Available online 27 March 2015
A network pharmacology approach to determine active ingredients and rationality of herb combinations of Modified-Simiaowan for treatment of gout
- Under a Creative Commons license
Modified Simiaowan (MSW) is a traditional Chinese medicine (TCM) formula and is widely used as a clinically medication formula for its efficiency in treating gouty diseases.To predict the active ingredients in MSW and uncover the rationality of herb combinations of MSW.
Materials and methods
Three drug-target networks including the “candidate ingredient-target network” (cI-cT) that links the candidate ingredients and targets, the “core ingredient-target-pathway network” connecting core potential ingredients and targets through related pathways, and the “rationality of herb combinations of MSW network”, which was derived from the cI-cT network, were developed to dissect the active ingredients in MSW and relationship between ingredients in herb combinations and their targets for gouty diseases. On the other hand, herbal ingredients comparisons were also conducted based on six physicochemical properties to investigate whether the herbs in MSW are similar in chemicals. Moreover, HUVEC viability and expression levels of ICAM-1 induced by monosodium urate (MSU) crystals were assessed to determine the activities of potential ingredients in MSW.
Predicted by the core ingredient-target-pathway network, we collected 30 core ingredients in MSW and 25 inflammatory cytokines and uric acid synthetase or transporters, which are effective for gouty treatment through some related pathways. Experimental results also confirmed that those core ingredients could significantly increase HUVEC viability and attenuate the expression of ICAM-1, which supported the effectiveness of MSW in treating gouty diseases. Moreover, heat-clearing and dampness-eliminating herbs in MSW have similar physicochemical properties, which stimulate all the inflammatory and uric acid-lowing targets respectively, while the core drug and basic prescription in MSW stimulate the major and almost all the core targets, respectively.
Our work successfully predicts the active ingredients in MSW and explains the cooperation between these ingredients and corresponding targets through related pathways for gouty diseases, and provides basis for an alternative approach to investigate the rationality of herb combinations of MSW on the network pharmacology level, which might be beneficial to drug development and applications.
- MSU, monosodium urate;
- NSAIDs, non-steroidal anti-inflammatory drugs;
- UA, uric acid;
- TCM, traditional Chinese medicine;
- MSW, Modified-Simiaowan;
- EM, Ermiao;
- SM, Simiao;
- AR, Atractylodes chinensis (DC.) Koidz.;
- PC, Phellodendron chinense Schneid.;
- AB, Achyranthes bidentata Bl.;
- CS, Coix lacryma-jobi L. var. mayuen (Roman.) Stapf;
- LJ, Lonicera japonica Thunb.;
- SG, Smilax glabra Roxb.;
- ICAM-1, intercellular cell adhesion molecule;
- OB, oral bioavailability;
- DL, drug-likeness;
- MW, molecular weight;
- nHDon, number of donor atoms for H-bonds;
- nHAcc, number of acceptor atoms for H-bonds;
- MLOGP, Moriguchi octanol–water partition coeff. (logP);
- DMEM, Dulbecco’s modified Eagle’s medium;
- FBS, fetal bovine serum;
- MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;
- SD, standard deviation;
- IL-1β, interleukin -1 beta;
- TNF-ɑ, tumor necrosis factor-alpha;
- PTGS2, prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase);
- COX, cyclooxygenase;
- P13K, phosphotylinosita l3 kinase;
- MAPK1, mitogen-activated protein kinase-1;
- TGFB1, transforming growth factor, beta 1;
- XOD, xanthine oxidase;
- OAT1, organic anion transporter 1;
- URAT1, urate-anion transporter1;
- GLUI9, glucose transporter 9;
- LEP, leptin;
- ABCG2, ATP-binding cassette sub-family G member 2;
- NF-ƙB, nuclear factor-kappa B;
- TLR, toll-like receptor.
- Modified Simiaowan;
- Network pharmacology;
- Active ingredients;
- Herb combinations
Hyperuricemia is predictive for the development of gout, renal dysfunction, hypertension and hyperlipidemia, which is one of the most common and extensive metabolic diseases in populations (Richette and Bardin, 2010). Gout is a common metabolic disorder in human that afflicts about 8.3 million people in the United States, 6.4 million in the European Union, and 2.9 million in Japan in 2008 (Miao et al., 2008, Doherty, 2009 and Zhu et al., 2011). The disease is also rapidly rising in China probably due to recent changes in dietary habits. Preventing and treating the occurrence of hyperuricemia and gout become imperative in contemporary clinical therapy, but there are lack of effective means to control acute gouty arthritis.
Gout is a type of inflammatory arthritis induced by deposition of monosodium urate (MSU) crystals in the joints and kidneys, where MSU crystals stimulate monocytesmacrophages and neutrophils to produce different pro-inflammatory cytokines, such as interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α (Tausche et al., 2004) and monocyte chemotactic factor (Inokuchi et al., 2006). Despite advances in the application of anti-gout drugs for the treatment, non-steroidal anti-inflammatory drugs (NSAIDs) such as indomethacin, as first-line agents, are commonly used for acute inflammation. However, according to epidemiological study, NSAIDs, which play an important role in terms of anti-inflammation by inhibiting the activity of cyclooxygenase (COX) (Yuan et al., 2000), show some adverse effects including renal toxicity, gastrointestinal toxicity and gastrointestinal bleeding (Sabina et al., 2011). Besides, Colchicine inhibits the propagation of inflammation by suppressing several genes encoding Caspase-1 such as phosphotylinosita l3 kinase (P13K) and mitogen-activated protein kinase-1 (MAPK1), which could reduce the release of mature IL-1 (Ben-Chetrit et al., 2005). Nevertheless, it is still limited and often poorly tolerated because of severe gastrointestinal reactions and toxicity (Borstad et al., 2004). Besides, underexcretion of urate has been implicated to produce gouty diseases (Rott and Agudelo, 2003 and Lioté, 2003). Ways to reduce uric acid (UA) levels can be classified into two types: decreasing UA synthesis with inhibitory activities of xanthine oxidase (XOD) and promoting UA excretion by stimulating some transporters such as organic anion transporter 1 (OAT1), urate-anion transporter 1 (URAT1), glucose transporter 9 (GLUI9) and leptin (LEP), which are important for drugs to treat Hyperuricemia (Sha and He, 2009 and Tong et al., 2013). Allopurinol is a frequently used XOD inhibitor in clinical use (Horiuchi et al., 2000 and Chen et al., 2005), and benzbromarone is an uricosuric agent by inhibiting the main renal UA transporter URAT1 (Kunishima et al., 2007). Nevertheless, both of them may result in allergic, sever hypersensitivity reactions, and nephropathy (Hande et al., 1984 and Kumar et al., 2005). Therefore, it underlines much impetus for urgent need of available anti-gout agents, especially herbal medicine (Ahmad et al., 2008 and An et al., 2010).
Traditional Chinese medicine (TCM) is a comprehensive medicinal system that has been used in clinical practice for thousands of years, whose applications have been raised dramatically around the world in recent decades because of their moderate treatment effects and lower side effects (Tang et al., 2009). Modified-Simiaowan (MSW) is widely used clinically as a formula for its prominent efficiency in terms of TCM for treating gouty diseases, such as gout, hyperuricemia and inflammatory arthritis (Yin and Si, 2006), which is a patented invention of the Chinese (Yin et al., 2008). It is made up of six herbs: Phellodendron chinense Schneid. (PC), Atractylodes chinensis (DC.) Koidz. (AR), Achyranthes bidentata Bl. (AB), Coix lacryma-jobi L. var. mayuen (Roman.) Stapf (CS), Smilax glabra Roxb. (SG) and Lonicera japonica Thunb. (LJ) and has been used in treating gouty diseases for its efficiency in anti-inflammation, analgesia and the reduction of UA levels (Shi et al., 2008).
In this formula, Ermiao (EM), also called as the core drug, which is composed of PC and AR, is a classical formula and recorded in State Pharmacopoeia of People’s Republic of China at all times for successful treatment of gout and hyperuricemia. According to Danxi’s China, EM is described to treat acute gout through clearing heat and eliminating dampness in terms of TCM. Simiao (SM), a classical formula, which is derived from EM with additions AB and CS. According to Cheng-Fang-Bian-Du (a Chinese medicine book named Conventional Prescriptions for Easily Reading), SM is commonly used to treat gout through clearing heat, eliminating dampness and strengthening the liver and kidney in terms of TCM (Shi et al., 2008; Hu et al., 2010; Hua et al., 2012).
Recent researches have demonstrated that MSW could inhibit the occurrence of acute gout by suppressing the expression of intercellular cell adhesion molecule (ICAM-1), protecting HUVECs by reducing cell apoptosis, increasing cell viability in MSU-induced HUVECs, and weakening adhesion between neutrophil and endothelial cells (Shi et al., 2013). Moreover, MSW could significantly reduce UA levels in serum and urine in hyperuricemic model, together with decreasing liver XOD activity and IL-1β, IL-6 levels (Zeng et al., 2014) and has a better anti-gouty inflammation effect compared with indomethacin in the clinical study (Shi et al., 2008). All the results indicate that MSW is a comprehensive therapeutic formula with multi-approaches for gouty diseases. However, the active ingredients and the rationality of herb combinations of MSW through integrated multiple pathways for gouty treatment are still unclear.
With the rapid progress of bioinformatics, systems biology and polypharmacology, network-based drug discovery is considered a promising approach toward more cost-effective drug development (Keith et al., 2005, Schadt et al., 2009, Jia et al., 2009 and Levinson, 2010). Network pharmacology can reveal the underlying complex relationship between a herbal formula and the whole targets. Besides, this sustainable development platform coupling with rich experience of TCM is hopeful of shifting the paradigm “one target, one drug” to the “network targets, multi-components” strategy (Li and Zhang, 2013).
Our previous studies have shown that “network target” as a key concept of TCM network pharmacology can help us to decipher active ingredients in MSW and rationality of herb combinations of MSW. In present study, we first manually collected the information of targets reported for main active ingredients in MSW and targets of gouty diseases from OMIM, and selected those gouty genes that are targeted by the corresponding ingredients as the candidate targets. After oral bioavailability (OB) screening and drug-likeness (DL) evaluation, potential ingredients and targets could be obtained. In order to predict the active ingredients in MSW and uncover the rationality of herb combinations of MSW, drug-target networks have constructed to this formula, which offers an opportunity for deep understanding of the efficiency and rationality of TCM in ways for the prevention of gouty diseases.
2. Material and methods
2.1. Chemical ingredients database building
A total of 631 chemical constituents of all six individual herbs in MSW were retrieved from Traditional Chinese Medicine Systems Pharmacology Database (TcmSP™, http://tcmspnw.com) and related literatures. TcmSP™ is a unique systems pharmacology platform designed for herbal medicines, from which 49 ingredients in AR, 58 in PC, 176 in AB, 38 in CS, 74 in SG, and 236 in LJ were all collected.
2.2. The properties of ingredients in MSW
In order to investigate whether the six individual herbs in MSW are similar or different in chemicals, herbal ingredients comparisons based on chemical properties were performed. The important pharmacology-related properties including molecular weight (MW), the number of donor atoms for H-bonds (nHDon), the number of acceptor atoms for H-bonds (nHAcc) and Moriguchi octanol–water partition coeff. (logP) (MLOGP) are also collected from TcmSP™ for each ingredient. These four parameters reflect the basic characteristic of a molecule ( Lipinski et al., 1997). Next, OB and DL properties of each herb ingredient were collected from the same database, which describe the physicochemical property and pharmacological feature of every ingredient. Therefore, the histograms of the physicochemical properties of all ingredients accompanying with t-test were carried out to analyze the variables in the property spaces.
2.3. Data preparing
2.3.1. Predicting target profiles of main active ingredients in MSW
Based on our pervious study and literature, the main ingredients in MSW were all obtained, such as the volatile oil in AR, alkaloids in PC, saponins in AB and flavonoids in LJ. We collected all their targets profiles from PubMed or SciFinder, and then removed the ones with no targeting information. After that, we extracted the relevant targets profiles of important ingredients. For the reason that some contain protein and gene files from different species, the demand for standardization of the information is necessary. We limited the species as “Homo sapiens” when entering the gene or protein target with the genetic search function in the NCBI database (http://www.ncbi.nlm.nih.gov/gene), which could obtain all the targets having revised to their official names, including multiple subtypes of genetic information. After the above retrieval and transformation, we gained distinct target files associated with main active ingredients.
2.3.2. Gout-associated targets
We collected different genes associated with gout from the following resources. (1) The Online Mendelian Inheritance in Man (OMIM) database, which catalogues all known diseases with a genetic component and when possible links them to the relevant genes in the human genome and provides references for further research and tools for genomic analysis of a catalogued gene (Hamosh et al., 2005). We searched the OMIM database with a keyword “gout” and found a set of genes, such as ABCG2, GLUT9, TNF-α, IL-8. (2) Genecards (http://www.genecards.org), it is a database about genes, their products and biomedical applications, which is maintained by Israel’s Weizmann Institute of science. (3) Literatures, we could collect some significant targets from gouty related literatures.
Based on the above three methods, we collected 277 distinct targets associated with gouty diseases.
2.4. Candidate ingredients and their corresponding targets prediction
Compared the target files of ingredients in MSW collected from literature with gouty genes from databases, we selected those gouty genes that could be targeted by the corresponding ingredients as the candidate targets. The candidate targets and corresponding ingredients are useful for the following study.
2.5. Oral bioavailability screening and drug-likeness evaluation
Generally, a TCM formula covers various chemicals, bioactive ingredients can contribute to its therapeutic effects. Hence, it is essential to conduct the OB screening and DL evaluation for the goal to identify the potential active ingredients in this formula.
2.5.1. OB screening
A robust in-house system OBioavail1.1 (Xu et al., 2012), is applied to calculate the OB value, which is efficient in screening out the potential ingredients. In this work, the OB threshold was defined as 30% and those ingredients with OB≧30% were selected as the ingredients for further analysis.
2.5.2. DL prediction
In this study, DL index of a new compound is calculated by Tanimoto similarity defined as
Briefly speaking, ingredients that meet both of the requirements OB≧30% and DL≧0.1 were selected as the potential ingredients.
2.6. Construction of network
In our present study, the content for construction of network was performed as following, (1) the “candidate ingredient-target network” was established by connecting the candidate ingredients and all their corresponding targets, (2) the “core potential ingredient-target-pathway network” was also constructed after OB screening and DL evaluation, by linking the potential ingredients and targets with high degree through related pathways, (3) the “rationality of herb combinations of MSW network”, which describes the complex relationship between herbs combined in MSW based on herbal ingredients comparisons. All the networks were created using network visualization software Cytoscape 2.8.2 (http://www.cytoscape.org/), which is an open source software project for integrating biomolecular interaction networks with high-throughput expression data and other molecular states into a unified conceptual framework, in order to facilitate scientific interpretation of complex relationships between medicines involved in formula orchestration (Shannon et al., 2003).
2.7. Experimental validation
2.7.1. Drugs and preparation
Chlorogenic Acid (no. 110753200413), Berberine (no. 713 9906), Astilbin (with a purity of 98% determined by HPLC), Caffeic acid and Ferulic acid were purchased from National Institute for the Control of Biological Products. Dulbecco’s modified Eagle’s medium (DMEM) (Gibco BRL, Grand Island, NY, USA), Fetal bovine serum (FBS) (Hangzhou Sijiqing Co., Ltd., China), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (AERESCO, USA), Uric acid (Sigma St. Louis, MO, USA), Indomethacin (no. 030901, Shanghai Yanan Vientiane Pharmaceutical Co., Ltd.) were used in this experiment. Other reagents were of analytical grade from commercial suppliers and manufactured in the People’s Republic of China.
Berberine (2.5 mg), Astilbin (2.5 mg), Chlorogenic Acid (2.5 mg), Ferulic acid (2.5 mg) and Caffeic acid (2.5 mg), DMSO was dissolved in every solution at concentration less than 0.02%, Serum-free DMEM culture medium was added into the five solutions at concentration of 2.5 µg/ml, 25 µg/ml and 250 µg/ml. Approximately 4 g of uric acid was dissolved and heated in 800 ml of H2O with NaOH (9 ml/0.5 N), adjusted to pH 8.9 at 60 1 C, cooled overnight in a cold room, washed, and dried. MSU crystals, needle-like crystals were recovered and were suspended in sterile saline (20 mg/ml) (Denko and Whitehouse, 1976).
2.7.2. Cells culture and treatment
HUVECs were provided by basic medical college from Nanjing university of Chinese medicine, and were isolated as described previously (De Martin et al., 1993). The cells were grown in DMEM culture medium containing 10% FBS at 37 °C in a humidified atmosphere of 5% CO2 and 95% air after digested by 0.25% trypsin, neutralized with the same DMEM, then centrifuged 6 min at 1000 r/min and discarded the supernate. Cells were then serially passaged with 0.25% trypsin/1 mM.
2.7.3. Detection on HUVECs activity stimulated by MSU
HUVECs were washed three times with DMEM culture medium containing 10% FBS after digested by 0.25% trypsin, centrifuged, and modulated the cell suspension at density of 4×104/ml with the same DMEM, then seeded onto a 96-well culture plate at density of 200 μl/well and incubated at 37 °C for 24 h. The medium was replaced with the following groups (8 well/group), then incubated at 37 °C in a humidified atmosphere of 5% CO2 and 95% air for 48 h, and collected the supernatant. The rest of endothelial cells were used to measure cell viability using MTT assay (Welde, 1992). MTT was added to cell cultures at concentration of 0.5 mg/ml. After 4 h, MTT was discarded and the formazan crystals were dissolved by adding 200 μl of dimethyl sulfoxide.
The absorbance at 490 nm was read on a microplate reader (Bio-Rad, Hercules, CA, USA). Absorbance, which was used as a measurement of cell viability, was normalized to cells incubated in the control medium. The cells were considered 100% viable. The percentage of cell viability was calculated as follows: cell viability %=b/a×100%. Here, a is the absorbance of the control, and b is the absorbance of cells with MSU crystals. Groups: blank group (DMEM culture medium 200 μl), model group (MSU 100 µg/ml), Indomethacin (MSU 100 µg/ml and Indomethacin 20 µg/ml), five low dosage groups (MSU 100 µg/ml and Berberine, Astilbin, Chilrogenic acid, Caffeic acid, Ferulic acid 2.5 µg/ml); five middle groups (MSU 100 µg/ml and Berberine, Astilbin, Chilrogenic acid, Caffeic acid, Ferulic acid 25 µg/ml), five large groups (MSU 100 µg/ml and Berberine, Astilbin, Chilrogenic acid, Caffeic acid, Ferulic acid 250 µg/ml)
2.7.4. Detection on expression of ICAM-1
HUVECs were seeded into tissue culture flask under logarithmic growth phase after digested by 0.25% trypsin, and prepared into cell suspension at the density of 5×109 l−1. After about 24 h, the supernatant was discarded. Groups: control group, model group (MSU 100 µg/ml), Indomethacin (MSU 100 µg/ml and Indomethacin 2.5 µg/ml), five drug groups (MSU 100 µg/ml and Berberine, Astilbin, Chilrogenic acid, Caffeic acid, Ferulic acid 2.5 µg/ml).
HUVECs were cultured for 24 h and collected by PBS. The supernatant was removed by centrifugation and then CD54 monoclonal antibody was added in. HUVECs were washed with PBS and resuspended after 30 min, Detect the percentage of positive cells by flow cytometry (FCM) and repeated for 3 times. The percentage of the expression of ICAM-1 was calculated as follows: Inhibition rate %=b/a×100%. Here, a is the value of the control, and b is the value of cells with MSU crystals.
3.1. Herbal ingredients comparisons in MSW
MSW consists of six herbs, including AR, PC, AB, CS, LJ and SG. In order to investigate whether the six herbs are similar or different in chemicals, herbal ingredients comparisons were conducted based on six significant properties: MW, nHDon, nHAcc, MLogP, OB and DL. (1) From the average number of MWs (Fig. 1 and Table 1), we could see that the values of MWs are similar (p=0.73) for AR (300.42) and CS (361.74), as well as the values (p=0.93) for PC (363.57) and SG (367.04), while AB (405.94) is significantly higher than that of PC (p=0.0026) and LJ (p=0.0212). (2) The ingredient in CS is most hydrophobic with the average number of MlogP of 7.16, which is higher than AR of 4.27, followed by AB, PC and SG, which share the similar values. While for LJ, its average number of MLogP exhibits the lowest degree. (3) The average number of H-bond donors of AR is similar with CS (p=0.053), while AB (3.80) is larger than that of other herbs, especially CS of 0.76 (p=0.00058). (4) The average of H-bond acceptors (HAcc) of AB (7.98) is significantly larger than AR of 2.51 (1.14E−15) and CS of 2.5 (2.12E−14). (5) The OB and DL analysis indicate much difference among these six herbs. For OB, we could realize that SG exhibits the highest value of 33.24, followed by AR and CS, which possess the similar OB values, and AB exhibits the lowest (22.28), which is significantly different from others. (6) For DL, unlike OB distribution, PC possesses the highest average value of 0.49, followed by CS of 0.32 and AB of 0.31. Whereas LJ shows the lowest average DL index (0.23). Besides, AR and SG exhibit the similar values (AR=0.29, SG=0.25), which display no significant difference (p=0.42).
Index AR PC AB CS LJ SG MW 300.42 363.57 (136.00#) 405.94 (200.10##,270.91*) 361.74 (157.94, 156.44) 307.7 (189.50, 190.22) 367.04 (129.68, 223.87) ALogP 4.27 3.29(2.37) 3.505 (2.63,2.63) 7.16 (2.59##, 3.78**) 2.5 (3.16##,3.11) 3.25 (2.93, 2.89) Hdon 1.31 1.62(1.85) 3.9 (3.27##, 3.27**) 0.76 (1.25, 1.70*) 2.38 (2.90#,2.92) 3.08 (2.19##, 3.20**) Hacc 2.51 5.03(3.08##) 7.98 (5.43##, 6.11**) 2.5 (2.16, 2.82**) 4.87 (5.12##,5.03) 6.08 (3.38##, 5.07) OB 32.90 32.3(14.51) 22.28 (17.32##, 17.71**) 32.90 (13.98, 15.43) 30.01 (20.02, 20.20) 33.24 (20.08, 20.49) DL 0.29 0.49(0.31##) 0.31 (0.30, 0.32**) 0.32 (0.28, 0.30**) 0.23 (0.26, 0.28**) 0.25 (0.27, 0.30**)
- SD, standard deviation; AR, Atractylodes chinensis (DC.) Koidz.; PC, Phellodendron chinense Schneid.; AB, Achyranthes bidentata Bl.; CS, Coix lacryma-jobi L. var. mayuen (Roman.) Stapf; LJ, Lonicera japonica Thunb.; SG, Smilax glabra Roxb
- p<0.05 when compared with AR.
- p<0.01 when compared with PC.
In general, all the results show that although there are diverse ingredients in six herbs, many of them still have similar chemical properties. To some extent, as dampness-eliminating herbs, the properties of AR are similar with CS, the same as PC and LJ, which play an important role in clearing heat. What’s more, SG has somewhat similar properties with AR.
3.2. Targets prediction in the cI-cT Network
It could be meaningful to bridge ingredients in MSW and gouty diseases via their common targets. We constructed the candidate ingredient-target network (cI-cT), which contains active ingredients in this formula and their corresponding targets, and then mapped these nodes onto the network. As depicted in Fig. 2, 44 candidate ingredients in this formula yielded 71 targets with anti-inflammatory and UA-lowering therapeutic effects. Targets in the outer circle show much less interactions with the candidate ingredients than those in the inner, which also indicate that many candidate targets are hit by only one candidate ingredient, but some could be modulated by multiple rather than single one. For example, some inflammatory cytokines such as IL-6, PTGS2, MAPK1, are activated by multiple ingredients including Wogonin, Berberine, Luteoline. Besides, some UA synthetase XOD and transporters OAT1, GLUT9, could also be modulated by more than one. Thus, we could have a rough observation on the relationships between candidate ingredients and corresponding targets from the cI-cT network.
3.3. OB prediction and DL evaluation of candidate ingredients in MSW
OB represents a subcategory of absorption and is the fraction of the orally-administered does that reaches the systemic circulation unchanged (Xu et al., 2012). It is one of the essential used pharmacokinetic parameters in drug screening cascades. While DL is a qualitative concept used in drug design for how “drug-like” a substance is with respect to factors like OB. It is estimated from the molecular structure before the substance is even synthesized and tested. In order to further screen out the potential active ingredients from the candidate ingredients in MSW, OB prediction and DL evaluation were conducted. As listed in Table 2, the OB and DL calculation show that 31 of 44 candidate ingredients in MSW possess proper values.
No. Compound OB DL Structure ARM1 Beta eudesmol 29.97 0.1 ARM2 Palmitic acid 19.3 0.1 ARM3 AtractylenolideI 37.37 0.15 ARM4 Atractylenolide III 31.66 0.17 ARM6 Wogonin 30.68 0.23 ARM8 Vanillic acid 35.47 0.04 PCM1 Syringin 14.64 0.32 PCM2 Berberine 36.86 0.78 PCM3 Jatrorrhizine 19.65 0.59 PCM5 Obacunone 43.29 0.77 PCM6 Tetrahydroberberine 53.83 0.77 ABM2 Beta sitosterol 36.91 0.75 ABM4 Ecdysterone 6.94 0.82 ABM5 Betaine 40.92 0.01 CSM1 Coixol 63.01 0.05 CSM2 Coixenolide 32.4 0.43 CSM3 Oleic acid 33.13 0.14 CSM4 Triglyceride 33.61 0.03 LJM1 Luteoline 36.16 0.25 LJM5 Chlorogenic acid 13.61 0.31 LJM6 Caffeic acid 54.97 0.05 LJM7 Ferulic acid 40.43 0.06 LJM8 Rutin 11.7 0.683 SGM1 Quercetin 46.43 0.28 SGM2 Astilbin 36.46 0.74 SGM3 Taxifolin 60.51 0.27 SGM4 Epicatechin 48.96 0.24 SGM6 Kaempferol 41.88 0.24 SGM7 Resveratrol 19.07 0.11 SGM8 Oxyresveratrol 109.29 0.13 SGM9 Dioscin 17.75 0.06
The rhizome of A. chinensis (DC.) Koidz. has been used widely in Chinese traditional medicine for various indications such as rheumatic diseases, digestive disorders, night blindness, and influenza. These traditional uses are explained by the compound’s ability to eliminate dampness, strengthen the spleen, expel wind-cold from the superficial parts of the body, and clear away the common cold ( Koonrungsesomboon et al., 2014). Of 11 ingredients with corresponding targets in which 70.45% have appropriate OB (≧30%) and DL values (DL≧0.1), including β-Eudesmol, Palmitic acid, Wogonin, Atractylenolide І, Atractylenolide III, Vanillic acid. As seen in Table 2, almost all the ingredients have relatively moderate bioavailability (OB≈30%). However, Vanillic acid, as an active ingredient in AR, exhibits low DL value (OB=35.47%, DL=0.04). Considering its UA-lowing effect through targeting uarte synthetase and transporters ( Oskoueian et al., 2011 and Wang and Sweet, 2012), it was also selected for the further study.
3.3.2. PCCortex Phellodendri, known as “Huang Bai”, is derived from the dried bark of P. chinense Schneid. or Phellodendron amurense Rupr. (Family Rutaceae) according to Chinese Pharmacopoeia Commission 2010. It is one of the most commonly used traditional Chinese medicinal plants, which is widely used to remove damp heat, quench fire, counteract toxicity, relieve consumptive fever and also effective in curing dysentery, diarrhea and other syndromes ( Yen, 1994). Among the six active ingredients in PC, five of them
have suitable OB and DL values, including Berberine, Jatrorrhizine, Obacunone, Tetrahydroberberine and Syringing. However, Jatrorrhizine and Syringing, the main active components in PC ( Zhang et al., 2012), exhibit low DL values (19.65%, 14.64%). Considering their potential pharmacological effects ( Hu et al., 2009 and Gong et al., 2014), they were still adopted for the further targeting.
A. bidentata Blume (Amaranthaceae family), a perennial herbaceous plant, is widely distributed and grown in China. The roots of A. bidentata Blume have proven to possess many pharmaceutical properties, including immunostimulant, anti-inflammatory, antitumor, analgestic, cognition-enhancing, anti-osteoporotic, anti-bacterial, uteri-excitant and antifertility activities ( Li et al., 2007). However, only three from the candidate ingredients could be used for further targeting. Although Ecdysterone has poor OB value (OB=6.94), it is one of the representative ingredients of AB ( Shen et al., 2011). As for Betaine, which exhibits low DL value (DL=0.01), it is also one of the major ingredients for lowering UA, and could protect the occurrence of inflammation by inhibiting many inflammatory cytokines ( Fan et al., 2014). In view of considerations above, we selected the two ingredients as potential ingredients.
CS is the dry and ripe seeds of C. lacryma-jobi L. var. mayuen (Roman.) Stapf, which first sets out in “Shen Nong’s Herb”, and has the effects of clearing damp and promoting dieresis, invigorating the spleen and stopping diarrhea, resolving toxin and dissipating binds. Modern pharmacology study shows that it is commonly used Chinese medicine in treatment of anti-cancer, boosting immunity, anti-hypertensive, anti-oxidation, anti-inflammatory ( Liu et al., 2010). The details were shown in Fig. 3, four ingredients in CS including Coixol, Coixenolide, Oleic acid and Triglyceride have suitable OB values. Although some of them exhibit low DL values, they were still included for further targeting, since they are the active ingredients in CS.
L. japonica Thunb. (family Caprifoliaceae) are perennial arching shrubs or twining vines that are commonly found as dried buds or early-opened flowers. It is commonly used in traditional Chinese medicine for the treatment of various diseases, including arthritis, diabetes mellitus, fever, infections, sores, and swelling. Pharmacological studies have shown that extracts of LJ flower buds have a broad spectrum of biological activity, including antibacterial, anti-inflammatory, antioxidant, antiangiogenic, antipyretic, antiviral, and hepatoprotective effects ( Qian et al., 2007). It has nine candidate ingredients in which over half show proper OB values (>30%). Interestingly, Caffeic acid and Ferulic acid, the representative active ingredients in LJ ( She and Zhu, 2008), exhibit low DL values, although their OB values meet the standards. They show potent antisepsis and anti-inflammatory effects ( Ma et al., 2010, Zhang et al., 2014 and Tang et al., 2014) and could be selected for further evaluation. Based on all these considerations, it was reasonable to believe that five could be listed as potential active ingredients.
SG, belonging to the Smilacaceae family, Smilax genus, is described to be effective in xeransis, detoxification, and easing joint movement in traditional Chinese medical literature, which include the Compendium of Materia Medica and the State Pharmacopoeia of the People’s Republic of China. Extract of SG has also been studied for multiple pharmacologic activities, such as immunomodulatory (Jiang and Xu, 2003), anti-inflammatory (Jiang et al., 1997). It has nine valid ingredients in which almost all of them have appropriate OB and DL values. Those ingredients that meet the selection criteria are selected for further study, including Quercetin, Astilbin, Taxifolin, Epicatechin, Oxyresveratrol, Kaempferol, Resveratrol, Dioscin. Notably, Oxyresveratrol has significantly high OB value (109.29%), which means it is more easily to be absorbed into our bodies. Another active ingredient Resveratrol, exhibits low OB value (19.07%). However, as the homolog of Oxyresveratrol, it is rather effective for anti-inflammation (Chen et al., 2011) and especially benefit for lowering UA by reducing the expression of UA synthetase XOD or transporters such as OAT1, GLUT9 (Shi et al., 2012). Therefore, it was added as potential ingredients for further analysis.
3.4. Network construction and analysis
A drug-target network is defined as a bipartite network for the drug-target associations consisting of two disjoint sets of nodes. We constructed the networks to find out the active ingredients in MSW, as well as to understand rationality of herb combinations of MSW.
3.4.1. The core potential ingredients-target-pathway network
Based on the cI-cT network which illustrates the relationships between candidate ingredients and targets, the potential ingredient-target network (pI-pT) was generated by connecting potential ingredients and corresponding targets. In order to clearly distinguish the relationship between ingredients and their targets, we constructed a core potential ingredients-target-pathway network (Fig. 3), that depicts the relationship between 30 core ingredients and 25 corresponding targets through related pathways. In this study, we selected those whose degree values are more than four as the core ingredients from the pI-pT network for further research.
Furthermore, multiple gouty related pathways were listed as follows to reveal possible mechanism involved in gouty treatment. A central feature of gout is inflammation, Table 3 includes several possible pathways related to inflammatory signaling, in which the most important one is the Toll-like receptor (TLR) signaling pathway. Some other pathways, such as NF-kappa B (NF-ƙB), TNF signaling pathways, have been known to be associated with gouty treatment. Another prominent feature of gout is enhanced uric acid, which implies that the synthesis and excretion of UA pathways are also associated with the function of lowing UA.
No. Target Degree Description Possible pathway G5 PTGS2 23 Prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase) NF-kappa B, TNF signaling pathways G56 TNF 23 Tumor necrosis factor Toll-like receptor, NF-kappa B, mTOR, MAPK, TNF signaling pathways G57 IL6 22 Interleukin 6 Toll-like receptor, P13K-Akt, TNF signaling pathways G46 MAPK1 17 Mitogen-activated protein kinase 1 Toll-like receptor, MAPK, mTOR, P13K-Akt, TNF signaling pathways G58 IL1B 16 Interleukin 1, beta Toll-like receptor, MAPK, NF-kappa B, TNF signaling pathways G8 BAX 11 BCL2-associated X protein p53 Signaling pathway G10 BCL2 11 B-cell CLL/lymphoma 2 NF-kappa B, P13K-Akt signaling pathways G47 MAPK3 10 Mitogen-activated protein kinase 3 MAPK signaling pathway G4 PIKC3G 9 Phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit gamma P13K-Akt signaling pathway G31 HSP90AA1 9 Heat shock protein 90 kDa alpha (cytosolic), class A member 1 P13K-Akt signaling pathway G50 MAPK14 9 Mitogen-activated protein kinase 14 MAPK signaling pathway G52 IL8 9 Interleukin 8 Toll-like receptor, NF-kappa B signaling pathways Rheumatoid arthritis G6 MTOR 8 Mechanistic target of rapamycin (serine/threonine kinase) mTOR, P13K-Akt signaling pathways G2 PIK3CA 6 Phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha P13K-Akt signaling pathway G3 PIK3CB 6 Phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit beta P13K-Akt signaling pathway G23 PPARA 5 Peroxisome proliferator-activated receptor alpha PPAR signaling pathway G7 RPS6KB1 4 Ribosomal protein S6 kinase, 70 kDa, polypeptide 1 MAPK, P13K-Akt, mTOR signaling pathways G15 TGFB1 4 Transforming growth factor, beta 1 MAPK signaling pathway Rheumatoid arthritis G37 JAK2 4 Janus kinase 2 P13K-Akt signaling pathway G55 IL10 4 Interleukin 10 JAK-STAT signaling pathway G59 IL4 4 Interleukin 4 P13K-Akt signaling pathway P1 XOD 17 Xanthine oxidase Uric acid synthesis P3 OAT1 7 Solute carrier family 22 (organic anion transporter), member 6 Uric acid excretion P5 GLUT9 5 Solute carrier family 2 (facilitated glucose transporter), member 9 Uric acid excretion P2 URAT1 4 Solute carrier family 22 (organic anion/urate transporter), member 12 Uric acid excretion
- Core targets are those whose degree values are more than four from the potential ingredient-target network. The possible pathways are collected from KEGG database.
As listed in Table 3, the expression of IL-6, TNF, PTGS2, one of the most core targets with highest degrees, could be inhibit by more than 20 distinct ingredients in this formula via TLR, NF-ƙB or mTOR signaling pathways. IL-1β, the major mediator that induces acute gouty arthritis (Schlesinger et al., 2012 and Terkeltaub et al., 2013), is also suppressed by 16 ingredients through TLR, NF-ƙB and MAPK signaling pathways. Moreover, XOD, a key enzyme to generate UA as well as the decisive factor for regulating generation of UA, is intimately connected with the potential ingredients. To be specific, over half potential ingredients are able to inhibit the expression of XOD. It indicates that XOD inhibitory activity might be one of anti-gout mechanisms, and ingredients inhibiting the expression of XOD are effective in reducing UA for gouty treatment. Besides, OAT1 is a potential molecular component in the first step of renal urate secretion, which can mediate the active uptake of organic anions and controls the final exit into the urine via ATP-powered transporters and bidirectional exchangers (EI-Sheikh et al., 2008). More than five potential ingredients in MSW could increase renal OAT1 expression, resulting in the increased urate secretion.
In conclusion, these anti-inflammatory and UA-lowing properties of MSW account, at least in most part, for its pharmacological efficacy against gouty diseases. However, their detailed mechanism involved and clinical significance activity require further study.
3.4.2. Explore the rationality of herb combinations of MSW
The core drug (EM) is descried to treat acute gout through clearing heat and eliminating dampness in terms of TCM (Kong et al., 2004). Basic prescription (SM), which is described in a famous TCM monograph Danxi Xinfa (comprehensive medical book), has been used for hundreds of years in Chinese medicine for treating gouty arthritis with eliminating dampness, clearing heat and promoting blood circulation (Hua et al., 2012 and Shi et al., 2013). Therefore, the network (Fig. 4) was displayed to reveal rationality of herb combinations between ingredients and corresponding targets.
EM has both anti-inflammatory and uricosuric effects through hitting most core targets including inflammatory cytokines such as IL-6, TNF-α, COX-2, IL-1β, and UA synthetase XOD or several transporters URAT1, OAT1, OAT3, ABCG2. SM is able to stimulate almost all the core targets, owing to the additional herbs AB and CS, which could hit more inflammatory cytokines and UA transporters, such as IL-10, TGFB1, IL-4, PPARA, GLUT9, hUAT and LEP. Mostly important, MSW, which is derived from SM, could activate all the inflammatory cytokines and UA-lowing targets. The results show that EM plays a key role, while SM plays an important role in MSW for the treatment of acute gout.
According to the nature of Chinese herbs, most of inflammatory cytokines such as IL-6, TNF-α, IL-1β, MAPK1, could be hit by those ingredients such as Berberine, Syringing, Luteoline, Chlorogenic acid, which are just important ingredients in PC and LJ. As antipyretics, PC and LJ play the major role of clearing heat on anti-inflammation for gouty treatment. While AR, CS and SG, used for eliminating dampness in TCM, could reduce UA levels through targeting UA synthetase and transporters. Equally important, AB, as a hemorheologic agent, promotes blood circulation and could be an adjuvant therapy for gouty diseases. These results preliminarily show that the nature of TCM is consistent with the clinical therapeutic effects.
3.5. Experimental validation for anti-gout effects of ingredients in MSW
Berberine, Astilbin, Chlorogenic Acid, Ferulic acid and Caffeic acid are the representative ingredients PC, LJ and SG, which actually play a crucial role in clearing heat on anti-inflammation. Besides, they are highly connected with the core targets. Therefore, they were selected to investigate therapeutic effects for treating acute gouty arthritis in MSU-induced HUVECs.
3.5.1. Effect on cell viability stimulated by MSU
To determine whether ingredients in MSW have protective effects on HUVECs damaged by MSU crystals, cell viability was assessed using MTT assay. Results (Fig. 5) confirmed that all ingredients exhibit significant protective effect on HUVECs compared with the model. To be specific, compared with the model, effect of Berberine has a significant difference (p<0.05) with dose-effect relationship. While effect of Astilbin exhibits an extremely significant difference (p<0.01) with does-effect relationship varying from low to middle dosage. The same as Caffeic Acid, Ferulic Acid and Chlorogenic Acid (p<0.01), and Chlorogenic Acid shows a dose-effect relationship.
3.5.2. Effect on expression of ICAM-1 stimulated by MSU
As depicted in Fig. 6, the expression level of ICAM-1 in HUVECs was a lot in untreated HUVECs and was augmented by stimulation with MSU crystals (100 μg/ml) for 24 h. Treatment of HUVECs with Berberine, Astilbin, Chlorogenic Acid, Caffeic Acid, Ferulic Acid (2.5 μg/ml) and Indomethacin (2.5 μg/ml) for 24 h blocked the increase of ICAM-1 expression induced by MSU crystals to different extents compared with the model.
In general, we could draw such a conclusion that Berberine, Astilbin, Chlorogenic Acid, Caffeic Acid and Ferulic Acid are the effective ingredients for acute Gout. This study has also demonstrated for the first time that ingredients in MSW exhibit significant does-dependent inhibitory effects in experimental gouty arthritis models in vivo induced by MSU crystals.
Gout is a type of inflammatory arthritis induced by the deposition of MSU crystals in the tissues or a joint, and is a polygenetic disorder induced by the increase of UA synthesis or decrease of excretion through UA synthetas XOD and transporters, accompanying with inflammation caused by inflammatory cytokines (Sha and He, 2009 and Tong et al., 2013).
Given the intensity of inflammatory reactions characterizing the acute attack, NSAIDs and Colchicine, as appropriate first-line agents in clinical use (Neogi, 2010), could stimulate several targets such as COX-2, MAPK1, in a” single-target” paradigm (Yuan et al., 2000 and Ben-Chetrit et al., 2005). However, Colchicine’s toxicity was wildly reported (Finkelstein et al., 2010). NSAIDs also present some adverse effects such as gastrointestinal toxicity, renal toxicity and gastrointestinal bleeding (Sabina et al., 2011). Besides, Benzbromarone, an uricosuric agent, has been used for more than 25 years to control hyperuricemia by inhibiting the main renal UA transporter URAT1, which could suppress the reabsorption of UA (Kunishima et al., 2007). It was withdrawn from the European market due to the risk of severe hepatotoxicity (Kumar et al., 2005). Allopurinol, as a frequently used XOD inhibitor in clinical use (Horiuchi et al., 2000 and Chen et al., 2005), could cause severe hypersensitivity and was restricted in patients with renal insufficiency (Hande et al., 1984).
Predicted by network pharmacology, many ingredients in MSW are able to stimulate MAPK1, COX-2, XOD, URAT1 through inflammatory and synthesis or secretion of UA pathways, which are just the targets of classical anti-gout drugs. Besides, those genes such as IL-8, TNF-α, IL-1β, OAT1, GLUI9 (Rott and Agudelo, 2003, Lioté, 2003 and Tausche et al., 2004), that may induce the occurrence of gout, could also be targeted by various ingredients in MSW. Experimental results show that MSW could play an important role in terms of anti-inflammation by inhibiting expression of ICAM-1, preventing neutrophil infiltration and apoptosis of HUVECs (Shi et al., 2013) and significantly reducing serum UA levels, together with decreasing liver XOD activity and IL-1β, IL-6 levels (Zeng et al., 2014). Thus, we consider that MSW may be effective in treating gouty diseases by cooperation of multiple targets and multiple pathways.
The study also indicates the rationality of herb combinations of EM and SM in MSW for gouty treatment. To be specific, EM is a classical formula used for clearing heat and eliminating dampness to treat gouty diseases through stimulating most core targets such as XOD, URAT1, IL-6, TNF-α, which are consistent with those collected from published literatures (Kong et al., 2004 and Kao and Dong, 2013). As a classic formula derived from EM, SM is commonly used to treat gout and hyperuricemia in clinical (Hu et al., 2010 and Hua et al., 2012), eliminating dampness, clearing heat and promoting blood circulation in terms of TCM through targeting UA synthetase, transporters and inflammatory cytokines. Literatures also suggest that SM could suppress inflammation and inhibit the release of IL-1β, TNF-α (Xu et al., 2013 and Liu et al., 2014). Besides, it could also significantly inhibit XOD activities in serum and liver, and reverse oxonate-induced alterations in renal URAT1, GLUT9, OAT1 mRNA and protein levels, resulting in the enhancement of renal urate excretion (Hua et al., 2012), which are consistent with those predicted from the gouty network. Mostly important, MSW, which develops based on TCM theory, clinical research, and pharmacodynamic findings (Yin and Si, 2006), is prepared with two additional agents on the basis of SM and does a more comprehensive and synergetic effect than EM and SM on gouty diseases.
Acute gouty arthritis is caused by MSU crystals in and around the joints, which causes acute inflammation that manifests as intense pain, swelling, and reddening of the skin (Chia et al., 2008). TCM believes that a formula with clearing heat, eliminating dampness and promoting blood circulation is useful for treating acute gout. Chemical ingredients in six herbs (AR, PC, AB, CS, LJ and SG) of MSW were compared based on those important drug-associated descriptors including MW, ALogP, Hdon, Hacc, OB and DL. Results from herbal ingredients comparisons (Table 1) and networks (Fig. 4) suggested that AR and CS have similar chemical properties on most of the descriptors except ALogP. Both of them are used for eliminating dampness by targeting most UA synthetase and transporters through synthesis or excretion of UA. Besides, PC and LJ, which are similar with each other on those descriptors including MW, ALogP, Hdon, Hacc and OB, play the role of clearing heat on anti-inflammation by targeting many inflammatory cytokines such as TNF-α, IL-6, IL-1β via TLR, NF-ƙB and other signaling pathways. While SG, having somewhat similar properties with AR and PC, is useful in both clearing heat and eliminating dampness. AB, a hemorheologic agent, which is different from others, could promote blood circulation by activating inflammatory cytokines and UA-lowing targets. The study based on network pharmacology confirms herb combinations of MSW including heat-clearing, dampness-eliminating and blood-activating drugs is rational for gouty treatment. Experimental results first demonstrated that core ingredients in MSW could protect HUVECs by reducing cell apoptosis and inhibiting expression of ICAM-1 in MSU-induced HUVECs for anti-acute gout. Besides, these ingredients such as Quercetin, Resveratrol, Palmitic acid, Astilbin, down-regulated hepatic XOD and enhanced renal urate excretion in hyperuricemic mice by up-regulating OAT1 and down-regulating URAT1 and GLUT9 mRNA and protein levels in the kidney (Wang and Sweet, 2012, Shi et al., 2012 and Hu et al., 2012). Researches confirm that various ingredients in MSW are effective for gouty treatment by targeting inflammatory cytokines and UA synthetas or transporters through different related pathways, which indicates a whole regulation with the paradigm of “multi-ingredient, multi-target, multi-pathway”.
Combination therapy is a fundamental principle of TCM, which is developed for the purpose of maximizing the efficacy and minimizing the adverse effects or toxicity (Sucher, 2013). An herbal formula is not a simple quantitative addition of different herbs (Jia et al., 2004), instead, all the herbs of a defined prescription should correspond exactly to the related diseases, showing a significantly better effect than the constituent herb used alone (Scholey and Kennedy, 2002). We integrate our previous methods into a TCM network pharmacology platform to illustrate network connections between multiple targets of ingredients in herbal formula and multiple targets of a specific disease such as gout. Our study has successfully predicted the active ingredients in MSW and uncovered the rationality of herb combinations of MSW. Therefore, such a network pharmacology strategy and platform is expected to make the systematical study of herbal formulae achievable and make the TCM drug discovery predictable.
Conflict of interest
All authors have no financial or scientific conflict of interest with regard to the research described in this manuscript.
This study is supported by Natural Science Foundation of Jiangsu Province, China (no. BK2002033), Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, China and Research Foundation of Nanjing University of Traditional Chinese Medicine, China.
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