甘石青黛膏对大鼠湿疹模型皮损作用机制研究
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摘要
目的
1观察中药甘石青黛膏对大鼠湿疹模型皮损作用的治疗效果。
2从湿疹皮损LTB4水平、CysLTR1和CysLTR2表达、角质形成细胞(KC)与淋巴细胞(LYM)的凋亡以及皮损Bcl-α与Bax蛋白调控表达方面探讨中药甘石青黛膏治疗湿疹的可能作用机制。
方法
本课题研究采用两部分进行：
1甘石青黛膏对大鼠湿疹模型皮损作用的疗效观察
大鼠湿疹模型造模成功后进行中药甘石青黛膏干预,并与安慰剂对比。皮损的评分采用赵辨EASI评分法,皮损总体疗效判定标准参照郑筱萸主编之《中药新药临床研究指导原则》(试行)执行,进一步观察湿疹模型红斑、水肿、表皮剥脱、苔藓化等变化。
2甘石青黛膏对大鼠湿疹模型皮损作用机制研究
①采用ELISA法,在实验0天、7天、14天测定治疗组和对照组大鼠湿疹模型皮损白三烯B4(LTB4)含量。
②采用Western blot法,检测治疗组和对照组大鼠湿疹模型干预前后皮损半胱氨酰白三烯受体(CysLTR1和CysLTR2)表达。
③采用TUNEL法检测治疗组和对照组大鼠湿疹模型皮损角质形成细胞(KC)与淋巴细胞(LYM)的凋亡情况。
④采用原位杂交技术检测治疗组和对照组大鼠湿疹模型皮损Bcl-α与Bax蛋白调控表达。
结果
1疗效：甘石青黛膏干预大鼠湿疹模型皮损,在皮损的红斑、水肿、表皮剥脱以及苔藓化方面,与干预前相比,差异极显著(P<0.01),干预后与安慰剂组相比差异极显著(P<0.000),各项指标疗效优于安慰剂组；对于两组大鼠湿疹模型皮损干预后总体疗效评价中,安慰剂组总有效率为42.10%,甘石青黛膏组总有效率为72.22%,经统计学处理,P<0.05,差异显著,甘石青黛膏总体疗效优于安慰剂组。
2机制：
①LTB4水平：空白组大鼠在实验前后皮损LTB4浓度基本持平,安慰剂组和甘石青黛膏组大鼠干预后皮损LTB4浓度较干预前明显降低(P<0.01),而甘石青黛膏组大鼠皮损LTB4浓度降低最明显,与安慰剂组疗后相比具有明显差异(P<0.01)；而且随用药时间延续,LTB4含量逐渐降低。研究结果表明甘石青黛膏能够抑制皮肤LTB4释放,而且对LTB4释放控制程度,随疗程增加而增加。
②CysLTR1和CysLTR2：在干预前模型组皮损的CysLTR含量明显高于空白大鼠皮肤CysLTR蛋白含量；干预2周以后,中药甘石青黛膏组和安慰剂组CysLTR含量均有减少,但甘石青黛膏组CysLTR含量减少最明显,二者相比具有明显差异(P<0.01)；而在干预后,应用Western-blot法测定受体蛋白表达,中药甘石青黛膏组CysLTR1和CysLTR2表达明显弱于空白组和安慰剂组。
③角质形成细胞(KC)与淋巴细胞(LYM)的凋亡：空白组大鼠皮肤在实验0天和14天均可见少量凋亡细胞,两者相比没有差异(P>0.05)；安慰剂组大鼠皮损干预前可见少量凋亡细胞,干预后凋亡细胞数量增加,与疗前相比较具有显著性差异,(P<0.05)；甘石青黛膏组大鼠皮损干预前可见少量凋亡细胞,与安慰剂组相比没有显著性差异,干预后凋亡细胞数量明显增加,与疗前相比较具有极显著性差异(P<0.001),具有统计学意义；安慰剂组和甘石青黛膏组疗后相比,具有极显著性差异(P<0.000),后者优于前者。
④Bcl-α与Bax蛋白表达：甘石青黛膏组大鼠皮损在0d角质形成细胞和淋巴细胞Bcl-α蛋白呈强阳性表达,在14d后阳性表达明显减弱,前后相比有极显著性差异(P<0.001)；安慰剂组和甘石青黛膏组在Od Bcl-α蛋白表达相比没有显著性差异(P>0.05),14d后Bcl-α蛋白表达相比有极显著性差异(F=11.79,P<0.001),甘石青黛膏组对Bcl-α蛋白抑制优于安慰剂组；甘石青黛膏组大鼠皮损在0d角质形成细胞和淋巴细胞Bax蛋白呈阳性表达,在14d后阳性表达增强,前后相比有极显著性差异(P<0.001)；安慰剂组和甘石青黛膏组在Od Bax蛋白表达相比没有显著性差异(P>0.05),14d后Bax蛋白表达相比有极显著性差异(F=-31.30,P<0.001)。甘石青黛膏组对Bax蛋白具有促进作用,其作用优于安慰剂组。
结论
中药甘石青黛膏对大鼠湿疹模型皮损具有控制红斑,消除水肿,促进破损的表皮修复,抑制苔藓化作用；中药甘石青黛膏能够抑制抑制LTB4释放、抑制或拮抗皮损中CysLTR、抑制角质形成细胞增殖和促进角质细胞和淋巴细胞凋亡、抑制角质形成细胞和淋巴细胞Bcl-α蛋白表达,促进角质形成细胞和淋巴细胞Bax蛋白表达作用。本课题创新点是首次从湿疹炎症因子与受体、细胞凋亡与凋亡调控蛋白角度对中药外治湿疹作用机制进行研究。
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 rats
①using 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.
1观察中药甘石青黛膏对大鼠湿疹模型皮损作用的治疗效果。
2从湿疹皮损LTB4水平、CysLTR1和CysLTR2表达、角质形成细胞(KC)与淋巴细胞(LYM)的凋亡以及皮损Bcl-α与Bax蛋白调控表达方面探讨中药甘石青黛膏治疗湿疹的可能作用机制。
方法
本课题研究采用两部分进行：
1甘石青黛膏对大鼠湿疹模型皮损作用的疗效观察
大鼠湿疹模型造模成功后进行中药甘石青黛膏干预,并与安慰剂对比。皮损的评分采用赵辨EASI评分法,皮损总体疗效判定标准参照郑筱萸主编之《中药新药临床研究指导原则》(试行)执行,进一步观察湿疹模型红斑、水肿、表皮剥脱、苔藓化等变化。
2甘石青黛膏对大鼠湿疹模型皮损作用机制研究
①采用ELISA法,在实验0天、7天、14天测定治疗组和对照组大鼠湿疹模型皮损白三烯B4(LTB4)含量。
②采用Western blot法,检测治疗组和对照组大鼠湿疹模型干预前后皮损半胱氨酰白三烯受体(CysLTR1和CysLTR2)表达。
③采用TUNEL法检测治疗组和对照组大鼠湿疹模型皮损角质形成细胞(KC)与淋巴细胞(LYM)的凋亡情况。
④采用原位杂交技术检测治疗组和对照组大鼠湿疹模型皮损Bcl-α与Bax蛋白调控表达。
结果
1疗效：甘石青黛膏干预大鼠湿疹模型皮损,在皮损的红斑、水肿、表皮剥脱以及苔藓化方面,与干预前相比,差异极显著(P<0.01),干预后与安慰剂组相比差异极显著(P<0.000),各项指标疗效优于安慰剂组；对于两组大鼠湿疹模型皮损干预后总体疗效评价中,安慰剂组总有效率为42.10%,甘石青黛膏组总有效率为72.22%,经统计学处理,P<0.05,差异显著,甘石青黛膏总体疗效优于安慰剂组。
2机制：
①LTB4水平：空白组大鼠在实验前后皮损LTB4浓度基本持平,安慰剂组和甘石青黛膏组大鼠干预后皮损LTB4浓度较干预前明显降低(P<0.01),而甘石青黛膏组大鼠皮损LTB4浓度降低最明显,与安慰剂组疗后相比具有明显差异(P<0.01)；而且随用药时间延续,LTB4含量逐渐降低。研究结果表明甘石青黛膏能够抑制皮肤LTB4释放,而且对LTB4释放控制程度,随疗程增加而增加。
②CysLTR1和CysLTR2：在干预前模型组皮损的CysLTR含量明显高于空白大鼠皮肤CysLTR蛋白含量；干预2周以后,中药甘石青黛膏组和安慰剂组CysLTR含量均有减少,但甘石青黛膏组CysLTR含量减少最明显,二者相比具有明显差异(P<0.01)；而在干预后,应用Western-blot法测定受体蛋白表达,中药甘石青黛膏组CysLTR1和CysLTR2表达明显弱于空白组和安慰剂组。
③角质形成细胞(KC)与淋巴细胞(LYM)的凋亡：空白组大鼠皮肤在实验0天和14天均可见少量凋亡细胞,两者相比没有差异(P>0.05)；安慰剂组大鼠皮损干预前可见少量凋亡细胞,干预后凋亡细胞数量增加,与疗前相比较具有显著性差异,(P<0.05)；甘石青黛膏组大鼠皮损干预前可见少量凋亡细胞,与安慰剂组相比没有显著性差异,干预后凋亡细胞数量明显增加,与疗前相比较具有极显著性差异(P<0.001),具有统计学意义；安慰剂组和甘石青黛膏组疗后相比,具有极显著性差异(P<0.000),后者优于前者。
④Bcl-α与Bax蛋白表达：甘石青黛膏组大鼠皮损在0d角质形成细胞和淋巴细胞Bcl-α蛋白呈强阳性表达,在14d后阳性表达明显减弱,前后相比有极显著性差异(P<0.001)；安慰剂组和甘石青黛膏组在Od Bcl-α蛋白表达相比没有显著性差异(P>0.05),14d后Bcl-α蛋白表达相比有极显著性差异(F=11.79,P<0.001),甘石青黛膏组对Bcl-α蛋白抑制优于安慰剂组；甘石青黛膏组大鼠皮损在0d角质形成细胞和淋巴细胞Bax蛋白呈阳性表达,在14d后阳性表达增强,前后相比有极显著性差异(P<0.001)；安慰剂组和甘石青黛膏组在Od Bax蛋白表达相比没有显著性差异(P>0.05),14d后Bax蛋白表达相比有极显著性差异(F=-31.30,P<0.001)。甘石青黛膏组对Bax蛋白具有促进作用,其作用优于安慰剂组。
结论
中药甘石青黛膏对大鼠湿疹模型皮损具有控制红斑,消除水肿,促进破损的表皮修复,抑制苔藓化作用；中药甘石青黛膏能够抑制抑制LTB4释放、抑制或拮抗皮损中CysLTR、抑制角质形成细胞增殖和促进角质细胞和淋巴细胞凋亡、抑制角质形成细胞和淋巴细胞Bcl-α蛋白表达,促进角质形成细胞和淋巴细胞Bax蛋白表达作用。本课题创新点是首次从湿疹炎症因子与受体、细胞凋亡与凋亡调控蛋白角度对中药外治湿疹作用机制进行研究。
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 rats
①using 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.
引文
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[7]Prieto JM, RecioMC, Giner RM, et al. Influence of traditional Chinese antiinflammatory medicinal plants on leukocyte and platelet functions. J Pharm Pharmacol,2003,55:1275-1282
[8]林撤谷,吴宜鸿,杨荣季,等.复方青黛油膏治疗银屑病的临床疗效评估[J].成都中医药大学学报,2006,29(2)：13-16
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[2]Hua HX, Bai L. Expression and significance of P53, Bcl-α and cmyc in lesions from patients with lichen planus. Chin J Dermatol,2007,40 (2):118
[3]Zhao ZQ, Li GT. The expression of Bcl-α and Fas in vulvar white lesion. J Qiqi Med Coll,2006,27 (10):1153-1155
[4]Smaili SS, Hsu YT, Sanders KM,et al. Bax translocation to mitochondria subsequent to arapid loss of mitochondrial membrane potential. Cell Death Differ,2001,8(9):909-920
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[6]祝青,刁庆春,白晋,等.聚焦超声对慢性湿疹模型豚鼠皮肤淋巴细胞和角质形成细胞凋亡的影响[J].中国医学影像技术,2008,24(2)：167-168