养血解毒法治疗斑块型银屑病血燥证疗效观察及分子机制研究

【作者】 姜春燕；

【导师】 李元文；

【作者基本信息】 北京中医药大学 ， 中医外科学， 2014， 博士

【摘要】 背景银屑病是以表皮细胞过度增殖、角化不全等表皮动力学紊乱为特征的慢性炎症性皮肤病,属于皮外科的疑难病证之一。斑块型银屑病是银屑病中的难治型,临床实践证明,具有整体调整功能的中药复方能够取得良好疗效。养血化斑汤是李元文教授在深入总结前人经验及长期临床验证、反复筛试的基础上拟定的具有养血解毒功能的治疗斑块型银屑病的中药复方。前期课题组已经对该复方治疗银屑病进行了初步临床研究,发现其安全性好、疗效可靠,但缺少循证医学的客观评价,且其作用机制尚不明确。根据系统生物学的观点,疾病的发生、发展与一系列相互作用的基因或蛋白相关,呈现出复杂的网络关系特征；药物是通过作用于疾病网络中的多个靶点,对各靶点的作用产生协同效应,从而对其发生、发展进行干预而达到治疗效果。中药复方具有多组分、多途径、多靶点协同作用特点,基于分子网络层面探索其效应机制,有助于更全面、更准确地把握其效应整体性,而且能为中药药理作用机制研究提供新思路。因此,本研究基于分子网络分析方法探索养血化斑汤治疗银屑病的机制。目的(1)科学地评价养血化斑汤治疗斑块型银屑病血燥证的疗效与安全性；(2)探讨养血解毒药对组合干预银屑病的作用靶点及分子网络机制。方法(1)临床安全性及疗效评价采用前瞻性、随机、对照的试验设计,观察养血化斑汤治疗斑块型银屑病血燥证的临床疗效及安全性。以2：1比例随机区组纳入120例斑块型银屑病血燥证患者：养血化斑汤治疗组80例,消银胶囊对照组40例,两组均配合白凡士林外涂,疗程8周。治疗后,通过比较银屑病皮损面积和严重程度指数(PASI)评分以及中医临床症状评估指标评定其疗效,记录不良反应发生情况,对安全性进行评价。(2)疗效靶点及分子网络机制研究参考《中药大辞典(第二版)》找到当归、鸡血藤、槐花、土茯芩的化学成分,应用PubChem数据库检索这些成分对应的靶蛋白；以"Psoriasis"为关键词,在PubMed gene数据库中检索银屑病的相关基因；然后将上述数据集导入IPA软件,分别构建中药靶蛋白和银屑病相关基因的分子网络及生物学通路；再通过网络映射、比较,可视化呈现养血药对、解毒药对及它们的组合干预银屑病的作用靶点和分子网络机制。结果(1)疗效及安全性观察结果养血化斑汤治疗斑块型银屑病血燥证临床痊愈9.0%,显效38.4%,有效30.8%,无效21.8%；消银胶囊对照组临床痊愈5.4%,显效16.2%,有效40.5%,无效37.8%。经统计分析发现：试验组在银屑病临床疗效、银屑病临床总有效率与对照组相当,其差异无统计学意义。而在银屑病临床愈显率方面试验组(47.4%)优于对照组(21.6%)。提示养血化斑汤虽然在治疗血燥型银屑病总疗效上与消银胶囊相当,但在痊愈及显效患者比例上多于对照组,其改善皮疹程度上优于对照组。试验组在中医证候疗效、中医证候愈显率疗效、中医证候总有效率方面均优于对照组(P<0.05)。由于血燥证银屑病中医证候评价主要由鳞屑、瘙痒、口干症状改善几方面构成,提示养血化斑汤对综合改善血燥型银屑病患者鳞屑、瘙痒程度、口干症状方面优于对照组(P<0.05)。对试验组患者疗前、疗后进行安全性实验室检查,经观察服用养血化斑汤治疗组患者全部实验室指标未出现异常。试验组在治疗过程中,未出现不良反应。对照组有1例出现皮疹一过性加重,未采取其他治疗方法,1周后症状逐渐缓解。(2)生物信息学分析结果通过检索靶蛋白和基因数据库查找到：当归靶蛋白44个、鸡血藤靶蛋白41个、槐花靶蛋白34个、土茯苓靶蛋白35个,银屑病相关基因165个。对上述各个中药的靶蛋白及银屑病基因分别进行生物功能分析、生物学通路分析及分子网络分析发现：①当归、鸡血藤、槐花和土茯苓各自相关的疾病不同；相关度最强的疾病分别为：Nutritional Disease,Neurological Disease,Cancer和Psychological Disorders; Dermatological diseases and conditions是它们共同对应的疾病；提示这4味中药治疗疾病的范围广泛而不同,但都可治疗皮肤相关疾病；银屑病对应的生物功能主要涉及Inflammatory Response,Immunological regulation和Cell-To-Cell Signaling and Interaction。②当归、鸡血藤、槐花和土茯苓各自相关的生物学通路不同；相关度最强的生物学通路分别为：Aryl Hydrocarbon Receptor Signaling, Aryl Hydrocarbon ReceptorSignaling, ATM Signaling和Gai Signaling;与银屑病最相关的生物学通路是Dendritic Cell Maturation。③当归、鸡血藤、槐花和土茯芩4个中药及银屑病各自的分子网络分别有5、4、4、2、16个亚网络；各个网络表现的功能不同：当归网络的主要功能涉及Lipid Metabolism、Molecular Transport、Small Molecule Biochemistry等,鸡血藤网络的主要功能涉及Neurological Disease、Psychological Disorders、Metabolic Disease等,槐花网络的主要功能涉及Cell Cycle、Organismal Development、DNA Replication、 Recombination and Repair等,土茯苓网络的主要功能涉及Metabolic Disease、 Neurological Disease、Organismal Injury and Abnormalities等,银屑病网络的主要功能涉及Cell-To-Cell Signaling and Interaction、Hematological System Development and Function、Cellular Movement等。把与中药相关的生物学通路和分子网络与银屑病相关的生物学通路和分子网络进行比较整合分析发现：①当归干预银屑病相关的特有生物学通路主要包括Renin-Angiotensin Signaling, IL-6Signaling和Inhibition of Matrix Metalloproteases等；鸡血藤干预银屑病相关的特有的生物学通路包括Cdc42Signaling和Superpathway of Citrulline Metabolism:槐花干预银屑病相关的特有生物学通路是FXR/RXR Activation:土茯苓干预银屑病相关的特有生物学通路包括Serotonin Receptor Signaling和Aldosterone Signaling in Epithelial Cells。②当归-鸡血藤养血药对干预银屑病相关的特有生物学通路主要包括Endothelin-1Signaling、RAR Activation、LPS/IL-1Mediated Inhibition of RXR Function等；槐花-土茯苓解毒药对干预银屑病相关的特有生物学通路是G-Protein Coupled Receptor Signaling。③当归-鸡血藤-槐花-土茯苓药对组合干预银屑病相关的特有生物学通路包括Aryl Hydrocarbon Receptor Signaling、Androgen Signaling、eNOS Signaling和PI3K/AKT Signaling。④当归干预银屑病特有的关键网络靶标分子主要包括Hsp27、PARP、PEPCK等；鸡血藤干预银屑病特有的关键网络靶标分子主要包括Gsk3、Interferon alpha、NOS2等；槐花干预银屑病特有的关键网络靶标分子主要包括NOTCH1、Alpha tubulin、CD3等；土茯苓干预银屑病特有的关键网络靶标分子包括Beta Arrestin、Gpcr和TNF；⑤当归-鸡血藤养血药对干预银屑病特有的关键网络靶标分子主要包括IL1、Pro-inflammatory Cytokine、P38MAPK等；槐花-土茯苓解毒药对干预银屑病特有的关键网络靶标分子主要包括LDL、IFN Beta、glutathione peroxidase等。⑥当归-鸡血藤-槐花一土茯苓药对组合干预银屑病特有的关键网络靶标分子包括26s Proteasome、ERK1/2、NFkB (complex)、UBC和Ubiquitin。结论(1)养血化斑汤治疗斑块型银屑病血燥证安全有效,值得临床推广应用。(2)当归-鸡血藤-槐花-土茯苓养血解毒药对组合通过调控Aryl Hydrocarbon Receptor Signaling、Androgen Signaling、eNOS Signaling和PI3K/AKT Signaling生物学通路及针对Ubiquitin、Proteasome。ERK1/2和NF-κB分子靶点对银屑病发挥疗效。以上经典通路及靶点的扰动也反映了斑块型银屑病血燥证的生物学基础。(3)分子网络分析技术是探索中药治疗疾病靶点及机制的有效方法。更多还原

【Abstract】 BackgroundPsoriasis is a common, chronic, inflammatory disease of the skin, whose typical clinical manifestions are increased cellular activity of the epidermis and dilated capillaries of the dermis. Psoriasis is one of the difficult miscellaneous diseases in dermasurgery. Plaque type psoriasis is a refractory type of psoriasis. Clinical practice has proved that Chinese herbal compound (CHM) treatment can achieve good curative effect on psoriasis relying on its overall adjustment function. Based on the in-depth summary of predecessors’experience, long-term clinical validation and repeated screening test, Li Yuanwen professor invented Yangxue Huaban decoction (YXHB) with functions of nourishing blood and detoxification (Yangxue and Jiedu). Preliminary clinical observation of YXHB treatment psoriasis showed that YXHB has a good safety and reliable curative effect. However there is lack of the objective evaluation on the basis of the evidence-based medicine and the mechanism of YXHB treatment psoriasis is unclear.According to systems biology viewpoint, the occurrence and development of diseases are associated with a series of interacting genes or proteins that show the characteristics of complex network. Drug produces the synergistic effect through its effects on the multiple targets in the disease network and thus intervenes to the occurrence and development of the disease. CHM is characterized by multi-component, multi-pathways and multi-targets synergy. Exploring CHM effect mechanism at the molecular network level not only is conducive to more comprehensive and more accurate decryption of CHM integrity effect but also provides new ideas for mechanism study pharmacology of Chinese medicine. Therefore using the molecular network analysis method is expected to explore YXHB mechanism for the treatment of psoriasis.Objective(1) To evaluate the efficacy and safety of YXHB on treatment of plaque type psoriasis.(2) To explore the possible acting targets and molecular network mechanism of Yangxue and Jiedu herbal pairs complex prescription (YJPC) on psoriasis. And thus to promote the clinical application and development of traditional Chinese medicine. Methods(1) Efficacy and safety of YXHBIn this prospective, randomized and control clinical trial of120patients with plaque type psoriasis,80were treated with YXHB and40were treated with Xiaoyin capsule (XYC) for8weeks. The therapeutic outcomes were evaluated by PASI score and clinical symptoms evaluation indicators in traditional Chinese medicine. And the adverse effects were analyzed.(2) Acting targets and molecular network mechanism of YJPCThe effective components of Angelica sinensis (DG), Caulis Spatholobi (JXT), Flos Sophorae (HH) and Rhizoma smilacis glabrae (TFL) were retrievalled from the Grand Dictionary of Chinese Medicine (Second Edition), the human target proteins of those effective components were retrievalled from Pubchem database, and genes related psoriasis were retrievalled from Pubmed gene database. Then the target proteins and genes were respectively uploaded Ingenuity Pathways Analysis (IPA) data analysis platform, constructing the molecular networks and biological pathways of each herb and psoriasis. Finally through the network comparison, acting targets and molecular network mechanism of YJPC were presented visually.Results(1) Efficacy and safety of YXHBEffective rates of YXHB group and XYC group respectively were78.2%and62.2%after treatment and there were no significant differences between the two groups. PASI score and clinical symptoms of YXHB group were significantly alleviated than those of XYC group (P<0.05). In the process of treatment, there were no significant adverse reactions and toxicity in YXHB group and one patient had a transient rash in XYC group.(2) Bioinformatics analysis resultsTarget proteins and genes were searched from corresponding database. The number of target proteins of DG, JXT, HH and TFL respectively is44,41,34and35. Psoriasis related genes are165. The results of biological function, biological pathways and molecular network analysis showed as follows:①each herb corresponds to different diseases; the most relevant disease corresponding to DG, JXT, HH and TFL respectively is nutritional disease, neurological disease, cancer and psychological disorders; Dermatological diseases and conditions are common diseases corresponding to each herb; psoriasis involved biological functions mainly includes inflammatory response, immunological regulation and cell-to-cell signaling and interaction.②ach herb corresponds to different biological pathways; the most relevant biological pathway corresponding to DG, JXT, HH, TFL and psoriasis respectively is aryl hydrocarbon receptor signaling, aryl hydrocarbon receptor signaling, ATM signaling and Gai signaling and dendritic cell maturation.③Molecular networks corresponding to DG, JXT, HH, TFL and psoriasis respectively have5,4,4,2,16sub-network; each network related biological functions are different; DG network functions mainly involve lipid metabolism, molecular transport, small molecule biochemistry, etc.; JXT network functions mainly involve neurological disease, psychological disorders, metabolic disease, etc.; HH network functions mainly involve cell cycle, organismal development, DNA replication, recombination and repair, etc.; TFL network functions mainly involve metabolic disease, neurological disease, organismal injury and abnormalities, etc.; psoriasis network functions mainly involve cell-to-cell signaling and interaction, hematological system development and function, cellular movement etc.Compared biological pathways and molecular networks of each herb with those of psoriasis, the results showed as follows:①the special pathways of DG to psoriasis mainly include renin-angiotensin signaling, IL-6signaling, inhibition of matrix metalloproteases, etc.; the special pathways of JXT to psoriasis include Cdc42signaling and superpathway of citrulline metabolism; the special pathway of HH to psoriasis is FXR/RXR activation; the special pathways of TFL to psoriasis include serotonin receptor signaling and aldosterone signaling in epithelial cells,②the special pathways of DG-JXT herbal pair to psoriasis mainly include endothelin-1signaling, RAR activation, LPS/IL-1mediated inhibition of RXR Function, etc.; the special pathway of HH-TFL herbal pair to psoriasis is G-protein coupled receptor signaling.③the special pathways of DG-JXT and HH-TFL herbal pairs (YJPC) to psoriasis include aryl hydrocarbon receptor signaling, androgen signaling, eNOS signaling and PI3K/AKT signaling.④the special key network target molecules of DG to psoriasis mainly include Hsp27, PARP, PEPCK, etc.; the special key network target molecules of JXT to psoriasis mainly include Gsk3, interferon alpha, NOS2, etc.; the special key network target molecules of HH to psoriasis mainly include NOTCH1, Alpha tubulin, CD3, etc.; the special key network target molecules of TFL to psoriasis include beta arrestin, Gpcr and TNF.⑤the special key network target molecules of DG-JXT herbal pair to psoriasis mainly include IL1, pro-inflammatory cytokine, p38MAPK, etc.; the special key network target molecules of HH-TFL herbal pair to psoriasis mainly include LDL, IFN Beta, glutathione peroxidase, etc. ⑥he special key network target molecules of DG-JXT and HH-TFL herbal pairs (YJPC) to psoriasis include26s proteasome, ERK1/2, NFkB (complex), UBC and Ubiquitin.Conclusion(1) YXHB is effective and safe on treatment of plaque type psoriasis and is worth clinical application.(2) YJPC treats psoriasis by regulating four biological pathways (aryl hydrocarbon receptor signaling, androgen signaling, eNOS signaling and PI3K/AKT signaling) and four network target molecules (Ubiquitin, Proteasome, ERK1/2and NF-κB). Perturbation of the biological pathways and molecules also explain the modern biological mechanisms of plaque type psoriasis Xuezao syndrome.(3) Molecular network analysis technique is an effective method to explore and predict targets and mechanisms of traditional Chinese medicine treating diseases.更多还原