凉血祛湿止痒汤治疗湿疹的临床观察及其作用机理实验研究

发布时间：2018-01-11 23:25

本文关键词：凉血祛湿止痒汤治疗湿疹的临床观察及其作用机理实验研究　出处：《北京中医药大学》2017年博士论文　论文类型：学位论文

【摘要】：目的 1.观察凉血祛湿止痒汤治疗急性湿疹的临床疗效。2.采用小鼠湿疹模型皮损,分别从皮损肿胀度及厚度差,IL-2、IL-4及LTB4水平,AQP3mRNA的基因及蛋白表达方面探究凉血祛湿止痒汤的可能作用机制。方法本课题研究采用两部分进行:1.凉血祛湿止痒汤的临床疗效观察。严格遵照制定急性湿疹(湿热内蕴证)诊断标准,设计随机、开放、平行对照实验,以氯雷他定片为对照药物,观察红斑、丘疹、水疱、渗出、靶皮损面积及瘙痒程度,评估临床总体疗效,并观察复发率及药物不良反应。2.探究凉血祛湿止痒汤的作用机理。(1)观察小鼠湿疹模型皮损的形态学变化。采用2,4-二硝基氯苯丙酮溶液诱导建立湿疹模型,进行凉血祛湿止痒汤药物干预,并设立阳性对照氯雷他定组及阴性对照模型组(生理盐水)、空白组,根据赵辨的EASI评分方法对皮损进行评分,参照《中药新药临床研究指导原则》(试行)进行疗效评定,观察红斑、水肿、渗出、表皮剥脱等皮损变化。(2)将药物干预后的小鼠进行皮损取材,测定凉血祛湿止痒汤组、氯雷他定组、模型组皮损厚度差及肿胀度。(3)采用ELISA方法,检测凉血祛湿止痒汤组、氯雷他定组、模型组和空白组小鼠湿疹模型皮损的IL-2、IL-4及LTB4的含量。(4)采用RT-PCR方法,检测凉血祛湿止痒汤组、氯雷他定组、模型组和空白组小鼠湿疹模型皮损AQP3mRNA的含量。(5)采用Western Blot方法,检测凉血祛湿止痒汤组、氯雷他定组、模型组和空白组AQP3的蛋白表达。结果 1.临床疗效:凉血祛湿止痒汤治疗急性湿疹(湿热内蕴证),在渗出、水疱方面明显优于氯雷他定组,差异具有统计学意义(P0.05),在红斑、丘疹方面与氯雷他定组疗效相当(P0.05);在靶皮损面积方面与氯雷他定组相比,有明显的统计学差异(P0.05);在瘙痒程度方面,明显优于氯雷他定组(P0.05)。对于两组总体疗效评价中,凉血祛湿止痒汤组总有效率为86.49%,氯雷他定组总有效率为80.56%,经统计分析,P0.05,凉血祛湿止痒汤组总体疗效优于氯雷他定组。2.基础实验研究(1)形态学观察:凉血祛湿止痒汤干预药物诱导小鼠湿疹模型,与氯雷他定组、模型组对照,在红斑、丘疹、渗出、表皮剥脱方面,具有明显统计学差异(P0.01),进行组间比较,凉血祛湿止痒汤组与氯雷他定组明显优于模型组,差异极其显著(P0.01),并且凉血祛湿止痒汤组各指标疗效明显优于氯雷他定组(P0.05)。在总体疗效评价方面,凉血祛湿止痒汤组的总有效率为88.89%,氯雷他定组为77.78%,模型组为27.78%,凉血祛湿止痒汤总体疗效优于氯雷他定组及模型组,差异具有统计学意义(P0.05)。(2)厚度差、肿胀度方面:本实验在小鼠模型皮损厚度差、肿胀度方面,四组小鼠具有明显统计学差异(P0.01)。与空白组比较,三组的皮损厚度与皮损重量具有显著差异(P0.01),与模型组比较,凉血祛湿止痒组与氯雷他定组具有显著差异(P0.05),并且凉血祛湿止痒汤在厚度差、肿胀度两方面明显优于氯雷他定组(P0.05),差异具有统计学意义。(3)IL-2、IL-4和LTB4 水平:凉血祛湿止痒汤组、氯雷他定组、模型组及空白组在IL-2、IL-4和LTB4水平方面具有显著差异(P0.01)。凉血祛湿止痒汤组、氯雷他定组的IL-2水平,明显高于模型组(P0.05),低于空白组(P0.05),凉血祛湿止痒汤组较氯雷他定组明显升高(P0.05)。在IL-4、LTB4水平,凉血祛湿止痒汤组、氯雷他定组低于模型组(P0.01),高于空白组(P0.01),凉血祛湿止痒汤组优于氯雷他定组(P0.05),差异具有统计学意义。(4)AQP3mRNA基因水平:四组在AQP3mRNA的2-△△CT值具有显著差异,(P0.01),凉血祛湿止痒汤组、氯雷他定组、空白组与模型组比较显著降低,(P0.01)。进行组间比较,凉血祛湿止痒汤组、氯雷他定组较空白组显著升高(P0.01),较模型组显著降低(P0.01)。与氯雷他定组比较,凉血祛湿止痒汤组显著降低(P0.05),差异具有统计学意义。(5)AQP3蛋白表达水平:四组条带清晰可辨,灰度差可见,模型组最强,依次是氯雷他定组、凉血祛湿止痒汤组、空白组,其中空白组灰度最低。对AQP3/β-actin值进行统计分析,与空白组对照,三组具有显著差异(P0.01);凉血祛湿止痒汤组、氯雷他定组明显低于模型组(P0.01);而凉血祛湿止痒汤组优于氯雷他定组(P0.05),差异具有统计学意义。结论凉血祛湿止痒汤临床疗效显著,具有明显的消除红斑、水疱、丘疹及减少渗出的作用,基础实验表明本药可以减轻小鼠皮损肿胀程度,减轻皮损红斑、渗出、表皮剥脱等炎症反应,有效的调节皮损中IL-2上升,下调IL-4的含量,抑制皮损中LTB4释放,有效抑制模型皮损中AQP3的代偿性增殖。考虑凉血祛湿止痒汤具有明显的抗炎作用及修复屏障功能的作用。由此可见,凉血祛湿止痒汤可以调节Th1/Th2的平衡,抗变态反应,抑制LTBs释放,减轻炎性症状。初步表明凉血祛湿止痒汤具有抗炎作用机制。本实验发现凉血祛湿止痒汤可以明显抑制AQP3的在急性湿疹中的高表达,从而达到修复屏障功能,减少渗出的作用,初步表明凉血祛湿止痒汤的作用靶点可能为修复皮肤屏障功能。
[Abstract]:Objective To observe the clinical curative effect of.2. 1. cooling dampness Zhiyang Decoction in treating acute eczema with eczema in mice skin lesions, respectively from the swelling degree and the thickness difference, IL-2, IL-4 and LTB4, gene and protein expression of AQP3mRNA may be the mechanism of cooling dampness Zhiyang decoction. The research methods two parts: observation the clinical efficacy of the 1. cooling dampness Zhiyang decoction. In strict accordance with the development of acute eczema (damp heat syndrome) diagnostic criteria, randomized, open, parallel controlled experiment, with Loratadine Tablets as the control drug, observation of erythema, papules, blisters, exudation, target lesion area and pruritus, evaluate the overall clinical efficacy, mechanism of action and adverse reactions were observed the recurrence rate of.2. and explore the drug cooling dampness Zhiyang decoction. (1) to observe the morphologic changes of skin lesions. Eczema in mice induced by 2,4- two nitro chloro benzene acetone solution Eczema model of cooling dampness Zhiyang Decoction drug intervention, and the establishment of positive control group and negative control loratadine model group (saline), control group, according to the EASI score method Zhao Bian wasassessed lesions, according to "Chinese medicine clinical research guiding principles" (Trial) was used to evaluate the curative effect observation, erythema, edema. Exudation, exfoliation and other skin changes. (2) the drug intervention mice lesions were measured cooling dampness itching Decoction group, loratadine group, model group and lesion thickness difference of swelling degree. (3) using the ELISA method to detect cooling dampness itching Decoction group, loratadine group, model group and the control group of mice eczema model lesions of IL-2, content of IL-4 and LTB4. (4) using the RT-PCR method to detect cooling dampness itching Decoction group, loratadine group, the content of the model group and the control group of mice model of eczema lesional AQP3mRNA. (5) by Western Blot Detection method, cooling dampness itching Decoction group, loratadine group, model group and blank group expression of AQP3 protein. Results: the clinical efficacy of the 1. cooling dampness Zhiyang Decoction in treating acute eczema (damp heat syndrome), the exudation was superior to loratadine group, blister, the difference was statistically significant (P0.05) in erythema, papules. The aspects of the effect of loratadine group (P0.05); in the target lesion area compared with loratadine group, there is a statistically significant difference (P0.05); the degree of itching, significantly better than the other two groups (P0.05). To evaluate the overall efficacy of the two groups, cooling dampness itching decoction group, the total efficiency of 86.49%, loratadine group efficiency is 80.56%, through statistical analysis, P0.05, the overall effect is better than loratadine group.2. based experimental study on removing dampness and relieving itching Liangxue Decoction group (1) morphological observation: Liangxue dispelling wet itching soup intervention drug induced eczema in mice model Type, and loratadine group, model control group, the papules, erythema, exudation, exfoliation, with significant difference (P0.01), comparison between groups, cooling dampness Zhiyang Decoction group and loratadine group was significantly better than the model group, significant difference (P0.01), and the cooling dampness Zhiyang Decoction groups curative effect obviously better than the other two groups (P0.05). The overall curative effect evaluation, cooling dampness itching Decoction group, the total efficiency is 88.89%, the loratadine group was 77.78%, 27.78% in model group, cooling dampness Zhiyang Decoction and the overall effect is better than loratadine group and model group, the difference was statistically significant (P0.05). (2) the difference in thickness, swelling the degree of this experiment in a mouse model of skin thickness difference, swelling degree, four groups of mice with significant difference (P0.01). Compared with the control group, the thickness and weight of three lesions lesions group with significant difference (P0.01), and die Type group, cooling dampness itching group and loratadine group with significant difference (P0.05), and the cooling dampness itching soup in the thickness difference, the swelling degree of two was better than loratadine group (P0.05), the difference was statistically significant. (3) IL-2, IL-4 and LTB4 levels: cooling dampness itching Decoction group, loratadine group, model in the IL-2 group and the control group, with significant difference between IL-4 and LTB4 levels (P0.01). Cooling dampness itching Decoction group, loratadine group IL-2 level was significantly higher than the model group (P0.05), lower than that of the control group (P0.05), blood dispelling wet itching soup was significantly higher than that of loratadine group increased (P0.05). In IL-4. The level of LTB4, cooling dampness itching Decoction group, loratadine group was lower than that of model group (P0.01), higher than the control group (P0.01), cooling dampness Zhiyang Decoction group was better than loratadine group (P0.05), the difference was statistically significant. (4) AQP3mRNA gene level: the four groups in the AQP3mRNA 2- Delta The CT value had significant difference (P0.01), cooling dampness itching Decoction group, loratadine group, blank group and model group decreased significantly (P0.01). Comparison between groups, cooling dampness itching Decoction group, loratadine group was significantly higher than that in blank group (P0.01), significantly lower than that in model group (P0.01). Compared with loratadine group, cooling dampness Zhiyang Decoction group was significantly lower (P0.05), the difference was statistically significant. (5) the expression level of AQP3 protein: the four groups with clear and visible difference, the strongest in model group, loratadine group, cold dampness itching blood decoction group, blank group, the blank control group the lowest gray for AQP3/ beta -actin value of statistical analysis, compared with the blank group, the three groups had significant difference (P0.01); cooling dampness itching Decoction group, loratadine group was significantly lower than the model group (P0.01); and the cooling dampness itching decoction was better than loratadine group (P0.05), with statistical difference Significance. Conclusion the curative effect of cooling dampness decoction has obvious itching significantly, eliminate erythema, blisters, papules and reduce the leakage effect, basic experiment showed that this treatment can reduce the swelling degree of mice skin, reduce skin erythema, exudation, exfoliation and inflammation, effectively regulate the rise in the lesions of IL-2, down-regulation of IL-4 content, inhibiting the release of LTB4 in lesional skin, effectively inhibit the proliferation of compensatory AQP3 model in lesions. Considering the cooling dampness itching soup has anti-inflammatory effect and obvious effect of repairing barrier function. Thus, cooling dampness Zhiyang decoction can regulate Th1/Th2 balance, anti allergy, inhibiting the release of LTBs, relieve the inflammatory symptoms showed that cooling dampness. Zhiyang decoction has anti-inflammatory effect mechanism. This experiment showed that high expression in acute eczema in cooling dampness itching decoction can obviously inhibit AQP3, so as to achieve the restoration of barrier function And reduce the leakage effect, suggested that the target cooling dampness Zhiyang decoction may repair the skin barrier function.

【学位授予单位】：北京中医药大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R275.9

【相似文献】