雷公藤与甲氨蝶呤、黄芪与当归联用治疗RA机制的生物信息学分析
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摘要
背景
类风湿关节炎(rheumatoid arthritis, RA)是一种以关节滑膜炎症为特征的慢性全身性自身免疫性疾病。全世界患病率为0.3%～1%,各年龄组人群均可发病,据有关资料统计我国发病率约为0.32%～0.36%,如不及时治疗,可引起严重的关节畸形,关节功能障碍,严重影响患者生活质量。
类风湿性关节炎的治疗目标是通过减轻关节炎带来的疼痛、防止关节的破坏及维持关节的机能来提高患者身体上、精神上以及在社会中的生活品质。类风湿性关节炎的核心治疗是药物治疗,西药包括抗类风湿药物、非甾体类抗炎药物及甾体类药物等。抗类风湿性药物是控制由于免疫机能异常而引起的类风湿性关节炎的药物,目前有免疫调节药、免疫抑制药及生物制剂等。剂量服用西药治疗本病,易出现胃肠道症状及间质性肾损害,临床常常结合中药治疗本病,中医药以辨证论治为主,强调整体性,可以从多层次、通过多途径来调节机体的免疫功能,具有较大的潜力与优势。多项临床研究证明某些中药制剂及与西药联合治疗的联合制剂有着比单用西药更令人满意的疗效可提高、巩固疗效,同时能降低药物的毒副作用。
文本挖掘是一个交叉的研究领域,它涉及到自然语言处理、机器学习、数据挖掘、信息检索等多个领域的内容,在商业、传媒、教育、政府、银行及生物技术、医疗卫生等行业领域都发挥着不可忽视的作用。中医药学包含着丰富的知识,经过近十多年各方面研究人员的努力,已经积累了大量以数据库存储为主的中医药数据,这些数据以文本型居多。文本挖掘能从海量的中医药文献中发现知识,以促进中医药的发展。
生物信息学是八十年代末随着人类基因组计划的启动而兴起的一门新的交叉学科是在生命科学的研究中以计算机为工具对生物信息进行储存检索和分析的科学。生物信息学用于中药的研究,可确定中药的作用靶位。中药及其复方有效世人共知,但迄今为止中药还没有进入国际医药市场的主流,其最根本的原因是中药理论无法与国际上普遍接受的现代医学理论接轨。通过人类基因组计划筛选和分离出各种疾病相应的致病基因,再以其作为药物的靶标来研究重要作用的分子机制,这可能是未来中药药理研究的主要方向之一。应用基因芯片技术和蛋白组相关分析技术研究中药复方对细胞基因表达谱和蛋白表达谱的影响,可以建立基因芯片及蛋白组学和生物信息学等技术平台,利用此平台将中药复方多组分、多靶点、多途径作用与基因、蛋白表达关联起来,比较各自不同的表达差异谱,确定不同配伍组方对应基因及蛋白表达靶点,并根据表达的器官特异性及表达水平与复方的君、臣、佐、使理论及用药剂量相关联,同时根据不同配伍组方对应基因及蛋白靶点的相互作用,分析各组成复方单药之间的密切关系,阐明药物作用的物质基础及内在的配伍规律性。这种方法对于中药及其复方的研究与开发及走向世界均具有重要的实际意义。
我们希望能依据现有的文献报道,利用生物信息学的分析方法,得到药物联合应用的网络,探索中西药间、中药配伍间的相互作用规律,以便为中西药联合、中药配伍合理应用,提供依据,逐步达到联合用药确实“安全、有效、经济、合理”。
目的
采用文本挖掘方法,得出常用治疗RA的中西药联合、中药配伍用药为研究对象,利用Pubchem查找到相应的人类靶标蛋白,将其导入IPA分析平台(Ingenuity Pathways Analysis, IPA)得到相应的network、pathway等,与RA患者与健康人差异基因导入IPA获得的相应network、pathway对比分析,探讨其生物通路网络的构建及其治疗RA的机制。
研究内容
本研究完成下列内容的研究工作：
1治疗RA常用中西药联合、中药配伍应用规律的挖掘。
利用文本挖掘技术：分别找到中西药联合、中药配伍应用频次最高的药物。
2雷公藤与甲氨蝶呤(methotrexate, MTX)、黄芪与当归药理网络的构建及其治疗RA机制的生物信息学分析,主要包括如下具体研究内容：
(1)应用Pubchem检索雷公藤及MTX的药物人类靶蛋白,将其导入IPA分析平台得到相应的network、pathway等,探讨其生物通路网络的构建；对比RA患者与健康人差异表达基因构建的生物通路网络,探讨其治疗RA的机制。
(2)应用Pubchem检索当归及黄芪的药物人类靶蛋白,将其导入IPA分析平台得到相应的network、pathway等,探讨其生物通路网络的构建；对比RA患者与健康人差异表达基因构建的生物通路网络,探讨其治疗RA的机制。
研究结果
1治疗RA常用中西药联合、中药配伍应用规律的挖掘。
中成药和西药的联用中,雷公藤多甙片和MTX联合应用明显较多。雷公藤多甙片的临床报告近年较多,居于中成药的首位,返查文献发现,临床联合应用时,文献大多报道可以起到协同作用,合用的疗效优于单用,依据大量文献中西医联合用药疗效比较确切。
从挖掘结果来看,中药桂枝、芍药、知母、黄芪、当归配伍频次较高,其中喻含着汤药桂枝芍药知母汤、当归补血汤,反查文献发现,对各证型的RA均可加减运用,值得进一步深入研究。
2雷公藤与MTX、黄芪与当归药理网络的构建及其治疗RA机制的生物信息学分析。
2.1蛋白质泛素化途径可能是雷公藤、MTX、雷公藤联合MTX、黄芪联合当归治疗RA的作用机制之一。
蛋白质泛素化途径与免疫系统疾病RA密切相关。根据生物分析的结果提示,药物雷公藤、MTX、雷公藤联合MTX、黄芪、当归联合黄芪都可能通过蛋白质泛素化途径来影响治疗RA。所以,推测蛋白质泛素化途径是雷公藤、MTX、雷公藤联合MTX、黄芪、当归联合黄芪能够治疗RA的作用途径之一。进一步分析上述药物作用与该途径的靶蛋白发现有共同点,这个共同点就是作用于E3RING靶点。
E3RING是泛素蛋白连接酶(E3)的两个主要的家庭之一,并且已有文献报道E3连接酶很可能是一种治疗RA的新的治疗方法。
2.2雷公藤与MTX联合应用,不仅作用于蛋白质泛素化途径,较单独应用MTX,增加了更广泛的生物通路。
生物分析结果提示,雷公藤、MTX联合应用可能通过影响粒细胞—巨噬细胞集落刺激因子通路、二十二碳六烯酸信号通路、酪氨酸蛋白激酶1和3细胞因子信号作用通路等几个生物通路,更有效,更全面的对RA患者进行治疗。
2.3热休克蛋白是黄芪与当归联合应用与RA患者及健康人的差异基因作用于蛋白质泛素化途径时的共同靶点之一。
生物信息学结果分析可以提示,除了结果分析出黄芪联合当归通过E3Ring影响蛋白质泛素化途径外,还可以通过HSP来影响蛋白质泛素化途径,是其治疗RA的重要机制之一。另我们生物学分析结果中,黄芪与健康人和RA患者差异基因有共同影响的几个生物通路,包括：蛋白质泛素化途径、凋亡信号、糖皮质激素受体信号等,并且在两者联用之后,共同影响的通路在治疗RA中权重变大,而当归与健康人和RA患者差异基因未有共同影响的生物通路。
结论
1大多数中成药都与MTX联合应用。其中,雷公藤多甙片与MTX联合应用频次最多。最常应用治疗RA的中药有桂枝、白芍、知母、黄芪、当归,在这样联合应用中,提示治疗RA的中药复方配伍包括两个中药经典方：桂枝芍药知母汤、当归补血汤(黄芪、当归)。
2雷公藤联合MTX、黄芪联合当归治疗RA的机制之一,可能是通过影响以下一系列的信号转导途径来实现的：E3RING→E3泛素连接酶→蛋白质泛素化→NF-κKB/TGF-β。
3雷公藤、MTX联合应用较单独应用MTX,可能通过影响粒细胞—巨噬细胞集落刺激因子通路、二十二碳六烯酸信号通路、酪氨酸蛋白激酶1和3细胞因子信号作用通路、凋亡信号通路等几个生物通路,更全面更有效的对RA患者进行治疗。
4黄芪联合当归治疗RA除了影响E3RING→E3泛素连接酶靶点进而影响蛋白质泛素化途径,还通过HSP来影响该途径从而治疗RA。另外,根据我们生物信息学分析的结果,印证了当归补血汤应用治疗RA时,黄芪是主药,当归是辅药,两者为“相使”关系。
Background
Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease characterized by joint synovitis. The prevalence rate of the disease is0.3-1%worldwide, and the prevalence rate is0.32-0.36%in china according to certain reference, with the appearance in different age groups. If not treated, the disease can induce severe joint deformity, joint dysfunction, which seriously affect the life quality of patients.
The goal of rheumatoid arthritis treatment is to improve the patient's physical, mental and quality of life in society by alleviating the pain caused by arthritis, prevent joint damage and maintain joint function. The core of rheumatoid arthritis treatment is medical treatment, which includes anti-rheumatic drugs, non-steroidal anti-inflammatory drugs and steroidal drugs as to western medicine. Anti-rheumatoid drugs, including immunomodulatory drugs, immunosuppressive drugs and biological agents, control rheumatoid arthritis induced by immune function abnormity. Western medicine treatment is inclined to bring gastrointestinal symptoms and interstitial renal damage, therefore traditional Chinese medicines is often employed in combination with western medicine in clinic. Treatment by traditional Chinese medicines put stress integrity, combating the diseases from multi-level, multi-channel to regulate the body immune function, which exhibits with great potential and advantages. Several clinical studies have also shown that the treatment of RA by some traditional Chinese medicines in combination with western medicines can produce more satisfactory curative effects and reduce more side effects than the treatment by western medicines only.
Text mining is an interdisciplinary research field relating to natural language processing, machine learning, data mining, information retrieval etc. It plays an unnegligible role in business, media, education, government, banking, biotechnology, medical and health industry. There is rich knowledge in traditional Chinese medicines and. Abundance of data on traditional Chinese medicines, the majority of which are text data, have been accumulated through the efforts of researchers in the past decade. New knowledge can be discovered from the medical literature by text mining to facilitate the development of traditional Chinese medicines.
Bioinformatics is an interdisciplinary science emerging in the late1980s with the launching of human genome project. It covers the storage, retrieval and analysis of bioinformation using computers in life science research. When used in TMC research, it helps to determine the target of drug action. While it is well known that TMC and its compound preparations are effective, TMC has not entered the mainstream international pharmaceutical market, essentially due to the fact that the TMC theories fail to integrate with the internationally accepted modern medical theories. It is likely that screening and isolation of disease-specific pathogenic genes as target for the study of molecular mechanisms of important actions will become one of the main directions of future TMC pharmacology studies. Study of the effects of TMC compound preparations on the cytogene expression profile and protein expression profile by using genetic chip technology and proteome assays promotes the establishment of technology platforms for genetic chips, proteomics and bioinformatics, which allow multi-component, multi-target and multi-path connections between compound TMC preparations and genetic and protein expressions, so as to compare the different expression profiles, determine the gene and protein expression targets for different TMC combinations, allow connections between the organ specificity&level of expression and the TMC theory of "principal,adjuvant,auxiliary and conductant ingredirnts" as well as the dosage, and to determine the relations between different components of compound preparations based on the interactions between gene and protein targets corresponding to different combinations, thus clarifying the material basis for drug action and the combination rules.This methodology will be of practical importance for the research, development and internalization of TMC and its compound preparations.
Using the analysis methods of bioinformatics, we intended to generate a network for drug combinations based on existing literature reports and to explore the rules for interactions between Chinese and Western medicines and between different TMC combinations, so as to provide evidence for rational use of integrated Chinese-Western medicine as well as TMC combinations, in an effort to achieve "safe, effective, economic and rational" drug combinations.
Purpose
To identify the common Chinese-Western or Chinese-Chinese medicine combinations for RA as the study subject by text mining, and, using Pubchem, identify the corresponding human target proteins, which were to be introduced to Ingenuity Pathways Analysis (IPA) platform to produce the corresponding networks and pathways, etc. for comparison with the networks and pathways obtained when RA differential gene (the gene difference between healthy population and RA patients) was introduced to IPA, so as to explore the structure of biopathway network as well as the mechanism for treatment of RA.
Study contents
The followings were completed for this study:
1Mining for the application rules for common Chinese-Western or Chinese-Chinese medicine combinations for RA
The most commonly used Chinese-Western or Chinese-Chinese medicine combinations were identified using text mining.
2Establishment of "pharmacology networks of tripterygium wilfordii/methotrexate (MTX) and radix/angelica, and bioinformatics analysis of their therapeutic mechanisms for RA. Specifically, the followings were studied:
(1) Human target proteins for tripterygium wilfordii and MTX were identified through Pubchem retrieval and introduced to IPA platform to obtain the corresponding networks and pathways, etc., in exploration of the construction of their biopathway networks; their therapeutic mechanisms for RA were explored by comparing with the biopathway network construction obtained when RA differential gene was introduced to IPA.
(2) Human target proteins for angelica and radix were identified through Pubchem retrieval and introduced to IPA platform to obtain the corresponding networks and pathways, etc., in exploration of the construction of their biopathway networks; their therapeutic mechanisms for RA were explored by comparing with the biopathway network construction obtained when RA differential gene was introduced to IPA.
Results
1Mining for application rules for common Chinese-Western or Chinese-Chinese medicine combinations for RA
Among the common Chinese-Western medicine combinations, the combination of tripterygium wilfordii polyglycoside tablet and MTX was predominant. There has been large number of clinical reports on ripterygium wilfordii polyglycoside tablet, which leads the TMC preparations. Search into the literatures found that most literatures reported synergic action with clinical combinations, which were superior over single agent. Large number of literatures support that Chinese-Western combinations have definite therapeutic effect.
It was observed through mining that the TMCs ramulus cinnamomi, paeonia, anemarrhenae, radix and angelica were the most frequent to be subject to combinations, which included Paeoniae and Anemarrhenae Decoction and Angelicae Sinensis Decoction for Supplementing Blood. Search into the literatures found that addition to or subtraction from the combinations were possible for each symptomatic type of RA, which warrants further investigation.
2Establishment of pharmacology networks of tripterygium wilfordii/methotrexate (MTX) and radix/angelica, and bioinformatics analysis of their therapeutic mechanisms for RA
2.1Protein ubiquitinoylation pathway is closely related to RA, an immunologic disease. Bioanalyses indicated that tripterygium wilfordii alone, MTX alone, tripterygium wilfordii/MTX combination, radix alone and radix/angelica combination have therapeutic effect for RA through protein ubiquitinoylation pathways. It was therefore presumed that protein ubiquitinoylation pathway is one of the courses of action for tripterygium wilfordii alone, MTX alone, tripterygium wilfordii/MTX combination, radix alone or radix/angelica combination to treat RA. Further analysis identified a target protein for protein ubiquitinoylation pathway common for these treatments, i.e., E3RING target.
E3RING is one of the two major families of ubiquitin protein ligase (E3), and has been reported to be a likely new treatment option for RA.
2.2The combination of tripterygium wilfordii and MTX not only acts on the protein ubiquitinoylation pathways, but also allows more extensive biopathways as compared to MTX alone.
Bioanalyses indicated that the combination of tripterygium wilfordii and MTX may provide more effective and more comprehensive treatment for RA patients by affecting the granulocyte-macrophage colony stimulating factor(GM-CSF) pathway, the docosahexaenoic acid (DHA) signal pathway, the tyrosine protein kinase (TPK)1and3cytokine signal pathway.etc.
2.3Heat shock protein (HSP) is one of the targets for protein ubiquitinoylation pathway common for radix/angelica combination and the differential gene for RA.
Bioinformactics results indicate that radix/angelica combination affects the protein ubiquitinoylation pathway through E3RING and HSP, which serves as one of its important mechanisms for treating RA. In addition, bioanalyses identified several biopathway targets common for radix and RA differential gene, including protein ubiquitinoylation pathway, apoptosis signals, and glucocorticoid receptor signals, etc. When radix and angelica were combined, the weighing of their common target pathways was increased in treating RA, while no common biopathway targets were identified for angelica and the RA differential gene.
Conclusions
1Most of the TMC preparations were used in combination with MTX. Among them, tripterygium wilfordii polyglycoside tablet was the most frequent to be combined with MTX. The TMCs ramulus cinnamomi, paeonia, anemarrhenae, radix and angelica were the mostly used for combinations, including two classic TMC compound preparations, Paeoniae and Anemarrhenae Decoction and Angelicae Sinensis Decoction for Supplementing Blood (radix ad angelica)
2One of the mechanisms for tripterygium wilfordii in combination with MTX or radix to treat RA was through affecting the following signal conduction pathway:E3RING→E3ubiquitin protein ligase→protein ubiquitinoylation→NF-κB/TGF-β.
3Compared to MTX alone, the combination of tripterygium wilfordii and MTX may provide more comprehensive and more effective treatment for RA patients by affecting the GM-CSF pathway, the DHA signal pathway, the TPK-1and-3cytokine signal pathway, and the apoptosis signal pathway, etc.
4Aside from acting on the E3RING→E3ubiquitin protein ligase target and thus the protein ubiquitinoylation pathway, the combination of radix and angelica provides therapeutic effect for RA also through affecting the HSP pathway. In addition, bioinformatics analysis indicated that when Angelicae Sinensis Decoction for Supplementing Blood is used for RA, radix is the principal agent while angelica is the adjuvant agent, contributing to their "mutual enhancing" relationship.
类风湿关节炎(rheumatoid arthritis, RA)是一种以关节滑膜炎症为特征的慢性全身性自身免疫性疾病。全世界患病率为0.3%～1%,各年龄组人群均可发病,据有关资料统计我国发病率约为0.32%～0.36%,如不及时治疗,可引起严重的关节畸形,关节功能障碍,严重影响患者生活质量。
类风湿性关节炎的治疗目标是通过减轻关节炎带来的疼痛、防止关节的破坏及维持关节的机能来提高患者身体上、精神上以及在社会中的生活品质。类风湿性关节炎的核心治疗是药物治疗,西药包括抗类风湿药物、非甾体类抗炎药物及甾体类药物等。抗类风湿性药物是控制由于免疫机能异常而引起的类风湿性关节炎的药物,目前有免疫调节药、免疫抑制药及生物制剂等。剂量服用西药治疗本病,易出现胃肠道症状及间质性肾损害,临床常常结合中药治疗本病,中医药以辨证论治为主,强调整体性,可以从多层次、通过多途径来调节机体的免疫功能,具有较大的潜力与优势。多项临床研究证明某些中药制剂及与西药联合治疗的联合制剂有着比单用西药更令人满意的疗效可提高、巩固疗效,同时能降低药物的毒副作用。
文本挖掘是一个交叉的研究领域,它涉及到自然语言处理、机器学习、数据挖掘、信息检索等多个领域的内容,在商业、传媒、教育、政府、银行及生物技术、医疗卫生等行业领域都发挥着不可忽视的作用。中医药学包含着丰富的知识,经过近十多年各方面研究人员的努力,已经积累了大量以数据库存储为主的中医药数据,这些数据以文本型居多。文本挖掘能从海量的中医药文献中发现知识,以促进中医药的发展。
生物信息学是八十年代末随着人类基因组计划的启动而兴起的一门新的交叉学科是在生命科学的研究中以计算机为工具对生物信息进行储存检索和分析的科学。生物信息学用于中药的研究,可确定中药的作用靶位。中药及其复方有效世人共知,但迄今为止中药还没有进入国际医药市场的主流,其最根本的原因是中药理论无法与国际上普遍接受的现代医学理论接轨。通过人类基因组计划筛选和分离出各种疾病相应的致病基因,再以其作为药物的靶标来研究重要作用的分子机制,这可能是未来中药药理研究的主要方向之一。应用基因芯片技术和蛋白组相关分析技术研究中药复方对细胞基因表达谱和蛋白表达谱的影响,可以建立基因芯片及蛋白组学和生物信息学等技术平台,利用此平台将中药复方多组分、多靶点、多途径作用与基因、蛋白表达关联起来,比较各自不同的表达差异谱,确定不同配伍组方对应基因及蛋白表达靶点,并根据表达的器官特异性及表达水平与复方的君、臣、佐、使理论及用药剂量相关联,同时根据不同配伍组方对应基因及蛋白靶点的相互作用,分析各组成复方单药之间的密切关系,阐明药物作用的物质基础及内在的配伍规律性。这种方法对于中药及其复方的研究与开发及走向世界均具有重要的实际意义。
我们希望能依据现有的文献报道,利用生物信息学的分析方法,得到药物联合应用的网络,探索中西药间、中药配伍间的相互作用规律,以便为中西药联合、中药配伍合理应用,提供依据,逐步达到联合用药确实“安全、有效、经济、合理”。
目的
采用文本挖掘方法,得出常用治疗RA的中西药联合、中药配伍用药为研究对象,利用Pubchem查找到相应的人类靶标蛋白,将其导入IPA分析平台(Ingenuity Pathways Analysis, IPA)得到相应的network、pathway等,与RA患者与健康人差异基因导入IPA获得的相应network、pathway对比分析,探讨其生物通路网络的构建及其治疗RA的机制。
研究内容
本研究完成下列内容的研究工作：
1治疗RA常用中西药联合、中药配伍应用规律的挖掘。
利用文本挖掘技术：分别找到中西药联合、中药配伍应用频次最高的药物。
2雷公藤与甲氨蝶呤(methotrexate, MTX)、黄芪与当归药理网络的构建及其治疗RA机制的生物信息学分析,主要包括如下具体研究内容：
(1)应用Pubchem检索雷公藤及MTX的药物人类靶蛋白,将其导入IPA分析平台得到相应的network、pathway等,探讨其生物通路网络的构建；对比RA患者与健康人差异表达基因构建的生物通路网络,探讨其治疗RA的机制。
(2)应用Pubchem检索当归及黄芪的药物人类靶蛋白,将其导入IPA分析平台得到相应的network、pathway等,探讨其生物通路网络的构建；对比RA患者与健康人差异表达基因构建的生物通路网络,探讨其治疗RA的机制。
研究结果
1治疗RA常用中西药联合、中药配伍应用规律的挖掘。
中成药和西药的联用中,雷公藤多甙片和MTX联合应用明显较多。雷公藤多甙片的临床报告近年较多,居于中成药的首位,返查文献发现,临床联合应用时,文献大多报道可以起到协同作用,合用的疗效优于单用,依据大量文献中西医联合用药疗效比较确切。
从挖掘结果来看,中药桂枝、芍药、知母、黄芪、当归配伍频次较高,其中喻含着汤药桂枝芍药知母汤、当归补血汤,反查文献发现,对各证型的RA均可加减运用,值得进一步深入研究。
2雷公藤与MTX、黄芪与当归药理网络的构建及其治疗RA机制的生物信息学分析。
2.1蛋白质泛素化途径可能是雷公藤、MTX、雷公藤联合MTX、黄芪联合当归治疗RA的作用机制之一。
蛋白质泛素化途径与免疫系统疾病RA密切相关。根据生物分析的结果提示,药物雷公藤、MTX、雷公藤联合MTX、黄芪、当归联合黄芪都可能通过蛋白质泛素化途径来影响治疗RA。所以,推测蛋白质泛素化途径是雷公藤、MTX、雷公藤联合MTX、黄芪、当归联合黄芪能够治疗RA的作用途径之一。进一步分析上述药物作用与该途径的靶蛋白发现有共同点,这个共同点就是作用于E3RING靶点。
E3RING是泛素蛋白连接酶(E3)的两个主要的家庭之一,并且已有文献报道E3连接酶很可能是一种治疗RA的新的治疗方法。
2.2雷公藤与MTX联合应用,不仅作用于蛋白质泛素化途径,较单独应用MTX,增加了更广泛的生物通路。
生物分析结果提示,雷公藤、MTX联合应用可能通过影响粒细胞—巨噬细胞集落刺激因子通路、二十二碳六烯酸信号通路、酪氨酸蛋白激酶1和3细胞因子信号作用通路等几个生物通路,更有效,更全面的对RA患者进行治疗。
2.3热休克蛋白是黄芪与当归联合应用与RA患者及健康人的差异基因作用于蛋白质泛素化途径时的共同靶点之一。
生物信息学结果分析可以提示,除了结果分析出黄芪联合当归通过E3Ring影响蛋白质泛素化途径外,还可以通过HSP来影响蛋白质泛素化途径,是其治疗RA的重要机制之一。另我们生物学分析结果中,黄芪与健康人和RA患者差异基因有共同影响的几个生物通路,包括：蛋白质泛素化途径、凋亡信号、糖皮质激素受体信号等,并且在两者联用之后,共同影响的通路在治疗RA中权重变大,而当归与健康人和RA患者差异基因未有共同影响的生物通路。
结论
1大多数中成药都与MTX联合应用。其中,雷公藤多甙片与MTX联合应用频次最多。最常应用治疗RA的中药有桂枝、白芍、知母、黄芪、当归,在这样联合应用中,提示治疗RA的中药复方配伍包括两个中药经典方：桂枝芍药知母汤、当归补血汤(黄芪、当归)。
2雷公藤联合MTX、黄芪联合当归治疗RA的机制之一,可能是通过影响以下一系列的信号转导途径来实现的：E3RING→E3泛素连接酶→蛋白质泛素化→NF-κKB/TGF-β。
3雷公藤、MTX联合应用较单独应用MTX,可能通过影响粒细胞—巨噬细胞集落刺激因子通路、二十二碳六烯酸信号通路、酪氨酸蛋白激酶1和3细胞因子信号作用通路、凋亡信号通路等几个生物通路,更全面更有效的对RA患者进行治疗。
4黄芪联合当归治疗RA除了影响E3RING→E3泛素连接酶靶点进而影响蛋白质泛素化途径,还通过HSP来影响该途径从而治疗RA。另外,根据我们生物信息学分析的结果,印证了当归补血汤应用治疗RA时,黄芪是主药,当归是辅药,两者为“相使”关系。
Background
Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease characterized by joint synovitis. The prevalence rate of the disease is0.3-1%worldwide, and the prevalence rate is0.32-0.36%in china according to certain reference, with the appearance in different age groups. If not treated, the disease can induce severe joint deformity, joint dysfunction, which seriously affect the life quality of patients.
The goal of rheumatoid arthritis treatment is to improve the patient's physical, mental and quality of life in society by alleviating the pain caused by arthritis, prevent joint damage and maintain joint function. The core of rheumatoid arthritis treatment is medical treatment, which includes anti-rheumatic drugs, non-steroidal anti-inflammatory drugs and steroidal drugs as to western medicine. Anti-rheumatoid drugs, including immunomodulatory drugs, immunosuppressive drugs and biological agents, control rheumatoid arthritis induced by immune function abnormity. Western medicine treatment is inclined to bring gastrointestinal symptoms and interstitial renal damage, therefore traditional Chinese medicines is often employed in combination with western medicine in clinic. Treatment by traditional Chinese medicines put stress integrity, combating the diseases from multi-level, multi-channel to regulate the body immune function, which exhibits with great potential and advantages. Several clinical studies have also shown that the treatment of RA by some traditional Chinese medicines in combination with western medicines can produce more satisfactory curative effects and reduce more side effects than the treatment by western medicines only.
Text mining is an interdisciplinary research field relating to natural language processing, machine learning, data mining, information retrieval etc. It plays an unnegligible role in business, media, education, government, banking, biotechnology, medical and health industry. There is rich knowledge in traditional Chinese medicines and. Abundance of data on traditional Chinese medicines, the majority of which are text data, have been accumulated through the efforts of researchers in the past decade. New knowledge can be discovered from the medical literature by text mining to facilitate the development of traditional Chinese medicines.
Bioinformatics is an interdisciplinary science emerging in the late1980s with the launching of human genome project. It covers the storage, retrieval and analysis of bioinformation using computers in life science research. When used in TMC research, it helps to determine the target of drug action. While it is well known that TMC and its compound preparations are effective, TMC has not entered the mainstream international pharmaceutical market, essentially due to the fact that the TMC theories fail to integrate with the internationally accepted modern medical theories. It is likely that screening and isolation of disease-specific pathogenic genes as target for the study of molecular mechanisms of important actions will become one of the main directions of future TMC pharmacology studies. Study of the effects of TMC compound preparations on the cytogene expression profile and protein expression profile by using genetic chip technology and proteome assays promotes the establishment of technology platforms for genetic chips, proteomics and bioinformatics, which allow multi-component, multi-target and multi-path connections between compound TMC preparations and genetic and protein expressions, so as to compare the different expression profiles, determine the gene and protein expression targets for different TMC combinations, allow connections between the organ specificity&level of expression and the TMC theory of "principal,adjuvant,auxiliary and conductant ingredirnts" as well as the dosage, and to determine the relations between different components of compound preparations based on the interactions between gene and protein targets corresponding to different combinations, thus clarifying the material basis for drug action and the combination rules.This methodology will be of practical importance for the research, development and internalization of TMC and its compound preparations.
Using the analysis methods of bioinformatics, we intended to generate a network for drug combinations based on existing literature reports and to explore the rules for interactions between Chinese and Western medicines and between different TMC combinations, so as to provide evidence for rational use of integrated Chinese-Western medicine as well as TMC combinations, in an effort to achieve "safe, effective, economic and rational" drug combinations.
Purpose
To identify the common Chinese-Western or Chinese-Chinese medicine combinations for RA as the study subject by text mining, and, using Pubchem, identify the corresponding human target proteins, which were to be introduced to Ingenuity Pathways Analysis (IPA) platform to produce the corresponding networks and pathways, etc. for comparison with the networks and pathways obtained when RA differential gene (the gene difference between healthy population and RA patients) was introduced to IPA, so as to explore the structure of biopathway network as well as the mechanism for treatment of RA.
Study contents
The followings were completed for this study:
1Mining for the application rules for common Chinese-Western or Chinese-Chinese medicine combinations for RA
The most commonly used Chinese-Western or Chinese-Chinese medicine combinations were identified using text mining.
2Establishment of "pharmacology networks of tripterygium wilfordii/methotrexate (MTX) and radix/angelica, and bioinformatics analysis of their therapeutic mechanisms for RA. Specifically, the followings were studied:
(1) Human target proteins for tripterygium wilfordii and MTX were identified through Pubchem retrieval and introduced to IPA platform to obtain the corresponding networks and pathways, etc., in exploration of the construction of their biopathway networks; their therapeutic mechanisms for RA were explored by comparing with the biopathway network construction obtained when RA differential gene was introduced to IPA.
(2) Human target proteins for angelica and radix were identified through Pubchem retrieval and introduced to IPA platform to obtain the corresponding networks and pathways, etc., in exploration of the construction of their biopathway networks; their therapeutic mechanisms for RA were explored by comparing with the biopathway network construction obtained when RA differential gene was introduced to IPA.
Results
1Mining for application rules for common Chinese-Western or Chinese-Chinese medicine combinations for RA
Among the common Chinese-Western medicine combinations, the combination of tripterygium wilfordii polyglycoside tablet and MTX was predominant. There has been large number of clinical reports on ripterygium wilfordii polyglycoside tablet, which leads the TMC preparations. Search into the literatures found that most literatures reported synergic action with clinical combinations, which were superior over single agent. Large number of literatures support that Chinese-Western combinations have definite therapeutic effect.
It was observed through mining that the TMCs ramulus cinnamomi, paeonia, anemarrhenae, radix and angelica were the most frequent to be subject to combinations, which included Paeoniae and Anemarrhenae Decoction and Angelicae Sinensis Decoction for Supplementing Blood. Search into the literatures found that addition to or subtraction from the combinations were possible for each symptomatic type of RA, which warrants further investigation.
2Establishment of pharmacology networks of tripterygium wilfordii/methotrexate (MTX) and radix/angelica, and bioinformatics analysis of their therapeutic mechanisms for RA
2.1Protein ubiquitinoylation pathway is closely related to RA, an immunologic disease. Bioanalyses indicated that tripterygium wilfordii alone, MTX alone, tripterygium wilfordii/MTX combination, radix alone and radix/angelica combination have therapeutic effect for RA through protein ubiquitinoylation pathways. It was therefore presumed that protein ubiquitinoylation pathway is one of the courses of action for tripterygium wilfordii alone, MTX alone, tripterygium wilfordii/MTX combination, radix alone or radix/angelica combination to treat RA. Further analysis identified a target protein for protein ubiquitinoylation pathway common for these treatments, i.e., E3RING target.
E3RING is one of the two major families of ubiquitin protein ligase (E3), and has been reported to be a likely new treatment option for RA.
2.2The combination of tripterygium wilfordii and MTX not only acts on the protein ubiquitinoylation pathways, but also allows more extensive biopathways as compared to MTX alone.
Bioanalyses indicated that the combination of tripterygium wilfordii and MTX may provide more effective and more comprehensive treatment for RA patients by affecting the granulocyte-macrophage colony stimulating factor(GM-CSF) pathway, the docosahexaenoic acid (DHA) signal pathway, the tyrosine protein kinase (TPK)1and3cytokine signal pathway.etc.
2.3Heat shock protein (HSP) is one of the targets for protein ubiquitinoylation pathway common for radix/angelica combination and the differential gene for RA.
Bioinformactics results indicate that radix/angelica combination affects the protein ubiquitinoylation pathway through E3RING and HSP, which serves as one of its important mechanisms for treating RA. In addition, bioanalyses identified several biopathway targets common for radix and RA differential gene, including protein ubiquitinoylation pathway, apoptosis signals, and glucocorticoid receptor signals, etc. When radix and angelica were combined, the weighing of their common target pathways was increased in treating RA, while no common biopathway targets were identified for angelica and the RA differential gene.
Conclusions
1Most of the TMC preparations were used in combination with MTX. Among them, tripterygium wilfordii polyglycoside tablet was the most frequent to be combined with MTX. The TMCs ramulus cinnamomi, paeonia, anemarrhenae, radix and angelica were the mostly used for combinations, including two classic TMC compound preparations, Paeoniae and Anemarrhenae Decoction and Angelicae Sinensis Decoction for Supplementing Blood (radix ad angelica)
2One of the mechanisms for tripterygium wilfordii in combination with MTX or radix to treat RA was through affecting the following signal conduction pathway:E3RING→E3ubiquitin protein ligase→protein ubiquitinoylation→NF-κB/TGF-β.
3Compared to MTX alone, the combination of tripterygium wilfordii and MTX may provide more comprehensive and more effective treatment for RA patients by affecting the GM-CSF pathway, the DHA signal pathway, the TPK-1and-3cytokine signal pathway, and the apoptosis signal pathway, etc.
4Aside from acting on the E3RING→E3ubiquitin protein ligase target and thus the protein ubiquitinoylation pathway, the combination of radix and angelica provides therapeutic effect for RA also through affecting the HSP pathway. In addition, bioinformatics analysis indicated that when Angelicae Sinensis Decoction for Supplementing Blood is used for RA, radix is the principal agent while angelica is the adjuvant agent, contributing to their "mutual enhancing" relationship.
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