The Study On Mechanism Of GanShiQingDai Cream Intervening Lesions Of Eczema Model In Rats

Objective:1 To observe the therapeutic effect of GanShiQingDai cream intervening lesions of eczema model in rats.2 To discuss the possible mechanism of GanShiQingDai cream treating lesions of eczema model in rats from LTB4 level, expression of CysLTR, and CysLTR2, apoptosis of keratinocyte cells (KC) and lymphocytes (LYM), and expression of Bcl-a and Bax regulation protein.Method:The research contains two parts:1 The observation of the therapeutic effect of GanShiQingDai cream intervening lesions of eczema model in rats.In the first place, GanShiQingDai cream intervened lesions of eczema model after Successfully making eczema model in rats, and was to compare wi th placebo. the EASI score method designed by ZHAO Bian was select. The overall efficacy criteria of lesions refered to "new drug clinical research of traditional Chinese medicine guiding principles" (the pilot)edited by Zheng Xiaoyu.The change of lesions from erythema, edema, skin exfoliation, and moss et al were further observed.2 The study of the mechanism of GanShiQingDai cream treating lesions of eczema model in rats①using ELISA to determine leukotriene B4 (LTB4) content coming from the treatment group's and control group's eczema model lesions in the experimental 0 days,7 days,14 days.②The method of Western blot was used to detect the expression of lesions cysteinyl leukotriene receptor (CysLTR1, and CysLTR2) deriving from treatment group and control group rats eczema model in the experimental Odays,14days.③The method of TUNEL was to be selected to observe the change of apoptosis of keratinocyte cells (KC) and lymphocytes (LYM) in the experimental 0 days, 14 days.④The expression of Bcl-αand Bax regulation was to be determined by the method of Hybridization-in-situ in the experimental 0 days,14 days.Results:1 Effect:The effects of erythema, edema, skin exfoliation, and moss from eczema model of skin lesions in rats treated by GanShiQingDai cream were very good, the difference of effect after treating compared with before treating was significant (P< 0.01). The effects of erythema, edema, skin exfoliation, and moss from eczema model of skin lesions in rats treated by GanShiQingDai cream were superior to ones of placebo group (P<0.000) in the experimental 14 days. The total effective rate of placebo group was 42.10%, the total effective rate of GanShiQingDai cream was 72.22%, by statistical analysis, the total effective rate of GanShiQingDai cream was better than one of placebo, and the difference was significant (P<0.05).2 Mechanisms:①the level of LTB4:The Concentration of LTB4 of the blank group was Almost no change before or after the experiment. The concentration of LTB4 of placebo group and GanShiQingDai group were diluter after the experiment than before (P<0.01), The reduce of the Concentration of LTB4 of GanShiQingDai group was very more than one of placebo group (P<0.01), and the Concentration of LTB4 decreased gradually with the medication time in GanShiQingDai group. The results showed that GanShiQingDai cream could inhibit skin LTB4 release,the degree of LTB4 controled was directly proportional to the medication time.②CysLTR1 and CysLTR2:The content of CysLTR protein of model group (placebo group, GanShiQingDai group) lesions was significantly higher than the blank group.After having been intervened for 2 weeks, The content of CysLTR protein of placebo group and GanShiQingDai group were lower after the experiment than before (P<0.01), The reduce of the content of CysLTR protein of GanShiQingDai group was more significant than ones of placebo group (P<0.01); At the same time, the CysLTR, and CysLTR2 protein expression of GanShiQingDai group were weaker than one of placebo group by means of Western-blot method,③keratinocyte cells (KC) and lymphocytes (LYM) apoptosis:A few apoptotic cells were shew in the experimental 0 days and 14 days, the results compared were no difference (P>0.05). Placebo rats lesions before intervention can be seen a small amount of apoptotic cells, the number of apoptot ic cells increased after the intervention, with significant difference compared to before intervention (P<0.05). GanShiQingDai group rats lesions before intervention could be seen a small amount of apoptotic cells, compared with placebo, there was no significant difference (P<0.05). The number of apoptotic cells increased significantly after the intervention, compared to before treatment, the difference was very significant(P<0.001). After intervened, The amount of of apoptotic cells of GanShiQingDai group was more than one of placebo group, their difference were very very significant(P< 0.000).④Bcl-αand Bax protein expression:The Bcl-αprotein expression of GanShiQingDai group rats lesions keratinocyte cells (KC) and lymphocytes (LYM) shew positive, after 14 days, the positive expression was significantly weakened, compared to before treatment, the difference was very significant (P <0.001). The Bcl-αprotein expression were not different at Od GanShiQingDai group compared with placebo group, after 14 days of Bcl-αprotein expression compared to the highly significant difference (F=11.79, P<0.001), The Bcl-αprotein inhibition of GanShiQingDai was superior to the placebo. The Bax protein expression of GanShiQingDai group rats lesions keratinocyte cells (KC) and lymphocytes (LYM) shew positive, after 14 days, the positive expression was significantly strengthened, compared to before treatment, the difference was a very s ignificant (P<0.001). The Bax protein express ion were not different at Od GanShiQingDai group compared wi th placebo group, after 14 days of Bax protein expression compared to the highly significant difference (F=-31.30, P< 0.001), The Bax protein stimulation of GanShiQingDai was superior to the placebo.Conclusion:Traditional Chinese medicine GanShiQingDai cream could control erythema, eliminate edema, repair the damaged epidermis, inhibit moss role of eczema model of skin lesions in rats; GanShiQingDai cream could inhibit skin LTB4 release, inhibit or antagonize the lesions CysLTR protein, inhibit lesions keratinocyte cells (KC) and lymphocytes (LYM) to proliferate, promote lesions keratinocyte cells (KC) and lymphocytes (LYM) to apoptosis, control the Bcl-αprotein expression and promote the Bax protein expression of lesions keratinocyte cells (KC) and lymphocytes (LYM). The innovation of this project, containing firstly inflammatory cytokines and receptors,apoptos is and apoptosis regulatory protein, was the mechanism of herb treating externally eczema.