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Tetramethylpyrazine identified by a network pharmacology approach ameliorates methotrexate-induced oxidative organ injury
Author’s Accepted Manuscript Tetramethylpyrazine identified by network pharmacology approaches ameliorates methotrexate-induced oxidative organ injury Bo Zhang, Cheng Lu, Ming Bai, Xiaojuan He, Yong Tan, Yanqin Bian, Cheng Xiao, Ge Zhang, Aiping Lu, Shao Li www.elsevier.com
PII: DOI: Reference:
S0378-8741(15)30159-8 http://dx.doi.org/10.1016/j.jep.2015.09.034 JEP9757
To appear in: Journal of Ethnopharmacology Received date: 28 May 2015 Revised date: 29 September 2015 Accepted date: 30 September 2015 Cite this article as: Bo Zhang, Cheng Lu, Ming Bai, Xiaojuan He, Yong Tan, Yanqin Bian, Cheng Xiao, Ge Zhang, Aiping Lu and Shao Li, Tetramethylpyrazine identified by network pharmacology approaches ameliorates methotrexate-induced oxidative organ injury, Journal of Ethnopharmacology, http://dx.doi.org/10.1016/j.jep.2015.09.034 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Title: Tetramethylpyrazine identified by network pharmacology approaches ameliorates methotrexate-induced oxidative organ injury
Author names: Bo Zhang 1, 5, #, Cheng Lu 2, 3, #, Ming Bai 1, Xiaojuan He 2, Yong Tan 2, Yanqin Bian 2, Cheng Xiao 4, Ge Zhang 3, Aiping Lu 2, 3, *, Shao Li1, * *: Corresponding author. #: These two authors contributed equally to this work.
Author’s affiliations: 1. MOE Key Laboratory of Bioinformatics and Bioinformatics Division, TNLIST / Department of Automation, Tsinghua University, Beijing 100084, China 2. Institute of Basic Research in Clinical Medicine, China Academy of Chinese Medical Sciences, 100700 Beijing, China 3. Institute for Advancing Translational Medicine in Bone & Joint Diseases, School of Chinese Medicine, Hong Kong Baptist University, 00852 Hong Kong SAR, China 4. China-Japan Friendship Hospital, Beijing 100030, China 5. Tianjin International Joint Academy of Biotechnology & Medicine, Tianjin 300457, China
E-mails: Bo Zhang: [email protected] Cheng Lu: [email protected] Ming Bai: [email protected] Xiaojuan He: [email protected] Yong Tan: [email protected]
Yanqin Bian: [email protected] Cheng Xiao: [email protected] Ge Zhang: [email protected] Aiping Lu: [email protected] Shao Li: [email protected]
Contact: * Address, Room 1-107, FIT Building, Tsinghua University. Phone/Fax: 86-10-62797035 / 86-10- 62773552 E-mail: [email protected]
Author contributions: S.L. and A.L. conceived the study. B.Z. and C.L. performed experiments, obtained and analysed data and did statistical analysis. M.B. performed target profile prediction and enrichment analysis. B.Z., C.L. and Y.T. performed animals and experimental design for toxicity studies. Y.T. and X.H. performed cAMP, MDA and GSH assay. Y.T. and Y.B. did histopathological preparation and analysis. B.Z., A.L. and S.L. conceptualized the project and supervised the work. All authors discussed the results and B.Z., C.L., A.L. and S.L. wrote the manuscript.
Sources of funding: This work was supported by the National Natural Science Foundation of China (Nos. 81225025, 91229201 and 81303139), the CATCM project, Interdisciplinary Research Matching Scheme (IRMS) of Hong Kong Baptist University (RC-IRMS/12-13/02) and Hong Kong Baptist University Strategic Development Fund (SDF13-1209-P01).
Disclosure of Potential Conflicts of interest: No potential conflicts of interest disclosed.
Abstract Ethnopharmacological relevance: Ligusticum wallichii Franchat, namely “Chuan-xiong” in traditional Chinese medicines, has been described in application for alleviating the toxicity of chemotherapy. Our studies showed that tetramethylpyrazine (TMP), the bioactive compound from Chuan-xiong, could be valuable for preventing the methotrexate (MTX) -induced oxidative injury. However, its mechanism remains unclear.
Aim of the study: The present study aimed to develop combination treatments that could reduce the toxicity of MTX from Chinese herbal medicine and characterize their mechanism of combinational actions.
Materials and methods: A network pharmacology-based screening strategy is applied for target profile prediction and pharmacological characterization of herbal compounds, which is used to guide the following in vitro and in vivo experiments. The effect of herbal compounds identified by network pharmacology approaches to reduce the toxicity of MTX was assessed by MTX-induced rat toxicity model. Malondialdehyde and glutathione levels in the liver and kidney homogenates were assayed using commercially available immune colorimetric assay kits. Cyclic adenosine monophosphate (cAMP) in the serum was assayed using a commercially available radioimmunoassay kit. Tissue specimens from the ileum, liver and kidney were stained with hematoxylin and eosin. The potential targets of herbal compound in this study were evaluated using standard protocols provided by Cerep, Inc.
Results: This strategy identified TMP from Ligusticum wallichii Franchat as a potent compound for ameliorating the oxidative organ injury of MTX. According to the predicted target profiles of TMP, a possible mechanism of the abrogation of MTX-induced toxicity is that TMP could upregulate cAMP by inhibiting phosphodiesterase (PDE) 10A2 activity. Another novel finding is that the competitive binding and antagonistic effects of TMP on adenosine receptor 2A and 2B appear to play important roles in the TMP-mediated reversal of MTX-induced hepatic injury.
Conclusion: TMP identified by network pharmacology approaches could ameliorate MTX-induced oxidative organ injury. Our results provide a rationale for the preclinical evaluation of TMP and MTX in patients with rheumatoid arthritis.
Keywords Tetramethylpyrazine; methotrexate; network pharmacology; oxidative organ injury 1. Introduction Developing treatments that might reduce the toxicity of clinical drugs is a well-recognized need (Cleeland et al., 2012; Laviano et al., 2012). The folate antagonist Methotrexate (MTX) is a extensively used and effective anti-rheumatoid arthritis and anti-tumor agent that functions by competitively inhibiting dihydrofolate reductase (DHFR), which interferes with nucleic acid or methionine synthesis (Cronstein, 2005; Cronstein et al., 1993). Long-term MTX administration causes substantial discomfort such as stomach upset, nausea, diarrhea, constipation, bloating, and stomach cramps and results in a cumulative dose-dependent toxicity that is characterized by oxidative stress-induced organ injury, including damage to the small intestine, liver and kidney (van Ede et al., 1998; Relling et al., 1994; Miyazono et al., 2004; Abraham et al., 2010; Tabassum et al., 2010). The depletion of glutathione is responsible for the enhanced cytotoxicity because it triggers oxidative stress-induced
injury (al Casey et al., 1995; Masutani, 2001). Additionally, the adenosine/adenosine receptor signaling axis is involved in the toxic effects induced by MTX. For instance, MTX-induced nodulosis in rheumatoid arthritis is mediated by adenosine through the adenosine A1 receptor because giant cell formation in an in vitro model is reversed by a specific adenosine A1 receptor antagonist (Merrill et al., 1997). MTX-induced hepatic fibrosis was also found to be mediated through an adenosine A2A receptor signaling pathway. In contrast to wild-type or A3 receptor-deficient mice, adenosine A2A receptor-deficient mice are protected from developing hepatic fibrosis in response to hepatoxin administration. Similarly, an adenosine A2A receptor-selective antagonist and nonselective adenosine receptor antagonist were shown to prevent hepatic fibrosis in mice following hepatotoxin treatment. These results demonstrate that hepatic adenosine A2A receptors play a critical role in the pathogenesis of MTX-induced hepatic injury (Chan et al., 2006).
To reduce this MTX-induced toxicity, combination therapy for clinical therapies of MTX has attracted increasing attention (Morgan et al., 1990; Rath and Rubbert, 2010; Tugwell et al., 1995). Toxicity from oxidative injury was recently shown to be prevented or alleviated by herbal compounds with antioxidative activity (Dalaklioglu et al., 2013; Bozkurt et al., 2014; Ali et al., 2014). Resveratrol may have a protective effect against MTX-induced hepatotoxicity by blocking oxidative stress-mediated lipid peroxidation (Dalaklioglu et al., 2013). Lutein reverses the MTX-induced apoptosis of IEC-6 cells (Chang et al., 2013). Curcumin can protect rats from MTX nephrotoxicity through its antioxidant and anti-inflammatory activities (Morsy et al., 2013). Chrysin has a beneficial effect against hepatotoxicity induced by MTX via the attenuation of oxidative stress and apoptosis (Ali et al., 2014). Thus previous studies have identified a large number of compounds that display detoxifying activities in the setting of MTX-induced oxidative injury. However, experimental screening methods for identifying combinations with MTX are still in their relative infancy. Therefore, an alternative approach for the rational design of MTX adjuvant therapies based on the mechanisms of MTX-induced toxicity may be required.
The recent availability of traditional Chinese medicine (TCM) network pharmacology provides the means to address this issue (Li and Zhang, 2013). By integrating target profile prediction with computational identification of pharmacological action, these network-based approaches have been used for determining active compounds or their combinations derived from Chinese herbs (Li et al., 2014; Li et al., 2011). In fact, we initially built a target profile prediction method based on network pharmacology, drugCIPHER, to comprehensively describe possible mechanism of action of small molecular compounds (Zhao and Li, 2010). In this study, using the predicted target profiles of herbal compounds, we identified tetramethylpyrazine (TMP), derived from a Chinese medicinal herb (Ligusticum wallichii Franchat, Chuan Xiong), as a rescue agent that may reduce the toxicity of MTX. We show that this compound counteracts MTX toxicity in a rat model by increasing the cAMP level after PDE10A2 inhibition. Competitive binding and antagonistic effects of TMP on the adenosine A2A and A2B receptor were determined, suggesting that the detoxifying mechanism of TMP is at least partly dependent on the blockage of the adenosine A2A receptor pathway.
2. Materials and methods 2.1. Target profile prediction and enrichment analysis In silico prediction of comprehensive target profiles of herbal compounds is the first step in network pharmacology. Compared with virtual screening based on docking analysis, the network-based computational approach for drug target identification is not restricted to the target protein structures. In this study, a network-based regression model (drugCIPHER) for target profile prediction was used. The drugCIPHER method scored the likelihood of drug target interactions by integrating structural similarities of drugs and protein-protein interactions in a heterogeneous network that correlated chemical and genomic spaces (Zhao and Li, 2010). Briefly, drugCIPHER was performed to predict the target profiles of each herbal compound in our library.
To identify the herbal compounds that ameliorate MTX toxicity, we used the functional enrichment tool of the DAVID database to evaluate the enrichment of the target profile of each ingredient in MTX toxicity-related pathways with a false discovery rate less than 0.05 by the Fisher exact test (Huang et al., 2009). We only selected herbal compounds with < 0.05 after Benjamini’s correction.
2.2. Drugs and chemical reagents All tissue storage solution and reagents was purchased from Miltenyi Biotec (Shanghai, China). MTX and TMP were obtained from Sigma-Aldrich (Shanghai, China). All other reagents were the highest quality that could be obtained.
2.3. Animals and experimental design for toxicity studies Forty Wistar rats, half of which were males and half of which were females, were obtained from the Center for Laboratory Animal Care, Chinese Academy of Medical Sciences. The rats (3 months old, 200-250 g body weight) that were used for this study were allowed unrestricted access to water and food. The Animal Ethics Committee of China Academy of Chinese Medical Science approved all animal procedures. To evaluate the effect of TMP on MTX-induced toxicity, all animals were randomly divided into four groups of ten rats each. The MTX treatment group was given MTX i.p. at 20 mg/kg as a single dose on day 4 of the experiment. Normal saline (NS) at 5 ml/kg was administered i.p. as a placebo every day until the rats were sacrificed. In the TMP and MTX treatment groups, TMP at 80 mg/kg was first given i.p. every day until the rats were sacrificed and then MTX at 20 mg/kg was given i.p. on day 4 of the experiment. TMP treatment group was given TMP i.p. at 80 mg/kg every day. On day 4 of the experiment, normal saline at 1 ml/kg was injected rather than MTX. The experimental control group was given normal saline i.p. at 1 ml/kg and received no additional treatment other than standard care and housing. Rats were killed at day 8 by cervical dislocation. Blood was collected for serum GSH, MDA and cAMP measurements, and the small intestine, kidney and liver were collected and rinsed in
PBS for histology.
2.4. Serum and tissue homogenate preparation At 24 h after the last treatment, all of the rats were anesthetized and blood was collected to obtain serum by centrifugation at 3,500×g for 15 min. The ileum, liver and kidney were removed and homogenized in Tris-buffered saline (pH=7.4). The homogenates were centrifuged at 6,000×g for 15 min, and then supernatants were collected and stored at −80°C until required.
2.5. Determination of cAMP, MDA and GSH Malondialdehyde (MDA) and glutathione (GSH) levels in the liver and kidney homogenates were assayed using commercially available immune colorimetric assay kits (Beijing North Institute of Biological Technology, Beijing, China). For the MDA kit, the sensitivity was 0.02 nmol/mL, and the mean intra- and interassay coefficients of variation were 7.3% and 10.0%, respectively (manufacturer's data). For the GSH kit, the sensitivity was 0.1 μmol/L, and the mean intra- and inter assay coefficients of variation were 7.3% and 10.0%, respectively (manufacturer's data). cAMP in the serum was assayed using a commercially available radioimmunoassay (RIA) kit (Beijing North Institute of Biological Technology, Beijing, China). The sensitivity was 0.1 pmol/mL. The mean intra- and interassay coefficients of variation were 5.8% and 6.9%, respectively (manufacturer's data).
2.6. Histopathological preparation and analysis For light-microscopic investigations, tissue specimens from the ileum, liver and kidney were fixed in 10% formaldehyde and processed routinely for embedding in paraffin. Tissue sections of 5 μm were stained with hematoxylin and eosin (H&E) and examined under a Leica DM3000B (Germany) light microscope.
2.7. Adenosine receptor binding assays The potential targets of TMP were evaluated for receptor binding selectivity against adenosine receptors (A1, A2A, A2B, A3) using standard receptor binding protocols provided by Cerep, Inc.
2.8. Agonist or antagonist effect assays of adenosine receptors The agonist or antagonist effect of TMP against different adenosine receptors was evaluated in a cellular-based assay (CHO cells for A1 and A3, PC12 cells for A2A, and HEK293 cells for A2B) using standard receptor binding protocols provided by Cerep, Inc.
2.9. In vitro PDE Assays. The PDE assays were performed using the standard procedures described by Cerep, Inc.
2.10.
Statistical analysis
Data are presented as the mean ± S.D. Significant differences were assessed by one-way analysis of variance (ANOVA). P<0.05 was accepted as representing a significant difference.
3. Results 3.1 Target interaction-based computational screen identifies TMP in combination with MTX Comprehensive target profiles are used to determine the biological activity of herbal compounds by mapping them on signaling and metabolic pathways (Rix and Superti-Furga, 2009; Besnard et al., 2012). To enable the rapid development of MTX-based combination therapies for combating its toxicities, we developed a target profile mapping-aided screening strategy that can be used to identify herbal compounds that counteract MTX toxicity and cooperate with MTX to improve the
therapeutic index of RA. Because MTX-induced toxicity is mediated by direct interference with the purine metabolism pathway through the inhibition of GAR transformylase
and
5-aminoimidazole-4-carboxamide
ribonucleotide
(AICAR)
transformylase (Morgan et al., 2004; Cutolo et al., 2001; Schalinske et al., 1996) and the indirect activation of the adenosine A1 or A2 receptor (Merrill et al., 1997; Chan et al., 2006), if a given target profile of an herbal compound is significantly enriched in the gene set defined by genes involved in purine metabolism and adenosine A1 or A2 receptor signaling, the potential effects of the herbal compound may be associated with the regulation of these two pathways (Berger et al., 2010). We can thus identify herbal compounds that specifically regulate the toxicity induced by MTX. This proof-of-principle screening approach differs from other recently performed experimental screens in MTX-based combination therapies because it can, in principle, predict the herbal compounds that cooperate with MTX to improve clinical safety.
We quantified the degree of enrichment when mapping the target profiles of herbal compounds into a gene set associated with drug efficacy and toxicity. We exploited a repository of the target profiles of 8,965 natural products from 621 Chinese herbs; drugCIPHER, a network-based target prediction approach, can be used to characterize the target profile of each compound with 3,840 druggable target genes. Each target profile was represented as a list of genes ranked according to the relevance of chemical similarity and targets based on a protein-protein interaction network (Zhao and Li, 2010). For the mechanism of toxicity induced by MTX, we extracted the purine metabolism pathway (map00230) and cAMP signaling pathway (map04024) from the KEGG database as a specific gene set for MTX-induced toxicity (Kanehisa et al., 2014). For each compound, we selected the first 100 druggable proteins with high precision at the top of the target profile as a candidate target set. We then presented the p-value of enrichment analysis as an evaluation score and computed it using the DAVID. A smaller p-value indicated a higher probability that the compound would interfere with the purine metabolism pathway. We computed the p-value for each target profile of the 8,965 herbal compounds enriched in the specific gene set
(data not shown).
The above screening procedure led to the identification of TMP derived from Ligusticum wallichii Franchat (Figure 1A). TMP is known to have protective effects against thioacetamide-induced acute hepatotoxicity and has anti-inflammatory activity (Ozaki, 1992; So et al., 2002). However, the mechanisms involved are unclear. The common genes involved in TMP target profile (top 100 genes in Supplementary 1) and MTX toxicity-related pathways are described in Figure 1B. As shown in Figure 1C, we hypothesized that TMP may have protective effects against the MTX toxicity by acting on phosphodiesterases (PDEs) or the adenosine pathway because the TMP target profile suggested that TMP may act on proteins in the PDE family and adenosine receptors (A1, A2A, A2B). It is also hypothesized that TMP was expected to decrease the content of nitric oxide (NO) by inhibiting the adenosine receptors A2A and A2B as well as induce a rise in the level of cAMP by activating the A1 or inhibiting the PDEs. Therefore, we speculated that TMP may act antagonistically with MTX to influence its clinical toxicity through reducing oxidative injury. To test these hypotheses, we explored the specific effects of TMP on the toxicity and therapeutic efficacy of MTX in experimental animal model.
3.2. TMP alleviates MTX-induced oxidative stress in vivo via up-regulation of cAMP primarily by inhibiting PDE10A2 Oxidative damage contributes to the pathogenesis of MTX-induced tissue injury in rats (Miyazono et al., 2004). To experimentally test our hypothesis, we evaluated the effect of TMP on high oxidative stress levels induced by MTX. We observed that MTX treatment decreased liver GSH levels significantly (P<0.05), almost to 50% of that in the NS group, whereas 80 mg/kg TMP treatment following MTX prevented this reduction in GSH (P<0.01) (Figure 2A). Similarly, kidney GSH levels were decreased (P < 0.001) in MTX-treated rats (Figure 2B). However, treatment with TMP abolished the MTX-induced GSH reduction. Additionally, liver MDA levels were also increased significantly (P<0.01) following the administration of MTX, and TMP
treatment exhibited a trend toward reduction of the MDA levels, although the difference did not reach significance (Figure 2C). The levels of MDA in the kidney as products of lipid peroxidation were increased after treatment of rats with MTX compared to untreated controls (P<0.001) (Figure 2D). Administration of TMP reversed kidney MDA levels back to the values in rats not treated with MTX (P<0.01) (Figure 2D).
Morphological changes of the ileum, liver and kidney exposed to the drugs were examined (Figure 3A-L). The ileum and liver tissues from MTX-treated rats showed severe pathological changes compared with those in the control group. The changes in ileum tissue included intestinal villus atrophy, degenerated surface epithelium and severe inflammatory cell infiltration (Figure 3B), whereas changes in liver tissue included dissolved cytoplasm, degenerated hepatocytes, lymphocytic infiltration, and dilatation and vascular congestion in sinusoids (Figure 3F). In TMP-treated rats, no obvious pathological damage was observed in the ileum, liver and kidney tissue (Figure 3C, 3G and 3K). The morphological injuries were significantly recovered in MTX+TMP treated rats, e.g., only moderate inflammatory cell infiltration was observed in the ileum (Figure 3D) and decreased numbers of lymphocytes were observed in most regions of the liver (Figure 3H). There were no obvious morphological changes in the kidney tissue of MTX, TMP and MTX+TMP treated rats (Figure 3I, 3J, 3K and 3L).
Increased oxidative stress by MTX impaired cAMP accumulation and suppressed cAMP levels, leading to MTX-related toxicity (Kreml et al., 1979). To determine whether MTX-induced changes in cAMP levels were regulated by TMP addition, we performed RIA to measure the concentration of cAMP. Consistent with previous studies, MTX effectively reduced the serum cAMP level in rats (P<0.01). Compared with MTX-treated rats, combined TMP and MTX treatment of rats led to recovery of the cAMP balance (Figure 4A). The target profiles of TMP suggested that TMP might counteract
the
MTX-induced
decreases
in
serum
cAMP
by
inhibiting
phosphodiesterases, which leads to cAMP accumulation. To validate our predictions in vitro, we used an enzyme activity assay to determine the inhibitory effect of TMP on PDE isozymes. 100 μM TMP had almost no effect on the PDE2, PDE3, PDE4, PDE5, PDE6, PDE7, PDE8 and PDE11; however, it affected PDE10A2 (Figure 4B). Notably, TMP at 10 μM to 1 mM concentration-dependently inhibited the metabolic activity of PDE10A2 with an IC50 value of 700 μM (Figure 4C).
3.3. Competitive binding and antagonistic effect of different concentrations of TMP on adenosine A2A and A2B receptors Although adenosine/adenosine receptor signaling plays a critical role in the anti-inflammatory effects of MTX, previous studies have investigated the role of the adenosine pathway in MTX-induced toxicity (Merrill et al., 1997; Chan et al., 2006). The predicted target profile suggests a possible role for TMP in the control of the adenosine signaling pathway. To determine whether the attenuation of MTX toxicity through combination with TMP resulted from modulation of A1, A2A and A2B, we examined the biochemical interaction of TMP with adenosine receptors and the biochemical effects (antagonist or agonist) for these receptors by in vitro assays. As illustrated in Figure 5A, 100 μM TMP had no binding activity with A1 as determined by an antagonist and agonist radioligand binding assay. TMP at concentrations of 10 μM — 1 mM inhibited specific agonist radioligand binding with A2A with a Ki of 4.6×10-4 M (n=3) (IC50: 5.6×10-4 M), indicating that TMP binds to A2A competitively. Similarly, a high concentration of TMP slightly inhibited antagonist radioligand binding with A2B (Figure 5B). In two types of biochemical assays, 100 μM TMP partially inhibited the activity of A2A and A2B (Figure 5D). In addition, we observed that TMP reduced the activation of A2B in a concentration-dependent manner (Figure 5E). We did not observe any changes in the agonist activity of A2A and A2B on treatment with 100 μM TMP (Figure 5C). These results suggest that attenuation of MTX toxicity by TMP may be correlated to the antagonism of A2A and A2B.
4. Discussion & Conclusions Recently, computational approaches have greatly increased the potential of identifying more effective and better combinational therapies (Al-Lazikani et al., 2012). Although some computational approaches have promise and have shifted the landscape to a new drug discovery paradigm, they have still not yielded breakthroughs. Network-based approaches will pave the way for rationally designed and hopefully optimized combinations of multi-target therapies. Our screening approach is performed based on the targeting of functional gene sets rather than the targeting of single molecules. This strategy meets the features of mechanisms of herbal compounds.
Oxidative stress has been identified as one of the major factors responsible for MTX toxicity (Miyazono et al., 2004; Kremer, 2004). A major challenge in MTX therapy for rheumatoid arthritis is to identify combinations that prevent the emergence of toxicity. In the present study, we computationally predicted a panel of herbal compounds with the potential ability to relieve MTX-induced oxidative damage using our network pharmacology approach. Critically, we experimentally demonstrated that a herbal compound derived from ligusticum chuanxiong hort, tetramethylpyrazine, effectively rebalances MTX-induced oxidative stress as a PDE10A2 inhibitor. Furthermore, we found that TMP elicits an antagonist effect against A2A and A2B by competitively binding them, leading to direct inhibition of the activation of A2A and A2B receptors by MTX-induced adenosine signaling, thus decreasing MTX-induced toxicity. Our data, in conjunction with previous studies (Mishima et al., 2006; Wu et al., 2004), suggest that cAMP in rat blood serum is up-regulated by TMP, which contributes to alleviation of MTX-induced oxidative organ injury.
Mechanisms of MTX toxicity often involve folate metabolism, purine metabolism, adenosine deaminase activity with accumulation of adenosine and deoxyadenosine, polyamine synthesis and homocysteine metabolism (van Ede et al., 1998). Disruption of metabolic homeostasis leads to continued oxidative stress that increases the
formation of reactive oxygen species (ROS) over antioxidant protection and subsequently induces organ DNA damage, lipid peroxidation, protein modification and other toxic effects (Pravenec et al., 2013; Garca et al., 2014; Harley et al., 1997). Increased ROS levels inhibit the protective effects of cAMP, rendering normal cells more vulnerable to oxidative damage (Ling et al., 2007), whereas increasing levels of cAMP are known to attenuate NAPDH oxidase activity in normal cells, resulting in decreased oxidative stress (Saha et al., 2011; Saha et al., 2008). In addition, G-protein coupled receptors of the P1 purinergic family, i.e., the A1, A2A, A2B, and A3 adenosine receptor subtypes, are expressed in various non-inflammatory cells, tissues and organs (Cronstein, 1997). If the signaling pathways through A1 or A2A/B are functionally intact in the same tissues, a preponderance of the A1 pathway is expected because of the higher affinity of adenosine for the A1 receptor. This would cause a decrease of cAMP levels, leading to the further enhancement of MTX-induced oxidative stress (Cutolo et al., 2001). Indeed, treatment with MTX may lead to reduced cAMP levels in cultured L5178Y cells (Kreml et al., 1979). Importantly, we also found that MTX decreased cAMP levels in rat serum, which may provide an explanation for the enhancement of MTX-induced oxidative stress by reduced levels of cAMP. Phosphodiesterase (PDE) inhibitors, which are used to maintain the cAMP levels and prevent the inactivation of cAMP, can ameliorate oxidative stress and DNA damage in normal organs (Dias et al., 2014). Moreover, adenosine A2A and A2B receptor pathway stimulation by MTX-induced adenosine may potentiate nitric oxide release that plays a pivotal role in oxidative organ injury (Saura et al., 2005; Olanrewaju and Mustafa, 2000).
We performed our strategy primarily based on our previous observation that purine metabolism and adenosine receptor pathway is a major causative factor in the development of MTX-induced toxicity. Here, adenosine induced by MTX as the endogenous ligand interacts with four known receptors, A1, A2A, A2B and A3 in the adenosine receptor pathway. A1 is coupled to a Gαi/o protein, which would lead to cAMP decrease; however, A2 is coupled to a Gαs protein, which would lead to cAMP
increase. Because the affinity of adenosine binding to these receptor subtypes is different: A1>A2A>A2B, MTX administration to rats resulted in a significant decrease in the level of serum cAMP, which could reflect intracellular cAMP level in certain degree through a significant release of cellular cAMP (Brown et al., 1977). Based on computational predictions, we propose that TMP may affect MTX-induced oxidative organ injury because its target profile falls within functional gene sets defined by network neighborhood of genes potentially related to MTX-induced toxicity. Subsequently, experimental evidence indicates that TMP has an inhibitory effect on PDE10A2, A2A and A2B, whereas TMP has no effect on A1. In agreement with our hypothesis, inhibition of PDE10A2 by TMP relieves MTX toxicity and increases the cAMP level in rat serum despite substantial inhibition of A2A and A2B and no inhibition of A1. Our inference for the increase of serum cAMP in TMP-treated group was the higher expression of PDE10A2 than A1 and the lower affinity of adenosine to A2A than A1 in non-inflammatory tissues. Additional evidence that TMP can ameliorate MTX-induced oxidative injury comes from literature report in which we analyzed antagonism of A2A by TMP may decrease the reported toxicity of nitric oxide released by the adenosine A2A receptor pathway.
Ligusticum Chuanxiong, one of the most commonly used herbs in traditional Chinese medicine, is included in the herbal formula, Ba-Wei-Di-Huang Wan. TMP is one of the primary herbal compounds from Ligusticum Chuanxiong and has a broad clinical application. Many compounds from herbal medicine affect the human body in a low-affinity and multi-target manner (Kitano, 2007). High-throughput experimental screening assays are used as a type of trial and error method to explore the target proteins of herbal compounds. In contrast to such experimental methods alone, our computational platform has the capacity to predict the target profiles of herbal compounds to comprehensively determine their pharmacological activity. For instance, TMP can reduce streptomycin-induced ototoxicity by increasing the amplitudes of calcium sensitive potassium channels of outer hair cells of guinea pig cochlea (Cui et al., 2007). Interestingly, six out of the top ten hits are cation channels. Furthermore,
based on our predicted results, we also found that adenylate cyclase 1 (ADCY1) and adenylate cyclase 2 (ADCY2) were ranked 80th and 91th for druggable target profiles of TMP, respectively. Adenylyl cyclase inhibitors have been used to treat neuropathic and inflammatory pain (Wang et al., 2011). Thus, we speculated that the antinociceptive mechanisms of TMP may be associated with adenylate cyclases regulation. Our study also reveals the availability of network-based identification of pharmacological action.
Network pharmacology approaches await further improvements. Nevertheless, the analyses reported here identify a combination therapy for MTX and reveal the mechanism of action, to some extent. TMP is a potential small molecular compound from herbal medicine that may be useful for the alleviation of MTX-induced oxidative organ injury with a mechanism of action that involves potent inhibition of PDE10A2 and A2A. We believe that the network pharmacology approach serves as a framework for combination therapy development and will provide an important method of determining the mechanisms underlying the activity of herbal compounds and an important paradigm for drug discovery.
Acknowledgement This work was supported by the National Natural Science Foundation of China (Nos. 81225025, 91229201 and 81303139), the CATCM project, Interdisciplinary Research Matching Scheme (IRMS) of Hong Kong Baptist University (RC-IRMS/12-13/02) and Hong Kong Baptist University Strategic Development Fund (SDF13-1209-P01).
References Abraham, P., Kolli, V.K., Rabi, S., 2010. Melatonin attenuates methotrexate-induced oxidative stress and renal damage in rats. Cell Biochemistry and Function 28, 426-433. al Casey, S., Brewster, D., Viau, C., Acosta, D., 1995. Effect of glutathione depletion and oxidative stress on the in vitro cytotoxicity of velnacrine maleate. Toxicology Letters 76, 257-265. Ali, N., Rashid, S., Nafees, S., Hasan, S.K., Sultana, S., 2014. Beneficial effects of Chrysin against Methotrexate-induced hepatotoxicity via attenuation of oxidative stress and apoptosis. Molecular and Cellular Biochemistry 385, 215-223. Al-Lazikani, B., Banerji, U., Workman, P., 2012. Combinatorial drug therapy for cancer in the post-genomic era. Nature Biotechnology 30, 679-692. Berger, S.I., Ma'ayan, A., Iyengar, R., 2010. Systems pharmacology of arrhythmias. Science Signaling 3, ra30. Besnard, J., Ruda, G.F., Setola, V., Abecassis, K., Rodriguiz, R.M., Huang, X.P., Norval, S., Sassano, M.F., Shin, A.I., Webster, L.A., Simeons, F.R., Stojanovski, L., Prat, A., Seidah, N.G., Constam, D.B., Bickerton, G.R., Read, K.D., Wetsel, W.C., Gilbert, I.H., Roth, B.L., Hopkins, A.L., 2012. Automated design of ligands to polypharmacological profiles. Nature 492, 215-220. Bozkurt, M., Em, S., Oktayoglu, P., Turkcu, G., Yuksel, H., Sarıyıldız, M.A., Caglayan, M., Batmaz, I., Nas, K., Bozkurt, Y., Kuyumcu, M., 2014. Carvacrol prevents methotrexate-induced renal oxidative injury and renal damage in rats. Clinical & Investigative Medicine 37, E19-E25. Brown, E.M., Carroll, R.J., Aurbach, G.D., 1977. Dopaminergic stimulation of cyclic AMP accumulation and parathyroid hormone release from dispersed bovine parathyroid cells. Proceedings of the National Academy of Sciences 74, 4210-4213.
Chan, E.S., Montesinos, M.C., Fernandez, P., Desai, A., Delano, D.L., Yee, H., Reiss, A.B., Pillinger, M.H., Chen, J.F., Schwarzschild, M.A., Friedman, S.L., Cronstein, B.N., 2006. Adenosine A(2A) receptors play a role in the pathogenesis of hepatic cirrhosis. British Journal of Pharmacology 148, 1144-1155. Chang, C.J., Lin, J.F., Chang, H.H., Lee, G.A., Hung, C.F., 2013. Lutein protects against methotrexate-induced and reactive oxygen species-mediated apoptotic cell injury of IEC-6 cells. PLoS One 8, e72553. Cleeland, C.S., Allen, J.D., Roberts, S.A., Brell, J.M., Giralt, S.A., Khakoo, A.Y., Kirch, R.A., Kwitkowski, V.E., Liao, Z., Skillings, J., 2012. Reducing the toxicity of cancer therapy: recognizing needs, taking action. Nature Reviews Clinical Oncology 9, 471-478. Cronstein, B.N., 1997. The mechanism of action of methotrexate. Rheumatic Disease Clinics of North America 23, 739–755. Cronstein, B.N., 2005. Low-dose methotrexate: a mainstay in the treatment of rheumatoid arthritis. Pharmacological Reviews 57, 163-172. Cronstein, B.N., Naime, D., Ostad, E., 1993. The antiinflammatory mechanism of methotrexate. Increased adenosine release at inflamed sites diminishes leukocyte accumulation in an in vivo model of inflammation. Journal of Clinical Investigation 92, 2675-2682. Cui, G.Y., Shi, L.J., Tang, H., 2007. Reduction of Streptomycin Ototoxicity by Tetramethylpyrazine and its Ionic Channel Mechanism. Journal of Audiology and Speech Pathology 15, 129-131. Cutolo, M., Sulli, A., Pizzorni, C., Seriolo, B., Straub, R.H., 2001. Anti-inflammatory mechanisms of methotrexate in rheumatoid arthritis. Annals of the Rheumatic Diseases 60, 729-735. Dalaklioglu, S., Genc, G.E., Aksoy, N.H., Akcit, F., Gumuslu, S., 2013. Resveratrol ameliorates methotrexate-induced hepatotoxicity in rats via inhibition of lipid peroxidation. Human & Experimental Toxicology 32, 662-671.
Dias, A.T., Rodrigues, B.P., Porto, M.L., Gava, A.L., Balarini, C.M., Freitas, F.P., Palomino, Z., Casarini, D.E., Campagnaro, B.P., Pereira, T.M., Meyrelles, S.S., Vasquez, E.C., 2014. Sildenafil ameliorates oxidative stress and DNA damage in the stenotic kidneys in mice with renovascular hypertension. Journal of Translational Medicine 12, 35. Garca, M.F., Demir, H., Turan, M., Bozan, N., Kozan, A., Belli, Ş.B., Arslan, A., Cankaya, H., 2014. Assessment of adenosine deaminase (ADA) activity and oxidative stress in patients with chronic tonsillitis. European archives of oto-rhino-laryngology 271, 1797-1802. Harley, E.H., Paglia, D., Weber, B., 1997. Oxidative damage and purine metabolism: Investigation of haemolytic anaemia in the black rhinoceros. Clinical Biochemistry 30, 259. Huang da, W., Sherman, B.T., Lempicki, R.A. 2009. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nature Protocols 4, 44-57. Kanehisa, M., Goto, S., Sato, Y., Kawashima, M., Furumichi, M., Tanabe, M., 2014. Data, information, knowledge and principle: back to metabolism in KEGG. Nucleic Acids Research 42(Database issue), D199-D205. Kitano, H., 2007. A robustness-based approach to systems-oriented drug design. Nature Reviews Drug Discovery 6, 202-210. Kremer, J.M., 2004. Toward a better understanding of methotrexate. Arthritis & Rheumatology 50, 1370-1382. Kreml, J.A., Hryniuk, W.M., Yamada, E.W., 1979. Effect of methotrexate on cyclic AMP levels in cultured L5178Y cells. Biochemical Medicine 22, 43-49. Laviano, A., Molfino, A., Rossi Fanelli, F., 2012. Cancer-treatment toxicity: can nutrition help? Nature Reviews Clinical Oncology 9. Li, H., Zhao, L., Zhang, B., Jiang, Y., Wang, X., Guo, Y. Liu, H., Li, S., Tong, X., 2014. A network pharmacology approach to determine active compounds and action mechanisms of ge-gen-qin-lian decoction for treatment of type 2 diabetes. Evidence-Based Complementary and Alternative Medicine 2014, 495840.
Ling, S., Li, Z., Borschukova, O., Xiao, L., Pumpens, P., Holoshitz, J., 2007. The rheumatoid arthritis shared epitope increases cellular susceptibility to oxidative stress by antagonizing an adenosine-mediated anti-oxidative pathway. Arthritis Research & Therapy 9, R5. Li, S., Zhang, B., 2013. Traditional Chinese medicine network pharmacology: theory, methodology and application. Chinese Journal of Natural Medicines 11, 110-120. Li, S., Zhang, B., Zhang, N., 2011. Network target for screening synergistic drug combinations with application to traditional Chinese medicine. BMC Systems Biology 5 Suppl 1, S10. Lu, Y., Zhu, M., Chen, W., Yin, L., Zhu, J., Chen, N., Chen, W., 2014. Tetramethylpyrazine improves oxazolone-induced colitis by inhibiting the NF-κB pathway. Clinical & Investigative Medicine 37, E1-E9. Masutani, H., 2001. Oxidative stress and redox imbalance in acetaminophen toxicity. Pharmacogenomics Journal 1, 165-166. Merrill, J.T., Shen, C., Schreibman, D., Coffey, D., Zakharenko, O., Fisher, R., Lahita, R.G., Salmon, J., Cronstein, B.N., 1997. Adenosine A1 receptor promotion of multinucleated giant cell formation by human monocytes: a mechanism for methotrexate-induced
nodulosis
in
rheumatoid
arthritis.
Arthritis
&
Rheumatology 40, 1308-1315. Mishima, K., Baba, A., Matsuo, M., Itoh, Y., Oishi, R., 2006. Protective effect of cyclic AMP against cisplatin-induced nephrotoxicity. Free Radical Biology & Medicine 40, 1564-1577. Miyazono, Y., Gao, F., Horie, T., 2004. Oxidative stress contributes to methotrexate-induced small intestinal toxicity in rats. Scandinavian Journal of Gastroenterology 39, 1119-1127. Morgan, S.L., Baggott, J.E., Vaughn, W.H., Young, P.K., Austin, J.V., Krumdieck, C.L. Alarcón, G.S., 1990. The effect of folic acid supplementation on the toxicity of low-dose methotrexate in patients with rheumatoid arthritis. Arthritis & Rheumatology 33, 9-18.
Morgan, S.L., Oster, R.A., Lee, J.Y., Alarcón, G.S., Baggott, J.E., 2004. The effect of folic
acid
and
folinic
acid
supplements
on
purine
metabolism
in
methotrexate-treated rheumatoid arthritis. Arthritis & Rheumatology 50, 3104-3111. Morsy, M.A., Ibrahim, S.A., Amin, E.F., Kamel, M.Y., Rifaai, R.A., Hassan, M.K., 2013. Curcumin ameliorates methotrexate-induced nephrotoxicity in rats. Advances in Pharmacological Sciences 2013, 387071. Olanrewaju, H.A., Mustafa, S.J., 2000. Adenosine A(2A) and A(2B) receptors mediated nitric oxide production in coronary artery endothelial cells. General Pharmacology 35, 171-177. Ozaki, Y., 1992. Antiinflammatory effect of tetramethylpyrazine and ferulic acid. Chemical & Pharmaceutical Bulletin (Tokyo) 40, 954-956. Pravenec, M., Kozich, V., Krijt, J., Sokolová, J., Zídek, V., Landa, V., Simáková, M., Mlejnek, P., Silhavy, J., Oliyarnyk, O., Kazdová, L., Kurtz, T.W., 2013. Folate deficiency is associated with oxidative stress, increased blood pressure, and insulin resistance in spontaneously hypertensive rats. American Journal of Hypertension 26, 135-140. Rath, T., Rubbert, A., 2010. Drug combinations with methotrexate to treat rheumatoid arthritis. Clinical and Experimental Rheumatology 28, S52-S57. Relling, M.V., Fairclough, D., Ayers, D., Crom, W.R., Rodman, J.H., Pui, C.H., Evans, W.E., 1994. Patient characteristics associated with high-risk methotrexate concentrations and toxicity. Journal of Clinical Oncology 12, 1667–1672. Rix, U., Superti-Furga, G., 2009. Target profiling of small molecules by chemical proteomics. Nature Chemical Biology 5, 616-624. Tabassum, H., Parvez, S., Pasha, S.T., Banerjee, B.D., Raisuddin, S., 2010. Protective effect of lipoic acid against methotrexate-induced oxidative stress in liver mitochondria. Food and Chemical Toxicology 48, 1973-1979.
Saha, S., Jia, Z.Q., Liu, D.M., Misra, H.P., 2011. The Roles of cAMP and G Protein Signaling in Oxidative Stress-Induced Cardiovascular Dysfunction. Studies on Experimental Models, Oxidative Stress in Applied Basic Research and Clinical Practice. Humana Press, 621-635. Saha, S., Li, Y., Anand-Srivastava, M.B., 2008. Reduced levels of cyclic AMP contribute to the enhanced oxidative stress in vascular smooth muscle cells from spontaneously hypertensive rats. Canadian Journal of Physiology and Pharmacology 86, 190-198. Saura, J., Angulo, E., Ejarque, A., Casadó, V., Tusell, J.M., Moratalla, R., Chen, J.F., Schwarzschild, M.A., Lluis, C., Franco, R., Serratosa, J., 2005. Adenosine A2A receptor stimulation potentiates nitric oxide release by activated microglia. Journal of Neurochemistry 95, 919-929. Schalinske, K.L., Steele, R.D., 1996. Methotrexate alters carbon flow through the hepatic folate-dependent one-carbon pool in rats. Carcinogenesis 17, 1695-1700. So, E.C., Wong, K.L., Huang, T.C., Tasi, S.C., Liu, C.F., 2002. Tetramethylpyrazine protects mice against thioacetamide-induced acute hepatotoxicity. Journal of Biomedical Science 9, 410-414. Tugwell, P., Pincus, T., Yocum, D., Stein, M., Gluck, O., Kraag, G., McKendry, R., Tesser, J., Baker, P., Wells, G., 1995. Combination therapy with cyclosporine and methotrexate in severe rheumatoid arthritis. The Methotrexate-Cyclosporine Combination Study Group. The New England Journal of Medicine 333, 137-141. van Ede, A.E., Laan, R.F., Blom, H.J., De Abreu, R.A., van de Putte, L.B., 1998. Methotrexate in rheumatoid arthritis: an update with focus on mechanisms involved in toxicity. Seminars in Arthritis and Rheumatism 27, 277-292. Wang, H., Xu, H., Wu, L.J., Kim, S.S., Chen, T., Koga, K., Descalzi, G., Gong, B., Vadakkan, K.I., Zhang, X., Kaang, B.K., Zhuo, M., 2011. Identification of an adenylyl cyclase inhibitor for treating neuropathic and inflammatory pain. Science Translational Medicine 3, 65ra3.
Wu, Y., Zheng, J., Linden, J., Holoshitz, J., 2004. Genoprotective pathways. Part I. Extracellular signaling through G(s) protein-coupled adenosine receptors prevents oxidative DNA damage. Mutation Research 546, 93-102. Zhao, S., Li, S., 2010. Network-based relating pharmacological and genomic spaces for drug target identification. PLoS One 5: e11764.
Figure legend Figure 1. Network target of TMP and possible molecular mechanisms involved in reducing MTX toxicity using TMP. A. Chemical structure of TMP. B. Functional sub-network perturbed by TMP in the context of biological mechanisms of MTX-induced toxicity. In the protein-protein interaction (edges) network, the target profile of TMP (blue nodes) perturbs the biological process (triangular nodes represent proteins in the adenosine receptor pathway; rectangle nodes represent proteins in purine metabolism) involved in MTX-induced oxidative organ injury. C. Models for a hypothetical mechanism that can explain the protective effect of TMP against MTX toxicity. The genes with a red symbol are involved in the predicted target profile of TMP. The dashed lines indicate that TMP could either increase or inhibit the activity of these target proteins.
Figure 2. Administration of 80 mg/kg TMP partly restores normal levels of GSH and MDA in the kidney and liver after 20 mg/kg MTX exposure. A and B. GSH levels in the liver and kidney of NS, MTX-treated, TMP-treated, and MTX+TMP-treated groups. C and D. MDA levels in the liver and kidney of NS, MTX-treated, TMP-treated, and MTX+TMP-treated groups. * P<0.05, ** P<0.01, *** P<0.001.
Figure 3. Morphological changes of ileum, liver and kidney tissues in TMP, MTX and MTX+TMP treated rats. H & E staining of formalin-fixed, paraffin-embedded sections. A, E and I: Normal morphology of the ileum (A), liver (E) and kidney (I) in control rats. B, F and J: Intestinal villus atrophy, degenerated surface epithelium and severe inflammatory cell infiltration in the ileum (B); dissolved cytoplasm, degenerated hepatocytes, lymphocytic infiltration, and dilatation and vascular congestion in sinusoids of liver (F); and no pathological damages in kidney (J) in MTX-treated group. C, G and K: No obvious pathological damage in the ileum (C), liver (G) and kidney (J) in TMP-treated groups. D, H and L: Milder histopathological changes observed in MTX+TMP treated rats compared to MTX-treated rats (B), only moderate inflammatory cell infiltration in the ileum (D) compared to the MTX-treated rats (F), and decreased numbers of lymphocytes in most regions of the liver (H). No pathological damage was observed in the kidney (L) in MTX+TMP treated rats. Original magnification: A, B, C and D, ×100; E, F, G, H, I, J, K and L, ×200.
Figure 4. TMP-mediated reversal of the reduction of serum cAMP levels caused by MTX may be associated with antagonism of the activity of PDE10A2. A. Serum radioimmunoassay showed that TMP increased the content of serum cAMP and relieved the reduction of serum cAMP caused by MTX. B. Enzyme assays and in vitro pharmacology confirmed that exposure to 100 μM TMP partly inhibited the activity of PDE10A2. C. The enzyme assay of PDE10A2 further demonstrated that TMP repressed PDE10A2 activity in a concentration-dependent manner.
Figure 5. TMP can competitively bind to A2A and A2B as an inhibitor of the adenosine A2 receptor. A. Binding assays indicated that TMP does not bind to A1. B. Binding assays demonstrated binding of TMP to A2A (IC50=5.6E-04 M) and A2B (1.2E-03 M) in a concentration-dependent manner. Cellular receptor functional assays further evaluated the agonist or antagonist effect of TMP on A2A and A2B. C. TMP at 100 μM had no agonist effect on A2A and A2B. D. TMP at 100 μM had an antagonist effect on A2A and A2B. E. Cellular receptor functional assays confirmed that
different concentrations of TMP had antagonist effects on A2A (IC50: 0.0005132 M) and A2B (IC50: 0.001715 M) in a concentration-dependent manner.
Figures Figure 1.
Figure 2.
Figure 3.
Figure 4.
Figure 5.
*Graphical Abstract-revised
PII: DOI: Reference:
S0378-8741(15)30159-8 http://dx.doi.org/10.1016/j.jep.2015.09.034 JEP9757
To appear in: Journal of Ethnopharmacology Received date: 28 May 2015 Revised date: 29 September 2015 Accepted date: 30 September 2015 Cite this article as: Bo Zhang, Cheng Lu, Ming Bai, Xiaojuan He, Yong Tan, Yanqin Bian, Cheng Xiao, Ge Zhang, Aiping Lu and Shao Li, Tetramethylpyrazine identified by network pharmacology approaches ameliorates methotrexate-induced oxidative organ injury, Journal of Ethnopharmacology, http://dx.doi.org/10.1016/j.jep.2015.09.034 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Title: Tetramethylpyrazine identified by network pharmacology approaches ameliorates methotrexate-induced oxidative organ injury
Author names: Bo Zhang 1, 5, #, Cheng Lu 2, 3, #, Ming Bai 1, Xiaojuan He 2, Yong Tan 2, Yanqin Bian 2, Cheng Xiao 4, Ge Zhang 3, Aiping Lu 2, 3, *, Shao Li1, * *: Corresponding author. #: These two authors contributed equally to this work.
Author’s affiliations: 1. MOE Key Laboratory of Bioinformatics and Bioinformatics Division, TNLIST / Department of Automation, Tsinghua University, Beijing 100084, China 2. Institute of Basic Research in Clinical Medicine, China Academy of Chinese Medical Sciences, 100700 Beijing, China 3. Institute for Advancing Translational Medicine in Bone & Joint Diseases, School of Chinese Medicine, Hong Kong Baptist University, 00852 Hong Kong SAR, China 4. China-Japan Friendship Hospital, Beijing 100030, China 5. Tianjin International Joint Academy of Biotechnology & Medicine, Tianjin 300457, China
E-mails: Bo Zhang: [email protected] Cheng Lu: [email protected] Ming Bai: [email protected] Xiaojuan He: [email protected] Yong Tan: [email protected]
Yanqin Bian: [email protected] Cheng Xiao: [email protected] Ge Zhang: [email protected] Aiping Lu: [email protected] Shao Li: [email protected]
Contact: * Address, Room 1-107, FIT Building, Tsinghua University. Phone/Fax: 86-10-62797035 / 86-10- 62773552 E-mail: [email protected]
Author contributions: S.L. and A.L. conceived the study. B.Z. and C.L. performed experiments, obtained and analysed data and did statistical analysis. M.B. performed target profile prediction and enrichment analysis. B.Z., C.L. and Y.T. performed animals and experimental design for toxicity studies. Y.T. and X.H. performed cAMP, MDA and GSH assay. Y.T. and Y.B. did histopathological preparation and analysis. B.Z., A.L. and S.L. conceptualized the project and supervised the work. All authors discussed the results and B.Z., C.L., A.L. and S.L. wrote the manuscript.
Sources of funding: This work was supported by the National Natural Science Foundation of China (Nos. 81225025, 91229201 and 81303139), the CATCM project, Interdisciplinary Research Matching Scheme (IRMS) of Hong Kong Baptist University (RC-IRMS/12-13/02) and Hong Kong Baptist University Strategic Development Fund (SDF13-1209-P01).
Disclosure of Potential Conflicts of interest: No potential conflicts of interest disclosed.
Abstract Ethnopharmacological relevance: Ligusticum wallichii Franchat, namely “Chuan-xiong” in traditional Chinese medicines, has been described in application for alleviating the toxicity of chemotherapy. Our studies showed that tetramethylpyrazine (TMP), the bioactive compound from Chuan-xiong, could be valuable for preventing the methotrexate (MTX) -induced oxidative injury. However, its mechanism remains unclear.
Aim of the study: The present study aimed to develop combination treatments that could reduce the toxicity of MTX from Chinese herbal medicine and characterize their mechanism of combinational actions.
Materials and methods: A network pharmacology-based screening strategy is applied for target profile prediction and pharmacological characterization of herbal compounds, which is used to guide the following in vitro and in vivo experiments. The effect of herbal compounds identified by network pharmacology approaches to reduce the toxicity of MTX was assessed by MTX-induced rat toxicity model. Malondialdehyde and glutathione levels in the liver and kidney homogenates were assayed using commercially available immune colorimetric assay kits. Cyclic adenosine monophosphate (cAMP) in the serum was assayed using a commercially available radioimmunoassay kit. Tissue specimens from the ileum, liver and kidney were stained with hematoxylin and eosin. The potential targets of herbal compound in this study were evaluated using standard protocols provided by Cerep, Inc.
Results: This strategy identified TMP from Ligusticum wallichii Franchat as a potent compound for ameliorating the oxidative organ injury of MTX. According to the predicted target profiles of TMP, a possible mechanism of the abrogation of MTX-induced toxicity is that TMP could upregulate cAMP by inhibiting phosphodiesterase (PDE) 10A2 activity. Another novel finding is that the competitive binding and antagonistic effects of TMP on adenosine receptor 2A and 2B appear to play important roles in the TMP-mediated reversal of MTX-induced hepatic injury.
Conclusion: TMP identified by network pharmacology approaches could ameliorate MTX-induced oxidative organ injury. Our results provide a rationale for the preclinical evaluation of TMP and MTX in patients with rheumatoid arthritis.
Keywords Tetramethylpyrazine; methotrexate; network pharmacology; oxidative organ injury 1. Introduction Developing treatments that might reduce the toxicity of clinical drugs is a well-recognized need (Cleeland et al., 2012; Laviano et al., 2012). The folate antagonist Methotrexate (MTX) is a extensively used and effective anti-rheumatoid arthritis and anti-tumor agent that functions by competitively inhibiting dihydrofolate reductase (DHFR), which interferes with nucleic acid or methionine synthesis (Cronstein, 2005; Cronstein et al., 1993). Long-term MTX administration causes substantial discomfort such as stomach upset, nausea, diarrhea, constipation, bloating, and stomach cramps and results in a cumulative dose-dependent toxicity that is characterized by oxidative stress-induced organ injury, including damage to the small intestine, liver and kidney (van Ede et al., 1998; Relling et al., 1994; Miyazono et al., 2004; Abraham et al., 2010; Tabassum et al., 2010). The depletion of glutathione is responsible for the enhanced cytotoxicity because it triggers oxidative stress-induced
injury (al Casey et al., 1995; Masutani, 2001). Additionally, the adenosine/adenosine receptor signaling axis is involved in the toxic effects induced by MTX. For instance, MTX-induced nodulosis in rheumatoid arthritis is mediated by adenosine through the adenosine A1 receptor because giant cell formation in an in vitro model is reversed by a specific adenosine A1 receptor antagonist (Merrill et al., 1997). MTX-induced hepatic fibrosis was also found to be mediated through an adenosine A2A receptor signaling pathway. In contrast to wild-type or A3 receptor-deficient mice, adenosine A2A receptor-deficient mice are protected from developing hepatic fibrosis in response to hepatoxin administration. Similarly, an adenosine A2A receptor-selective antagonist and nonselective adenosine receptor antagonist were shown to prevent hepatic fibrosis in mice following hepatotoxin treatment. These results demonstrate that hepatic adenosine A2A receptors play a critical role in the pathogenesis of MTX-induced hepatic injury (Chan et al., 2006).
To reduce this MTX-induced toxicity, combination therapy for clinical therapies of MTX has attracted increasing attention (Morgan et al., 1990; Rath and Rubbert, 2010; Tugwell et al., 1995). Toxicity from oxidative injury was recently shown to be prevented or alleviated by herbal compounds with antioxidative activity (Dalaklioglu et al., 2013; Bozkurt et al., 2014; Ali et al., 2014). Resveratrol may have a protective effect against MTX-induced hepatotoxicity by blocking oxidative stress-mediated lipid peroxidation (Dalaklioglu et al., 2013). Lutein reverses the MTX-induced apoptosis of IEC-6 cells (Chang et al., 2013). Curcumin can protect rats from MTX nephrotoxicity through its antioxidant and anti-inflammatory activities (Morsy et al., 2013). Chrysin has a beneficial effect against hepatotoxicity induced by MTX via the attenuation of oxidative stress and apoptosis (Ali et al., 2014). Thus previous studies have identified a large number of compounds that display detoxifying activities in the setting of MTX-induced oxidative injury. However, experimental screening methods for identifying combinations with MTX are still in their relative infancy. Therefore, an alternative approach for the rational design of MTX adjuvant therapies based on the mechanisms of MTX-induced toxicity may be required.
The recent availability of traditional Chinese medicine (TCM) network pharmacology provides the means to address this issue (Li and Zhang, 2013). By integrating target profile prediction with computational identification of pharmacological action, these network-based approaches have been used for determining active compounds or their combinations derived from Chinese herbs (Li et al., 2014; Li et al., 2011). In fact, we initially built a target profile prediction method based on network pharmacology, drugCIPHER, to comprehensively describe possible mechanism of action of small molecular compounds (Zhao and Li, 2010). In this study, using the predicted target profiles of herbal compounds, we identified tetramethylpyrazine (TMP), derived from a Chinese medicinal herb (Ligusticum wallichii Franchat, Chuan Xiong), as a rescue agent that may reduce the toxicity of MTX. We show that this compound counteracts MTX toxicity in a rat model by increasing the cAMP level after PDE10A2 inhibition. Competitive binding and antagonistic effects of TMP on the adenosine A2A and A2B receptor were determined, suggesting that the detoxifying mechanism of TMP is at least partly dependent on the blockage of the adenosine A2A receptor pathway.
2. Materials and methods 2.1. Target profile prediction and enrichment analysis In silico prediction of comprehensive target profiles of herbal compounds is the first step in network pharmacology. Compared with virtual screening based on docking analysis, the network-based computational approach for drug target identification is not restricted to the target protein structures. In this study, a network-based regression model (drugCIPHER) for target profile prediction was used. The drugCIPHER method scored the likelihood of drug target interactions by integrating structural similarities of drugs and protein-protein interactions in a heterogeneous network that correlated chemical and genomic spaces (Zhao and Li, 2010). Briefly, drugCIPHER was performed to predict the target profiles of each herbal compound in our library.
To identify the herbal compounds that ameliorate MTX toxicity, we used the functional enrichment tool of the DAVID database to evaluate the enrichment of the target profile of each ingredient in MTX toxicity-related pathways with a false discovery rate less than 0.05 by the Fisher exact test (Huang et al., 2009). We only selected herbal compounds with < 0.05 after Benjamini’s correction.
2.2. Drugs and chemical reagents All tissue storage solution and reagents was purchased from Miltenyi Biotec (Shanghai, China). MTX and TMP were obtained from Sigma-Aldrich (Shanghai, China). All other reagents were the highest quality that could be obtained.
2.3. Animals and experimental design for toxicity studies Forty Wistar rats, half of which were males and half of which were females, were obtained from the Center for Laboratory Animal Care, Chinese Academy of Medical Sciences. The rats (3 months old, 200-250 g body weight) that were used for this study were allowed unrestricted access to water and food. The Animal Ethics Committee of China Academy of Chinese Medical Science approved all animal procedures. To evaluate the effect of TMP on MTX-induced toxicity, all animals were randomly divided into four groups of ten rats each. The MTX treatment group was given MTX i.p. at 20 mg/kg as a single dose on day 4 of the experiment. Normal saline (NS) at 5 ml/kg was administered i.p. as a placebo every day until the rats were sacrificed. In the TMP and MTX treatment groups, TMP at 80 mg/kg was first given i.p. every day until the rats were sacrificed and then MTX at 20 mg/kg was given i.p. on day 4 of the experiment. TMP treatment group was given TMP i.p. at 80 mg/kg every day. On day 4 of the experiment, normal saline at 1 ml/kg was injected rather than MTX. The experimental control group was given normal saline i.p. at 1 ml/kg and received no additional treatment other than standard care and housing. Rats were killed at day 8 by cervical dislocation. Blood was collected for serum GSH, MDA and cAMP measurements, and the small intestine, kidney and liver were collected and rinsed in
PBS for histology.
2.4. Serum and tissue homogenate preparation At 24 h after the last treatment, all of the rats were anesthetized and blood was collected to obtain serum by centrifugation at 3,500×g for 15 min. The ileum, liver and kidney were removed and homogenized in Tris-buffered saline (pH=7.4). The homogenates were centrifuged at 6,000×g for 15 min, and then supernatants were collected and stored at −80°C until required.
2.5. Determination of cAMP, MDA and GSH Malondialdehyde (MDA) and glutathione (GSH) levels in the liver and kidney homogenates were assayed using commercially available immune colorimetric assay kits (Beijing North Institute of Biological Technology, Beijing, China). For the MDA kit, the sensitivity was 0.02 nmol/mL, and the mean intra- and interassay coefficients of variation were 7.3% and 10.0%, respectively (manufacturer's data). For the GSH kit, the sensitivity was 0.1 μmol/L, and the mean intra- and inter assay coefficients of variation were 7.3% and 10.0%, respectively (manufacturer's data). cAMP in the serum was assayed using a commercially available radioimmunoassay (RIA) kit (Beijing North Institute of Biological Technology, Beijing, China). The sensitivity was 0.1 pmol/mL. The mean intra- and interassay coefficients of variation were 5.8% and 6.9%, respectively (manufacturer's data).
2.6. Histopathological preparation and analysis For light-microscopic investigations, tissue specimens from the ileum, liver and kidney were fixed in 10% formaldehyde and processed routinely for embedding in paraffin. Tissue sections of 5 μm were stained with hematoxylin and eosin (H&E) and examined under a Leica DM3000B (Germany) light microscope.
2.7. Adenosine receptor binding assays The potential targets of TMP were evaluated for receptor binding selectivity against adenosine receptors (A1, A2A, A2B, A3) using standard receptor binding protocols provided by Cerep, Inc.
2.8. Agonist or antagonist effect assays of adenosine receptors The agonist or antagonist effect of TMP against different adenosine receptors was evaluated in a cellular-based assay (CHO cells for A1 and A3, PC12 cells for A2A, and HEK293 cells for A2B) using standard receptor binding protocols provided by Cerep, Inc.
2.9. In vitro PDE Assays. The PDE assays were performed using the standard procedures described by Cerep, Inc.
2.10.
Statistical analysis
Data are presented as the mean ± S.D. Significant differences were assessed by one-way analysis of variance (ANOVA). P<0.05 was accepted as representing a significant difference.
3. Results 3.1 Target interaction-based computational screen identifies TMP in combination with MTX Comprehensive target profiles are used to determine the biological activity of herbal compounds by mapping them on signaling and metabolic pathways (Rix and Superti-Furga, 2009; Besnard et al., 2012). To enable the rapid development of MTX-based combination therapies for combating its toxicities, we developed a target profile mapping-aided screening strategy that can be used to identify herbal compounds that counteract MTX toxicity and cooperate with MTX to improve the
therapeutic index of RA. Because MTX-induced toxicity is mediated by direct interference with the purine metabolism pathway through the inhibition of GAR transformylase
and
5-aminoimidazole-4-carboxamide
ribonucleotide
(AICAR)
transformylase (Morgan et al., 2004; Cutolo et al., 2001; Schalinske et al., 1996) and the indirect activation of the adenosine A1 or A2 receptor (Merrill et al., 1997; Chan et al., 2006), if a given target profile of an herbal compound is significantly enriched in the gene set defined by genes involved in purine metabolism and adenosine A1 or A2 receptor signaling, the potential effects of the herbal compound may be associated with the regulation of these two pathways (Berger et al., 2010). We can thus identify herbal compounds that specifically regulate the toxicity induced by MTX. This proof-of-principle screening approach differs from other recently performed experimental screens in MTX-based combination therapies because it can, in principle, predict the herbal compounds that cooperate with MTX to improve clinical safety.
We quantified the degree of enrichment when mapping the target profiles of herbal compounds into a gene set associated with drug efficacy and toxicity. We exploited a repository of the target profiles of 8,965 natural products from 621 Chinese herbs; drugCIPHER, a network-based target prediction approach, can be used to characterize the target profile of each compound with 3,840 druggable target genes. Each target profile was represented as a list of genes ranked according to the relevance of chemical similarity and targets based on a protein-protein interaction network (Zhao and Li, 2010). For the mechanism of toxicity induced by MTX, we extracted the purine metabolism pathway (map00230) and cAMP signaling pathway (map04024) from the KEGG database as a specific gene set for MTX-induced toxicity (Kanehisa et al., 2014). For each compound, we selected the first 100 druggable proteins with high precision at the top of the target profile as a candidate target set. We then presented the p-value of enrichment analysis as an evaluation score and computed it using the DAVID. A smaller p-value indicated a higher probability that the compound would interfere with the purine metabolism pathway. We computed the p-value for each target profile of the 8,965 herbal compounds enriched in the specific gene set
(data not shown).
The above screening procedure led to the identification of TMP derived from Ligusticum wallichii Franchat (Figure 1A). TMP is known to have protective effects against thioacetamide-induced acute hepatotoxicity and has anti-inflammatory activity (Ozaki, 1992; So et al., 2002). However, the mechanisms involved are unclear. The common genes involved in TMP target profile (top 100 genes in Supplementary 1) and MTX toxicity-related pathways are described in Figure 1B. As shown in Figure 1C, we hypothesized that TMP may have protective effects against the MTX toxicity by acting on phosphodiesterases (PDEs) or the adenosine pathway because the TMP target profile suggested that TMP may act on proteins in the PDE family and adenosine receptors (A1, A2A, A2B). It is also hypothesized that TMP was expected to decrease the content of nitric oxide (NO) by inhibiting the adenosine receptors A2A and A2B as well as induce a rise in the level of cAMP by activating the A1 or inhibiting the PDEs. Therefore, we speculated that TMP may act antagonistically with MTX to influence its clinical toxicity through reducing oxidative injury. To test these hypotheses, we explored the specific effects of TMP on the toxicity and therapeutic efficacy of MTX in experimental animal model.
3.2. TMP alleviates MTX-induced oxidative stress in vivo via up-regulation of cAMP primarily by inhibiting PDE10A2 Oxidative damage contributes to the pathogenesis of MTX-induced tissue injury in rats (Miyazono et al., 2004). To experimentally test our hypothesis, we evaluated the effect of TMP on high oxidative stress levels induced by MTX. We observed that MTX treatment decreased liver GSH levels significantly (P<0.05), almost to 50% of that in the NS group, whereas 80 mg/kg TMP treatment following MTX prevented this reduction in GSH (P<0.01) (Figure 2A). Similarly, kidney GSH levels were decreased (P < 0.001) in MTX-treated rats (Figure 2B). However, treatment with TMP abolished the MTX-induced GSH reduction. Additionally, liver MDA levels were also increased significantly (P<0.01) following the administration of MTX, and TMP
treatment exhibited a trend toward reduction of the MDA levels, although the difference did not reach significance (Figure 2C). The levels of MDA in the kidney as products of lipid peroxidation were increased after treatment of rats with MTX compared to untreated controls (P<0.001) (Figure 2D). Administration of TMP reversed kidney MDA levels back to the values in rats not treated with MTX (P<0.01) (Figure 2D).
Morphological changes of the ileum, liver and kidney exposed to the drugs were examined (Figure 3A-L). The ileum and liver tissues from MTX-treated rats showed severe pathological changes compared with those in the control group. The changes in ileum tissue included intestinal villus atrophy, degenerated surface epithelium and severe inflammatory cell infiltration (Figure 3B), whereas changes in liver tissue included dissolved cytoplasm, degenerated hepatocytes, lymphocytic infiltration, and dilatation and vascular congestion in sinusoids (Figure 3F). In TMP-treated rats, no obvious pathological damage was observed in the ileum, liver and kidney tissue (Figure 3C, 3G and 3K). The morphological injuries were significantly recovered in MTX+TMP treated rats, e.g., only moderate inflammatory cell infiltration was observed in the ileum (Figure 3D) and decreased numbers of lymphocytes were observed in most regions of the liver (Figure 3H). There were no obvious morphological changes in the kidney tissue of MTX, TMP and MTX+TMP treated rats (Figure 3I, 3J, 3K and 3L).
Increased oxidative stress by MTX impaired cAMP accumulation and suppressed cAMP levels, leading to MTX-related toxicity (Kreml et al., 1979). To determine whether MTX-induced changes in cAMP levels were regulated by TMP addition, we performed RIA to measure the concentration of cAMP. Consistent with previous studies, MTX effectively reduced the serum cAMP level in rats (P<0.01). Compared with MTX-treated rats, combined TMP and MTX treatment of rats led to recovery of the cAMP balance (Figure 4A). The target profiles of TMP suggested that TMP might counteract
the
MTX-induced
decreases
in
serum
cAMP
by
inhibiting
phosphodiesterases, which leads to cAMP accumulation. To validate our predictions in vitro, we used an enzyme activity assay to determine the inhibitory effect of TMP on PDE isozymes. 100 μM TMP had almost no effect on the PDE2, PDE3, PDE4, PDE5, PDE6, PDE7, PDE8 and PDE11; however, it affected PDE10A2 (Figure 4B). Notably, TMP at 10 μM to 1 mM concentration-dependently inhibited the metabolic activity of PDE10A2 with an IC50 value of 700 μM (Figure 4C).
3.3. Competitive binding and antagonistic effect of different concentrations of TMP on adenosine A2A and A2B receptors Although adenosine/adenosine receptor signaling plays a critical role in the anti-inflammatory effects of MTX, previous studies have investigated the role of the adenosine pathway in MTX-induced toxicity (Merrill et al., 1997; Chan et al., 2006). The predicted target profile suggests a possible role for TMP in the control of the adenosine signaling pathway. To determine whether the attenuation of MTX toxicity through combination with TMP resulted from modulation of A1, A2A and A2B, we examined the biochemical interaction of TMP with adenosine receptors and the biochemical effects (antagonist or agonist) for these receptors by in vitro assays. As illustrated in Figure 5A, 100 μM TMP had no binding activity with A1 as determined by an antagonist and agonist radioligand binding assay. TMP at concentrations of 10 μM — 1 mM inhibited specific agonist radioligand binding with A2A with a Ki of 4.6×10-4 M (n=3) (IC50: 5.6×10-4 M), indicating that TMP binds to A2A competitively. Similarly, a high concentration of TMP slightly inhibited antagonist radioligand binding with A2B (Figure 5B). In two types of biochemical assays, 100 μM TMP partially inhibited the activity of A2A and A2B (Figure 5D). In addition, we observed that TMP reduced the activation of A2B in a concentration-dependent manner (Figure 5E). We did not observe any changes in the agonist activity of A2A and A2B on treatment with 100 μM TMP (Figure 5C). These results suggest that attenuation of MTX toxicity by TMP may be correlated to the antagonism of A2A and A2B.
4. Discussion & Conclusions Recently, computational approaches have greatly increased the potential of identifying more effective and better combinational therapies (Al-Lazikani et al., 2012). Although some computational approaches have promise and have shifted the landscape to a new drug discovery paradigm, they have still not yielded breakthroughs. Network-based approaches will pave the way for rationally designed and hopefully optimized combinations of multi-target therapies. Our screening approach is performed based on the targeting of functional gene sets rather than the targeting of single molecules. This strategy meets the features of mechanisms of herbal compounds.
Oxidative stress has been identified as one of the major factors responsible for MTX toxicity (Miyazono et al., 2004; Kremer, 2004). A major challenge in MTX therapy for rheumatoid arthritis is to identify combinations that prevent the emergence of toxicity. In the present study, we computationally predicted a panel of herbal compounds with the potential ability to relieve MTX-induced oxidative damage using our network pharmacology approach. Critically, we experimentally demonstrated that a herbal compound derived from ligusticum chuanxiong hort, tetramethylpyrazine, effectively rebalances MTX-induced oxidative stress as a PDE10A2 inhibitor. Furthermore, we found that TMP elicits an antagonist effect against A2A and A2B by competitively binding them, leading to direct inhibition of the activation of A2A and A2B receptors by MTX-induced adenosine signaling, thus decreasing MTX-induced toxicity. Our data, in conjunction with previous studies (Mishima et al., 2006; Wu et al., 2004), suggest that cAMP in rat blood serum is up-regulated by TMP, which contributes to alleviation of MTX-induced oxidative organ injury.
Mechanisms of MTX toxicity often involve folate metabolism, purine metabolism, adenosine deaminase activity with accumulation of adenosine and deoxyadenosine, polyamine synthesis and homocysteine metabolism (van Ede et al., 1998). Disruption of metabolic homeostasis leads to continued oxidative stress that increases the
formation of reactive oxygen species (ROS) over antioxidant protection and subsequently induces organ DNA damage, lipid peroxidation, protein modification and other toxic effects (Pravenec et al., 2013; Garca et al., 2014; Harley et al., 1997). Increased ROS levels inhibit the protective effects of cAMP, rendering normal cells more vulnerable to oxidative damage (Ling et al., 2007), whereas increasing levels of cAMP are known to attenuate NAPDH oxidase activity in normal cells, resulting in decreased oxidative stress (Saha et al., 2011; Saha et al., 2008). In addition, G-protein coupled receptors of the P1 purinergic family, i.e., the A1, A2A, A2B, and A3 adenosine receptor subtypes, are expressed in various non-inflammatory cells, tissues and organs (Cronstein, 1997). If the signaling pathways through A1 or A2A/B are functionally intact in the same tissues, a preponderance of the A1 pathway is expected because of the higher affinity of adenosine for the A1 receptor. This would cause a decrease of cAMP levels, leading to the further enhancement of MTX-induced oxidative stress (Cutolo et al., 2001). Indeed, treatment with MTX may lead to reduced cAMP levels in cultured L5178Y cells (Kreml et al., 1979). Importantly, we also found that MTX decreased cAMP levels in rat serum, which may provide an explanation for the enhancement of MTX-induced oxidative stress by reduced levels of cAMP. Phosphodiesterase (PDE) inhibitors, which are used to maintain the cAMP levels and prevent the inactivation of cAMP, can ameliorate oxidative stress and DNA damage in normal organs (Dias et al., 2014). Moreover, adenosine A2A and A2B receptor pathway stimulation by MTX-induced adenosine may potentiate nitric oxide release that plays a pivotal role in oxidative organ injury (Saura et al., 2005; Olanrewaju and Mustafa, 2000).
We performed our strategy primarily based on our previous observation that purine metabolism and adenosine receptor pathway is a major causative factor in the development of MTX-induced toxicity. Here, adenosine induced by MTX as the endogenous ligand interacts with four known receptors, A1, A2A, A2B and A3 in the adenosine receptor pathway. A1 is coupled to a Gαi/o protein, which would lead to cAMP decrease; however, A2 is coupled to a Gαs protein, which would lead to cAMP
increase. Because the affinity of adenosine binding to these receptor subtypes is different: A1>A2A>A2B, MTX administration to rats resulted in a significant decrease in the level of serum cAMP, which could reflect intracellular cAMP level in certain degree through a significant release of cellular cAMP (Brown et al., 1977). Based on computational predictions, we propose that TMP may affect MTX-induced oxidative organ injury because its target profile falls within functional gene sets defined by network neighborhood of genes potentially related to MTX-induced toxicity. Subsequently, experimental evidence indicates that TMP has an inhibitory effect on PDE10A2, A2A and A2B, whereas TMP has no effect on A1. In agreement with our hypothesis, inhibition of PDE10A2 by TMP relieves MTX toxicity and increases the cAMP level in rat serum despite substantial inhibition of A2A and A2B and no inhibition of A1. Our inference for the increase of serum cAMP in TMP-treated group was the higher expression of PDE10A2 than A1 and the lower affinity of adenosine to A2A than A1 in non-inflammatory tissues. Additional evidence that TMP can ameliorate MTX-induced oxidative injury comes from literature report in which we analyzed antagonism of A2A by TMP may decrease the reported toxicity of nitric oxide released by the adenosine A2A receptor pathway.
Ligusticum Chuanxiong, one of the most commonly used herbs in traditional Chinese medicine, is included in the herbal formula, Ba-Wei-Di-Huang Wan. TMP is one of the primary herbal compounds from Ligusticum Chuanxiong and has a broad clinical application. Many compounds from herbal medicine affect the human body in a low-affinity and multi-target manner (Kitano, 2007). High-throughput experimental screening assays are used as a type of trial and error method to explore the target proteins of herbal compounds. In contrast to such experimental methods alone, our computational platform has the capacity to predict the target profiles of herbal compounds to comprehensively determine their pharmacological activity. For instance, TMP can reduce streptomycin-induced ototoxicity by increasing the amplitudes of calcium sensitive potassium channels of outer hair cells of guinea pig cochlea (Cui et al., 2007). Interestingly, six out of the top ten hits are cation channels. Furthermore,
based on our predicted results, we also found that adenylate cyclase 1 (ADCY1) and adenylate cyclase 2 (ADCY2) were ranked 80th and 91th for druggable target profiles of TMP, respectively. Adenylyl cyclase inhibitors have been used to treat neuropathic and inflammatory pain (Wang et al., 2011). Thus, we speculated that the antinociceptive mechanisms of TMP may be associated with adenylate cyclases regulation. Our study also reveals the availability of network-based identification of pharmacological action.
Network pharmacology approaches await further improvements. Nevertheless, the analyses reported here identify a combination therapy for MTX and reveal the mechanism of action, to some extent. TMP is a potential small molecular compound from herbal medicine that may be useful for the alleviation of MTX-induced oxidative organ injury with a mechanism of action that involves potent inhibition of PDE10A2 and A2A. We believe that the network pharmacology approach serves as a framework for combination therapy development and will provide an important method of determining the mechanisms underlying the activity of herbal compounds and an important paradigm for drug discovery.
Acknowledgement This work was supported by the National Natural Science Foundation of China (Nos. 81225025, 91229201 and 81303139), the CATCM project, Interdisciplinary Research Matching Scheme (IRMS) of Hong Kong Baptist University (RC-IRMS/12-13/02) and Hong Kong Baptist University Strategic Development Fund (SDF13-1209-P01).
References Abraham, P., Kolli, V.K., Rabi, S., 2010. Melatonin attenuates methotrexate-induced oxidative stress and renal damage in rats. Cell Biochemistry and Function 28, 426-433. al Casey, S., Brewster, D., Viau, C., Acosta, D., 1995. Effect of glutathione depletion and oxidative stress on the in vitro cytotoxicity of velnacrine maleate. Toxicology Letters 76, 257-265. Ali, N., Rashid, S., Nafees, S., Hasan, S.K., Sultana, S., 2014. Beneficial effects of Chrysin against Methotrexate-induced hepatotoxicity via attenuation of oxidative stress and apoptosis. Molecular and Cellular Biochemistry 385, 215-223. Al-Lazikani, B., Banerji, U., Workman, P., 2012. Combinatorial drug therapy for cancer in the post-genomic era. Nature Biotechnology 30, 679-692. Berger, S.I., Ma'ayan, A., Iyengar, R., 2010. Systems pharmacology of arrhythmias. Science Signaling 3, ra30. Besnard, J., Ruda, G.F., Setola, V., Abecassis, K., Rodriguiz, R.M., Huang, X.P., Norval, S., Sassano, M.F., Shin, A.I., Webster, L.A., Simeons, F.R., Stojanovski, L., Prat, A., Seidah, N.G., Constam, D.B., Bickerton, G.R., Read, K.D., Wetsel, W.C., Gilbert, I.H., Roth, B.L., Hopkins, A.L., 2012. Automated design of ligands to polypharmacological profiles. Nature 492, 215-220. Bozkurt, M., Em, S., Oktayoglu, P., Turkcu, G., Yuksel, H., Sarıyıldız, M.A., Caglayan, M., Batmaz, I., Nas, K., Bozkurt, Y., Kuyumcu, M., 2014. Carvacrol prevents methotrexate-induced renal oxidative injury and renal damage in rats. Clinical & Investigative Medicine 37, E19-E25. Brown, E.M., Carroll, R.J., Aurbach, G.D., 1977. Dopaminergic stimulation of cyclic AMP accumulation and parathyroid hormone release from dispersed bovine parathyroid cells. Proceedings of the National Academy of Sciences 74, 4210-4213.
Chan, E.S., Montesinos, M.C., Fernandez, P., Desai, A., Delano, D.L., Yee, H., Reiss, A.B., Pillinger, M.H., Chen, J.F., Schwarzschild, M.A., Friedman, S.L., Cronstein, B.N., 2006. Adenosine A(2A) receptors play a role in the pathogenesis of hepatic cirrhosis. British Journal of Pharmacology 148, 1144-1155. Chang, C.J., Lin, J.F., Chang, H.H., Lee, G.A., Hung, C.F., 2013. Lutein protects against methotrexate-induced and reactive oxygen species-mediated apoptotic cell injury of IEC-6 cells. PLoS One 8, e72553. Cleeland, C.S., Allen, J.D., Roberts, S.A., Brell, J.M., Giralt, S.A., Khakoo, A.Y., Kirch, R.A., Kwitkowski, V.E., Liao, Z., Skillings, J., 2012. Reducing the toxicity of cancer therapy: recognizing needs, taking action. Nature Reviews Clinical Oncology 9, 471-478. Cronstein, B.N., 1997. The mechanism of action of methotrexate. Rheumatic Disease Clinics of North America 23, 739–755. Cronstein, B.N., 2005. Low-dose methotrexate: a mainstay in the treatment of rheumatoid arthritis. Pharmacological Reviews 57, 163-172. Cronstein, B.N., Naime, D., Ostad, E., 1993. The antiinflammatory mechanism of methotrexate. Increased adenosine release at inflamed sites diminishes leukocyte accumulation in an in vivo model of inflammation. Journal of Clinical Investigation 92, 2675-2682. Cui, G.Y., Shi, L.J., Tang, H., 2007. Reduction of Streptomycin Ototoxicity by Tetramethylpyrazine and its Ionic Channel Mechanism. Journal of Audiology and Speech Pathology 15, 129-131. Cutolo, M., Sulli, A., Pizzorni, C., Seriolo, B., Straub, R.H., 2001. Anti-inflammatory mechanisms of methotrexate in rheumatoid arthritis. Annals of the Rheumatic Diseases 60, 729-735. Dalaklioglu, S., Genc, G.E., Aksoy, N.H., Akcit, F., Gumuslu, S., 2013. Resveratrol ameliorates methotrexate-induced hepatotoxicity in rats via inhibition of lipid peroxidation. Human & Experimental Toxicology 32, 662-671.
Dias, A.T., Rodrigues, B.P., Porto, M.L., Gava, A.L., Balarini, C.M., Freitas, F.P., Palomino, Z., Casarini, D.E., Campagnaro, B.P., Pereira, T.M., Meyrelles, S.S., Vasquez, E.C., 2014. Sildenafil ameliorates oxidative stress and DNA damage in the stenotic kidneys in mice with renovascular hypertension. Journal of Translational Medicine 12, 35. Garca, M.F., Demir, H., Turan, M., Bozan, N., Kozan, A., Belli, Ş.B., Arslan, A., Cankaya, H., 2014. Assessment of adenosine deaminase (ADA) activity and oxidative stress in patients with chronic tonsillitis. European archives of oto-rhino-laryngology 271, 1797-1802. Harley, E.H., Paglia, D., Weber, B., 1997. Oxidative damage and purine metabolism: Investigation of haemolytic anaemia in the black rhinoceros. Clinical Biochemistry 30, 259. Huang da, W., Sherman, B.T., Lempicki, R.A. 2009. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nature Protocols 4, 44-57. Kanehisa, M., Goto, S., Sato, Y., Kawashima, M., Furumichi, M., Tanabe, M., 2014. Data, information, knowledge and principle: back to metabolism in KEGG. Nucleic Acids Research 42(Database issue), D199-D205. Kitano, H., 2007. A robustness-based approach to systems-oriented drug design. Nature Reviews Drug Discovery 6, 202-210. Kremer, J.M., 2004. Toward a better understanding of methotrexate. Arthritis & Rheumatology 50, 1370-1382. Kreml, J.A., Hryniuk, W.M., Yamada, E.W., 1979. Effect of methotrexate on cyclic AMP levels in cultured L5178Y cells. Biochemical Medicine 22, 43-49. Laviano, A., Molfino, A., Rossi Fanelli, F., 2012. Cancer-treatment toxicity: can nutrition help? Nature Reviews Clinical Oncology 9. Li, H., Zhao, L., Zhang, B., Jiang, Y., Wang, X., Guo, Y. Liu, H., Li, S., Tong, X., 2014. A network pharmacology approach to determine active compounds and action mechanisms of ge-gen-qin-lian decoction for treatment of type 2 diabetes. Evidence-Based Complementary and Alternative Medicine 2014, 495840.
Ling, S., Li, Z., Borschukova, O., Xiao, L., Pumpens, P., Holoshitz, J., 2007. The rheumatoid arthritis shared epitope increases cellular susceptibility to oxidative stress by antagonizing an adenosine-mediated anti-oxidative pathway. Arthritis Research & Therapy 9, R5. Li, S., Zhang, B., 2013. Traditional Chinese medicine network pharmacology: theory, methodology and application. Chinese Journal of Natural Medicines 11, 110-120. Li, S., Zhang, B., Zhang, N., 2011. Network target for screening synergistic drug combinations with application to traditional Chinese medicine. BMC Systems Biology 5 Suppl 1, S10. Lu, Y., Zhu, M., Chen, W., Yin, L., Zhu, J., Chen, N., Chen, W., 2014. Tetramethylpyrazine improves oxazolone-induced colitis by inhibiting the NF-κB pathway. Clinical & Investigative Medicine 37, E1-E9. Masutani, H., 2001. Oxidative stress and redox imbalance in acetaminophen toxicity. Pharmacogenomics Journal 1, 165-166. Merrill, J.T., Shen, C., Schreibman, D., Coffey, D., Zakharenko, O., Fisher, R., Lahita, R.G., Salmon, J., Cronstein, B.N., 1997. Adenosine A1 receptor promotion of multinucleated giant cell formation by human monocytes: a mechanism for methotrexate-induced
nodulosis
in
rheumatoid
arthritis.
Arthritis
&
Rheumatology 40, 1308-1315. Mishima, K., Baba, A., Matsuo, M., Itoh, Y., Oishi, R., 2006. Protective effect of cyclic AMP against cisplatin-induced nephrotoxicity. Free Radical Biology & Medicine 40, 1564-1577. Miyazono, Y., Gao, F., Horie, T., 2004. Oxidative stress contributes to methotrexate-induced small intestinal toxicity in rats. Scandinavian Journal of Gastroenterology 39, 1119-1127. Morgan, S.L., Baggott, J.E., Vaughn, W.H., Young, P.K., Austin, J.V., Krumdieck, C.L. Alarcón, G.S., 1990. The effect of folic acid supplementation on the toxicity of low-dose methotrexate in patients with rheumatoid arthritis. Arthritis & Rheumatology 33, 9-18.
Morgan, S.L., Oster, R.A., Lee, J.Y., Alarcón, G.S., Baggott, J.E., 2004. The effect of folic
acid
and
folinic
acid
supplements
on
purine
metabolism
in
methotrexate-treated rheumatoid arthritis. Arthritis & Rheumatology 50, 3104-3111. Morsy, M.A., Ibrahim, S.A., Amin, E.F., Kamel, M.Y., Rifaai, R.A., Hassan, M.K., 2013. Curcumin ameliorates methotrexate-induced nephrotoxicity in rats. Advances in Pharmacological Sciences 2013, 387071. Olanrewaju, H.A., Mustafa, S.J., 2000. Adenosine A(2A) and A(2B) receptors mediated nitric oxide production in coronary artery endothelial cells. General Pharmacology 35, 171-177. Ozaki, Y., 1992. Antiinflammatory effect of tetramethylpyrazine and ferulic acid. Chemical & Pharmaceutical Bulletin (Tokyo) 40, 954-956. Pravenec, M., Kozich, V., Krijt, J., Sokolová, J., Zídek, V., Landa, V., Simáková, M., Mlejnek, P., Silhavy, J., Oliyarnyk, O., Kazdová, L., Kurtz, T.W., 2013. Folate deficiency is associated with oxidative stress, increased blood pressure, and insulin resistance in spontaneously hypertensive rats. American Journal of Hypertension 26, 135-140. Rath, T., Rubbert, A., 2010. Drug combinations with methotrexate to treat rheumatoid arthritis. Clinical and Experimental Rheumatology 28, S52-S57. Relling, M.V., Fairclough, D., Ayers, D., Crom, W.R., Rodman, J.H., Pui, C.H., Evans, W.E., 1994. Patient characteristics associated with high-risk methotrexate concentrations and toxicity. Journal of Clinical Oncology 12, 1667–1672. Rix, U., Superti-Furga, G., 2009. Target profiling of small molecules by chemical proteomics. Nature Chemical Biology 5, 616-624. Tabassum, H., Parvez, S., Pasha, S.T., Banerjee, B.D., Raisuddin, S., 2010. Protective effect of lipoic acid against methotrexate-induced oxidative stress in liver mitochondria. Food and Chemical Toxicology 48, 1973-1979.
Saha, S., Jia, Z.Q., Liu, D.M., Misra, H.P., 2011. The Roles of cAMP and G Protein Signaling in Oxidative Stress-Induced Cardiovascular Dysfunction. Studies on Experimental Models, Oxidative Stress in Applied Basic Research and Clinical Practice. Humana Press, 621-635. Saha, S., Li, Y., Anand-Srivastava, M.B., 2008. Reduced levels of cyclic AMP contribute to the enhanced oxidative stress in vascular smooth muscle cells from spontaneously hypertensive rats. Canadian Journal of Physiology and Pharmacology 86, 190-198. Saura, J., Angulo, E., Ejarque, A., Casadó, V., Tusell, J.M., Moratalla, R., Chen, J.F., Schwarzschild, M.A., Lluis, C., Franco, R., Serratosa, J., 2005. Adenosine A2A receptor stimulation potentiates nitric oxide release by activated microglia. Journal of Neurochemistry 95, 919-929. Schalinske, K.L., Steele, R.D., 1996. Methotrexate alters carbon flow through the hepatic folate-dependent one-carbon pool in rats. Carcinogenesis 17, 1695-1700. So, E.C., Wong, K.L., Huang, T.C., Tasi, S.C., Liu, C.F., 2002. Tetramethylpyrazine protects mice against thioacetamide-induced acute hepatotoxicity. Journal of Biomedical Science 9, 410-414. Tugwell, P., Pincus, T., Yocum, D., Stein, M., Gluck, O., Kraag, G., McKendry, R., Tesser, J., Baker, P., Wells, G., 1995. Combination therapy with cyclosporine and methotrexate in severe rheumatoid arthritis. The Methotrexate-Cyclosporine Combination Study Group. The New England Journal of Medicine 333, 137-141. van Ede, A.E., Laan, R.F., Blom, H.J., De Abreu, R.A., van de Putte, L.B., 1998. Methotrexate in rheumatoid arthritis: an update with focus on mechanisms involved in toxicity. Seminars in Arthritis and Rheumatism 27, 277-292. Wang, H., Xu, H., Wu, L.J., Kim, S.S., Chen, T., Koga, K., Descalzi, G., Gong, B., Vadakkan, K.I., Zhang, X., Kaang, B.K., Zhuo, M., 2011. Identification of an adenylyl cyclase inhibitor for treating neuropathic and inflammatory pain. Science Translational Medicine 3, 65ra3.
Wu, Y., Zheng, J., Linden, J., Holoshitz, J., 2004. Genoprotective pathways. Part I. Extracellular signaling through G(s) protein-coupled adenosine receptors prevents oxidative DNA damage. Mutation Research 546, 93-102. Zhao, S., Li, S., 2010. Network-based relating pharmacological and genomic spaces for drug target identification. PLoS One 5: e11764.
Figure legend Figure 1. Network target of TMP and possible molecular mechanisms involved in reducing MTX toxicity using TMP. A. Chemical structure of TMP. B. Functional sub-network perturbed by TMP in the context of biological mechanisms of MTX-induced toxicity. In the protein-protein interaction (edges) network, the target profile of TMP (blue nodes) perturbs the biological process (triangular nodes represent proteins in the adenosine receptor pathway; rectangle nodes represent proteins in purine metabolism) involved in MTX-induced oxidative organ injury. C. Models for a hypothetical mechanism that can explain the protective effect of TMP against MTX toxicity. The genes with a red symbol are involved in the predicted target profile of TMP. The dashed lines indicate that TMP could either increase or inhibit the activity of these target proteins.
Figure 2. Administration of 80 mg/kg TMP partly restores normal levels of GSH and MDA in the kidney and liver after 20 mg/kg MTX exposure. A and B. GSH levels in the liver and kidney of NS, MTX-treated, TMP-treated, and MTX+TMP-treated groups. C and D. MDA levels in the liver and kidney of NS, MTX-treated, TMP-treated, and MTX+TMP-treated groups. * P<0.05, ** P<0.01, *** P<0.001.
Figure 3. Morphological changes of ileum, liver and kidney tissues in TMP, MTX and MTX+TMP treated rats. H & E staining of formalin-fixed, paraffin-embedded sections. A, E and I: Normal morphology of the ileum (A), liver (E) and kidney (I) in control rats. B, F and J: Intestinal villus atrophy, degenerated surface epithelium and severe inflammatory cell infiltration in the ileum (B); dissolved cytoplasm, degenerated hepatocytes, lymphocytic infiltration, and dilatation and vascular congestion in sinusoids of liver (F); and no pathological damages in kidney (J) in MTX-treated group. C, G and K: No obvious pathological damage in the ileum (C), liver (G) and kidney (J) in TMP-treated groups. D, H and L: Milder histopathological changes observed in MTX+TMP treated rats compared to MTX-treated rats (B), only moderate inflammatory cell infiltration in the ileum (D) compared to the MTX-treated rats (F), and decreased numbers of lymphocytes in most regions of the liver (H). No pathological damage was observed in the kidney (L) in MTX+TMP treated rats. Original magnification: A, B, C and D, ×100; E, F, G, H, I, J, K and L, ×200.
Figure 4. TMP-mediated reversal of the reduction of serum cAMP levels caused by MTX may be associated with antagonism of the activity of PDE10A2. A. Serum radioimmunoassay showed that TMP increased the content of serum cAMP and relieved the reduction of serum cAMP caused by MTX. B. Enzyme assays and in vitro pharmacology confirmed that exposure to 100 μM TMP partly inhibited the activity of PDE10A2. C. The enzyme assay of PDE10A2 further demonstrated that TMP repressed PDE10A2 activity in a concentration-dependent manner.
Figure 5. TMP can competitively bind to A2A and A2B as an inhibitor of the adenosine A2 receptor. A. Binding assays indicated that TMP does not bind to A1. B. Binding assays demonstrated binding of TMP to A2A (IC50=5.6E-04 M) and A2B (1.2E-03 M) in a concentration-dependent manner. Cellular receptor functional assays further evaluated the agonist or antagonist effect of TMP on A2A and A2B. C. TMP at 100 μM had no agonist effect on A2A and A2B. D. TMP at 100 μM had an antagonist effect on A2A and A2B. E. Cellular receptor functional assays confirmed that
different concentrations of TMP had antagonist effects on A2A (IC50: 0.0005132 M) and A2B (IC50: 0.001715 M) in a concentration-dependent manner.
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*Graphical Abstract-revised