Abstract
Ischemic attack is the third leading cause of exit in the world. Our previous study originate that cynandione A (CYNA), the absolute component from the root of Cynanchum bungei, exhibits anti-ischemic rub gently activity. In this work, we investigated the curative mechanisms of CYNA to ischemic pat at protein network level. First, PC12 cells and cerebellar small particle neurons were prepared to validate the personal estate of CYNA against glutamate injury. Our experiments suggested that CYNA could disagreeable lot-dependently mitigate glutamate-induced neurons neurotoxicity and obstruct glutamate-induced upregulation of KHSRP and HMGB1, farther on confirming the neuroprotective effects of CYNA in vivo. Then, on the pathway sub-networks, which existing biological processes that can be impacted absolutely or in periphery nodes by drugs via their targets, we found that CYNA regulates 11 pathways associated with the biological process of thrombotic or embolic occlusion of a cerebral artery. Meanwhile, ~ the agency of defining a network-based anti-ischemic rap effect score, we showed that CYNA has a significantly higher fact score than random counterparts, which suggests a synergistic event of CYNA to ischemic stroke. This study may put off new lights on the study of netting based pharmacology.
Citation: Fang H, Yue R, Ga Y, Zhang Y, Shan L, Zhao J (2015) Analysis of Cynandione A’s Anti-Ischemic Stroke Effects from Pathways and Protein-Protein Interactome. PLoS ONE 10(5): e0124632. doi:10.1371/magazine.pone.0124632
Academic Editor: Giuseppe Pignataro, Federico II University of Naples, ITALY
Received: November 1, 2014; Accepted: March 16, 2015; Published: May 8, 2015
Copyright: © 2015 Fang et al. This is an open access article distributed under the stipulations of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the pristine author and source are credited
Data Availability: All fit data are within the paper and its Supporting Information files.
Funding: This examination was supported by the National Natural Science Foundation of China (61372194, 81260672, and 81230090) and FP7-PEOPLE-IRSES-2008 (TCMCANCER Project 230232).
Competing interests: The authors be the subject of declared that no competing interests continue.
Introduction
Stroke, also known as cerebrovascular affront (CVI) and cerebrovascular accident (CVA), is the brain malfunction resulted from deficient blood supply to the brain [1]. This malfunction is brought near by either ischemia or hemorrhage. Ischemic stroke, featuring the abrupt interrupt of life-~ transportation to an portion of the brain, leads to the neurologic malfunction, which can cause permanent neurological damage or end of life and earns itself the title of the third killer of health worldwide in 2010 [2,3]. Ischemic shock is caused by thrombotic or embolic occlusion of a cerebral artery [4, 5].
Currently, the important treatment for ischemic stroke could be summarized to three categories: mild hypothermia therapy [6, 7], thrombolysis [8–10] and automatic thrombectomy [11, 12]. Thrombolysis is the and nothing else approbatory therapy for acute ischemic knock in North America [13]. Nonetheless, it excepting that takes effects in 3 hours. Beyond this yoke of animals cerebral hemorrhage and edema are besides risky. Although numerous potential treatment strategies bear been investigated, most of them regard been proven inefficacious in humans in a spirited trial design [14, 15]. Therefore, there is an urgent demand to inquire into new or alternative anti-ischemic sudden effort agents.
Cynanchum bungei is a shape of Polygonum multiflorum. As the Apocynaceae Cynanchum plants, it is widely distributed in China. CYNA, every acetophenone molecule, is a main ingredient from the root of Cynanchum bungei and other class. Fig 1 shows its chemical mode of building. Earlier studies have demonstrated that CYNA possesses neuroprotective and hepatoprotectives goods [16, 17].
Fig 1. The chemical form of CYNA.
doi:10.1371/periodical.pone.0124632.g001
A series of empirical studies by one of our laboratories be under the necessity been conducted on CYNA’s movables on ischemic stroke in rats. Evidence has shown that CYNA manifests itself taken in the character of effective antioxidant vigor and behaves neuroprotectively the couple in vitro and in a rat imitation of transient focal cerebral ischemia with diminished neurological scarcity scores and infarct largeness [18]. These phenomena suggested that CYNA be further exploited as a therapy to fortify nerves in ischemic stroke treatment. On the other pass by ~, it has been realized that network based analysis is suitable for unanimity the action mode of natural compounds that exhibit low affinity inhibition on multiple targets [19–21].
In this ~ hangings, we studied the anti-ischemic knock effects of CYNA from two aspects, pathways and PPI networks. We in the beginning collected genes associated with ischemic hit and putative targets of CYNA. Then, in standing rule to identify pathways significantly regulated ~ dint of. CYNA, we constructed pathway sub-networks and scored the press close together of CYNA on these sub-networks. At in conclusion, the network based anti-ischemic shock effect score was defined based up~ the body the random walk with restart to quantitatively decompose the anti-ischemic stroke effect of CYNA. Experiments were in like manner carried out in PC12 cells and cerebellar little grain neurons to confirm the neuroprotective movables of CYNA in vivo and validate brace targets of CYNA identified by our judged by comparison proteomic experiment.
Materials and Methods
Experimental tests and validation
Reagents.
Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY). Antibodies in spite of KHSRP (#13398), HMGB1 (#6893), and β-actin were obtained from Cell Signaling Technology (Beverly, MA).
Cells cultures and medicine treatments.
The high differentiated rat pheochromocytoma tumefaction cell line PC12 (Cell Bank of the Shanghai Institute of Biochemistry & Cell Biology, Shanghai Institute in the place of Biological Sciences, Chinese Academy of Sciences, Shanghai, China, http://www.cellbank.org.cn) was maintained in DMEM containing 10% violence-inactivated equine serum and 5% FBS supplemented with 100 U/mL penicillin and 100 μg/mL streptomycin in a humidified atmosphere containing 5% CO2 at 37°C. For neuronal cultures, original cultures of mouse (C57BL/6) cerebellar small particle neurons were prepared from 6- to 8-daytime -old postnatal mice (Shanghai SLAC Lab. Animal Co., Ltd.). Briefly, cerebella were dissected later decapitation and cleaned free of meninges, and soon afterward treated with 0.025% w/v trypsin liquefaction for 30 min at 37°C. A trypsin inhibitor was therefore added to block the enzyme, and 0.05% w/v DNase was added to shatter DNAs from dead cells. After a concatenation of trituration and mild centrifugation steps, the cells were plating in DMEM and 10% FBS supplemented through KCI to a final concentration of 30 mM. Cells were plated onto 24-well dishes containing poly-L-lysine-coated coverslips at a closeness of 6 × 105 per well as antidote to 7 days. CYNA was dissolved in dimethyl sulfoxide (DMSO) (Sigma. St. Louis, MO) and were freshly prepared cropped land time before use (DMSO final civilization concentration < 0.1%). For neuroprotection analysis, cells were treated with 1, 10 and 100 μM CYNA since 6 h before exposure to 5 mM glutamate in opposition to PC12 cells or 100 μM glutamate on the side of cerebellar granule neurons and then maintained despite 24 h. At the end of the management period, protein lysates were prepared and toward the west blot analysis was performed.
The study protocol was approved ~ dint of. the local institutional review board at the authors’ affiliated institutions and denizen of the deep experiments were carried out in conformation with the established institutional guidelines because animal care and use at the Second Military Medical University.
Cell viability assays.
Cell viability was uniform by the 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-Disulfophen-yl)-2H-tetrazoliummonosodium taste method using Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) according to the manufacturer’s instructions. Absorbance was know fully at 450 nm by a BioTek Synergy 2 silverware reader (BioTek Instruments, Inc., Winooski, Vt, USA).
Western blotting.
Cells were collected ~ the agency of centrifugation at 1000 g for 3 min. The small room pellets were then washed with PBS, resuspended in lysis guard containing 150 mM NaCl, 50 mM Tris (pH 8.0), 0.02% NaN3, 0.01% PMSF, 0.2% aprotinin, 1% tritonX-100 supplemented by protease inhibitor cocktail (Thermo Scientific), and centrifuged at 12,000 g as antidote to 10 min. The concentration of undivided proteins was determined by a BCA small pail (Pierce, Rockford, IL). 30 μg proteins by lane was electrophoresed on 10% SDS polyacrylamide gels rear boiling for 5 min and transferred to nitrocellulose membranes. Nonspecific reactivity was blocked ~ means of 5% nonfat milk prepared in TBST (10 mM Tris, 150 mM NaCl, 0.05% Tween-20, pH 7.5) at space temperature for 1 h. The membranes were incubated by antibodies diluted according to the manufacturers’ instructions. Blots were washed three times in TBST, followed by incubation by the appropriate horseradish peroxidase-linked delegate antibodies for 1 h at opportunity temperature. The proteins in the blots were visualized using the ECL more system (Amersham Pharmacia Biotech, Buckinghamshire UK).
Statistical Analysis.
Data were analyzed using GraphPad Prism software version 5 (Graph Pad software Inc., San Diego, CA, USA). Multiple comparisons were compared ~ means of one-way ANOVA analysis of change of condition followed by Tukey post hoc exhibition. The quantitative data were reported of the same kind with means ± SEM from at in the smallest degree three independent experiments. Statistical significance was determined being of the kind which P < 0.05.
Data combination
Ischemic stroke associated genes.
The ischemic stroke associated genes were collected from databases OMIM (The Online Mendelian Inheritance in Man database) and GAD (Genetic Association Database).
OMIM[22] is a database concerning human gene and genetic disarrays. It classifies altogether the known diseases with a genetic composing and connects them to the interrelated genes in the human genome, through text information and reference information, series records, human genome and other premises contained. With the keyword “Ischemic stroke”, we searched the OMIM database and place 5 associated genes, ALOX5AP, F2, F5, NOS3 and PRKCH.
GAD[23] is a database of human genetic combination researches of complicated diseases. It embraces temporary data distilled from published papers in equal reviewed journals on candidate gene and GWAS researches. With the similar keyword, we searched the GAD and ground 60 genes whose association with ischemic dash was not “N”.
Based up~ the body the above two databases, 61 separate ischemic stroke associated genes were obtained. Among them, four genes in the OMIM database are moreover contained in the GAD, which are ALOX5AP, F2, F5 and NOS3. The appointment to special service information could be seen in S1 Table.
Putative targets because of CYNA.
We obtained 17 putative targets of CYNA using couple methods as follows.
Comparative proteomic analytics: In our earlier study, we carried fully comparative proteomic analysis by matrix-assisted laser desorption/ionization-time of volley (MALDI-TOF) MS/MS of the pheochromocytoma tumor cell line PC12 cells treated with CYNA[18].
Here we briefly represent the process of the matrix-assisted laser desorption/ ionization-time of volitation (MALDI-TOF) MS/MS. Each specimen was suspended in 0.7 μL matrix disjunction containing a-cyano-4-hydroxycinnamic tart in acetonitrile/water (1:1, v/v) acidified through 0.1% trifluoroacetic acid. Then the salmagundi was immediately spotted onto the MALDI target. Analyses were performed on a 4700 Proteomics Analyzer equipped with a 355 nm Nd:YAG laser. The proteins were identified by peptide mass fingerprinting and tandem mass spectrometry using the program MASCOT v. 1.9 (Matrix Science, London, UK) facing the SWISS-PROT database with the GPS explorer software (Applied Biosystems). MASCOT protein scores (based ~ward combined MS and MS/MS spectra) of greater than 64 were considered statistically weighty (P < 0.05).
The exact trial identified 11 differentially expressed proteins in PC12 cells caused by the treatment of 10μM CYNA, in that only one protein (heterogeneous nuclear ribonucleoprotein H1HNRNPH1) was up-regulated. We considered this protein for the re~on that a side-effect target and excluded it from our study. We mapped the 10 etc.-regulated rat proteins to human genome ~ means of homologous analysis and got 10 putative protein targets of CYNA. See Table 1 because detail information.
Table 1. The CYNA’s targets obtained from able to compare proteomic analysis, where pt and pt, comp exhibit a rough estimate of the substance of pulled-down protein before and later treatment by CYNA, respectively.
doi:10.1371/journal.pone.0124632.t001
Similarity search method: We used chemical similarity search tool to cloak similar drugs of CYNA through the structural analogy comparison with the condition that the homogeneous score is higher than 0.8 from TTD[24] (curative target database). Totally, we obtained 2 homogeneous drugs and 8 target proteins. See Table 2 with regard to detail.
Table 2. CYNA’s targets obtained from likeness search method.
doi:10.1371/journal.pone.0124632.t002
Protein-Protein Interaction Data.
Protein-protein interactions betwixt human proteins were downloaded from the translation 9.05 of STRING[25]. STRING includes material and operational interplays congregated from large sources, including experimental archives, computational foresee algorithms, and public text collections. An evaluation system is used to weigh the make clear of each interaction. The interaction scores were normalized to the space of time [0, 1]. It contains 16886 nodes and 1520927 edges.
Data of way gene sets and construction of footway sub-networks.
The pathway gene sets were downloaded from the C2: CP pile of MSigDB[26] database which were curated from several online pathway databases including bioCarta[27], KEGG[28], reactome[29] and for a like rea~n on. A total of 4722 pathways were included in this collection.
Then for each pathway gene solidify, we mapped its genes to the human PPI reticulated and extracted the sub-network including wholly the genes and their interactions. In this course, we obtained all the pathway sub-networks because of the CP collection of MSigDB database. We be able to see that a pathway sub-network is a connected fraction of the human protein-protein interaction netting, in which all the genes discharge the same cell function
Scoring the impression of CYNA on the pathway sub-network
Recent study found that a way sub-network can be impacted ~ dint of. drug’s targets through the following pair ways [30]:
A node of the footway sub-network is acted on ~ means of a drug directly.
A periphery swelling of the pathway sub-network, what one. interacts with the pathway sub-reticulated, is acted on. This case also should be included in our decomposition.
We apply the score s to regard how strong a pathway sub-network is affected by CYNA. The s-score is defined by the combination of various features of the pathway sub-network as follows [30]: (1) Where nnet denotes the calculate of genes on the pathway sub-reticulated, ndis,net denotes the number of ischemic rub gently associated genes on the pathway sub-network. Hence represents the ratio of ischemic touch associated genes to the total bulk of the affected pathway sub-network, i.e., how frequently genes of this indisposition are present in the sub-netting. Similarly, ntar,net is the tell off of CYNA’s targets on the course of life sub-network and its periphery nodes, though puts the impact of CYNA in reference to the size of the sub-reticulated. Besides the number of the target on the pathway sub-network, the artificial strength of CYNA to ischemic stroke, i.e., binding affinity also should exist considered [31]. The affinity measure is derived from chemical proteomics data directly and obtained by the following equation: (2) Where pt and pt,comp delineate a rough estimate of the result of pulled-down protein before and subsequently treatment by CYNA, respectively. Since in chemical proteomics the unsalable article is always presented at a broad excess of constant concentration, ln(pt) is used to in a descending course weigh parameter influence. Therefore, and typify the sum of affinities for the CYNA’s targets and targets up~ the body the pathway sub-network and its surface nodes, respectively. Hence, the last turn of expression could be interpreted as the rate of CYNA’s affinities on the track sub-network to overall affinity of CYNA’s targets used from comparative proteomic analysis.
Network scoring anti-ischemic hit of CYNA
Scoring network effect of a group of seed nodes.
In order to be established CYNA’s effect to all the genes in successi~ the PPI network, we applied the algorithm of chance walk with restart, which is used in various areas, such as identifying of functional modules, modeling the descent by continuous differentiation of social networks and so up~ [32,33]. The algorithm can number all the nodes’ score of the network based on a group of sperm nodes. In this paper, we used a weighted PPI netting as the network and ischemic thump associated genes or protein targets of CYNA viewed like the seed nodes.
The algorithm could be described as follows: A seed nodosity is chosen from the seed stake S before the random walk starting.
At harvested land step, the random walker either moves to a chosen neighbor u∊N of the current knot v, randomly, or it restarts at individual of the nodes in the matured ovule set S. The probability of restarting at a given time step is a fixed parameter, what one. is denoted by r. For each restart, the probability of restarting at v∊S suggests the class of association between v and the embryo set S. For each move, the presumption of moving to interacting partner u of the current process v is proportional to the reliability of the interaction between u and v. This step could be represented as follows: (3) where P is the adjacency matrix of the weighted PPI netting, representing the coupling strength of nodes in the reticulated; r ∊ [o,1] is a parameter denoting the restart likelihood which needs to be calibrated through real data; xt is a vector in which xt(v) denotes the probability that the prominence will be at node v at time t; x0 is a vector representing the energy of seed nodes. After a sufficiently for a ~ time time, the probability of being at nodosity v at a random time step provides a mete of the functional association between v and the genes in grain set S, hence, the effect force of seed set S to every one nodes in the network is defined ~ means of steady-state probability vector x∞ while xt+1 = xt.
Scoring ischemic stroke’s power on the human PPI network.
Taking ischemic stroke associated genes as the seed nodes. Although it be able to be assumed that the initial vigor values x0(v) of different kernel nodes are different as the associated interval of different ischemic stroke genes to ischemic rap is varying, for simplicity, all ischemic knock associated genes are treated equally in this algorithm, and beginning vector x0 could thus be defined while x0(v) = 1 if v is a matured ovule otherwise x0(v) = 0.
Then ischemic hardship effect score of each node in the human network was computed by random walk with restart and an ischemic stroke’s import vector xis was obtained.
Scoring CYNA’s weight on the human PPI network.
In this instance, CYNA’s effect on the human PPI netting is studied. The seed nodes are defined considered in the state of CYNA’s targets. Similarly, the effected fortitude of the CYNA’s targets to the ischemic dash is set as the initial brilliance values x0(v) of seed nodes. The affinities of CYNA’s targets obtained from judged by comparison proteomic experiment are known, and could have ~ing used to define initial strength worth of a seed node [34].
For CYNA, its drift score on each node in the human network was computed by random walk by restart and its drug effect vector xca was obtained.
Scoring the anti-ischemic calamity effects of CYNA.
The inner effect between the vectors of disease force and CYNA effect was applied to allotment how CYNA impacts the human interactome in the influence of ischemic stroke [34]. In this notes, E = < xis,xca >is defined like the anti- ischemic stroke effect charge of CYNA. The effect score of CYNA was therefore compared with that of its fortuitous contracts by Z-score.
Results and Discussion
CYNA protected in requital for glutamate-induced neurotoxicity in PC12 cells and cerebellar small particle neurons
We first validated whether CYNA could countenance against oxidative glutamate cytotoxicity in PC12 cells and cerebellar granule neurons. As shown in Fig 2A, CYNA could drench-dependently mitigate 5 mM glutamate-induced neurotoxicity from 10 to 100 μM in PC12 cells. Similar tools and materials were also obtained in cerebellar little grain neurons exposed to 100 μM glutamate from 10 to 100 μM (Fig 2B).
Fig 2. CYNA protected over ~ glutamate-induced neurotoxicity in PC12 cells and cerebellar granule neurons.
(A) Effects of CYNA without ceasing 5 mM glutamate-induced PC12 small cavity injury. (B) Effects of CYNA adhering 100 μM glutamate-induced cerebellar little grain neurons. Cells were treated with 1, 10, and 100 μM CYNA and at that time co-incubated with or without glutamate in quest of 24 h, and cell viability was determined ~ the agency of CCK8 assay. Data are presented as mean ± SEM (n = 6). ** P < 0.01 against glutamate by one-way ANOVA analytics of variance with Tukey’s HSD announce hoc test. (C) Proteins extracted from glutamate-treated cerebellar small particle neurons either with or without pre-incubation by CYNA were subjected to western blotting to descry changes in KHSRP and HMGB1 statement. 10 μM CYNA prevented the upregulation of KHSRP and HMGB1 in relation to 100 μM glutamate-treated. (D) Data shown are the results of three different experiments and are represented as the relative pronoun densities of protein bands normalized to β-actin. Results are presented to the degree that means ± SEM of three assays. ** Significant dissimilitude compared with glutamate by one-device ANOVA analysis of variance with Tukey’s HSD put in the ledger hoc test (P < 0.01).
doi:10.1371/diary.pone.0124632.g002
Then, we verified CYNA’s impulse on KHSRP and HMGB1, two targets of CYNA identified by our earlier comparative proteomic analysis (pay attention Table 1) whose antibodies are commercially serviceable. Proteins extracted from glutamate-treated cerebellar little grain neurons either with or without pre-incubation through CYNA were subjected to western blotting study to study the changes of KHSRP and HMGB1’s mode of speech (Fig 2C). 10 μM CYNA significantly prevented the upregulation of KHSRP and HMGB1 rear 100 μM glutamate-treated (Fig 2D).
Together, these results confirmed that CYNA protected in requital for glutamate-induced neurotoxicity in PC12 cells and cerebellar granule neurons and inhibited glutamate-induced upregulation of KHSRP and HMGB1.
The contact of CYNA on the pathway sub-networks
We further extracted data related to ischemic stroke’s pathogenesis and treatment—ischemic attack-associated genes and CYNA’s targets to make evident its anti-ischemic stroke effect in successi~ the pathway sub-networks. For both pathway sub-network, ischemic stroke associated genes and CYNA mark genes on this pathway and its superficies were obtained. And the target side face is weighted with respect to its relationship . In order to remove the pack close of network itself, we only deliberate the pathway sub-networks whose figure of ischemic stroke associated gene is again than 3 and the number of CYNA’s mark genes is more than 2. A full of 182 pathway sub-networks satisfied these criteria. Then, whole affected pathway sub-networks are scored using Eq (1), See S2 Table on this account that detail. Finally, 25 sub-networks were obtained whose s-scores are higher than 2 cot average value (average value is 0.0049). See Table 3 on account of the detail.
Table 3. Pathways whose sub-reticulated score s is higher than 0.0098, whither GN represents sub-network gene number, DN and TN represent disease gene digit and target gene number on the sub-reticulated, respectively.
doi:10.1371/journal.pone.0124632.t003
As shown in Table 3, there are 25 pathways whose scores are further significant than the others. Among them, the cut of pathways P130CAS LINKAGE TO MAPK SIGNALING FOR INTEGRINS and GRB2 SOS PROVIDES LINKAGE TO MAPK SIGNALING FOR INTERGRINS level reach 0.0539.
It is establish that 11 of the 25 pathways are associated by the biological process of thrombotic or embolic occlusion of a cerebral artery, that is the main cause of ischemic reverse. Specifically, the pathways P130CAS LINKAGE TO MAPK SIGNALING FOR INTEGRINS and GRB2 SOS PROVIDES LINKAGE TO MAPK SIGNALING FOR INTERGRINS act the same role, in which the platelets were stimuli activated by bioactive molecules such as thrombin, ADP, collagen, fibrinogen and thrombospondin, and the activated platelet integrin alphaIIbbeta3 interacts with the fibrinogen and links platelets simultaneously in an aggregate to form a platelet quid [35–40]. In the process of FIBRINOLYSIS PATHWAY, overabundance or increased activity of the plamsminogen activator inhibitors or reduced appearance or function of tissue-type plasminogen activator (tPA) or urokinase plasminogen activator (uPA) can cause an increase in fibrin removal or the formation of a thrombus, which further result in atherosclerotic disease and venose thrombosis [41,42]. As for the way of PLATELET ADHESION TO EXPOSED COLLAGEN, below acute vascular trauma, vasoconstrictor mechanisms prevail and the endothelium becomes prothrombotic, procoagulatory and proinflammatory in intelligent being. The chief trigger for the make different in endothelial function that leads to the making up of a haemostatic thrombus is the failure to win of the endothelial cell barrier between blood and extracellular matrix components [43,44]. The course of life of INTEGRIN ALPHAIIB BETA3 SIGNALING causes the instruction of platelet adhesion which could end in the platelet plug [45,46]. The other 6 pathways are too associated with platelet plug [47–52]. These results indicate that CYNA performs anti-ischemic blow effect by regulating several biological processes associated with ischemic stroke.
Fig 3 shows 8 of the 11 ischemic blow associated pathway sub-networks. It be possible to be seen that disease genes VWF, F2, ITGB3, FGA and FGB open to the view on most sub-networks, suggesting their influential roles in ischemic stroke. Although the CYNA’s targets strike few nodes on these sub-networks, their outside nodes contain most of CYNA’ targets, implying that the targets act forward the pathways through interactions on PPI netting.
Fig 3. Eight ischemic stroke associated track sub-networks regulated by CYNA, in what place red circles represent ischemic stroke associated genes, green circles represent CYNA’s targets, black and white circles represent other genes on the footway.
doi:10.1371/journal.pone.0124632.g003
On the other artisan, other pathways activated or inhibited through CYNA are not directly correlated by ischemic stroke pathogenesis. Some of them accept indirect relationship with the disease pathogenesis. Taken IL5 PATHWAY during the time that an example, IL-5 is an inflammatory signaling molecule that primarily stimulates eosinophil proliferation, maturation and activation [53]. Secreted IL-5 stimulates productions that migrate to tissues in replication to eotaxin and release factors that detriment tissues, causing some of the inadvisable consequences of inflammation. Once inflammation infects some cerebral artery, it may cause thrombotic or embolic occlusion. Another prototype is INTEGRIN2 PATHWAY [54]. Integrins are confined apartment surface receptors that interact with the extracellular matrix and middle intracellular signals in response to the extracellular matrix including cellular shape, mobility, and progression through the simplest organism cycle. Growth factor signaling pathways and the caveolin receptor set forth important cross talk with integrin receptors in alveolate responses like activation of map kinase, proliferation and motility. If some disorders occur in the process of INTEGRIN2 PATHWAY, family vessel cell proliferation and motility may subsist destroyed and lead to thrombotic or embolic occlusion. Hence these pathways may give new suggestions to identify other targets in rub gently.
Anti-ischemic stroke effects of CYNA ~ dint of. network scores
Calculating the optimal r for the CYNA.
The algorithm of casual walk with restart has been luckily used in the prioritization of candidate disease genes and r = 0.3 appeared to have existence a robust choice [55]. Thus we took r = 0.3 to charge ischemic stroke’s effect on the human PPI netting in this study. Since r = 0.3 was got ~ the agency of fitting real data of disease genes, it may not subsist optimum for estimating the impact of the trivial molecule CYNA on the network. Therefore, we tried to light upon the optimal r value by the following action:
Defining targets obtained from comparative proteomic test as seed nodes, and then defining targets obtained from similarity search method as test set P;
Taking r = 0 and far-sighted the score for the anti-ischemic sudden effect effects of CYNA on the human PPI network by Eq (3);
Descendingly ranking the genes according their scores;
Calculating the medium ranking score RS of genes in the ground of admission set P;
Seting r = r+0.05, and using the too magnanimous for procedure to obtain the corresponding RS utility;
Continuing to implement the above proceeding, we use different r values to subsist the corresponding RS values. Fig 4 shows the kinship between r and RS. It can be see that the curve of the correlation betwixt r value and RS value is a notching bend.. RS value decreases first and in that case increases. It reaches minimum when r = 0.1. Therefore, the optimal r regard for CYNA was taken as 0.1.
Fig 4. The curve of the correlation between r prize and RS value, where triangle presents a r rate for a RS value.
Red trigon represents the optimal r and its responding RS.
doi:10.1371/daily register.pone.0124632.g004
Network scoring anti-ischemic hardship of CYNA.
The network score was computed in require to explain the anti-ischemic attack effect of CYNA quantitatively. The targets obtained from comparative proteomics experiment and similarity search mode were combined as the group of descendants nodes (17 nodes). As a naturally-occurring material, inhibition potency of CYNA on targets could be much weaker, unlike that of specifically designed medicine molecules. Therefore, we defined the components of the first letter vector x0 corresponding to targets of CYNA obtained ~ dint of. comparative proteomics experiment as the normalized at, and the components of the first letter vector x0 corresponding to targets of CYNA by similarity search method as the medium of all the normalized at rate, otherwise x0(v) = 0.
Score vector xis of ischemic stroke’s import on the human PPI network and furrow vector xca of the anti-ischemic sudden effect effects of CYNA were respectively obtained based on Eq (3) and associated data. Then CYNA’s event on the human PPI network was calculated ~ dint of. E = < xis, xca >. The efficiency score is 0.0589.
Then 1000 stray target sets, containing same number of proteins to the degree that CYNA’s targets, were generated to detect out whether CYNA’s effect sake suggests significant anti-ischemic stroke force. The mean effect score and the support deviation of the 1000 random counterparts were calculated, and the z-notch of CYNA’s anti-ischemic visitation effect score was obtained. The independent value of z-score greater than 3 indicates a statistically forcible deviation between the actual value and the chance ones. Thus the z-score 3.803 of CYNA suggests its weighty anti-ischemic stroke effect. In incident, earlier study has reported the goods of CYNA on ischemic stroke [15, 18, 56–58].
Conclusions
This study demonstrates the anti-ischemic calamity effect of CYNA from a reticulated perspective.
First, we validated the neuroprotective personal estate of CYNA and found that it could house against glutamate-induced neurotoxicity in PC12 cells and cerebellar little grain neurons. Furthermore, we have extracted premises related to ischemic stroke’s pathogenesis and treatment—ischemic knock-associated genes from various databases and CYNA’s targets, particularly. Then two network methods were applied to adorn with pictures CYNA’s effect to ischemic attack. On the pathway sub-networks, every one score of the impact of CYNA to ischemic knock was obtained. Half of the despotic score pathways were associated with the biological course of thrombotic or embolic occlusion of a cerebral artery, which is the main cause of ischemic rap. In addition, we also quantitatively analyzed the anti-ischemic calamity effect of CYNA. We got the anti-ischemic hardship effect score of CYNA as 0.0589, which is significantly higher than that of its chance counterparts, suggesting significant anti-ischemic affliction effect of CYNA. This work applied network approach to explain CYNA’s anti-ischemic visitation effect from two aspects, respectively, which may give an inspiration to study tangle diseases’ pathogenesis and treatments.
Supporting Information
S1 Table. Genes associated by ischemic stroke from two resources.
doi:10.1371/daily register.pone.0124632.s001
(DOCX)
S2 Table. The mark of the impact of CYNA up~ the body the pathway sub-network.
doi:10.1371/periodical.pone.0124632.s002
(DOCX)
Author Contributions
Conceived and designed the experiments: JZ LS. Performed the experiments: HF RY. Analyzed the premises: HF YG YZ. Contributed reagents/materials/analysis tools: HF RY. Wrote the ~ hangings: HF JZ.
References
1. World Health O (1989) Stroke-1989: common fame of the WHO Task Force up~ the body Stroke and Other Cerebrovascular Disorders. Relation 20: 1407–1431.
2. Coyle JT, Puttfarcken P (1993) Oxidative emphasis, glutamate, and neurodegenerative disorders. Science 262: 689–695. pmid:7901908
3. Lloyd-Jones D, Adams RJ, Brown TM, Carnethon M, Dai S, De Simone , et al. Heart sickness and stroke statistics-2010 update A repercussion from the American Heart Association. Circulation 121: e46–e215. doi: 10.1161/CIRCULATIONAHA.109.192667. pmid:20019324
4. Del zoppo GJ, Poeck K, Pessin MS, Wolpert SM, Furlan AJ, Ferbert A, et al. (1992) Recombinant woven stuff plasminogen activator in acute thrombotic and embolic knock. Annals of neurology 32: 78–86. pmid:1642475
5. Ay H, Furie KL, Singhal A, Smith WS, Sorensen AG, Koroshetz WJ, et al. (2005) An evidence-based causative classification system for clear-sighted ischemic stroke. Annals of neurology 58: 688–697. pmid:16240340
6. Krieger DW, Michael A, Abou-Chebl A, Andrefsky JC, Sila CA, Katzan IL, et al. (2001) Cooling during the term of acute ischemic brain damage (COOL AID) An spread pilot study of induced hypothermia in severe ischemic stroke. Stroke 32: 1847–1854. pmid:11486115
7. Schwab S, Schwarz S, Spranger M, Keller E, Bertram M, Hacke W, et al. (1998) Moderate hypothermia in the management of patients with severe middle cerebral artery infarction. Stroke 29: 2461–2466. pmid:9836751
8. Hackam DG, Spence JD (2007) Combining Multiple Approaches instead of the Secondary Prevention of Vascular Events After Stroke A Quantitative Modeling Study. Stroke 38: 1881–1885. pmid:17431209
9. Clark WM, Wissman S, Albers GW, Jhamandas JH, Madden KP, Hamilton S, et al. (1999) Recombinant structure-type plasminogen activator (alteplase) for ischemic hardship 3 to 5 hours after token onset: the ATLANTIS study: a randomized controlled suit. Jama 282: 2019–2026. pmid:10591384
10. Dubinsky R, Lai SM (2006) Mortality of rap patients treated with thrombolysis: analysis of nationwide inpatient specimen. Neurology 66: 1742–1744. pmid:16769953
11. Flint AC, Duckwiler GR, Budzik RF, Liebeskind DS, Smith WS (2007) Mechanical thrombectomy of intracranial inner carotid occlusion pooled results of the MERCI and Multi MERCI Part I trials. Stroke 38: 1274–1280. pmid:17332445
12. Smith WS, Sung G, Starkman S, Saver JL, Kidwell CS, Gobin YP, et al. (2005) Safety and effectiveness of mechanical embolectomy in acute ischemic knock results of the MERCI trial. Stroke 36: 1432–1438. pmid:15961709
13. Adams HP Jr, Del Zoppo G, Alberts MJ, Bhatt DL, Brass L, Furlan A, et al. (2007) Guidelines on the side of the Early Management of Adults With Ischemic Stroke A Guideline From the American Heart Association/American Stroke Association Stroke Council, Clinical Cardiology Council, Cardiovascular Radiology and Intervention Council, and the Atherosclerotic Peripheral Vascular Disease and Quality of Care Outcomes in Research Interdisciplinary Working Groups: The American Academy of Neurology affirms the cost of this guideline as an educational tool ~ the sake of neurologists. Circulation 115: e478–e534. pmid:17515473
14. Hankey GJ (2006) Neuroprotection against acute ischaemic stroke: hope reignited. The Lancet Neurology 5: 287–288. pmid:16545741
15. Shuaib A, Lees KR, Lyden P, Grotta J, Davalos A, Davis SM, et al. (2007) NXY-059 in spite of the treatment of acute ischemic blow. New England Journal of Medicine 357: 562–571. pmid:17687131
16. Lee MK, Yeo H, Kim J, Markelonis GJ, Oh TH, Kim YC, et al. (2000) Cynandione A From Cynanchum wilfordii Protects Cultured Cortical Neurons From Toxicity Induced by H2O2, L-Glutamate, and Kainate. Journal of neuroscience study 59: 259–264. pmid:10650884
17. Lee MK, Yeo H, Kim J, Kim YC (2000) Protection of Rat Hepatocytes Exposed to CCl4 In-vitro ~ dint of. Cynandione A, a Biacetophenone from Cynanchum wilfordii. Journal of pharmacy and pharmacology 52: 341–345. pmid:10757424
18. Yue R, Yuan X, Liu X, Zhang J, Jiang P, He C, et al.(2012) Cynandione A mitigates ischemic injuries in rats with cerebral ischemia. Journal of neurochemistry 121: 451–464. doi: 10.1111/j.1471-4159.2012.07682.x. pmid:22309483
19. Zhao J, Jiang P, Zhang W (2012) Molecular networks conducive to the study of TCM pharmacology. Briefings in Bioinformatics 11: 417–430.
20. Zhao J, Yang P, Li F, Tao L, Ding H, Rui Y, et al. (2012) Therapeutic Effects of Astragaloside IV attached Myocardial Injuries: Multi-Target Identification and Network Analysis. PLoS One 7: e44938. doi: 10.1371/periodical.pone.0044938. pmid:23028693
21. Li S, Zhang B (2013) Traditional Chinese healing art network pharmacology: theory, methodology and perseverance. Chinese Journal of Natural Medicines 11: 110–120. doi: 10.1016/S1875-5364(13)60037-0. pmid:23787177
22. Hamosh A, Scott AF, Amberger JS, Bocchini CA, McKusick VA (2005) Online Mendelian Inheritance in Man (OMIM), a knowledgebase of human genes and genetic disorders. Nucleic acids inquiry 33: D514–D517. pmid:15608251
23. Becker KG, Barnes KC, Bright TJ, Wang SA (2004) The Genetic Association Database. Nat Genet 36: 431–432. pmid:15118671
24. Chen X, Ji ZL, Chen YZ (2002) TTD: Therapeutic Target Database. Nucleic Acids Res 30: 412–415. pmid:11752352
25. von Mering C, Jensen LJ, Snel B, Hooper SD, Krupp M, Foglierini M, et al. (2005) STRING: known and predicted protein-protein associations, integrated and transferred from one side of to the other organisms. Nucl Acids Res 33: D433–437. pmid:15608232
26. Liberzon A, Subramanian A, Pinchback R, Thorvaldsdóttir H, Tamayo P, Mesirov JP, et al. (2011) Molecular signatures database (MSigDB) 3.0. Bioinformatics 27: 1739–1740. doi: 10.1093/bioinformatics/btr260. pmid:21546393
27. Nishimura D (2001) BioCarta. Biotech Software & Internet Report: The Computer Software Journal with regard to Scient 2: 117–120.
28. Kanehisa M, Goto S (2000) KEGG: kyoto encyclopedia of genes and genomes. Nucleic acids exploration 28: 27–30. pmid:10592173
29. Joshi-Tope G, Gillespie M, Vastrik I, D’Eustachio P, Schmidt E, de Bono B, et al. (2005) Reactome: a knowledgebase of biological pathways. Nucleic acids exploration 33: D428–D432. pmid:15608231
30. Burkard TR, Rix U, Breitwieser FP, Superti-Furga G, Colinge J (2010) A computational carry toward to analyze the mechanism of influence of the kinase inhibitor bafetinib. PLoS computational biology 6: e1001001. doi: 10.1371/journal.pcbi.1001001. pmid:21124949
31. Ishihama Y, Oda Y, Tabata T, Sato T, Nagasu T, Rappsilber J, et al. (2005) Exponentially modified protein exuberance index (emPAI) for estimation of unconditional protein amount in proteomics by the contain of sequenced peptides per protein. Molecular & Cellular Proteomics 4: 1265–1272.
32. Tong H, Faloutsos C, Pan J-Y (2008) Random walk through restart: fast solutions and applications. Knowledge and Information Systems 14: 327–346.
33. Fang H, Wang Y, Yang T, Ga Y, Zhang Y, Liu R, et al. (2013) Bioinformatics Analysis with a view to the Antirheumatic Effects of Huang-Lian-Jie-Du-Tang from a Network Perspective. Evidence-Based Complementary and Alternative Medicine 2013. doi: 10.1155/2013/590718. pmid:24363768
34. Colinge J, Rix U, Superti-Furga G (2013) Novel global reticulated scores to analyze kinase inhibitor profiles. China: Suzhou 13: 305–313.
35. Calderwood DA (2004) Integrin activation. Journal of organic unit science 117: 657–666. pmid:14754902
36. Parise LV (1999) Integrin α< sub> IIb β< sub> 3 signaling in platelet adhering and aggregation[J]. Current opinion in small room biology, 1999, 11(5): 597–601. Current judgment in cell biology 11: 597–601. pmid:10508650
37. Shattil SJ, Newman PJ (2004) Integrins: dynamic scaffolds because of adhesion and signaling in platelets. Blood 104: 1606–1615. pmid:15205259
38. AnaKasirer A F (2007) Platelet integrins and immunoreceptors. Immunological reviews 218: 247–264. pmid:17624957
39. Clark EA, Brugge JS (1995) Integrins and remarkable transduction pathways: the road taken. Science 268: 233–239. pmid:7716514
40. Howe A, Aplin AE, Alahari SK, Juliano RL (1998) Integrin signaling and confined apartment growth control. Current opinion in enclosed space biology 10: 220–231. pmid:9561846
41. Zacharski LR, Wojtukiewicz MZ, Costantini V, Ornstein DL, Memoli VA. Pathways of coagulum/fibrinolysis activation in malignancy; 1992. pp. 104–116. pmid:1574711
42. Plow EF, Edgington TS (1975) An other pathway for fibrinolysis. I. The cleavage of fibrinogen through leukocyte proteases at physiologic pH. Journal of Clinical Investigation 56: 30. pmid:237938
43. Packham MA, Mustard JF (1984) Platelet adhesion. Progress in hemostasis and thrombosis 7: 211. pmid:6397767
44. Baumgartner HR, Muggli R, Tschopp TB, Turitto VT (1976) Platelet clinging, release and aggregation in flowing life-~: effects of surface properties and platelet employment. Thrombosis and haemostasis 35: 124–138. pmid:989184
45. Watson SP, Auger JM, McCarty OJT, Pearce AC (2005) GPVI and integrin αIIbβ3 signaling in platelets. Journal of Thrombosis and Haemostasis 3: 1752–1762. pmid:16102042
46. O’Toole TE, Mandelman D, Forsyth J, Shattil SJ, Plow EF, Ginsberg MH. (1991) Modulation of the connection. of integrin alpha IIb beta 3 (GPIIb-IIIa) ~ dint of. the cytoplasmic domain of alpha IIb. Science 254: 845–847. pmid:1948065
47. Carter LL, Fouser LA, Jussif J, Fitz L, Deng B, Wood CR, et al. (2002) PD‐1: PD‐L prohibitory pathway affects both CD4+ and CD8+ T cells and is surmount by IL‐2. European journal of immunology 32: 634–643. pmid:11857337
48. Chao J-R, Wang J-M, Lee S-F, Peng H-W, Lin Y-H, Chou C-H, et al. (1998) mcl-1 is some immediate-early gene activated by the granulocyte-macrophage colony-stimulating divisor (GM-CSF) signaling pathway and is any component of the GM-CSF viability response. Molecular and cellular biology 18: 4883–4898. pmid:9671497
49. Smith JB, Ingerman C, Kocsis JJ, Silver MJ (1973) Formation of prostaglandins during the aggregation of human blood platelets. Journal of Clinical Investigation 52: 965. pmid:4693658
50. Gailani D, Renne T (2007) The inherent pathway of coagulation: a target as far as concerns treating thromboembolic disease? Journal of Thrombosis and Haemostasis 5: 1106–1112. pmid:17388803
51. Mondino A, Blasi F (2004) uPA and uPAR in fibrinolysis, immunity and pathology. Trends in immunology 25: 450–455. pmid:15275645
52. Lee KR, Betz AL, Kim S, Keep RF, Hoff JT (1996) The role of the coagulation cascade in brain edema formation rear intracerebral hemorrhage. Acta neurochirurgica 138: 396–401. pmid:8738389
53. Egan R W, Umland S P, Cuss F M, Chapman RW.(1996) Biology of interleukin‐5 and its relevance to allergic malady. Allergy 51: 71–81. pmid:8738511
54. Conrad M E, Umbreit J N. (1993) A pointed review: Iron absorption—The mucin‐mobilferrin‐integrin pathway. A competitive pathway for metal absorbing.. American journal of hematology42: 67–73. pmid:8416300
55. Erten MS (2009) Network Based Prioritization of Disease Genes: Case Western Reserve University.
56. Xiong X-X, Gu L-J, Shen J, Kang X-H, Zheng Y-Y, Yue S-B, et al. (2014) Probenecid Protects Against Transient Focal Cerebral Ischemic Injury by Inhibiting HMGB1 Release and Attenuating AQP4 Expression in Mice. Neurochemical research39: 216–224. doi: 10.1007/s11064-013-1212-z. pmid:24317635
57. Zhang Z, Ottens AK, Sadasivan S, Kobeissy FH, Fang T, Hayes RL, et al. (2007) Calpain-mediated collapsin response mediator protein-1,-2, and-4 proteolysis later neurotoxic and traumatic brain injury. Journal of neurotrauma 24: 460–472. pmid:17402852
58. Jang YP, Kim SR, Choi YH, Kim J, Kim SG, Markelonis GJ, et al. (2002) Arctigenin protects cultured cortical neurons from glutamate-induced neurodegeneration ~ means of binding to kainate receptor. Journal of neuroscience exploration 68: 233–240. pmid:11948668
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