled, reduced sensitivity to stimulation, slow response;lassitude somnolence, frequent arousals, more diet, more drink, forming stool, cervine urine on the 21th day. The percentage of CD4+ and the percentage of CD8+decreased (P < 0.01), the CD4+/CD8+ratio decreased (P<0.05);in peripheral blood:the contents of IFN-γand IL-2 decreased significantly in serum (P<0.01), TNF-αand IL-6 increased significantly (P<0.01), IgM and IgG increased significantly (P < 0.01), however, IgA decreased significantly (P<0.01), CRH?ACTH and CORT increased significantly (P<0.01). Twi-factor group showed lavender apex of tongue; fur lackluster, curled, tiredness indifferent, reduced sensitivity to stimulation, slow to react, more diet, more drink, forming and stool wet, pale colour of urine on the 21th day. The percentage of CD4+decreased (P<0.01), The percentage of CD8+ had no significant difference (P>0.05), CD4+/CD8+ratio decreased (P<0.01) in peripheral blood; compared to normal control group, the content of IL-2 in serum decreased significantly (P<0.01), IL-6 had no significant difference (P>0.05), INF-γdecreased significantly (P<0.01), TNF-αincreased significantly (P<0.01), IgM and IgG increased significantly (P<0.01), IgA decreased significantly (P<0.01), however, CRH?ACTH and CORT increased significantly (P<0.01). Compared to tri-factor group, the percentage of CD4+of twi-factor group in peripheral blood had no significant difference, the percentage of CD8+increased (P<0.01), CD4+/CD8+ratio decreased (P<0.05). The content of IL-2 increased in serum (P<0.05), IL-6 decreased significantly (P<0.01), IFN-γand TNF-αhad no significant difference (P>0.05), and CRH?ACTH and CORT decreased significantly (P<0.01). Conclusions:⑴Compared to normal control group, the changes in the biological characterization of tri-factor group were closer to the clinical manifestations of abnormal savda syndrome in humans, tri-factor group had not only the changes of biological characterization, but also the changes of immune parameters and Hypothalamus-pituitary- adrenal axis function disorder simultaneously.⑵The extent of the changes of biological characterization showed by twi-factor model group was lower than that of tri-factor model group during the same period, and the changes of biological characterization was not entirely consistent with the clinical manifestations of abnormal savda syndrome in humans. At the same time, the extent of Hypothalamus-pituitary-adrenal axis hyperfunction, as well as cellular and humoral immunity dysfunction was lower than that of tri-factor group.⑶The model established by co-effect of multi-factor of dry and cool environment, hostile psych-stimulation, dry and cool food and so on was not only consistent with the clinical manifestations of abnormal savda syndrome in humans, but also stable and reliable.
引文
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