地黄饮通过调控JAK1/STAT3信号通路治疗特应性皮炎小鼠的作用机制

Mechanism of Dihuangyin in Treatment of Mice with Atopic Dermatitis by Regulating JAK1/STAT3 Signaling Pathway

目的:探讨地黄饮对2,4-二硝基氯苯(DNCB)诱导的特应性皮炎(AD)模型小鼠的作用及潜在机制。方法:通过DNCB反复刺激小鼠背部皮肤建立小鼠AD模型,造模成功后,随机分为模型组、润燥组(0.78 g·kg^(-1))、地黄饮高、中、低剂量组(40.30、20.15、10.08 g·kg^(-1)),每组12只,空白组12只,共72只。给药组按剂量灌胃给予相应药液,空白组和模型组灌胃给予同体积纯净水,1次/d,连续给药2周后观察小鼠皮损及搔抓次数;取小鼠背部皮损进行苏木素-伊红(HE)染色和甲苯胺蓝染色观察病理情况;酶联免疫吸附测定法(ELISA)检测小鼠血清免疫球蛋白E(IgE)、白细胞介素-4(IL-4)、白细胞介素-6(IL-6)、干扰素-γ(IFN-γ)含量;实时荧光定量聚合酶链式反应(Real-time-PCR)检测皮损组织中IFN-γ、IL-4、IL-6、Janus激酶1(JAK1)、转录激活因子3(STAT3)mRNA的表达水平;蛋白免疫印迹法(Western blot)检测皮损组织JAK1、磷酸化(p)-JAK1、STAT3、p-STAT3蛋白的表达。结果:与空白组比较,模型组小鼠背部皮肤可见大量结痂、干燥、糜烂、肥厚伴搔抓,皮损组织可见表皮增生,伴角化过度和角化不全,棘层肥厚伴水肿,真皮层可见大量肥大细胞浸润,部分处于脱颗粒状态,小鼠血清中IgE、IL-4、IL-6、IFN-γ含量显著升高(P<0.01),皮损组织中p-JAK1、STAT3、p-STAT3蛋白表达水平及IL-4、IL-6、IFN-γ、JAK1、STAT3 mRNA表达显著升高(P<0.01);与模型组比较,各给药组小鼠背部皮肤仅见少量干燥、脱屑,细胞水肿减轻,炎性浸润明显减轻,肥大细胞浸润数明显降低,小鼠血清中IgE、IL-4、IL-6、IFN-γ均有不同程度降低(P<0.05,P<0.01),皮损组织中p-JAK1、STAT3、p-STAT3蛋白表达水平及IL-4、IL-6、IFN-γ、JAK1、STAT3 mRNA表达明显降低,其中以地黄饮高剂量组效果最佳(P<0.01)。结论:地黄饮能改善AD小鼠的皮损及瘙痒症状,其作用机制可能与干预JAK1/STAT3信号通路有效调节辅助性T细胞(Th)1/Th2轴上的细胞因子,影响皮肤屏障功能有关。

Objective:To investigate the effect and potential mechanism of Dihuangyin on 2,4-dinitrochlorobenzene(DNCB)-induced model mice with atopic dermatitis(AD).Method:A mouse model with AD was established by repeatedly stimulating the back skin of mice with DNCB.After successful modeling,the mice were randomly divided into model group,Runzao group(0.78 g·kg^(-1)),and high,medium,and low dose(40.30,20.15,and 10.08 g·kg^(-1))groups of Dihuangyin,with 12 mice in each group,and the blank group consisted of 12 mice,72 in total.The administration groups were given the corresponding liquid by dose,and the blank group and model group were given the same dose of pure water by intragastric administration,once a day.The skin lesions and scratching times of mice were observed after continuous administration for two weeks.The back skin lesions of mice were stained with hematoxylin-eosin(HE)and toluidine blue to observe the pathology.The contents of serum immunoglobulin E(IgE),interleukin-4(IL-4),interleukin-6(IL-6),and interferon-γ(IFN-γ)were detected by enzyme-linked immunosorbent assay(ELISA).The mRNA expression levels of IFN-γ,IL-4,IL-6,Janus kinase 1(JAK1),and transcriptional activator 3(STAT3)in skin lesion tissue were detected by Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR).The expressions of JAK1,phosphorylation(p)-JAK1,STAT3,and p-STAT3 proteins in skin lesion tissue were detected by Western blot.Result:Compared with the blank group,the back skin of the model group showed large-scale scab,dryness,erosion,hypertrophy with scratching,epidermal hyperplasia with hyperkeratosis and parakeratosis,hyperacanthosis with edema,and a large number of mast cell infiltration in the dermis,some of which were degranulated.The contents of IgE,IL-4,IL-6,and IFN-γ in the serum of mice were significantly increased(P<0.01),and the protein expression levels of p-JAK1,STAT3,and p-STAT3 and mRNA expressions of IL-4,IL-6,IFN-γ,JAK1,and STAT3 in skin lesion tissue were significantly increased(P<0.01).Compared with the model group,only a small amount of dryness and desquamation were observed in the back skin of mice in each administration group,and cell edema was reduced.The inflammatory infiltration was significantly reduced,and the number of mast cell infiltration was significantly decreased.The serum IgE,IL-4,IL-6,and IFN-γof mice were decreased to varying degrees(P<0.05,P<0.01).The protein expression levels of p-JAK1,STAT3,and p-STAT3 and mRNA expressions of IL-4,IL-6,IFN-γ,JAK1,and STAT3 in skin lesion tissue were significantly decreased,and the effect of high dose group of Dihuangyin was the best(P<0.01).Conclusion:Dihuangyin can improve skin lesions and pruritus in mice with AD,and its mechanism may be related to the effective regulation of cytokines on the helper T cells(Th1)/Th2 axis by interfering with the JAK1/STAT3 signaling pathway and affecting skin barrier function.