Background
Traditional Chinese medicine (TCM), as a system of ancient medical practice, has been widely used in treating various diseases in Eastern Asia for many years. TCM continues to play a critical role in maintaining health for the peoples of China, and is growing in popularity in Western countries [
1]. Notably, modern medical researchers have adopted ideas from TCM, using combinations of drugs to treat complex diseases such as cancer and diabetes.
In last decades, researchers have made great efforts to investigate TCMs and to analyze their components. To date, numerous bioactive ingredients of TCMs have been isolated and identified [
2]. This has furthermore led to the discovery of a variety of single compound-based therapeutics in TCM, such as the anti-cancer compound salvicine [
3]. Indeed, these data/database can provide important hints/information for further biological systematic studies, however, TCM protocols should take a more holistic method; the TCMs always works as the result of synergistic interactions by multiple ingredients [
4]. Thus, to truly investigate TCMs, biological systematic methods should be applied, all the ingredients of herbs or formulae in TCM should be taken into consideration simultaneously [e.g.
5‐
7].
Currently, the most applied strategy in drug discovery is ‘one gene–one drug–one disease’ paradigm, involving the screening of potential compounds for individual disease-causing targets. In this case, the drug’s efficacy is often impaired, because the robustness of the protein interactions in the treated objectives was ignored. Therefore, the biological systems-oriented approaches, such as combinations of effective drugs with multiple targets, were called in drug discovery [
8]. The TCM presents a perfect example, as it treats diseases in a holistic way. In the formulae of TCM, over hundreds compounds were always be treated simultaneously to rebalance the organism. Therefore, TCM formulae with multi-components/targets should be analyzed by the same strategy as combination therapies of multi-component drugs.
IgA Nephropathy (IgAN), a primary glomerular disease, is the most common type of Chronic Kidney Disease (CKD) which is a common condition affecting up to 11 % of the population. This disease is now recognized to elevate the risk of cardiovascular disease as well as kidney failure and other complications [e.g.
9,
10]. It is a leading cause of end-stage kidney disease in China [
11]. Effective control of proteinuria may be a key strategy for treating CKD [reviewed in
12]. Reninangiotensin-aldosterone system blockers, glucocorticoids, and immunosuppressants have often been used for primary glomerular diseases. Immunosuppressive therapies have usually been applied to treat patients with heavy proteinuria, but are not entirely suitable for patients with non–nephrotic-range proteinuria [
13]. Moreover, treatments with glucocorticoids and immune-suppressants are usually long term, which can result in severe adverse effects and increase the risk of rebound [e.g.
13,
14]. TCM has promising effects on the control of proteinuria, protection of kidney function, and improvement in patients’ clinical symptoms [e.g.
15,
16]. For example,
Abelmoschus manihot, a traditional Chinese herb, has increasingly been used to treat a wide range of types of CKD, such as immunoglobulin A (IgA), nephropathy (IgAN) and diabetic nephropathy [e.g.
17,
18]. Clinical studies have proven that
A. manihot can reduce proteinuria and thus protect kidney function [e.g.
19]. However, TCM formulae are multi-component and multi-target agents, and it is therefore necessary to investigate the combination therapy of multi-component drugs.
Gubentongluo (GBTL) decoction, a classic TCM formula from Chinese medical sage Zhang Zhongjing, is prepared from a basic formula of nine herbs, including Rhizoma imperatae (BAI MAO GEN), Ramulus euonymi (GUI JIAN YU), Yerbadetajo herb (HAN LIAN CAO), Fructus ligustri lucidi (NU ZHEN ZI), Astragali radix (HUANG QI), Semen persicae (TAO REN), Rumex madaio (YANG TI GEN), Herba lycopi (ZE LAN YE) and Radix salvia miltiorrhizae (DAN SENG). It is widely used in China in accordance with the China Pharmacopoeia standard of quality control. In TCM theory, the multiple agents contained in a single formula must work synergistically. With regard to GBTL, Rhizoma imperatae is the primary herbs and is believed to be a very effective antioxidant, whereas Ramulus euonymi acts primarily as an anti-inflammatory. For the most part, however, it’s the pharmacological/molecular mechanisms of GBTL have not yet been fully elucidated.
In this study, we have developed a comprehensive systematic approach for understanding the pharmacological mechanisms of GBTL acting on IgAN; an overview of our approach is shown in Additional file
1: Figure S1. Three steps were taken to achieve this objective: (1) prediction of potential targets for GBTL; (2) collection of IgAN associated molecules and construction of an IgAN associated regulation network; (3) study of the relationships of GBTL potential targets with the network and corresponding signal pathways and (4) examine whether the treatment of GBTL induces changes in potential targets expression. This procedure would enhance our understanding of the pharmacological mechanisms of GBTL and its limitations.
Discussion
In the present study, by using a systematic biology method, we investigated the molecular mechanisms of GBTL on treating the IgAN. Moreover, by using animal model, we found GBTL induced a significant increase in the levels of two target genes: TNF and NOS2. This provide an example for understanding the multiple targets of herbs in Traditional Chinese Medicine formulae and their interaction in the context of a molecular network [reviewed in
46]. Our work also indicated that a single TCM formula could be applied to the treatment of a given disease, since only parts of disease-based associated proteins were covered.
This work is the first study to show that the potential targets of GBTL could cover 16 of the 25 proteins which are believed to be associated with IgAN. This explains why GBTL is most effective for IgAN [e.g. [
47‐
49]. Moreover, 6 of the 9 herbs of GBTL were determined to act on the 16 associated proteins of IgAN. HUANG QI is the most important component in the network, as it interacts with 14 IgAN associated proteins. Many previous studies have shown that HUANG QI can enhance myocardial contractility, improve circulation, protect myocardial cells and regulate immunity [e.g.
50,
51]. Our findings, in good agreement with a large number of other studies, indicate that HUANG QI has anti-viral, anti-inflammatory and immunoenhancing effects [e.g.
52‐
54]. Furthermore, by DGAT1and DGAT2, YANG DI GEN participates in the regulation of renin synthesis, which is associated with fat metabolism. In addition, NU ZHEN ZI, TAO REN and HUANG QI can be involved in T cell receptor signaling regulation though the TNF and JUN pathways. Since GBTL combines these herbs, it can provide an effective treatment to IgAN.
Out of 25 selected biomarkers of IgAN, 9 proteins were not overlapped with the potential targets of GBTL. Enrichment analysis of these 9 proteins suggests that biological processes such as G1/S transition of mitotic cell cycle, regulation of platelet activation, regulation of actin filament are also related to IgAN pathogenesis, however, GBTL may not able to regulate these processes, at least not directly as can be seen from the data. It suggests that this formula might have some shortcomings in the treatment of IgAN and could be improved in the future.
Moreover, many studies have already suggested that some of the herbs used in Chinese medicine can interact with drugs, and can have serious side effects [e.g.
55,
56]. In this study, more than 3800 proteins were not associated with IgAN directly. Although we showed that some of processes such as innate immune response and inflammatory response should associated with IgAN according to previous studies, processes such as synaptic transmission and response to drug could be related to potential side effects of GBTL. In contrast, the aforementioned 9 IgAN associated proteins are involved in several key biological processes, especially related to proteasome machinery. Most importantly, we have shown that in the pathway of the EBV, key genes/proteins (PSMB1 and SYK) related to the proteasome and downstream IgA1 production, were not targeted by GBTL directly.
Three herbs, BAI MAO GEN, ZE LAN YE and HAN LIAN CAO, were not associated directly with the IgAN, according to the current protein datasets. However, we analyzed these herbs separately and revealed that Bai Mao Gen is mainly associated with pathogenic
E. Coli infection, gap junction and phagosome, Ze Lan Ye regulate mainly galactose metabolism, whereas, Han Lian Cao is related with NOD-like receptor signaling, TLR receptor signaling and TNF signaling (data not shown). These pathways were believed to be correlated with IgAN [e.g.
57‐
59], indicating that these herbs should have additive or complementary effect together with other herbs in GBTL.
Conclusions
In this study we have shown the molecular mechanism of GBTL acting on IgAN. The GBTL potential protein-IgAN associated protein interactions showed that 6 herbs in GBTL acted on 16 IgAN associated proteins, mainly through the renin-angiotensin system, regulate the leukocyte proliferation and hypoxia, which are responsible for epithelial cell damage and leukocyte infiltration. DAN SHEN and YANG TI GEN have potential power to regulate the triglycerides level via DGAT1 and DGAT2. This demonstrates the basic therapeutic mechanisms of GBTL in treating IgAN. Our study also indicated that GBTL could not cover all IgAN associated biomarkers, such as Syk, a key mediator relevant to IgA1 stimulation. Overall, our study was the first to explore the molecular network of GBTL acting on IgAN, and further studies are called for to develop the formula of GBTL and to enhance its effectiveness on IgAN.
Acknowledgements
The authors would like to thank all of the colleagues who contributed to this study.