葛根芩连汤抑制特应性皮炎小鼠模型炎症反应及恢复皮肤屏障功能的实验研究

Study of Gegen Qinlian Decoction on inhibiting inflammatory reaction and restoring skin barrier function in mouse model of atopic dermatitis

目的 观察葛根芩连汤对2,4-二硝基氟苯(2,4-dinitrofluorobenzene,DNFB)诱导的特应性皮炎(atopic dermatitis,AD)模型小鼠的干预作用及潜在作用机制。方法 24只雄性BALB/c小鼠随机分为正常组、模型组、中药组(葛根芩连汤治疗)和对照组(糠酸莫米松治疗)。DNFB反复刺激皮肤致敏诱导AD小鼠模型。观测各组小鼠皮损评估、皮肤组织病理学、皮肤p H值、经皮水分流失、搔抓行为及血清总免疫球蛋白E (immunoglobulin E,Ig E)、白细胞介素6(interleukin-6,IL-6)、白细胞介素10(interleukin-10,IL-10)含量。逆转录—聚合酶链式反应(quantitative Real-time PCR,q RT-PCR)法测定丝聚合蛋白、密封蛋白1的m RNA的表达;蛋白免疫印迹法(Weston blot,WB)检测丝聚合蛋白、密封蛋白1的蛋白表达水平。结果 (1)治疗前,与正常组相比,模型组背部皮肤有明显的红斑水肿,并可见结痂、鳞屑、皮肤肥厚增生;搔抓次数明显增多,经皮失水及皮肤p H值显著升高;经治疗,与模型组比较,中药组与对照组皮肤损伤情况改善,搔抓次数、经皮失水、皮肤p H值显著降低(P <0.05);(2)治疗前,模型组小鼠血清总Ig E、IL-6、IL-10水平均与正常组有显著差异;经治疗,中药组、对照组总Ig E、IL-6水平与模型组相比含量降低(P <0.05);(3)与正常组比较,模型组q RT-PCR显示丝聚合蛋白、密封蛋白1的表达水平降低;WB显示丝聚合蛋白、密封蛋白1蛋白的含量降低;经治疗,中药组、对照组丝聚合蛋白、密封蛋白1的基因表达水平增高;丝聚合蛋白、密封蛋白1蛋白含量增高(P <0.05);(4)皮肤病理学显示与正常组相比,模型组表皮层存在不完全角化,颗粒层和棘层皮肤增厚,表皮、真皮层均有大量炎细胞浸润;经治疗,中药组及对照组小鼠表皮层角化不全程度较轻,颗粒层、棘层皮肤轻微增厚,炎细胞浸润减轻。结论 葛根芩连汤可以减轻AD模型小鼠皮损程度,减少搔抓次数,有效降低经皮失水、皮肤p H值,下调小鼠血清Ig E、IL-6水平,上调丝聚合蛋白、密封蛋白1的表达,抑制炎症反应和改善皮肤屏障,为中医“皮应大肠”理论和皮肤病从肠论治提供了实验依据。

Objective To observe the intervention effect and anti-inflammatory mechanism of Gegen Qinlian decoction on atopic dermatitis(AD) in mice.Methods The AD mouse model was induced by repeated skin stimulation with dinitrofluorobenzene(DNFB).24 male BALB/c mice were randomly divided into normal group,model group,control group(momethasone furoate) and traditional Chinese medicine treatment group(Gegen Qinlian decoction).The skin lesion evaluation,skin histopathology,skin pH value,percutaneous water loss(TWEL),scratching behavior and the contents of serum IgE,IL-6 and IL-10 were observed.The mRNA expressions of Filaggrin and Claudin-1 were detected by reverse transcription-polymerase chain reaction(qRT-PCR),and the protein expressions of Filaggrin and Claudin1 were detected by Western blotting.Results(1) Before treatment,compared with the normal group,the back skin of the model group had obvious erythema and edema,and scabs,scales,and skin hypertrophy were seen;the number of scratches were increased significantly;transdermal water loss and skin pH were significantly increased After treatment,compared with the model group,the skin damage in the traditional Chinese medicine group and the control group was improved,and the number of scratches,transdermal water loss,and skin pH were significantly reduced(P <0.05).(2) Before treatment,the serum levels of total IgE,IL-6 and IL-10 in the model group were significantly different from those in the normal group;after treatment,the levels of total IgE and IL-6 in serum were lower than those in the model group(P <0.05).(3) Before treatment,compared with the normal group,the results of qRT-PCR showed that the expressions of Filaggrin and Claudin-1 were decreased;the results of WB showed that the expressions of Filaggrin and Claudin1 were decreased;after treatment,the expressions of Filaggrin and Claudin-1 were increased(P<0.05).(4) Before treatment,compared with the normal group,the results of skin pathology showed that the model group had incomplete keratinization in the epidermis,thickening of the granular layer and acanthus layer,and a large number of inflammatory cells infiltrated in the epidermis and dermis;after treatment,the degree of parakeratosis in the epidermis of the mice in the control group was milder,the skin of the granular layer and the acanthus layer was slightly thickened,and the infiltration of inflammatory cells was reduced.Conclusion Gegen Qinlian decoction can reduce the degree of skin damage,reduce the number of scratches,effectively reduce transdermal water loss and skin pH value,dowr-regulate the levels of IgE and IL-6 in the serum,and up-regulate the expression of Filaggrin and Claudin-1 in AD model mice.This may be related to that,Gegen Qinlian decoction can improve the skin barrier and reduce the immune response in mice,which provides an experimental basis for the theory of "The skin is connected with the large intestine" in traditional Chinese medicine and the treatment of skin diseases from the intestine.