健脾养血祛风方对脾虚证慢性湿疹小鼠模型作用的实验研究
发布时间：2018-09-01 20:54
【摘要】：目的:探讨健脾养血祛风方对脾虚证慢性湿疹小鼠模型皮肤屏障功能及免疫机制的影响,为健脾养血祛风方治疗脾虚证慢性湿疹提供实验依据,从而进一步论证将“健脾养血祛风法”作为脾虚证慢性湿疹中医治疗手段的可行性和重要性。材料与方法:1材料:1.1实验动物及分组:健康雄性昆明种SPF级小鼠50只,随机分为空白对照组,脾虚证慢性湿疹模型组,脾虚证慢性湿疹西药治疗组,脾虚证慢性湿疹中药高剂量组,脾虚证慢性湿疹中药低剂量组。1.2实验用药:灌胃方式给药。中药治疗组采用健脾养血祛风方水煎剂;西药对照组采用盐酸左西替利嗪片,用蒸馏水溶解制成混浊液;空白组及模型组予等量生理盐水灌胃。2方法:2.1造模方法:先予小剂量反复DNCB涂抹建立慢性湿疹模型,后加入番泻叶致脾虚证模型,形成脾虚证慢性湿疹的病证结合动物模型。2.2实验方法:第一部分:健脾养血祛风方对脾虚证慢性湿疹小鼠模型炎症反应的影响(1)健脾养血祛风方对脾虚证慢性湿疹小鼠模型耳肿胀度、耳厚度差的影响:末次给药后24h,处死小鼠,剪下小鼠左右耳廓备检。用6mm打孔器取同部位的左右两耳片,用微测量卡尺测定小鼠耳中部的厚度,计算左右耳厚度差;电子天平称重耳片重量,左右耳片重量差为耳肿胀度。(2)健脾养血祛风方对脾虚证慢性湿疹小鼠模型脾脏指数、胸腺指数的影响:处死小鼠后,取出完整脾脏,去除筋膜、脂肪组织,电子天平称重得小鼠脾脏重量。每鼠的脾脏重量除以每鼠的体质量得脾脏指数。胸腺指数计算同脾脏指数。(3)健脾养血祛风方对脾虚证慢性湿疹小鼠模型耳部病变组织病理的影响:于处死小鼠耳部病变组织取皮肤组织块,固定,HE常规染色,光学显微镜下观察各组动物耳部皮肤组织病理学改变。第二部分:健脾养血祛风方对脾虚证慢性湿疹小鼠模型皮肤屏障功能的影响(1)健脾养血祛风方对脾虚证慢性湿疹小鼠模型背部皮肤组织中水通道蛋白3表达的影响:采用免疫组化方法检测小鼠背部皮肤组织AQP3表达水平,应用北京航空大学图像采集和分析系统,测定阳性细胞在单位面积表达的平均光密度值判定其在组织中表达情况。(2)健脾养血祛风方对脾虚证慢性湿疹小鼠模型背部皮肤组织中博来霉素水解酶表达的影响:采用免疫组化方法检测小鼠背部皮肤组织BH(博来霉素水解酶)的表达水平。具体方法同水通道蛋白3检测。第三部分:健脾养血祛风方对脾虚证慢性湿疹小鼠模型免疫机制的调节(1)健脾养血祛风方对脾虚证慢性湿疹小鼠模型脾T细胞亚群CD4+、CD8+及其比值的影响:采用流式细胞仪检测小鼠脾细胞CD4+、CD8+水平并计算其比值。(2)健脾养血祛风方对脾虚证慢性湿疹小鼠模型耳部皮肤组织中细胞间粘附分子-1(I C A M-1)及血管细胞间粘附分子-1(V C A M-1)表达的影响:采用免疫组化方法观察小鼠耳部皮肤组织中细胞间粘附分子-1(ICAM-1)及血管细胞间粘附分子-1(VCAM-1)的表达。(3)健脾养血祛风方对脾虚证慢性湿疹小鼠模型背部皮肤组织中半胱氨酰白三烯受体1(Cys LTR1)、半胱氨酰白三烯受体2(Cys LTR2)蛋白表达的影响:采用免疫组化方法(WB法)检测小鼠背部皮肤组织中半胱氨酰白三烯受体Cys LTRl和Cys LTR2的表达。(4)健脾养血祛风方对脾虚证慢性湿疹小鼠模型趋化因子CCL17、CCL18血清水平及背部皮肤组织中表达的影响:采用ELISA法检测小鼠血清嗜酸性细胞活化趋化因子CCLl7、CCLl8水平;采用RT-PCR法检测小鼠背部皮肤组织中CCLl7、CCLl8的表达。结果:第一部分:健脾养血祛风方对脾虚证慢性湿疹小鼠模型炎症反应的抑制(1)健脾养血祛风方可减轻小鼠模型耳肿胀度,缩小耳厚度差。与空白组比较,模型组及各给药组小鼠均发生不同程度的耳肿胀,耳厚度差增加,差异具有统计学意义(P0.05);与模型组比较,各治疗组能明显减轻小鼠耳肿胀,使耳厚度差减少,差异具有统计学意义(P0.01);西药组与中药各剂量组比较,耳肿胀度及耳厚度差无显著性差异(P0.05)。(2)健脾养血祛风方可降低小鼠模型的脾脏指数及胸腺指数。与空白组比较,模型组及各给药组小鼠脾脏脂数及胸腺指数均有不同程度的升高,差异具有统计学意义(P0.01);与模型组比较,各治疗组脾脏指数及胸腺指数均明显下降,差异具有统计学意义(P0.05);西药组与中药各剂量组比较,脾脏指数及胸腺指数下降程度无显著性差异(P0.05)。(3)健脾养血祛风方可抑制小鼠模型耳部皮损处的炎症反应,能改善慢性湿疹皮损的病理结构。空白组:表皮无角化过度、增厚及海绵形成,真皮层无炎细胞浸润,无血管扩张。模型组:表皮角化过度,棘层肥厚,细胞间水肿不明显,无表皮内水疱,真皮血管周围炎性浸润明显。西药组:与中药高剂量组结构变化基本相似。中药高剂量组:表皮角化过度不明显,棘层肥厚明显减轻,真皮内炎性浸润显著减少,内皮细胞肿胀减轻。中药低剂量组:表皮角化过度及棘层肥厚,真皮内炎性浸润减少,内皮细胞肿胀减轻。第二部分:健脾养血祛风方对脾虚证慢性湿疹小鼠模型皮肤屏障功能的修复(1)健脾养血祛风方对小鼠模型皮损中过度表达的AQP3具有显著的下调作用。AQP3在各组表皮细胞的细胞浆中均有不同程度的表达(浅褐色至棕褐色)。正常皮肤组织中表达呈弱阳性。与空白组比较,模型组AQP3表达明显增强,平均光密度值明显增高,差异具有统计学意义(P0.01);与模型组比较,各治疗组能下调AQP3在小鼠皮肤组织中的过度表达,平均光密度值明显下降,差异具有统计学意义(P0.05)。西药组与中药高剂量组比较,差异无统计学意义(P0.05)。(1)健脾养血祛风方对小鼠模型皮损中弱表达的BH(博来霉素水解酶)具有显著上调作用。BH在各组表皮细胞的细胞浆中均有不同程度的表达(浅褐色至棕褐色)。正常皮肤组织中表达呈强阳性。与空白组比较,模型组BH表达明显减弱,平均光密度值明显下降,差异具有统计学意义(P0.01);与模型组比较,各治疗组能上调BH在小鼠皮肤组织中的表达,平均光密度值显著增高,差异具有统计学意义(P0.05)。西药组与中药高剂量组比较,差异无统计学意义(P0.05)。第三部分:健脾养血祛风方对脾虚证慢性湿疹小鼠模型免疫机制的调节(1)健脾养血祛风方对小鼠模型脾T细胞亚群CD4+水平及CD4+/CD8+比值具有显著上调作用。与空白组比较,模型组CD4+水平、CD4+/CD8+显著下降,差异具有统计学意义(P0.01)。与模型组比较,西药组与中药高剂量组CD4+水平、CD4+/CD8+显著升高,差异具有统计学意义(P0.01);中药低剂量组较模型组亦有显著升高,差异具有统计学意义(P0.05)。西药组与中药高剂量组间比较,差异无统计学意义(P0.05)。与空白组比较,模型组及各治疗组CD8+水平变化不显著,差异无统计学意义(P0.05)。(2)健脾养血祛风方对小鼠模型皮损中细胞间粘附分子-1(I C A M-1)及血管细胞间粘附分子-1(V C A M-1)具有明显抑制作用。与空白组比较,模型组耳部病变组织ICAM-1及VCAM-1均有不同程度的阳性表达,而正常皮肤组织中基本无表达或偶见弱阳性表达,平均光密度值结果比较,差异有统计学意义(P0.01);与模型组比较,中药治疗高剂量组及西药组小鼠耳部病变组织ICAM-1和VCAM-1的表达均受到显著抑制,差异具有统计学意义(P0.01)。西药组与中药高剂量组比较,差异无统计学意义(P0.05)。(3)健脾养血祛风方对小鼠模型皮损中Cys LTR1、Cys LTR2的蛋白表达具有显著抑制作用。与空白组比较,Cys LTR1及Cys LTR2在模型组皮损中表达强度明显增强,差异具有统计学意义(P0.01)。与模型组相比,各治疗组Cys LTR1及Cys LTR2在皮肤组织中的表达强度明显减弱,差异具有统计学意义(P0.01)。与西药组比较,中药低剂量组表达较强,差异具有统计学意义(P0.01),而中药高剂量组表达强度与西药组无显著性差异(P0.05)。各组中Cys LTR1与Cys LTR2间的表达强度差异均无统计学意义(P0.05)。(4)健脾养血祛风方对小鼠模型血清中趋化因子CCL17、CCL18具有显著下调作用,并能抑制其在皮损中的高表达。与空白组比较,模型组血清中CCL17、CCL18水平显著升高,差异具有统计学意义(P0.01)。与模型组相比,各治疗组小鼠血清中CCL17、CCL18水平显著下降,差异具有统计学意义(P0.01)。与西药组相比,中药低剂量组血清中CCL17、CCL18水平明显高于西药组,差异具有统计学意义(P CCL170.05,P CCL180.01);中药高剂量组血清中CCL17、CCL18水平与西药组无显著性差异(P0.05)。与空白组比较,模型组皮损中CCL17、CCL18表达显著增强,差异具有统计学意义(P0.01)。与模型组相比,各治疗组小鼠皮损中CCL17、CCL18的表达较模型组明显减弱,差异具有统计学意义(P0.01)。与西药组相比,中药低剂量组皮损中CCL17、CCL18的表达明显强于西药组,差异具有统计学意义(P0.01);中药高剂量组表达强度与西药组无显著性差异(P0.05)。结论:1.采用复合因素造模方法建立脾虚证慢性湿疹病证结合的动物模型,说明病证结合的动物模型在皮肤病实验研究中切实可行。2.健脾养血祛风方能够显著减轻脾虚证慢性湿疹小鼠模型耳肿胀度,缩小耳厚度差;下调脾虚证慢性湿疹小鼠模型增高的脾脏指数及胸腺指数;抑制病变组织炎性反应,改善慢性湿疹皮损的病理结构。提示:健脾养血祛风方对脾虚证慢性湿疹小鼠模型具有较好的抗炎、抗过敏作用。3.健脾养血祛风方能够显著下调脾虚证慢性湿疹小鼠模型皮损处水通道蛋白3的过度表达、增强博来霉素水解酶在表皮层的表达。提示:健脾养血祛风方对脾虚证慢性湿疹小鼠模型皮肤屏障功能具有较好的修复作用。4.健脾养血祛风方能够显著上调脾虚证慢性湿疹小鼠模型脾细胞CD4+水平及其比值;抑制脾虚证慢性湿疹小鼠模型皮损中细胞间粘附分子-1(ICAM-1)及血管细胞间粘附分子-1(VCAM-1)的过度表达;抑制脾虚证慢性湿疹小鼠模型皮损中Cys LTR1、Cys LTR2的蛋白表达;下调脾虚证慢性湿疹小鼠模型趋化因子CCL17、CCL18血清水平、抑制其在病变组织中的表达。提示:健脾养血祛风方对脾虚证慢性湿疹小鼠模型失衡状态下的免疫功能具有较好的调节作用。
[Abstract]:Objective: To explore the effect of Jianpi Yangxue Qufeng Recipe on skin barrier function and immune mechanism of mice with chronic eczema of spleen deficiency syndrome, so as to provide experimental basis for the treatment of chronic eczema of spleen deficiency syndrome by Jianpi Yangxue Qufeng Recipe, and further demonstrate the feasibility and importance of "Jianpi Yangxue Qufeng Recipe" as a traditional Chinese medicine treatment for chronic eczema of spleen deficiency syndrome. Materials and Methods: 1 Material: 1.1 Experimental animals and groups: 50 healthy male Kunming SPF mice were randomly divided into blank control group, chronic eczema model group with spleen deficiency syndrome, western medicine treatment group with spleen deficiency syndrome, high dose group with spleen deficiency syndrome, low dose group with spleen deficiency syndrome. The treatment group was treated with Decoction of invigorating spleen, nourishing blood and removing wind; the control group was treated with levocetirizine hydrochloride tablets, which were dissolved in distilled water to make turbid solution; the blank group and the model group were given the same amount of normal saline intragastric administration. 2. Methods: Part 1: Effect of Jianpi Yangxue Qufeng Recipe on Inflammatory Reaction of Chronic Eczema Mice with Spleen Deficiency Syndrome (1) Effect of Jianpi Yangxue Qufeng Recipe on Ear Swelling and Ear Thickness of Chronic Eczema Mice with Spleen Deficiency Syndrome The left and right auricles of the mice were prepared for examination.The thickness of the middle ears of the mice was measured with a 6 mm perforator and the difference of the thickness between the left and right ears was calculated with a micrometer caliper. The spleen weight of each mouse was divided by the body weight of each mouse. The thymus index was calculated with the spleen index. (3) The effect of Jianpi Yangxue Qufeng Prescription on the histopathological changes of ear in mice with chronic eczema of spleen deficiency syndrome. The effect of Jianpi Yangxue Qufeng Recipe on skin barrier function of mice with chronic eczema of spleen deficiency syndrome (1) Jianpi Yangxue Qufeng Recipe on mice with chronic eczema of spleen deficiency syndrome Effect of aquaporin 3 expression in the skin tissue of the back of the model: AQP3 expression in the skin tissue of the back of the mice was detected by immunohistochemical method, and the average optical density value of the positive cells expressed per unit area was determined by the image acquisition and analysis system of Beijing University of Aeronautics. (2) The expression of AQP3 in the skin tissue of the spleen, blood-nourishing and wind-dispelling mice was determined. Effect of prescription on the expression of BH in the back skin of mice with chronic eczema due to spleen deficiency syndrome: The expression of BH in the back skin of mice with spleen deficiency syndrome was detected by immunohistochemical method. (1) Effect of Jianpi Yangxue Qufeng Recipe on spleen T lymphocyte subsets CD4 +, CD8 + and their ratio in mice with chronic eczema of spleen deficiency syndrome: The levels of CD4 +, CD8 + in spleen cells of mice were detected by flow cytometry and the ratio was calculated. (2) Jianpi Yangxue Qufeng Recipe was used to treat the ear skin tissue of mice with chronic eczema of spleen deficiency syndrome. The expression of ICAM-1 and VCAM-1 in the ear skin of mice was observed by immunohistochemistry. (3) Jianpi Yangxue Qufeng Prescription on the back skin of mice with chronic eczema of spleen deficiency syndrome Effects of cysteinyl leukotriene receptor 1 (Cys LTR1) and cysteinyl leukotriene receptor 2 (Cys LTR2) on the expression of Cys LTRl and Cys LTR2 in the back skin of mice were detected by immunohistochemical method (WB). (4) Effect of Jianpi Yangxue Qufeng Prescription on chemokines CC in chronic eczema mice with spleen deficiency syndrome The serum levels of L17 and CCL18 and the expression of CCLl8 in the back skin were detected by ELISA, and the expression of CCLl7 and CCLl8 in the back skin of mice were detected by RT-PCR. Results: Part I: Jianpi Yangxue Qufeng Fang had anti-inflammatory effect on chronic eczema mice with spleen deficiency syndrome. Corresponding inhibition (1) Jianpi Yangxue Qufeng Decoction can reduce the degree of ear swelling and ear thickness difference in mice model group. Compared with the blank group, the model group and each drug group mice have different degrees of ear swelling, ear thickness difference increased, the difference is statistically significant (P 0.05); Compared with the model group, each treatment group can significantly reduce ear swelling and make ear thickness difference (P 0.05). The difference was statistically significant (P 0.01). There was no significant difference in the degree of ear swelling and ear thickness between the western medicine group and the traditional Chinese medicine group (P 0.05). (2) Jianpi Yangxue Qufeng Decoction could reduce the spleen index and thymus index of the mouse model. Compared with the blank group, the spleen lipid number and thymus index of the model group and the medication group were not different. Compared with the model group, the spleen index and thymus index of each treatment group decreased significantly (P 0.05). Compared with the traditional Chinese medicine group, the spleen index and thymus index decreased significantly (P 0.05). (3) Jianpi Yangxue Qufeng Prescription could inhibit the decrease of spleen index and thymus index. Blank group: epidermis no hyperkeratosis, thickening and sponge formation, dermis no inflammatory cell infiltration, no vasodilation. Model group: epidermis hyperkeratosis, acanthosis layer hypertrophy, no obvious interstitial edema, no epidermal blisters, perivascular inflammatory immersion Western medicine group: the structure of the high-dose group was similar to that of the traditional Chinese medicine group. The second part: Jianpi Yangxue Qufeng Prescription restores the skin barrier function of mice with chronic eczema of spleen deficiency syndrome (1) Jianpi Yangxue Qufeng Prescription has a significant down-regulation effect on the over-expression of AQP3 in the skin lesions of mice model. Compared with the blank group, the expression of AQP3 in the model group was significantly increased, and the average optical density value was significantly increased (P 0.01); Compared with the model group, the treatment groups could down-regulate the overexpression of AQP3 in the skin tissue of mice, the average optical density value was significantly decreased, the difference was statistically significant (P 0.05). There was no significant difference between the two groups (P 0.05). (1) Jianpi Yangxue Qufeng Prescription could significantly up-regulate BH (bleomycin hydrolase) in the skin lesions of mice. BH was expressed in the cytoplasm of epidermal cells in different degrees (light brown to brown). The expression was strong in normal skin tissues. Compared with the blank group, the expression of BH in the model group was significantly decreased, and the average optical density value was significantly decreased, the difference was statistically significant (P 0.01); Compared with the model group, each treatment group could up-regulate the expression of BH in mouse skin tissue, the average optical density value was significantly increased, the difference was statistically significant (P 0.05). There was no significant difference between the two groups (P 0.05). Part III: Regulation of Jianpi Yangxue Qufeng Recipe on the immune mechanism of mice with chronic eczema of spleen deficiency syndrome (1) Jianpi Yangxue Qufeng Recipe significantly increased the levels of CD4 + and the ratio of CD4 + / CD8 + in spleen T cell subsets of mice. The difference was statistically significant (P 0.01). Compared with the model group, the levels of CD4 + and CD4 + / CD8 + in the western medicine group and the high dose group were significantly higher (P 0.01); the low dose group was also significantly higher than the model group, the difference was statistically significant (P 0.05). There was no significant difference between the Western medicine group and the high dose group (P (2) Jianpi Yangxue Qufeng Fang had significant inhibitory effect on I C A M-1 and V C A M-1 in mouse model lesions. Compared with the blank group, the changes of CD8+ levels in the model group and each treatment group had no significant difference (P 0.05). (2) Jianpi Yangxue Qufeng Fang had significant inhibitory effect on I C A M-1 and V C A M-1 in mouse model lesions. The expression of ICAM-1 and VCAM-1 in the ear lesions of mice treated with high dose of Chinese medicine and Western medicine was significantly higher than that of model group (P 0.01). The expression of Cys LTR1 and Cys LTR2 was significantly inhibited by Jianpi Yangxue Qufeng Recipe in the skin lesions of mice model. Compared with the protein group, the expression of Cys LTR1 and Cys LTR2 was significantly inhibited. Compared with the model group, the expression intensity of Cys LTR1 and Cys LTR2 in the skin tissue of each treatment group was significantly weakened, and the difference was statistically significant (P 0.01). Compared with the western medicine group, the expression intensity of the low dose group was stronger, and the difference was statistically significant (P 0.01). There was no significant difference in the expression of Cys LTR1 and Cys LTR2 between the two groups (P 0.05). (4) Jianpi Yangxue Qufeng Prescription could significantly down-regulate the expression of chemokines CCL17 and CCL18 in the serum of mice model and inhibit the high expression of CCL17 and CCL18 in the skin lesions. Compared with the model group, the levels of CCL17 and CCL18 in the serum of mice in each treatment group decreased significantly, and the difference was statistically significant (P 0.01). Compared with the western medicine group, the levels of CCL17 and CCL18 in the serum of the low dose group were significantly higher than those of the western medicine group, and the difference was statistically significant (P CCL170.05, P CCL180.01). Compared with the blank group, the expression of CCL17 and CCL18 in the skin lesions of the model group was significantly increased, and the difference was statistically significant (P 0.01). Compared with the model group, the expression of CCL17 and CCL18 in the skin lesions of mice in the treatment groups was significantly decreased, and the difference was statistically significant. Compared with the western medicine group, the expression of CCL17 and CCL18 in the skin lesions of the low dose group was significantly stronger than that of the western medicine group, and the difference was statistically significant (P 0.01). There was no significant difference in the expression intensity between the high dose group and the western medicine group (P 0.05). Conclusion: 1. The animal model of chronic eczema with spleen deficiency syndrome was established by the method of compound factor modeling. 2. Jianpi Yangxue Qufeng Decoction can significantly reduce the ear swelling and ear thickness difference in mice with chronic eczema due to spleen deficiency syndrome; reduce the spleen index and thymus index of mice with chronic eczema due to spleen deficiency syndrome; inhibit inflammatory reaction of pathological tissue and improve chronic eczema. The pathological structure of eczema skin lesions suggests that Jianpi Yangxue Qufeng Recipe has better anti-inflammatory and anti-allergic effects on mice with chronic eczema of spleen deficiency syndrome.3.Jianpi Yangxue Qufeng Recipe can significantly reduce the over-expression of aquaporin-3 and enhance the expression of bleomycin hydrolase in the epidermis of chronic eczema mice with spleen deficiency syndrome. Explanation: Jianpi Yangxue Qufeng Recipe has a good repair effect on skin barrier function of mice with chronic eczema of spleen deficiency syndrome. 4. Jianpi Yangxue Qufeng Recipe
【学位授予单位】：辽宁中医药大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R285.5;R-332
本文编号：2218321
[Abstract]:Objective: To explore the effect of Jianpi Yangxue Qufeng Recipe on skin barrier function and immune mechanism of mice with chronic eczema of spleen deficiency syndrome, so as to provide experimental basis for the treatment of chronic eczema of spleen deficiency syndrome by Jianpi Yangxue Qufeng Recipe, and further demonstrate the feasibility and importance of "Jianpi Yangxue Qufeng Recipe" as a traditional Chinese medicine treatment for chronic eczema of spleen deficiency syndrome. Materials and Methods: 1 Material: 1.1 Experimental animals and groups: 50 healthy male Kunming SPF mice were randomly divided into blank control group, chronic eczema model group with spleen deficiency syndrome, western medicine treatment group with spleen deficiency syndrome, high dose group with spleen deficiency syndrome, low dose group with spleen deficiency syndrome. The treatment group was treated with Decoction of invigorating spleen, nourishing blood and removing wind; the control group was treated with levocetirizine hydrochloride tablets, which were dissolved in distilled water to make turbid solution; the blank group and the model group were given the same amount of normal saline intragastric administration. 2. Methods: Part 1: Effect of Jianpi Yangxue Qufeng Recipe on Inflammatory Reaction of Chronic Eczema Mice with Spleen Deficiency Syndrome (1) Effect of Jianpi Yangxue Qufeng Recipe on Ear Swelling and Ear Thickness of Chronic Eczema Mice with Spleen Deficiency Syndrome The left and right auricles of the mice were prepared for examination.The thickness of the middle ears of the mice was measured with a 6 mm perforator and the difference of the thickness between the left and right ears was calculated with a micrometer caliper. The spleen weight of each mouse was divided by the body weight of each mouse. The thymus index was calculated with the spleen index. (3) The effect of Jianpi Yangxue Qufeng Prescription on the histopathological changes of ear in mice with chronic eczema of spleen deficiency syndrome. The effect of Jianpi Yangxue Qufeng Recipe on skin barrier function of mice with chronic eczema of spleen deficiency syndrome (1) Jianpi Yangxue Qufeng Recipe on mice with chronic eczema of spleen deficiency syndrome Effect of aquaporin 3 expression in the skin tissue of the back of the model: AQP3 expression in the skin tissue of the back of the mice was detected by immunohistochemical method, and the average optical density value of the positive cells expressed per unit area was determined by the image acquisition and analysis system of Beijing University of Aeronautics. (2) The expression of AQP3 in the skin tissue of the spleen, blood-nourishing and wind-dispelling mice was determined. Effect of prescription on the expression of BH in the back skin of mice with chronic eczema due to spleen deficiency syndrome: The expression of BH in the back skin of mice with spleen deficiency syndrome was detected by immunohistochemical method. (1) Effect of Jianpi Yangxue Qufeng Recipe on spleen T lymphocyte subsets CD4 +, CD8 + and their ratio in mice with chronic eczema of spleen deficiency syndrome: The levels of CD4 +, CD8 + in spleen cells of mice were detected by flow cytometry and the ratio was calculated. (2) Jianpi Yangxue Qufeng Recipe was used to treat the ear skin tissue of mice with chronic eczema of spleen deficiency syndrome. The expression of ICAM-1 and VCAM-1 in the ear skin of mice was observed by immunohistochemistry. (3) Jianpi Yangxue Qufeng Prescription on the back skin of mice with chronic eczema of spleen deficiency syndrome Effects of cysteinyl leukotriene receptor 1 (Cys LTR1) and cysteinyl leukotriene receptor 2 (Cys LTR2) on the expression of Cys LTRl and Cys LTR2 in the back skin of mice were detected by immunohistochemical method (WB). (4) Effect of Jianpi Yangxue Qufeng Prescription on chemokines CC in chronic eczema mice with spleen deficiency syndrome The serum levels of L17 and CCL18 and the expression of CCLl8 in the back skin were detected by ELISA, and the expression of CCLl7 and CCLl8 in the back skin of mice were detected by RT-PCR. Results: Part I: Jianpi Yangxue Qufeng Fang had anti-inflammatory effect on chronic eczema mice with spleen deficiency syndrome. Corresponding inhibition (1) Jianpi Yangxue Qufeng Decoction can reduce the degree of ear swelling and ear thickness difference in mice model group. Compared with the blank group, the model group and each drug group mice have different degrees of ear swelling, ear thickness difference increased, the difference is statistically significant (P 0.05); Compared with the model group, each treatment group can significantly reduce ear swelling and make ear thickness difference (P 0.05). The difference was statistically significant (P 0.01). There was no significant difference in the degree of ear swelling and ear thickness between the western medicine group and the traditional Chinese medicine group (P 0.05). (2) Jianpi Yangxue Qufeng Decoction could reduce the spleen index and thymus index of the mouse model. Compared with the blank group, the spleen lipid number and thymus index of the model group and the medication group were not different. Compared with the model group, the spleen index and thymus index of each treatment group decreased significantly (P 0.05). Compared with the traditional Chinese medicine group, the spleen index and thymus index decreased significantly (P 0.05). (3) Jianpi Yangxue Qufeng Prescription could inhibit the decrease of spleen index and thymus index. Blank group: epidermis no hyperkeratosis, thickening and sponge formation, dermis no inflammatory cell infiltration, no vasodilation. Model group: epidermis hyperkeratosis, acanthosis layer hypertrophy, no obvious interstitial edema, no epidermal blisters, perivascular inflammatory immersion Western medicine group: the structure of the high-dose group was similar to that of the traditional Chinese medicine group. The second part: Jianpi Yangxue Qufeng Prescription restores the skin barrier function of mice with chronic eczema of spleen deficiency syndrome (1) Jianpi Yangxue Qufeng Prescription has a significant down-regulation effect on the over-expression of AQP3 in the skin lesions of mice model. Compared with the blank group, the expression of AQP3 in the model group was significantly increased, and the average optical density value was significantly increased (P 0.01); Compared with the model group, the treatment groups could down-regulate the overexpression of AQP3 in the skin tissue of mice, the average optical density value was significantly decreased, the difference was statistically significant (P 0.05). There was no significant difference between the two groups (P 0.05). (1) Jianpi Yangxue Qufeng Prescription could significantly up-regulate BH (bleomycin hydrolase) in the skin lesions of mice. BH was expressed in the cytoplasm of epidermal cells in different degrees (light brown to brown). The expression was strong in normal skin tissues. Compared with the blank group, the expression of BH in the model group was significantly decreased, and the average optical density value was significantly decreased, the difference was statistically significant (P 0.01); Compared with the model group, each treatment group could up-regulate the expression of BH in mouse skin tissue, the average optical density value was significantly increased, the difference was statistically significant (P 0.05). There was no significant difference between the two groups (P 0.05). Part III: Regulation of Jianpi Yangxue Qufeng Recipe on the immune mechanism of mice with chronic eczema of spleen deficiency syndrome (1) Jianpi Yangxue Qufeng Recipe significantly increased the levels of CD4 + and the ratio of CD4 + / CD8 + in spleen T cell subsets of mice. The difference was statistically significant (P 0.01). Compared with the model group, the levels of CD4 + and CD4 + / CD8 + in the western medicine group and the high dose group were significantly higher (P 0.01); the low dose group was also significantly higher than the model group, the difference was statistically significant (P 0.05). There was no significant difference between the Western medicine group and the high dose group (P (2) Jianpi Yangxue Qufeng Fang had significant inhibitory effect on I C A M-1 and V C A M-1 in mouse model lesions. Compared with the blank group, the changes of CD8+ levels in the model group and each treatment group had no significant difference (P 0.05). (2) Jianpi Yangxue Qufeng Fang had significant inhibitory effect on I C A M-1 and V C A M-1 in mouse model lesions. The expression of ICAM-1 and VCAM-1 in the ear lesions of mice treated with high dose of Chinese medicine and Western medicine was significantly higher than that of model group (P 0.01). The expression of Cys LTR1 and Cys LTR2 was significantly inhibited by Jianpi Yangxue Qufeng Recipe in the skin lesions of mice model. Compared with the protein group, the expression of Cys LTR1 and Cys LTR2 was significantly inhibited. Compared with the model group, the expression intensity of Cys LTR1 and Cys LTR2 in the skin tissue of each treatment group was significantly weakened, and the difference was statistically significant (P 0.01). Compared with the western medicine group, the expression intensity of the low dose group was stronger, and the difference was statistically significant (P 0.01). There was no significant difference in the expression of Cys LTR1 and Cys LTR2 between the two groups (P 0.05). (4) Jianpi Yangxue Qufeng Prescription could significantly down-regulate the expression of chemokines CCL17 and CCL18 in the serum of mice model and inhibit the high expression of CCL17 and CCL18 in the skin lesions. Compared with the model group, the levels of CCL17 and CCL18 in the serum of mice in each treatment group decreased significantly, and the difference was statistically significant (P 0.01). Compared with the western medicine group, the levels of CCL17 and CCL18 in the serum of the low dose group were significantly higher than those of the western medicine group, and the difference was statistically significant (P CCL170.05, P CCL180.01). Compared with the blank group, the expression of CCL17 and CCL18 in the skin lesions of the model group was significantly increased, and the difference was statistically significant (P 0.01). Compared with the model group, the expression of CCL17 and CCL18 in the skin lesions of mice in the treatment groups was significantly decreased, and the difference was statistically significant. Compared with the western medicine group, the expression of CCL17 and CCL18 in the skin lesions of the low dose group was significantly stronger than that of the western medicine group, and the difference was statistically significant (P 0.01). There was no significant difference in the expression intensity between the high dose group and the western medicine group (P 0.05). Conclusion: 1. The animal model of chronic eczema with spleen deficiency syndrome was established by the method of compound factor modeling. 2. Jianpi Yangxue Qufeng Decoction can significantly reduce the ear swelling and ear thickness difference in mice with chronic eczema due to spleen deficiency syndrome; reduce the spleen index and thymus index of mice with chronic eczema due to spleen deficiency syndrome; inhibit inflammatory reaction of pathological tissue and improve chronic eczema. The pathological structure of eczema skin lesions suggests that Jianpi Yangxue Qufeng Recipe has better anti-inflammatory and anti-allergic effects on mice with chronic eczema of spleen deficiency syndrome.3.Jianpi Yangxue Qufeng Recipe can significantly reduce the over-expression of aquaporin-3 and enhance the expression of bleomycin hydrolase in the epidermis of chronic eczema mice with spleen deficiency syndrome. Explanation: Jianpi Yangxue Qufeng Recipe has a good repair effect on skin barrier function of mice with chronic eczema of spleen deficiency syndrome. 4. Jianpi Yangxue Qufeng Recipe
【学位授予单位】：辽宁中医药大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R285.5;R-332
【参考文献】
相关期刊论文 前1条
1 曲长江,刘劲,林庶如,夏淑杰;不同造模方法脾虚小鼠免疫学改变的比较研究[J];中国中医基础医学杂志;1999年04期
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