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  本文關鍵詞：涼血祛濕止癢湯治療濕疹的臨床觀察及其作用機理實驗研究　出處：《北京中醫(yī)藥大學》2017年博士論文　論文類型：學位論文

  更多相關文章： 濕疹 抗炎機制 屏障功能

【摘要】：目的 1.觀察涼血祛濕止癢湯治療急性濕疹的臨床療效。2.采用小鼠濕疹模型皮損,分別從皮損腫脹度及厚度差,IL-2、IL-4及LTB4水平,AQP3mRNA的基因及蛋白表達方面探究涼血祛濕止癢湯的可能作用機制。方法 本課題研究采用兩部分進行:1.涼血祛濕止癢湯的臨床療效觀察。嚴格遵照制定急性濕疹(濕熱內(nèi)蘊證)診斷標準,設計隨機、開放、平行對照實驗,以氯雷他定片為對照藥物,觀察紅斑、丘疹、水皰、滲出、靶皮損面積及瘙癢程度,評估臨床總體療效,并觀察復發(fā)率及藥物不良反應。2.探究涼血祛濕止癢湯的作用機理。(1)觀察小鼠濕疹模型皮損的形態(tài)學變化。采用2,4-二硝基氯苯丙酮溶液誘導建立濕疹模型,進行涼血祛濕止癢湯藥物干預,并設立陽性對照氯雷他定組及陰性對照模型組(生理鹽水)、空白組,根據(jù)趙辨的EASI評分方法對皮損進行評分,參照《中藥新藥臨床研究指導原則》(試行)進行療效評定,觀察紅斑、水腫、滲出、表皮剝脫等皮損變化。(2)將藥物干預后的小鼠進行皮損取材,測定涼血祛濕止癢湯組、氯雷他定組、模型組皮損厚度差及腫脹度。(3)采用ELISA方法,檢測涼血祛濕止癢湯組、氯雷他定組、模型組和空白組小鼠濕疹模型皮損的IL-2、IL-4及LTB4的含量。(4)采用RT-PCR方法,檢測涼血祛濕止癢湯組、氯雷他定組、模型組和空白組小鼠濕疹模型皮損AQP3mRNA的含量。(5)采用Western Blot方法,檢測涼血祛濕止癢湯組、氯雷他定組、模型組和空白組AQP3的蛋白表達。結果 1.臨床療效:涼血祛濕止癢湯治療急性濕疹(濕熱內(nèi)蘊證),在滲出、水皰方面明顯優(yōu)于氯雷他定組,差異具有統(tǒng)計學意義(P0.05),在紅斑、丘疹方面與氯雷他定組療效相當(P0.05);在靶皮損面積方面與氯雷他定組相比,有明顯的統(tǒng)計學差異(P0.05);在瘙癢程度方面,明顯優(yōu)于氯雷他定組(P0.05)。對于兩組總體療效評價中,涼血祛濕止癢湯組總有效率為86.49%,氯雷他定組總有效率為80.56%,經(jīng)統(tǒng)計分析,P0.05,涼血祛濕止癢湯組總體療效優(yōu)于氯雷他定組。2.基礎實驗研究(1)形態(tài)學觀察:涼血祛濕止癢湯干預藥物誘導小鼠濕疹模型,與氯雷他定組、模型組對照,在紅斑、丘疹、滲出、表皮剝脫方面,具有明顯統(tǒng)計學差異(P0.01),進行組間比較,涼血祛濕止癢湯組與氯雷他定組明顯優(yōu)于模型組,差異極其顯著(P0.01),并且涼血祛濕止癢湯組各指標療效明顯優(yōu)于氯雷他定組(P0.05)。在總體療效評價方面,涼血祛濕止癢湯組的總有效率為88.89%,氯雷他定組為77.78%,模型組為27.78%,涼血祛濕止癢湯總體療效優(yōu)于氯雷他定組及模型組,差異具有統(tǒng)計學意義(P0.05)。(2)厚度差、腫脹度方面:本實驗在小鼠模型皮損厚度差、腫脹度方面,四組小鼠具有明顯統(tǒng)計學差異(P0.01)。與空白組比較,三組的皮損厚度與皮損重量具有顯著差異(P0.01),與模型組比較,涼血祛濕止癢組與氯雷他定組具有顯著差異(P0.05),并且涼血祛濕止癢湯在厚度差、腫脹度兩方面明顯優(yōu)于氯雷他定組(P0.05),差異具有統(tǒng)計學意義。(3)IL-2、IL-4和LTB4 水平:涼血祛濕止癢湯組、氯雷他定組、模型組及空白組在IL-2、IL-4和LTB4水平方面具有顯著差異(P0.01)。涼血祛濕止癢湯組、氯雷他定組的IL-2水平,明顯高于模型組(P0.05),低于空白組(P0.05),涼血祛濕止癢湯組較氯雷他定組明顯升高(P0.05)。在IL-4、LTB4水平,涼血祛濕止癢湯組、氯雷他定組低于模型組(P0.01),高于空白組(P0.01),涼血祛濕止癢湯組優(yōu)于氯雷他定組(P0.05),差異具有統(tǒng)計學意義。(4)AQP3mRNA基因水平:四組在AQP3mRNA的2-△△CT值具有顯著差異,(P0.01),涼血祛濕止癢湯組、氯雷他定組、空白組與模型組比較顯著降低,(P0.01)。進行組間比較,涼血祛濕止癢湯組、氯雷他定組較空白組顯著升高(P0.01),較模型組顯著降低(P0.01)。與氯雷他定組比較,涼血祛濕止癢湯組顯著降低(P0.05),差異具有統(tǒng)計學意義。(5)AQP3蛋白表達水平:四組條帶清晰可辨,灰度差可見,模型組最強,依次是氯雷他定組、涼血祛濕止癢湯組、空白組,其中空白組灰度最低。對AQP3/β-actin值進行統(tǒng)計分析,與空白組對照,三組具有顯著差異(P0.01);涼血祛濕止癢湯組、氯雷他定組明顯低于模型組(P0.01);而涼血祛濕止癢湯組優(yōu)于氯雷他定組(P0.05),差異具有統(tǒng)計學意義。結論 涼血祛濕止癢湯臨床療效顯著,具有明顯的消除紅斑、水皰、丘疹及減少滲出的作用,基礎實驗表明本藥可以減輕小鼠皮損腫脹程度,減輕皮損紅斑、滲出、表皮剝脫等炎癥反應,有效的調(diào)節(jié)皮損中IL-2上升,下調(diào)IL-4的含量,抑制皮損中LTB4釋放,有效抑制模型皮損中AQP3的代償性增殖�？紤]涼血祛濕止癢湯具有明顯的抗炎作用及修復屏障功能的作用。由此可見,涼血祛濕止癢湯可以調(diào)節(jié)Th1/Th2的平衡,抗變態(tài)反應,抑制LTBs釋放,減輕炎性癥狀。初步表明涼血祛濕止癢湯具有抗炎作用機制。本實驗發(fā)現(xiàn)涼血祛濕止癢湯可以明顯抑制AQP3的在急性濕疹中的高表達,從而達到修復屏障功能,減少滲出的作用,初步表明涼血祛濕止癢湯的作用靶點可能為修復皮膚屏障功能。
[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.

【學位授予單位】：北京中醫(yī)藥大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R275.9

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