养血消风饮对变应性接触性皮炎的作用及免疫学机制研究

【作者】 芦源；

【导师】 关洪全；

【作者基本信息】 辽宁中医药大学 ， 中西医结合基础， 2009， 博士

【摘要】 目的:利用小鼠急、慢性变应性接触性皮炎(ACD)模型,通过动物的病理改变、组织形态学改变评价养血消风饮对急、慢性ACD的药理作用;并通过对相关细胞因及其mRNA、细胞凋亡等的检测,从细胞水平、分子水平对其免疫学机制进行探索,为临床更好地应用该方防治急、慢性ACD提供客观依据。材料与方法1.实验材料(1)实验动物:继康雄性昆明种小鼠。(2)实验用药:均采用灌胃给药的方法。中药治疗组采用养血消风饮水煎剂;西药对照组采用氯雷他定片,用蒸馏水溶解制成混浊液;空白组、模型组用等量蒸馏水灌胃。2.研究方法(1)造模方法:采用DNCB皮肤涂抹的方法分别制备小鼠急、慢性ACD动物模型。(2)通过观察养血消风饮对急、慢性ACD小鼠病变耳肿胀度、耳肿胀抑制百分率及脾指数和胸腺指数的影响,初步评价其对小鼠ACD的抑制。(3)通过光镜及透射电镜观察养血消风饮对急性ACD小鼠病变局部组织病理以及超微结构的影响,进一步分析养血消风饮对急性ACD病变组织形态学的影响。(4)采用ELISA法测定慢性ACD模型小鼠血清中IFN-γ、IL-4的含量,采用RT-PCR方法检测病变组织局部IFN-γmRNA、IL-4mRNA的表达情况,并进行血清中细胞因子的水平与病变组织中mRNA表达的相关分析,从Th1/Th2细胞因子网络平衡的角度进一步深入探讨养血消风饮抑制ACD的免疫学机制。(5)采用免疫组化的方法,检测慢性ACD模型小鼠病变组织中细胞因子TNF-α、NF-κBp50、Fas/FasL含量,通过研究慢性ACD发病过程中皮损部位上述细胞因子表达的变化,探求养血消风饮可能的作用机制和环节。(6)采用Annexin V-FITC细胞凋亡检测方法,用流式细胞仪对慢性ACD小鼠脾淋巴细胞早期凋亡百分率进行研究,从细胞水平探索养血消风饮能否通过诱导淋巴细胞凋亡下调免疫应答强度,进而抑制ACD的发生。结果1.养血消风饮可下调急、慢性ACD模型小鼠病变耳的重量差和肿胀度急、慢性ACD模型组小鼠的耳重量差值均明显高于空白组(P＜0.01),养血消风饮低、中、高剂量组和氯雷他定组的耳重量差与模型组相比均明显减小(P＜0.01或P＜0.05),在本实验所采用药量范围内,养血消风饮对急性ACD模型的作用无明显量-效依赖关系,对慢性ACD模型的作用呈一定的量-效依赖关系。2.养血消风饮可下调急、慢性ACD模型小鼠的脾指数及胸腺指数急、慢性ACD模型组小鼠的脾指数、胸腺指数均明显升高(P＜0.01);养血消风饮组和氯雷他定均可显著下调急、慢性ACD模型小鼠的脾指数及胸腺指数(P＜0.01);养血消风饮对二者的影响均呈现一定的量-效依赖关系。3.养血消风饮可减轻急性ACD模型小鼠病变局部组织病理及超微结构的损伤程度ACD模型组小鼠背部皮肤的组织病理切片显示:表皮部分坏死缺失,细胞间、细胞内水肿;真皮乳头水肿,真皮内大量炎细胞浸润;胶原纤维间隙增宽;血管扩张,内皮细胞肿胀。用养血消风饮干预后,其皮肤全层结构基本完整,细胞内和细胞间水肿明显减轻,真皮内炎性细胞浸润明显减少。各层损害均较ACD模型组明显减轻。氯雷他定组真皮内炎性细胞浸润数目较养血消风饮组高,其他改变与养血消风饮组大致相同。在皮肤超微结构的研究中,ACD模型组小鼠病变皮肤基底细胞、棘细胞均出现萎缩,表皮角质细胞电子密度略增强,基底细胞间连接消失,胞质内细胞器空泡化,细胞核内异染色质浓缩成团块状并趋向核周边化分布;养血消风饮组小鼠病变皮肤基底细胞与棘细胞结构完整,细胞轻度萎缩,细胞间以桥粒连结,核内染色质基本呈均匀分布,线粒体和粗面内质网结构基本完整,空泡化不明显,细胞损伤较模型组明显减轻。氯雷他定组核内染色质部分呈团块状分布,其他改变与养血消风饮组大致相同。4.养血消风饮可上调ACD模型小鼠血清中IL-4的含量及下调IFN-γ的含量,同时可上调耳组织中IL-4mRNA的表达水平,下调IFN-γmRNA的表达水平慢性ACD模型小鼠血清中IFN-γ的水平明显升高而IL-4的水平明显降低(P＜0.01),病变耳组织中有IFN-γmRNA的高表达而IL-4mRNA的表达减少(P＜0.01),模型组小鼠的血清IFN-γ/IL-4比值与耳组织中IFN-γmRNA/IL-4mRNA比值均明显升高(P＜0.01);养血消风饮或氯雷他定均可明显下调ACD小鼠血清中IFN-γ水平以及耳组织中IFN-γmRNA的表达水平(P＜0.01);养血消风饮还可明显上调血清中IL-4水平(P＜0.05)以及耳组织中IL-4mRNA的表达水平(P＜0.01),氯雷他定则上调血清中IL-4水平而对病变耳组织中IL-4mRNA的表达水平无影响;养血消风饮或氯雷他定均可显著降低IFN-γ/IL-4以及IFN-γmRNA/IL-4mRNA的比值(P＜0.01);养血消风饮对IFN-γmRNA/IL-4mRNA比值的影响优于氯雷他定组,而对IFN-γ/IL-4比值的影响二者无显著差异。5.养血消风饮可明显下调慢性ACD小鼠病变皮肤组织中TNF-α、NF-κBp50的表达水平,并明显上调Fas和FasL的表达慢性ACD小鼠耳组织TNF-α、NF-κBp50表达均明显增加(P＜0.01);养血消风饮可以明显下调组织中TNF-α、NF-κBp50表达(P＜0.01);氯雷他定组对组织中TNF-α表达的影响不明显(P＞0.05),但可以明显下调NF-κBp50表达(P＜0.05);养血消风饮组的作用明显优于氯雷他定组(P＜0.05或P＜0.01)。慢性ACD小鼠耳组织Fas、FasL表达均明显增加(P＜0.01);养血消风饮可以明显上调组织中Fas、FasL表达(P＜0.01):氯雷他定组对二者的影响均不明显(P＞0.05)。小鼠耳廓皮肤组织中TNF-α与NF-κBp50、Fas与FasL的表达分别呈正相关(相关系数分别为:0.783,P＜0.01:0.747,P＜0.01)。6.养血消风饮可明显促进慢性ACD小鼠脾淋巴细胞早期凋亡模型小鼠脾淋巴细胞早期凋亡的百分率明显增加(P＜0.01);养血消风饮或氯雷他定均可明显上调小鼠脾淋巴细胞早期凋亡的百分率,养血消风饮组的作用强于氯雷他定组(P＜0.01)。小鼠脾淋巴细胞早期凋亡的百分率与小鼠耳组织Fas、FasL的表达分别呈正相关(相关系数分别为:0.783,P＜0.01;0.785,P＜0.01)。结论1.养血消风饮对小鼠急、慢性ACD均有抑制作用,能减轻其耳廓的肿胀度,并能够抑制病变过程中免疫器官的肿大反应,下调胸腺指数和脾指数;2.养血消风饮能够减轻急性ACD小鼠病变局部组织病理及超微结构损伤的程度;3.养血消风饮可抑制慢性ACD小鼠Th1型细胞因子、促进Th2型细胞因子的分泌,从而调节Th1/Th2网络平衡:4.养血消风饮可明显下调慢性ACD小鼠病变皮肤组织中TNF-α、NF-κBp50的表达水平,可能在转录水平抑制炎症的发生,抑制免疫反应;5.养血消风饮可以明显上调慢性ACD小鼠病变皮肤组织中Fas和FasL的表达,促进小鼠脾淋巴细胞凋亡。更多还原

【Abstract】 Objective:This thesis evaluates the pharmacological effects of experienced prescription of the wind Yangxue drink on acute and chronic ACD from the perspective of pathological changes and histomorphology by adopting mice of acute and chronic allergic contact dermatitis(ACD) model,probes immunological mechanism on the level of cells and molecules by examines the relevant cells and its mRNA,so that to provide objective basis for the application of this experience experienced prescription’s better prevention and treatment of acute and chronic ACD clinically.Materials and Methods1.Experimental materials(1) Experimental animals:Healthy male Kunming mice.(2) Experimental medicine:The method of oral drug delivery is used.The experienced prescription of Yangxue Xiaofeng Drink is adopted in Chinese medical treatment and the control group of Western medicine used loratadine tablets,dissolved with distilled water made turbid liquid;blank group,model group gavage with distilled water contour.2.Research Methods(1)Model approach:To use the methods of DNCB skin to prepare acute and chronic ACD animal models of mice.(2)To make preliminary evaluation of effect of ACD- inhibition by observing the Yangxue Xiaofeng Drink on acute and chronic ACD ear swelling disease in mice,the percentage of swelling inhibition,and spleen index and thymus index.(3)To analyze the effects of Yangxue Xiaofeng Drink on acute acute lesions histomorphology through the light microscope and transmission electron microscopy observation of Yangxue Xiaofeng Drink lesions of acute ACD in mice and the pathological changes in tissue ultrastructure.(4)To determine the levels of IFN-γand IL-4 in the serum of ACD mouse model by using ELISA method,using RT-PCR used to detect lesions local IFN-γmRNA,IL-4mRNA expression and serum cytokine protein expression and mRNA expression in lesions of the correlation analysis,from the Th1 / Th2 cytokine balance in the perspective of the network to explore further in-depth Yangxue Xiaofeng Drink ACD drink immunological inhibition mechanism.(5)To detect content of TNF-α,NF-κBp50,Fas / Fas-L of chronic lesions of cytokines in ACD mouse model by using immunohistochemical methods,and explore the possible role of drinking wind mechanisms and links by studying the pathogenesis of chronic skin lesions of ACD positions cytokine expression in the above-mentioned changes in consumer Yangxue.(6)To study the percentage of early apoptosis by Annexin V-FITC apoptosis detection method. and using flow cytometry in chronic ACD mouse spleen lymphocytes,and to explore whether drinking Yangxue Xiaofeng Drink reduced intensity of immune response,thereby inhibiting the occurrence of ACD from cell level by inducing lymphoid ways of apoptosis.Results1.Yangxue Xiaofeng Drink can reduce acute and chronic ear disease model mice ACD weight difference and swelling degree.Acute and chronic ACD model of the ears of mice were significantly higher than the weight difference between the blank group(P＜0.01),Yangxue Wind-drink low-,medium-, high-dose group and the group of loratadine ear weight difference with the model group were significantly reduced compared to(P＜0.01 or P＜0.05).With the range of this experiment dose used,Yangxue Xiaofeng Drink on the role of acute ACD model no significang amount- effect dependence on the role of chronic ACD model was a certain amount - effect dependence.2.Yangxue Xiaofeng Drink can be reduced acute and chronic mouse model of ACD spleen index and thymus index.Acute and chronic ACD model group mice spleen index,thymus index were significantly higher(P＜0.01):Yangxue Xiaofeng Drinking group and loratadine can be significantly reduced acute and chronic mouse model of ACD and the spleen index Thymus index(P＜0.01);Yangxue Xiaofeng Drink on the impact of both showed a certain amount - effect dependence.3.Yangxue Xiaofeng Drinking mice reduce acute ACD partial lesion histopathology and ultrastructure of the extent of injuryThe mice back skin biopsy of ACD model group showed:loss of partial necrosis of epidermal cells,the cells edema;dermal papilla edema,dermal infiltration of inflammatory cells in large numbers;gap widened collagen fibers;vasodilator,endothelial cell swelling. After the intervention of Yangxue Xiaofeng Drink,the basic structure of full-thickness skin integrity,cells and intercellular edema significantly reduced and inflammatory cell infiltration in the dermis decreased significantly.All levels of damage are less than those of the ACD model group.The number of dermal inflammatory cell infiltration in Loratadine group is more than that of Yangxue Xiaofeng Drink group,and other changes is about the same with Yangxue Xiaofeng Drink group.In the study of ultrastructure of the skin,basal cell skin lesions and spine cells of ACD mouse model shrink and the electron density of epidermal keratinocyte slightly enhanced, inter-connection of epidermal basal cells disappear and within the cell,cytoplasm was vacuolated changes nuclear heterochromatin condensed into a mass distribution to the nuclear periphery.The lesions basal cell skin and spine cells of Yangxue Xiaofeng Drinking mice kept structural integrity,cells with mild atrophy,intercellular is liked with desmosomes and the nuclear chromatin basically uniform distribution.Mitochondria and rough endoplasmic reticulum are of structure integrity,little vacuolization,and cell injury is less than the model group.Loratadine group part of the nuclear chromatin was mass distribution,and other changes is about the same with Yangxue Xiaofeng Drink group.4.Yangxue Xiaofeng Drink can raised level of IL-4 and reduced levels of IFN-γin serum of ACD model,while increase expression level of of IL-4mRNA reduced the expression level of IFN-γmRNA in ear tissue.Levels of IFN-γsignificantly increased in serum of chronic ACD mouse model and the level of IL-4 decreased significantly(P＜0.01),there is high expression level of IFN-γmRNA in ear lesions organizations and expression level of IL-4mRNA reduced expression(P＜0.01).The ratio of serum IFN-γ/IL-4 ear tissues were significantly higher IFN-γmRNA/IL-4mRNA ratio(P＜0.01) in model mice group;Both Yangxue Xiaofeng Drink and loratadine ACD in mice significantly reduced serum levels of IFN-γas well as the ear tissue of IFN-γmRNA expression level(P＜0.01);Yangxue Xiaofeng Drink can significantly increase serum IL-4 levels(P＜0.05) as well as the ears tissue expression of IL-4mRNA level (P＜0.01),while loratadine increase serum levels of IL-4 on the ear lesion tissue expression of IL-4mRNA the level of no effect;Yangxue Xiaofeng Drink or loratadine group,while the impact of the ratio of IFN-γ/IL-4 is of no significant difference between the two groups.5.Yangxue Xiaofeng Drink ACD significantly reduced expression levels of TNF-α.and NF-κBp50 in chronic skin lesions of mice,and markedly increase that of Fas and FasL. TNF-αand NF-κBp50 expression were significantly increased(P＜0.01) in chronic ear organizations of ACD mouse model;Yangxue Xiaofeng Drink organizations can significantly lower TNF-α,NF-κBp50 expression(P＜0.01);TNF-αexpression was not obvious(P＞0.05) in tissue of Loratadine group,but can significantly reduced NF-κBp50 expression(P＜0.05); and the interference role of Yangxue Xiaofeng Drink is superior to that of loratadine group(P＜0.05 or P＜0.01).Fas and FasL expression were significantly increased(P＜0.01) in chronic ear organizations of ACD mouse model;Yangxue Xiaofeng Drink organizations can significantly increase Fas and FasL expression(P＜0.01);and loratadine group has no significant effects on the two(P＞0.05).TNF-αand NF-κBp50,Fas and Fas-L expression in ear skin of mice were positively correlated(correlation coefficients were:0.783,P＜0.01;0.747,P＜0.01).6.Yangxue Xiaofeng Drink chronic drinking can significantly promote the ACD mouse spleen T lymphocytes in early apoptosis.Percentage of early apoptosis of lymphocytes is significantly increased in the spleen of model of mouse(P＜0.01);Yangxue Xiaofeng Drink or loratadine may significantly increase percentage of early apoptosis of lymphocytes in the mouse spleen,Yangxue Xiaofeng Drinking group was stronger than the loratadine group(P＜0.01).Percentage of early apoptosis of lymphocytes in Mouse spleen and Fas.Fas-L expression in mouse ears organizations were positively correlated(correlation coefficients were:0.783.P＜0.01;0.785,P＜0.01).Conclusion:1.Yangxue Xiaofeng Drink has inhibition effect on acute and chronic ACD of mice and can reduce the swelling of the ear,inhibit the swelling response of immune organs in the course of changes and reduce thymus index and spleen index.2.Yangxue Xiaofeng Drink can effectively reduce the injury degree of ultrastructure and local tissue pathological lesions of acute ACD group in mice.3.Yangxue Xiaofeng can lower the cytokine of Th1 and promote the secreting of the cytokine of Th2,so that adjust network Th1/Th2 balance of mouse model of chronic ACD.4.Yangxue Xiaofeng Drink can significantly lower the TNF-α,NF-κBp50 expression levels in skin lesions of chronic ACD mice,and may inhibit inflammation at the transcriptional level and suppress immune response.5.Yangxue Xiaofeng Drink can significantly promote Fas / FasL expression level in the Fas / FasL expression level and promote apoptosis of lymphocytes in mouse spleen.更多还原