Zheng: A systems biology approach to diagnosis and treatments
Traditional Chinese medicine (TCM) is an ancient medical practice system which emphasizes regulating the integrity of the human body and its interrelationship with natural environments. As a key concept in TCM, Zheng (meaning syndrome or pattern) is the overall physiological and/or pathological pattern of the human body in response to a given internal and external condition, which usually is an abstraction of internal disharmony defined by a comprehensive analysis of the clinical symptoms and signs gathered by a practitioner using inspection, auscultation, olfaction, interrogation, and palpation of the pulses . Correctly identifying the Zheng is fundamental for the diagnosis and treatment of diseases.
Moreover, Zheng has been historically applied as the key pathological principle guiding the prescription of herbal formulas . A lack of research on Zheng has left us with little understanding of its underlying biology or the relationships between different Zhengs, diseases, and drugs. Moreover, there have been attempts to integrate Zheng differentiation with modern biomedical diagnostic methods, though these efforts have not achieved the desired results . Many well-known herbal recipes, such as Liu Wei Di Huang Wan and Jin Kui Shen Qi Wan, have long been used for the clinical treatment of Zheng disorders; however, Zheng-guided treatments are still scarce due to the lack of evidence-based interpretations of syndromes and treatment efficacies. Thus, investigating the biological basis of Zhengs from a molecular to systems level is important for advancing the identification and treatment of these syndromes, and for providing more objective and quantitative diagnostic criteria.
Zheng-guided disease research
In Western medicine, a disease is a particular abnormal and pathological condition that affects part or all of the human body and is often construed as a medical condition associated with specific symptoms. By contrast, Zheng puts forth a very different definition of a disease and encompasses all of the symptoms a patient presents. Because of the highly interconnected nature of the human interactome, it is difficult to study different diseases at the molecular level completely independent of one another , and this issue also applies to Zhengs. Moreover, Zhengs are dynamic with changing boundaries, overlapping symptoms, and a multiscale nature, which makes them difficult to under- stand at a biological and mechanistic level. Thus, we propose that a comprehensive Zheng map be constructed that links together all the Zhengs based on their molecular and cellular relationships. Further, we suggest creating the “Zhengome” as a new 'omics field, in which a network is the basic research unit used to investigate the hierarchy present in the human body, from the molecular to the systems level. A comprehensive understanding of the Zhengome requires us to bring together multiple sources of evidence, from shared genes to protein- protein interactions, shared environmental factors, common treatments, and phenotypic and clinical manifestations, in order to capture the relationships between the different Zhengs. Zheng uses the Yin-Yang, exterior-interior, cold-heat, and deficiency-excess definitions to describe patients’ conditions, which are then managed by Zheng-specific recipes . Modern 'omics techniques combined with bioinformatics and bionetwork models through a systems biology approach have been applied to investigate the differences between Zhengs and to identify novel biomarkers. For instance, rheumatoid arthritis (RA) patients differentiated on the basis of “hot” and “cold” Zhengs have been shown to be associated with different underlying genomic and metabolomic profiles, with the RA hot group showing more apoptotic activity than the cold group . Additionally, Li et al. used a network-based com- putational model to understand Zheng in the context of the neuro-endocrine-immune network and found that cold and hot Zhengs were closely related to a metabolism-immune imbalance . Wang and colleagues investigated the urine metabolome of patients with jaundice syndrome and its two subtypes of Yang Huang (acute) and Yin Huang (chronic), and identified several biomarker metabolites . However, most of the current studies have relied on only one or two approaches for molecular profiling and have lacked an efficient method to integrate data obtained at different 'omic levels. These studies also did not look at combining the analysis of molecular data with clinical variables, possibly missing an opportunity to generate more convincing conclusions. Considering the limitations of past studies, future efforts should integrate an analysis for all levels of 'omics (e.g., genomics, transcriptomics, epigenomics, and proteomics) data from a large number of patient samples for different Zhengs and include an investigation of the prognostic and therapeutic utilities of the data as a whole. Moreover, combining these molecular data with patients’ clinical information could pro- vide evidence-based theoretical interpretations for Zhengs and enable an assessment of Zheng-based therapeutic a proaches.
Zhengs may change dynamically during disease progression. Differentiating the specific Zheng involved in each stage of a disease could provide valuable guidance for prescribing a dynamic therapeutic recipe. Using dynamic network modeling, a disease process can be conceptualized as spatio-temporal changes in network structures. The changes associated with a Zheng under dynamic therapy can be used to identify the key factors in the dynamic biological networks. Appropriate network perturbation models and subsequent robustness and topology analysis could help unveil potential disease-related genes or therapeutic targets involved in a disease’s progression or evolution . The relationships between the different aspects of a disease (e.g., main symptoms versus complications) in a specific Zheng as well as the psychological, social, and even environmental factors should be taken into account during the modeling and simulation process in order to uncover the dynamic nature of complex diseases. Combining a Zhengome approach with dynamic modeling has the potential for establishing an accurate and quantitative Zheng research model, as well as for creating a new system for performing disease research.
Zheng-driven drug discovery
Despite considerable progress in genome, transcriptome, proteome, and metabolome-based high throughput screening methods and in rational drug design, drug discovery often encounters considerable costly failures that challenge the fidelity of the modern drug discovery system. Zheng-driven drug discovery has shown tremendous success for traditional drug discovery throughout Chinese medicine’s history. However, since this concept is completely new to Western medicine, it is no easy task to incorporate Zheng-driven drug discovery into modern drug discovery workflows.
Here, we propose the “Zheng to TCM” and “TCM to Zheng” strategies within the framework of systems pharmacology to investigate biological systems and develop new therapeutics . The first strategy, Zheng to TCM, proposes developing a pipeline from Zheng diagnoses to TCM drugs, including differentiating Zhengs, identifying Zheng-related diseases and the associated genes and proteins, reverse targeting of drug effects, constructing and analyzing network/systems, and finally identifying effective herbal medicines . In effect, this strategy can be considered a reverse targeting and screening approach that is designed to uncover drugs from natural products that can target multiple Zhengs or related diseases. The goal of this method is to help researchers identify the active components within medicinal plants and multi-ingredient synergistic herbal formulas or drug combinations . In fact, this novel strategy has already been successfully applied in a qi-blood study, where we identified the active compounds in the qi-enriching and blood-tonifying herbs, their targets, and the corresponding pathways involved in the treatment of qi and blood deficiency syndromes (8).
The second strategy, TCM to Zheng, consists of a whole- system evaluation process starting with herbs or herbal formulas and culminating in identifying the Zhengs. This process includes the initial collection and classification of herbal medicines; screening the ingredients for absorption, distribution, metabolism, excretion, and toxicity (ADME/T); performing targeted drug screenings and tissue localization; constructing and analyzing networks; and finally identifying Zhengs/diseases . Using this strategy, it is possible to identify novel multitarget drugs in natural products . One particularly striking example is the systematic analysis of blood stasis and qi deficiency syndrome in coronary heart disease and the herbal drugs used to treat the syndromes. The results indicate that the herbs for eliminating blood stasis have pharmacological activity that acts to dilate blood vessel, improve the microcirculation, reduce blood viscosity, and regulate blood lipid, while qi-enhancing herbs have the potential for enhancing energy metabolism and anti- inflammatory activity . The TCM to Zheng strategy can also help to elucidate the pharmacological effectiveness of herbs and formulas.
In our ongoing work investigating Pi-deficiency syndrome (PDS) in the context of Zheng, we are analyzing patient samples using the sequencing alternative polyadenylation sites (SAPAS) method, RNA sequencing, lipid metabolomics, proteomics, and transcriptomics in order to decipher the pathogenesis and complex responses of the human body to PDS. From a drug development perspective, we plan to systematically investigate the Si Jun Zi decoction, a widely used herbal recipe for PDS, within the framework of the “TCM to Zheng” strategy, so as to understand why this recipe can regulate the immune response, stimulate blood circulation, and adjust gastrointestinal digestive functions. Despite the progress in Zheng-guided drug discovery, its future success requires the integration of multidisciplinary technologies, together with further innovations in these technologies, to facilitate the understanding of multifactorial diseases and the development of new therapies.
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证：用系统生物学手段进行诊断与治疗
中医是一种古老的医学体系，它强调从整体来调节人体内部及人与自然的相互联系。作为中医的一个核心概念，“证”（meaning syndrome or pattern）是人体在一定内外因素影响下做出反应的全部生理与/或病理模式。它通常是代表人体内在失调的一个抽象概念，是医生运用四诊（望、闻、问、切）对病人的症状和体征进行综合分析后作出的诊断。正确地辩证是诊断和治疗疾病的基础。此外，证从古至今都是指导中药处方的关键病理依据.
现代对于“证”的研究的匮乏，让我们对其深层次的生物学机制和不同证、病、药之间联系的了解少之又少。而虽然此前有人尝试将辩证与现代生物医学诊断手段整合，但并没有取得预期的结果。许多有名的方子，像六味地黄丸和金匮肾气丸，一直被用于临床相应证的治疗;但是，以“证”为指导的现代治疗手段还是相当少的，因为缺少循证的、对症状和治疗效果的阐释。因此，从分子水平到系统水平整体地研究“证”的生物学基础对区分症状、对症下药和为诊断提供大量客观的标准都有重要意义。
以“证”为指导的疾病研究
在西医中，疾病特指不正常的病理现象，它会对人体造成局部或整体的影响，并常伴随着一系列特定症状。相比之下，“证”对疾病有着不同的定义，并囊括了病人身上所表现的所有症状。
由于人体相互作用组的高度相关性，将不同疾病相互完全分离地进行分子水平上的研究是很困难的。同样的情况对“证”也适用。而且，因为“证”具有界限模糊、症状重叠、多重尺度的特性，所以是动态变化的，这就很难从生物学和机械论的水平去理解。因此，我们提出构建一张综合的“证”的关系图，它根据分子间与细胞间关系，将所有证联系在一起。进一步地，我们提出创造“证组”，作为一个新的组学领域，以关系网为基本研究单位，从分子到整体地研究人体的不同层级。对“证组”的整体理解需要我们将许多依据集合起来，从共有基因到蛋白间反应，从共有环境因素到常见治疗，还有表型和临床的表现,为的就是获取不同“证”之间的联系。
“证”用阴阳、表里、寒热、虚实来描述患者的病情，然后医生再对证下药进行治疗。现代分子技术经系统生物学手段与生物信息学和生物网络模型结合了起来，并被用于区分不同的“证”和辨识特异的生物标记。例如，风湿性关节炎患者分为寒证和热证的不同，经研究发现与潜在的基因和代谢状况有关，患风湿性关节炎热证的患者细胞凋亡要比患寒证的患者活跃。除此之外，Li et al用基于网络的计算模型从神经-内分泌-免疫系统的角度来理解“证”，发现寒证和热证与新陈代谢-免疫系统失衡密切相关。
王和同事研究了黄疸患者的尿代谢和黄疸的两个亚型：阳黄（急性）和阴黄（慢性），并辨认了几种生物标记的代谢物。但是，大部分现代研究只仅仅依赖一到两种手段进行分子描述，缺少整合不同分子水平数据的有效率的方法。这些研究也没有将分子数据分析和临床可变因素相结合，错过了得出有力可信结论的机会。考虑到过去研究中的限制，未来努力的方向应朝着整合分析各个水平的分子组（如：基因组学、转录组学、表观基因组学、蛋白质组学）数据，搜集大量不同“证”的患者样本，并应分析这些数据在病情预后、疗法方面的有效性。此外，将这些分子数据与患者临床信息结合，能为“证”提供循证的理论阐释，并为基于证的治疗手段提供途径。
证在疾病的发展过程中会发生动态变化。在疾病的不同阶段准确辩证能有效地指导处方的动态变更。使用动态网络模型能使疾病的进程概念化为时空的变化。动态治疗下与“证”相关的变化能被用于确定动态生物网中的关键因素。适当的网络动态模型，后继的鲁棒性，和拓扑分析法能帮助揭示疾病发展的过程中潜在的与疾病相关的基因或治疗靶向。
对于某一特定的证，疾病不同层面之间的关系（如：主要症状对并发症）和情志、社会甚至环境因素都应被考虑在内，这样有助于在建模与模拟的过程中揭示复杂疾病变化的本质。建立“证组”的手段与动态造模的结合有可能建立一个精确而庞大的“证”研究模型，并创造一个新的疾病研究体系。
“证”推动的药物发现
尽管基因组、转录组、蛋白质组、基于代谢组的高生产量筛选手段、合理的药物设计取得了可观的进步，药物的发现常常遭遇大量损失惨重的失败，使现代药物生产体系的可信度到挑战。当中医从古至今都沿用传统的发现药物的方法，以“证”为指导的药物发现取得了极大的成功。然而，这一概念对于西医是全新的，循证制药的方法汇入现代药物发现的大流并非易事。
这里，我们提出“从证到药”和“从药到证”的策略，在系统药理学框架内研究其生物学体系并发展新的疗法。第一种策略，“从证到药”，提出建立从立证到用药的通路，包括辩证、辨病和相关的基因和蛋白质，反向定位药物疗效，建立网络并分析，最终确定有效的中药。在治疗效果上，这一策略可被看成反向定位和筛选手段，从能作用于多种证或相关疾病的自然产物中提取药物。其目标是为研究者确定药用植物或复方的活性成分。实际上，这一新奇的策略已被成功应用在气血的研究中，寻找益气补血药物中的活性成分，它们的靶向和它们在气虚、血虚治疗中相应的通路。
第二种策略，“从药到证”，以中草药和方剂为开始，以确认证候为结束，包括了对整个系统的分析过程。这一过程包括最初的草药挑选和分类，分析药物成分的吸收、分布、代谢、排泄和毒性情况来进行筛选，进行目标药物审查和组织定位，构建网络并分析，最终确认证候/疾病。通过这一策略，我们能在自然产物中找到新的多靶药物。一个尤其显著的例子就是对冠心病气虚血瘀证候及其草药处方的系统分析。研究结果指出，其中祛瘀的草药有扩张血管、促进微循环、降低血粘稠度和调节血脂的药理活性。而补气的中草药有增强能量代谢和抗炎的效果。“从药到证”的策略也能帮助阐明中草药与方剂的有效性。
在我们正在进行的从证的角度研究脾虚的工作中，我们用测序替代多聚腺苷酸位点技术（SAPAS）、RNA测序、类脂代谢学、蛋白质组学技术对患者样本进行分析，解释脾虚的病机和人体对其复杂的反应。从药物发展的角度，我们计划在“从药到证”的框架内系统地研究四君子汤——一首广泛用于治疗脾虚的方剂，从而了解这一组方为什么能调节免疫反应，促进血液循环并调节胃肠消化功能。尽管循证制药的研究取得了成效，它未来的成功需要多门学科技术的综合和这些技术的进一步创新来促进对多因素疾病的了解和新疗法的发展。