摘要
目的:利用超高效液相色谱-四极杆-飞行时间串联质谱(UPLC-Q-TOF-MS/MS)结合网络药理学与分子对接探究二妙四土汤对湿热型湿疹大鼠的药效影响及作用机制。方法:通过人工气候箱干预结合二硝基氯苯(DNCB)致敏诱导建立湿热型湿疹大鼠模型,随机分为正常组,模型组,二妙四土汤低、高剂量组(3.40、6.80 g·kg^(-1)),醋酸泼尼松组(2.51 mg·kg^(-1)),每组8只,共46只,其中含药血清实验6只,采用UPLC-Q-TOF-MS/MS对大鼠进行含药血清化学分析,结合网络药理学对关键成分、核心靶点及信号通路进行预测,CB-Dock2在线网站进行分子对接实验;通过表观指标结合病理组织染色法考察二妙四土汤治疗湿热型湿疹的药理作用;采用酶联免疫吸附测定法(ELISA)检测血清中胃泌素(GAS)、白细胞介素-4(IL-4)、白细胞介素-13(IL-13)含量水平;采用蛋白免疫印迹法(Western bolt)检测白细胞介素-6(IL-6)、Janus激酶1(JAK1)、磷酸化(p)-JAK1、信号转导与转录激活因子3(STAT3)、p-STAT3蛋白表达水平。结果:大鼠含药血清样品中共检测到19个活性成分,经预测,作用于198个靶点治疗湿热型湿疹,其中关键成分包括大黄素、黄柏碱、槲皮苷等,主要的核心靶点包括STAT3、肿瘤坏死因子(TNF)、IL-6等,主要涉及到癌症信号通路、磷脂酰肌醇3-激酶(PI3K)/蛋白激酶(Akt)信号通路、辅助性T细胞17(Th17)细胞分化信号通路、JAK/STAT信号通路等。分子对接结果提示,关键成分与JAK/STAT信号通路中核心靶点IL-6、JAK1、STAT3结合活性较强。动物实验结果表明,与正常组比较,模型组大鼠精神萎靡、毛发疏松、大便溏泻,表皮渗液、糜烂并生成厚厚的痂皮呈鳞屑状,体质量降低、肛温和饮水量升高、脾、胸腺、胃指数增大(P<0.05,P<0.01),皮损组织可见角化不全、炎性细胞聚集、表皮与真皮分离界限不明显,胃黏膜变薄欠缺、结构紊乱、可见明显的炎性细胞聚集,血清中GAS、IL-4、IL-13含量水平明显降低(P<0.05,P<0.01),皮损组织中IL-6、JAK1、p-JAK1、p-STAT3的蛋白表达水平明显升高(P<0.05,P<0.01);与模型组相比较,各给药组大鼠精神状态稳定、粪便成型、肛周洁净,表皮基本恢复正常、仅存在少量的鳞屑状痂皮,体质量升高、肛温和饮水量降低、脾、胸腺、胃指数降低(P<0.05,P<0.01),表皮厚度降低、表皮与真皮分离界限明显、但仍可见角化过度和炎症细胞聚集,胃黏膜厚度增加,结构有不同程度的恢复,大鼠血清中GAS、IL-4、IL-13含量水平均有不同程度地升高,皮损组织中IL-6、JAK1、p-JAK1、p-STAT3的蛋白表达水平明显下降(P<0.05,P<0.01)。结论:二妙四土汤可能通过调控JAK/STAT信号通路发挥治疗湿热型湿疹大鼠的作用。
Objective:Ultra performance liquid chromatography-quadrupole-time of flight tandem mass spectrometry(UPLC-Q-TOF-MS/MS)coupled with network pharmacology and molecular docking was utilized to explore the efficacy and mechanism of action of Ermiao Situ decoction on rats with damp-heat eczema.Methods:A rat model of damp-heat eczema was established by artificial climate chamber intervention combined with sensitization induction by dinitrochlorobenzene(DNCB),and it was randomly divided into the normal group,the model group,the medium-and high-dose groups of Ermiao Situ decoction(3.40 g·kg^(-1)and 6.80 g·kg^(-1)),and the prednisone acetate group(2.51 mg·kg^(-1)),with eight rats in each group,totalling 46 rats,of which six rats were tested with the drug-containing serum.The chemical analysis of drug-containing serum from rats was carried out by UPLC-Q-TOF-MS/MS,combined with network pharmacology for the prediction of key components,core targets,and signaling pathways,and molecular docking experiments were performed by CB-Dock2 online website.The pharmacological effects of Ermiao Situ decoction in the treatment of damp-heat eczema were investigated by epitaxial indexes combined with the pathologic tissue staining method.The serum levels of gastrin(GAS),interleukin-4(IL-4),and interleukin-13(IL-13)were measured by enzyme-linked immunosorbent assay(ELISA).Interleukin-6(IL-6),Janus kinase 1(JAK1),phosphorylated(p)-JAK1,signal transduction and activation of transcription factor 3(STAT3),and p-STAT3 protein expression level was determined by Western bolt.Results:A total of 19 active ingredients were detected in drug-containing serum samples of rats,which were predicted to act on 198 targets for the treatment of damp-heat eczema,among which the key ingredients included rhodopsin,huangpai alkaloids,and quercetin,and the main core targets included STAT3,tumor necrosis factor(TNF),and IL-6,which were mainly involved in the cancer signaling pathway,phosphatidylinositol 3-kinase(PI3K)/protein kinase(Akt)signaling pathway,T helper 17(Th17)cell differentiation signaling pathway,and JAK/STAT signaling pathway.The molecular docking results suggested that the key components had strong binding activities with the core targets IL-6,JAK1,and STAT3 in the JAK/STAT signaling pathway.The results of animal experiments showed that compared with those in the normal group,rats in the model group were depressed.They had loose hair,loose stools,epidermal oozing,vesiculation,and generation of thick scabs in the form of scales,decreased body weight,increased anus temperature and water intake,and increased indexes of the spleen,thymus gland,and stomach(P<0.05,P<0.01),and the lesion tissue could be seen to be hyperkeratotic,with the aggregation of inflammatory cells and nonsignificant separation of epidermis and dermis.The gastric mucosa was thinned,deficient,and structurally disorganized,and obvious inflammatory cell aggregation was seen.The levels of GAS,IL-4,and IL-13 in serum were significantly reduced(P<0.05,P<0.01),and the protein expression levels of IL-6,JAK1,p-JAK1,and p-STAT3 in the lesion tissue were significantly increased(P<0.05,P<0.01).Compared with those in the model group,rats in each administration group had stable mental states,formed feces,a clean perianal area,and basically normal epidermis.Only a small amount of scaly scabs existed,and the rats had body weight increased,with decreased anal temperature and water intake,as well as decreased spleen,thymus,and gastric indexes(P<0.05,P<0.01).Epidermal thickness was decreased,and epidermal and dermal separation boundaries were obvious,but hyperkeratotic and accumulation of inflammatory cells could still be seen.The thickness of gastric mucosa increased,and the structure was restored to varying degrees.The levels of GAS,IL-4,and IL-13 content in the serum of rats were increased to varying degrees,and the protein expression levels of IL-6,JAK1,p-JAK1,and p-STAT3 in the dermal lesion tissue were significantly decreased(P<0.05,P<0.01).Conclusion:Ermiao Situ decoction may exert therapeutic effects on rats with damp-heat eczema by modulating the JAK/STAT signaling pathway.
作者
韩康宁
胡俊杰
李娟
章敏
周贤
刘松林
陈新
HAN Kangning;HU Junjie;LI Juan;ZHANG Min;ZHOU Xian;LIU Songlin;CHEN Xin(Hubei Key Laboratory of Resources and Chemistry of Chinese Medicine,School of Pharmacy,Hubei University of Chinese Medicine,Wuhan 430065,China;Clinical College of Chinese Medicine,Hubei University of Chinese Medicine,Wuhan 430060,China)
出处
《中国实验方剂学杂志》
北大核心
2025年第9期37-47,共11页
Chinese Journal of Experimental Traditional Medical Formulae
基金
国家自然科学基金项目(82004253)
湖北省自然科学基金项目(2023AFD146)
湖北省中医药重点学科建设项目(鄂中医通[2023]2号)
湖北省卫生健康委重点科研项目(WJ2023Z002)。
