Indian Journal of Experimental Biology Vol. 52, September 2014, pp. 849-859

Prophylactic effect of pruriens Linn (velvet ) seed extract against experimental sputatrix envenomation: Gene expression studies

Shin Yee Funga*, Si Mui Sima, Kandiah, Jeyaseelanb, Arunmozhiarasi Armugamb, c a John Chinyere Aguiyi & Nget Hong Tan aFaculty of Medicine and CENAR, University of Malaya, Kuala Lumpur, Malaysia, bDepartment of Biochemistry, National University of Singapore, cDepartment of Pharmacology, University of Jos, Jos, Nigeria

Received 23 August 2013; revised 25 June 2014

Mucuna pruriens is widely used in traditional medicine for treatments of various diseases. In certain region of Nigeria, the seed is used as oral prophylactics for snakebite. Rats pretreated with the aqueous extract from M. pruriens seed (MPE) were protected against the lethal effects of Naja sputatrix (Javan spitting cobra) venom [Tan et al., J Ethnopharmacol, 123 (2009) 356]. The pretreatment also protected against venom-induced histopathological changes in rat heart. To contribute to the understanding of the mechanism of cardio-protective action, the present study examined the effects of MPE-pretreatment on gene expression profile of rat heart as well as effect of MPE-pretreatment on N. sputatrix venom-induced gene expression alterations in rat heart. The gene expression profiles were examined by microarray analysis and verified by real time PCR. The results showed that pretreatment with MPE caused 50 genes in the rat heart substantially up-regulated of which 19 were related to immune responses, 7 were related to energy production and metabolism. The up-regulation of genes related to energy metabolism probably plays a role in maintaining the viability of the heart. Four other genes that were up-regulated (alpha synuclein, natriuretic peptide precursor, calsequestrin and triadin) were involved in the maintenance of homeostasis of the heart or maintaining its viability, thereby contributing to the direct protective action. The results demonstrated that protective effect of MPE pretreatment against snake venom poisoning may involve a direct action on the heart.

Keywords: Cobra envenomation, Gene expression, Mucuna pruriens, Prophylactic effect

Mucuna pruriens (L) DC (family: , that the MPE immunogen generating the antibodies subfamily: Papilionoideae) is known as velvet bean that cross-react with E.carinatus venom is a or cowhage. It is commonly found in Asia, America multiform glycoprotein5. Anti-MPE antibodies were and Africa and has been used widely in traditional demonstrated to effectively neutralize the lethalities medicine to treat Parkinson’s disease, impotence, of several Asiatic cobra venoms3. Recent studies, intestinal gas and worms. It is used in Ayurvedic however, indicated that the protective effect of MPE medicine in India1. In Nigeria, the have been against venoms may involve mechanisms other than prescribed by traditional practitioners as an oral immunological neutralization. A proteomic analysis prophylactic for snake envenomations. The protective indicated that the MPE pre-treatment elicited the effect of the Mucuna pruriens aqueous seed extract activation of counterbalancing processes to (MPE) has been demonstrated in rodents; mice compensate for the imbalances caused by E.carinatus pretreated with the extract (21 mg/kg ip, three venom6. Subsequently, it was demonstrated that the weekly injections prior to venom challenge) were direct protective effect of MPE against the venom was protected against the lethal effect of Echis carinatus due to a Kunitz-type trypsin inhibitor7,8. On the other (saw-scaled viper) and Naja sputatrix (Javan spitting hand, it was demonstrated that the protective effect of cobra) venoms2-4. It was suggested that the protective MPE-pretreatment against cobra venom lethality action of MPE-pretreatment involves immunological involved a direct cardio-protective action, in addition neutralization of venom toxins by antibodies formed to the known immunological mechanism9. Further, against seed proteins2. Subsequently, it was reported MPE pretreatment in rats prevented the N. sputatrix venom-induced histopathological changes in hearts10. —————— The nature of the cardio-protective action, however, is *Correspondent author yet to be established. In the present study, the effects Telephone: +60379674958 Fax: +60379674957 of MPE pretreatment have been examined on gene E-mail: [email protected] expression profile of rat heart as well as effect of 850 INDIAN J EXP BIOL, SEPTEMBER 2014

MPE-pretreatment on N. sputatrix venom-induced Ethics statement and welfare considerations—All gene expression alterations in rat heart. The results experiments were handled according to guidelines will shed light on the molecular events that take place given by the Council for International Organization of in rat heart as a result of MPE-pretreatment and Medical Sciences (CIOMS) on animal contribute to understanding of the mechanism of experimentation11 and approved by the University of cardio-protective action of the MPE-pretreatment. Malaya Animal Experimentation Ethics Committee (Ethical clearance letter No. PM/03/03/2010/FSY(R). Materials and Methods The experiment was designed to result in the deaths of Materials—Naja sputatrix venom (NS) was as few animals as possible (n=6) per group. The obtained from Latoxan (Valence, France). dosage used (1.5 LD50, 1.25 µg/g body weight) was Mucuna pruriens seeds were obtained from Rukuba sufficiently high to cause a prompt death (as to not area in Jos, Nigeria, with the aid of a traditional delay death that may increase animal distress) and is healer, and authenticated by Professor S.W.H. Hussini conducted over the shortest time practicable (24 h). of the Department of Botany, University of Jos. As soon as the animals develop signs of severe pain Voucher specimen Number A102 has been deposited or distress, showing signs that death was inevitable in the Pharmacy Herbarium of the University of Jos. (among signs shown were : sudden appearance of Genome 230 2.0 Array was obtained from Affymetrix diarrhoea, and the concomitant rapid weight loss and (Santa Clara, USA), random hexamers were obtained dehydration, laboured breathing, abnormal posture, ® from Alpha DNA (Montreal, Canada), SYBR green lack of red light response, all which points to and Mastermix was obtained from Applied Biosystems confirmed that the animal is becoming severely (USA). Gene specific primers and probes were physiologically compromised and do not recover) , st designed and synthesized by 1 Base (Singapore). the animal was promptly killed humanely and without ® Ethidium bromide, TRIzol reagent, DNase delay since the pain cannot be alleviated. There was and reagents for oligonucleotide microarray no withholding or severe restriction of food or water TM cDNA synthesis were obtained from Invitrogen , on the animals.

Life Technologies (Carlsbad, CA, USA). Method of sacrifice—The animals were Deoxyribonucleotides (dATP, dCTP, dGTP, dTTP, anesthetized with urethane (1.4 g/kg, ip and dUTP) were from Boehringer Manheim, Germany. subsequently by cervical dislocation (AVMA RNase-free DNase I was from Pharmacia (USA). Guidelines on Euthanasia, 2007). The animals were Herring Sperm DNA was purchased from Promega killed in a quiet, clean environment and away from Corporation (USA). In vitro transcription reaction other animals and there was no disposal of the carcass reagents were from BioArray, Enzo Diagnostics Inc. until death was established. (USA). R-Phycoerythrin Streptividin (SAPE) was Male Sprague Dawley rats (200-250 g) were from Molecular Probes Inc. (USA). RNeasy spin maintained on an ad libitum intake of standard columns and Phase Lock Gel (PLG) were from laboratory chow and drinking water prior to and after Qiagen (Germany). Other reagents were analytical treatments. Rats were divided into following four grade and were obtained from Sigma (USA). groups of 6 each: Gr. I: negative control Preparation of M. pruriens seed extract (MPE)— (‘negative’group), Gr. II: MPE pretreated group Sun dried seeds of M. pruriens were ground to a paste (‘MPE’ group), Gr. III: N. sputatrix venom-challenge of uniform consistency. Paste (50 g) was soaked in group (‘NS’ group) and Gr. IV: N. sputatrix 100 mL distilled water and allowed to stand for 24 h venom-challenge to MPE pretreated animals group at 4 °C. The suspension was centrifuged at 10,000 × g (‘MPE-NS’ group). for 20 min, and the resultant supernatant lyophilized In Gr. II, rats were injected with MPE at a dose of to a powder (yield: 22% w/w) termed MPE, which was 21 mg/kg, ip, on day 0, 7 and 14, and sacrificed on then stored at -4 °C until use. Phytochemical screening day 21. In Gr. I, the rats were injected with saline of shows the presence of saponins, tannins and proteins. the same volume and sacrificed also on day 21. Hearts

Experimental animals and design of experiments— were then harvested immediately. Sprague Dawley rats were provided by Laboratory In Gr. III, untreated rats were challenged with Animals Center, Faculty of Medicine, University 1.5 LD50 (1.25 µg/g) of N. sputatrix venom of Malaya. whereas in the venom challenge to MPE pretreated FUNG et al.: MUCUNA PRURIENS SEED EXTRACT AGAINST NAJA SPUTATRIX ENVENOMATION 851

animals group (Gr. IV), MPE pretreated rats were (MD) or No Change (NC). Only the ‘I’ and ‘D’ challenged with 1.5 LD50 (1.25 µg/g) of N. sputatrix transcript level calls were taken into consideration venom, both on day 21. For Gr. III and Gr. IV, for further analyses in all microarray experiments. the rats were observed for 24 h after venom Also, only genes whose expression altered by 2-foldor challenged and hearts were harvested as soon as death greater in the pair-wise comparison (between occurred or 24 h after the venom injection, whichever venom-treated and controls) were selected. These occurred first. genes were then classified according to their 14 Total RNA isolation from tissues using TRIzol® biological functions as described by Liu et al. , using reagent—The tissue sample (50-100 mg) was NetAffix Analysis Center and PubMed databases. homogenized using 1 mL of TRIzol® reagent and The gene expression data were also subjected to extracted according to the modified methods of hierarchical clustering and principle component Chomczynski and Sacchi12 and Chomczynski 13. analysis using Genesis 1.6.0 Beta 1 Software Quantitation of RNA was carried out by reading the (Graz University of Technology, Austria). absorbance at 260 nm and 280 nm using a The data set was deposited to Gene Expression spectrophotometer and water was used as blank. Omnibus (GEO) with the accession number GSE46610. The quality of RNA was checked by using agarose gel electrophoresis. Reverse transcription and real-time quantitative

Microarray gene chip analysis—Total RNA PCR—Total RNA (300 ng) was reverse transcribed in a isolated from the harvested rat hearts was pooled prior 15 µL reaction mixture containing random hexamers (Alpha DNA, Montreal, Quebec, Canada) at 25 oC for to microarray probe preparation and hybridization to o o minimize inter-individual variation. The RNA 10 min, followed by 37 C for 60 min and 95 C for 5 preparation were processed and hybridized to each min. Gene-specific primers (forward and reverse) array of the Rat Genome 230 2.0 Array (Affymetrix, used for subsequent polymerase chain reaction (PCR) Santa Clara, CA, USA) according to the protocols amplification are shown in Table 1. Ribosomal RNA described in the Gene ChipTM expression analysis was used as the internal calibrator. The final PCR reactions contained complementary DNAs (cDNAs), technical manual (Affymetrix, Santa Clara, CA, ® USA). Each gene chip represents 28,700 genes and primers (100 nM) and SYBR green Master Mix (Applied Biosystems, Foster City, CA, USA). expressed sequence tags (ESTs). In the initial phase of o analysis, a grid was automatically placed over the .dat Amplification was performed for 1 cycle at 50 C for 2 min, followed by 40 cycles at 94 oC for 15s and file demarcating each probe cell. One of the probe o library files, the .cif file was used by GeneChip® 60 C for 1 min per cycle. All reactions were Operating Software to determine the appropriate grid carried out in triplicate using the ABI Prism 7000 SDS size to be used. The alignment of the grid was (Applied Biosystems, Foster City, CA, USA). confirmed by zooming in on each of the four corners A dissociation protocol was carried out at the end and on the centre of the image. If the grid was not of each experiment to ensure that the amplifications were specific. properly aligned, the alignment was adjusted by placing the cursor on an outside edge or the corner of Statistical analysis—Statistical analysis of data the grid. The cursor image was changed to a small obtained from quantitative real-time PCR was double headed arrow and the grid was adjusted. The performed using the unpaired Student’s t-test. Results software then analyzed the hybridization intensity were expressed as mean ± SE. P< 0.05 was considered from the GeneChip® expression probe arrays where statistically significant. data from each array were scaled to an average intensity of 800. The gene expression levels from Results venom-treated and control samples were compared Intravenous injection of the N. sputatrix venom using GeneChip® Operating Software and an (1.25 µg/g body weight) in rats caused paralysis and expression comparison analysis which calculates a set respiratory distress beginning approximately 120 min of comparison metrics. Some of the metrics were used after venom administration, with death usually by a decision matrix to determine a difference call occurred within the next hour. The dosage 1.25 µg/g for each transcript: Increased (I), Decreased (D), body weight was thus deemed an appropriate Marginally Increased (MI), Marginally Decreased challenge dose for the present studies. 852 INDIAN J EXP BIOL, SEPTEMBER 2014

Hierarchical clustering and principle component Real-time quantitation of gene expression—The analysis of expression data—To understand the expression of 23 genes was selected to be quantitated molecular events involved in the protection of MPE by real time PCR in order to validate results from pretreatment against the toxic action of Naja sputatrix microarray analysis. The 23 genes selected were venom on rat heart, gene expression studies were shown in Table 1. carried out using microarray analysis and real-time The relative expression of each gene was PCR techniques. obtained after normalizing against an internal control RNA isolated from Gr. I, II, III and IV were pooled (18 S ribosomal RNA) and a calibrator, in this case, before microarray probe preparation and hybridization the heart of saline-treated control (negative) to minimize inter-individual variation. Expression (relative gene expression =1). The results are shown analysis was carried out using Gene Chips from the in Table 1. These results are then compared with the Rat Genome 230 2.0 Array, with each chip results obtained from microarray analysis (Fig. 2, representing ~28,700 genes and expressed sequence Table 2 and Table S1). tags (ESTs). Probe sets designated as ‘absent’ by the analysis software were discarded, and only the genes Table 1—Validation of microarray data of selected genes using whose expression changed by 2-fold or greater in one real-time PCR respective pair-wise comparison were deemed [Values are mean ± SE] significant. The paired samples were as follows: Gene NS vs MPE vs MPENS Gr. IV versus Gr. III, Gr. III versus Gr. I and Gr.II negative negative vs NS versus Gr. I. 1. acid phosphatase 1 -0.7 ± 0.1 3.9 ± 0.0 42.9 ± 0.1 A total of 374 genes were of significance and 2. actin alpha cardiac 1 -0.2 ± 0.0 1.3 ± 0.0 8.6 ± 0.2 differently expressed. These probe sets were subjected 3. alpha synuclein -0.6 ± 0.0 3.2 ± 0.0 56.7 ± 0.2 to hierarchical clustering using Genesis, Version 1.6.0 4. basigin -0.5 ± 0.1 7.6 ± 0.0 27.0 ± 0.1 Beta 115 where expression patterns between pair-wise comparison and amongst genes within each respective 5. beta globin -0.2 ± 0.1 1.1 ± 0.1 28.8 ± 0.0 pair-wise comparison are represented as an expression 6. calreticulin -0.3 ± 0.1 2.4 ± 0.0 29.8 ± 0.2 image (data not shown). Hierarchical clustering is a 7. calsequestrin -0.3 ± 0.1 5.9 ± 0.1 12.1 ± 0.4 method of grouping objects into clusters and 8. cardiac myosin specifying relationships among objects in a cluster, light chain 2 -0.3 ± 0.0 1.4 ± 0.0 9.4 ± 0.1 16 resembling a phylogenetic tree . 9. caspase 3 -0.4 ± 0.0 2.8 ± 0.0 0.10 ± 0.1 Of the 374 genes observed to be differentially 10. cathepsin B -0.4 ± 0.1 2.5 ± 0.0 39.1 ± 0.1 expressed, 285 genes were differentially expressed in 11. cathepsin L -0.9 ± 0.1 3.3 ± 0.0 51.5 ± 0.0 Gr. III vs Gr. I pair wise comparison (Table S1); 12. creatine kinase -0.9 ± 0.0 2.0 ± 0.1 4.0 ± 0.7 59 genes were differentially expressed in Gr. II vs 13. defender against Gr. I comparison (Table 1), and 288 genes were cell death -0.9 ± 0.0 2.5 ± 0.1 13.5 ± 0.2 differentially expressed in Gr. IV vs Gr. III 14. desmin -0.4 ± 0.0 9.7 ± 0.2 5.9 ± 0.2 comparison (Table S1). 15. glutamine synthetase -0.7 ± 0.0 1.6 ± 0.0 2.4 ± 0.2 In the Gr. III vs Gr. I pair wise comparison, 272 genes (95%) were down-regulated. In the Gr. IV 16. myosin light polypeptide 3 -0.9 ± 0.0 3.2 ± 0.0 3.4 ± 0.0 vs Gr. III pair wise comparison, of the 288 genes altered in expression, only 12 were down-regulated. 17. natriuretic peptide precursor -0.3 ± 0.1 5.6 ± 0.0 28.3 ± 0.0 In the Gr. II vs Gr. I, of the 59 genes differently expressed, 76% were up-regulated. 18. neuropeptide Y -0.6 ±0.0 2.0 ± 0.1 11.1 ± 0.1 Principal Component Analysis showed that Gr. IV 19. phospholamban -0.4 ± 0.0 4.3 ± 0.1 27.2 ± 0.1 vs Gr. III pair wise comparison and Gr. II vs Gr. I 20. phospholipase A2, -0.7 ± 0.1 1.9 ± 0.0 22.8 ± 0.0 pair wise comparison are nearer to each other in group IIA the principal component space. On the other hand, 21. platelet factor 4 -0.2 ± 0.1 3.5 ± 0.1 2.9 ± 0.3 the Gr. IV vs Gr. III pair and Gr. III vs Gr. I pairs 22. pyruvate are rather far apart from each other in the 3D-space dehydrogenase kinase -0.8 ± 0.1 2.4 ± 0.1 5.2 ± 0.0 (Fig. 1). 23. triadin -0.9 ± 0.1 5.8 ± 0.1 6.7 ± 0.1 FUNG et al.: MUCUNA PRURIENS SEED EXTRACT AGAINST NAJA SPUTATRIX ENVENOMATION 853

In the Gr. III vs Gr. I pair comparison, two techniques, the absolute values of fold-change real-time PCR studies demonstrated that, all 23 genes did not necessarily correspond17(the same applies to were down-regulated. This is consistent with the the other comparisons). data from microarray analysis. However, due to In the Gr. II vs Gr. I pair, real-time PCR the different specificities and sensitivities of the demonstrated that the tested genes were all up-regulated. In microarray analysis, only alpha synuclein and glutamine synthetase were found to be up-regulated (> 2-fold). The rest of the tested genes were negative (unchanged). Evidently, lowly expressed genes (< 2-fold) that were determined to be unchanged in microarray analysis can be detected by the highly sensitive real-time -PCR as up-regulated. In the Gp. IV vs Gp. III pair, real-time PCR studies showed that only caspase 3 were slightly down-regulated, all the other 22 genes were up-regulated. This is also generally consistent with the microarray analysis.

Fig. 1—Principal component analysis clustered experiments in Thus, alterations of gene expression quantitated by three-dimensional space according to differences in the overall real-time PCR correlated well with the microarray expression of genes between sample sets determined by the spatial analysis data. The expression profiles obtained in the distance between the representative points. ‘MPENS vs NS’ and s‘NS v negative’ are two points that are rather far apart from each above cases were similar, thereby validating the other in the 3D-space (represented by axis X, Y and Z ) results of the microarray analysis.

Fig. 2—Expression pattern of both microarray and qPCR or correlation assay based on fold change for MPE vs control 854 INDIAN J EXP BIOL, SEPTEMBER 2014

Table 2—Functional clustering of genes from pair-wise comparison of MPE vs control (negative) exhibiting a 2-fold or greater change in expression in the heart. [Values preceded by the symbol (-) are fold of down-regulation, whereas values without the symbol are fold of up-regulation] Category of gene Description MPE Description MPE Description MPE vs neg vs neg vs neg Nervous system epidermal growth factor 4.9 protein kinase, cAMP dependent 2.8 sodium channel, 2.1 receptor regulatory, type II β voltage-gated, type 1, α-polypeptide glutamine synthetase 1 8.1 phosphatidylethanolamine N- -2.6 synuclein, alpha 3.5 methyltransferase Inflammatory procollagen, type I, α 2 2.6 collagen, type III, alpha 1 2.0 procollagen, type I, alpha 2 2.3 response fibronectin 1 2.0 interleukin 1 beta 2.0 Cholesterol complement component 4a, 8.6 biosynthesis and other complement component 4, steroid metabolism gene 2 Eicosanoid synthesis prostaglandin D2 synthase 3.7 transglutaminase 2, C polypeptide 2.3 arachidonate 12- 10.6 lipoxygenase prostaglandin-endoperoxide 2.0 synthase 1 Apoptosis BCL2/adenovirus E1B 19 6.5 GTP cyclohydrolase 1 4.6 perforin 1 (pore forming 2.1 kDa-interacting protein 3- protein) like death-associated kinase 2 3.7 Urea cycle and other branched chain 5.7 asparagine synthetase 2.6 amino acid aminotransferase 1, metabolism cytosolic Receptor signaling microtubule-associated 5.7 pathways protein 2 Protein biosynthesis lysyl oxidase 4.6 and modification Blood and wounding fibrinogen, gamma 2.1 tissue factor pathway inhibitor 2.0 glycoprotein Ib, beta 2.0 responses polypeptide polypeptide Integrin mediated cell integrin alpha 7 -2.1 mitogen-activated protein kinase 12 -2.0 adhesion Iron ferritin light chain 1 2.8 transferrin signal recognition 2.0 particle receptor, B subunit Cell cycle DNA primase, p49 subunit 6.5 cell division cycle 25 homolog B 2.0 (S. cerevisiae) Immune response bone marrow stromal cell 2.1 RT1 class Ib, locus Aw2 RT1 class I, 2.0 RT1 class Ib, locus Aw2 -294.1 antigen 1 CE12 RT1 class Ia, locus A2 cathepsin E 2.6 immunoglobulin heavy chain -3.5 major histocompatibility 2.0 (epsilon polypeptide) complex, class II,DM beta RT1 class II, locus Bb 48.5 RT1 class Ib, locus S3 9.8 defensin RatNP-3 32.0 precursor RT1 class II, locus Db1 19.7 RT1 class II, locus Ba 2.3 Fc receptor, IgG, low 3.5 affinity III, Fc gamma receptor II β Heme biosynthesis aminolevulinic acid synthase 2 8.0 Cardiac muscle Kv channel-interacting -2.5 protein 2 Statin pathway myosin heavy chain, -2.5 cytochrome P450, family 7, -2.0 apolipoprotein E 2.5 polypeptide 6 subfamily a, polypeptide 1 Energy production 3-hydroxybutyrate -2.3 dihydrolipoamide S- -2.1 pyruvate dehydrogenase -2.5 and metabolism dehydrogenase acetyltransferase kinase, isoenzyme 2 cytochrome b5, outer -2.6 enoyl-Coenzyme A, hydratase/3- -4.6 protein kinase, AMP- -2.0 mitochondrial membrane hydroxyacyl coenzyme A activated, alpha 2 isoform dehydrogenase catalytic subunit uncoupling protein 1 13.0 phosphofructokinase, liver, B 5.3 Others lectin, galactose binding, 7.5 soluble 5 , lectin, galactose binding, soluble 9 FUNG et al.: MUCUNA PRURIENS SEED EXTRACT AGAINST NAJA SPUTATRIX ENVENOMATION 855

Discussion oxidative stress, proteosome degradation, cardiac Cobra envenomation is an extremely complex muscle and statin pathway. The results are generally process that induces multiple-organ failure. Cher consistent with data reported18; even though the et al.18 reported that N. sputatrix venom injection animals, dosage and mode of venom administration induced major gene expression changes in the heart are different from the present studies. This wide and the genes altered were related to immune spectrum of gene down-regulation encompassing a response, apoptosis, ion-transport and binding, signal wide variety of biochemical pathways shows that the transduction, hypotension and energy metabolism. N. sputatrix venom affected the metabolism and Their results suggested that cardiotoxicity of the cobra structure of the heart extensively. Of particular venom is an important contributory cause in cases of interest, is the large number of down-regulated genes N. sputatrix envenomation. Recent studies showed related to nervous system (34), protein biosynthesis that MPE-pretreatment in rats prevented the N. sputatrix and modification (31), energy production and venom-induced histopathological changes in hearts10. metabolism (91) and cardiac muscles/smooth In an effort to understand molecular events that may muscle/striated muscle (12). take place in rat heart as a result of MPE-pretreatment The gene expression profile in the heart of MPE and the mechanism of the cardio-protective action of pretreated rat injected with N. sputatrix venom was, MPE-pretreatment, the effects of various treatments were however vastly different from that of without MPE investigated on the gene expression profile in rat hearts. pretreatment. Here, there were 288 genes differentially expressed, only 12 were down-regulated, the Principal component analysis—Altogether, three remaining 276 genes were up-regulated. There was a sets of pair-wise comparison that were deemed massive decrease in the number of genes down-regulated. important for understanding of the molecular events This shows that MPE pretreatment effectively of the cardio-protective action of MPE against prevented the detrimental effects of the venom on N. sputatrix venom were made. The ‘MPE versus heart. Much of these could well be the result of negative’ and ‘NS versus negative’ comparisons give immunological neutralizations of the venom information on the alterations of genetic expression in phospholipases A2 and neurotoxins by the antibodies rat heart as a result of the MPE pretreatment in rat and induced by MPE3. However, the fact that in the N. sputatrix venom injection into rat, respectively. ‘MPENS vs NS’ comparison, the majority of the The ‘MPENS versus NS’ comparison, on the other differentially expressed genes were up-regulated hand, provides information on the effects of MPE indicates that the MPE pretreatment has a direct effect pretreatment on the N. sputatrix venom-induced on heart, altering the genetic expression of certain genetic expression changes. genes that presumably renders the heart more resistant Principal component analysis indicates that the to the harmful effects of the venom, in particular the MPE pretreatment greatly altered the changes in rat’s reduction in atrial contractibility and rate19. Among heart gene expression profile induced by the injection the genes up-regulated were genes encoding proteins of N. sputatrix venom, as the ‘MPENS versus NS’ and enzymes related to energy production and point and the ‘NS versus negative’ point are far apart metabolism (77), nervous system (31), inflammatory from each other in the 3D space. response (1), apoptosis (16), eicosanoid synthesis (8), Alterations in gene expression induced by immune response (18), blood and wounding response N. sputatrix venom and the protective effect of MPE— (4) and cardiac muscle (5). The up-regulation of genes A total of 285 genes were differentially expressed in related to immune response is not surprising as the the heart of untreated rat injected with N. sputatrix MPE pretreatment certainly resulted in the induction venom. The vast majority of the genes were of immune response. And since the establishment of down-regulated including genes encoding immunity is inadvertently coupled to inflammation proteins and enzymes involved in nervous system, and hence, eicosanoid synthesis, one would expect genes apoptosis, urea cycle and amino acid metabolism, related to these processes would also be up-regulated. protein biosynthesis and modification, cholesterol The up-regulation of blood and wounding response biosynthesis, eicosanoid synthesis, receptor signaling might well be a homeostasis response. Nevertheless, the pathways, integrin mediated call adhesion, immune up-regulation of many other genes, in particular those response, blood and wounding responses, atrial related to energy production and metabolism did natriuretic factor, iron transport, cell cycle, energy suggest the possibility of direct protective action of production and metabolism, heme biosynthesis, MPE on rat heart against the cobra venom. 856 INDIAN J EXP BIOL, SEPTEMBER 2014

Data from microarray and real-time PCR on the genes were also up-regulated by at least 3-fold: alpha ‘MPE vs negative’ comparison gave a clearer picture synuclein, aminolevulinic acid synthase 2, BCL-2, on the direct protective action of MPE on heart. These branched chain aminotransferase I, cell division cycle data represent the alterations of gene expression in the 25 homolog B, death-associated kinase 2, epidermal rat heart as the result of MPE pretreatment. Analysis growth factor receptor, glutamine synthetase I, GTP of the gene expression profile in the heart revealed cyclohydrolase I, lysyl oxidase, microtubule-associated that 45 genes were up-regulated and 14 down-regulated. protein 2, phosphofructokinase (B type) and uncoupling Of the genes up-regulated, 10 are involved in immune protein 2. Examination of the quantitative real time-PCR responses; 5 in inflammatory response and 4 in eicosanoid results indicated that the following 5 genes: basigin, synthesis. Thus, the total number of genes up-regulated calsequestrin, desmin, natriuretic peptide precursor that may be associated with immune response is 19. and triadin; were up-regulated by at least 5-fold, even The up-regulation of genes involved in immune though the expression of these genes were below 2- responses is expected as the MPE pretreatment fold when examined by microarray analysis. The amounts to an immunization process. The up-regulation functions, systems or the physiological processes of genes involved in inflammatory response and involved of these 18 genes (13 from microarray eicosanoid synthesis might be related to the immune analysis and 5 from real-time PCR analysis) and also responses. Besides these genes, the following 13 their main functions are shown in Table 3.

Table 3—The functions, systems or physiological processes involved 18 selected up-regulated genes in the ‘MPE pretreatment’ versus ‘negative control’ pair wise comparison No Gene up-regulated System or physiological Functions process(es) involved 1 Calsequestrin Nervous system Involves in the modulation of calcium release process 23, 24 2 Basigin Lactate transport May play a role in providing more energy for heart 29, 30 (Energy metabolism) 3 Desmin Cardiac muscle The intermediate filament protein in muscle. May be involved in the maintenance of muscle mitochondria and responsible for the elastic properties of muscle 25, 26 4 Natriuretic peptide Atrial natriuretic factor Acts as vasodilators of cardiovascular tissues, maintenance of cardiovascular precursor homeostasis 21,34 – 37 5 Triadin Nervous system Involved in muscle contraction, calcium release for contraction 22 6 Alpha synuclein Nervous system Probably involves in muscle contraction, calcium release for contraction 20 7 Aminolevulinic acid Heme biosynthesis Increases hemoglobin and hence oxygen supply. May play a role in providing synthase 2 more energy for heart 32 8 BCL-2 Apoptosis, Immune Anti-apoptosis 38. Plays a pivotal role in the regulation of mitochondrial response homeostasis and cell death 39 9 Branched chain Amino acid metabolism May help to provide more energy for heart 28 aminotransferase I 10 Cell division cycle 25 Cell division, mitosis Involved in the start of the cell cycle40 homolog B 11 Death-associated Apoptosis Positive regulator of apoptosis (UniProt database) kinase 2 12 Epidermal growth factor Cell division Promotes cell proliferation and differentiation 41. May play a role in neural receptor development and regeneration42,43 13 Glutamine synthetase I Amino acid metabolism May have a role in regulation of neurotransmitter levels 44 14 GTP cyclohydrolase I Apoptosis Induction of apoptosis, for synthesis of tetrahydrobiopterin. May increase vasodilation 45 15 Lysyl oxidase Collagen and elastin Blood vessel development and collagen fibril organization. Improve fibre synthesis mechanical strength in dermal wounds during healing 31 16 Microtubule-associated Receptor signaling Regulates and organizes microtubules. Allows dendrite development and protein 2 pathway microtubule bundle formation 46 17 Phosphofructokinase Energy metabolism May help to provide more energy for heart via accelerated glycolysis 27,47 (B type) 18 Uncoupling protein 2 Energy metabolism Heat generation 33 FUNG et al.: MUCUNA PRURIENS SEED EXTRACT AGAINST NAJA SPUTATRIX ENVENOMATION 857

Of particular interest are the four genes that are examination of the microarray analysis of gene involved in muscle contraction (alpha synuclein, expression in rat heart as a result of MPE pretreatment natriuretic peptide precursor, calsequestrin and alone showed that, in addition to eliciting immune triadin) as well as the genes related to energy response, MPE may exert a direct protective effect on metabolism (desmin, phosphofructokinase B type, the heart, as the pre-treatment resulted in the branched chain aminotransferase I, lysyl oxidase and up-regulation of 7 genes related to energy production basigin). Recently, it has been suggested that alpha and metabolism, some of which may play a role in synuclein may be involved in muscle contraction and maintaining the viability of the heart. Thus, gene calcium release for contraction20. The natriuretic expression studies together with pharmacological peptides contribute to the maintenance of studies using isolated spontaneously beating rat atrial cardiovascular homeostasis. Increase in triadin and preparation9 do support the possibility that the calsequestrin expression in MPE pretreated rats would protective effect of MPE pretreatment against snake implicate an increase in calcium movement, to enable venom poisoning may involve a direct action on the the chambers of the heart to contract and relax heart. However, further experiments including (excitation-contraction coupling). proteomic studies and identification of the M. pruriens The 7 genes related to energy supply that were seed constituents that cause these gene expression up-regulated include: desmin, phosphofructokinase changes are necessary to prove this hypothesis. (B type), branched chain amino transferase I, basigin, lysyl oxidase, aminolevulinic acid synthase and Supplementary Data uncoupling protein 2. The up-regulation of genes Supplementary data (Table S1) associated with this related to energy metabolism probably plays a role in article may be obtained from the correspondent author maintaining the viability of the heart (e.g. resisting the on request. drop in contractibility induced by venom). It is Acknowledgement interesting to note that animals pretreated with MPE This study was supported by RG076/12BIO by in the present studies were also observed to be more University of Malaya. active and energetic.

The significance of the up-regulation of the genes Conflict of interests related to neurotransmitter release (glutamine None declared. synthetase I), microtubule development (microtubule- associated protein 2), cell division (epidermal growth References factor receptor, cell division cycle 25 homolog B), 1 Sathiyanarayanan L & Arulmozhi S, Mucuna pruriens Linn. apoptosis (BCL-2, death-associated kinase 2, – A comprehensive review, Pharmacogn Rev, 1 (2007) 157. GTP cyclohydrolase I) are not clear. 2 Guerranti R, Aguiyi JC, Neri S, Leoncini R, Pagani R & Marinello E, Proteins from Mucuna pruriens and enzymes Thus, results of gene expression studies support from Echis carinatus venom: Characterization and cross strongly the suggestion that the protective effect of reactions, J Biol Chem, 277 (2002) 17072. MPE pretreatment includes a direct protective action 3 Tan NH, Fung SY, Sim SM, Marinello E, Guerranti R & on rat heart in additional to the immunological Aguiyi JC, The protective effect of Mucuna pruriens seeds mechanism. However, further studies are necessary to against snake venom poisoning, J Ethnopharmacol, 123 (2009) 356. firmly establish the molecular basis of the directly 4 Tan NH, Fung SY & Sim SM, Extracts of Cowhage (Mucuna protective action of M. pruriens pretreatment. Pruriens) Seeds and anti-snake venom effects, in Nuts and seeds in health and disease prevention, edited by Preedy VR, Conclusions Watson, RR, Patel VB (Elsevier, USA) 2011. Microarray analysis demonstrated that N. sputatrix 5 Guerranti R, Aguiyi JC, Ogueli IG, Onorati G, Neri S, Rosati venom injection into rats resulted in the F, Del Buono F, Lampariello R, Pagani R & Marinello E, Protection of Mucuna pruriens seeds against Echis carinatus down-regulation of a vast number of genes that affect venom is exerted through a multiform glycoprotein whose the metabolism and structure of the rat heart resulting oligosaccharide chains are functional in this role, Biochem in its dysfunction. The present results show that Biophy Res Commun, 323 (2004) 484. these detrimental effects were prevented by 6 Guerranti R, Ogueli IG, Bertocci E, Muzzi C, Aguiyi JC, MPE-pretreatment. The protection is presumably Cianti R, Armini A, Bini L, Leoncini R, Marinello E & Pagani R, Proteomic analysis of the pathophysiological related to the immunological neutralization of the process involved in the anti-snake venom effect of Mucuna main cobra toxins by anti-MPE antibodies. However, pruriens extract, Proteomics, 8 (2008) 402. 858 INDIAN J EXP BIOL, SEPTEMBER 2014

7 Hope-Onyekwere NS, Ogueli GI, Cortelazzo A, Cerutti H, 24 Fan GC, Gregory KN, Zhao W, Park WJ & Kranias EG, Cito A, Aguiyi JC & Guerranti R, Effects of Mucuna Regulation of myocardial function by histidine-rich pruriens protease inhibitors on Echis carinatus venom, calcium-binding protein, Am J Physiol - Heart C, 287 Phytother Res, 26 (12) (2012) 1913. (2004) H1705. 8 Scirè A, Tanfani F, Bertoli E, Furlani E, Nadozie HO, 25 Milner DJ, Mavroidis M, Weisleder N & Capetanaki Y, Cerutti H, Cortelazzo A, Bini L & Guerranti R, Desmin cytoskeleton linked to muscle mitochondrial The belonging of gpMuc, a glycoprotein from Mucuna distribution and respiratory function, J Cell Biol, 150 pruriens seeds, to the Kunitz-type trypsin inhibitor family (2000) 1283. explains its direct anti-snake venom activity, Phytomedicine, 26 Shah SB, Davis J, Weisleder N, Kostavassili I, McCulloch 18 (2011) 887. AD, Ralston E, Capetanaki Y & Lieber RL, Structural and 9 Fung SY, Tan NH, Sim SM & Aguiyi JC, Effects of Mucuna functional roles of desmin in mouse skeletal muscle during pruriens seed extract pretreatment on the responses of passive deformation, Biophy J, 86 (2004) 2993. spontaneously beating rat atria and aortic ring to 27 Goldstein BN & Maevsky AA, Critical switch of the Naja sputatrix (Javan spitting cobra) venom, Evid Based metabolic fluxes by phosphofructo-2-kinase: fructose-2,6- Complement Alternat Med, 2012 (2012) 486390. bisphosphatase. A kinetic model, FEBS Lett, 532 (2002) 295. 10 Fung SY, Tan NH, Liew SH, Sim SM & Aguiyi JC, The 28 Bixel MG, Hutson SM & Hamprecht B, Cellular distribution protective effects of Mucuna pruriens seed extract against of branched-chain amino acid aminotransferase isoenzymes histopathological changes induced by Malayan cobra among rat brain glial cells in culture, J Histochem Cytochem, (Naja sputatrix) venom in rats, Trop Biomed, 26 (2009) 80. 45 (1997) 685. 11 Howard-Jones NA, A CIOMS ethical code for animal 29 Kirk P, Wilson MC, Heddle C, Brown MH, Barclay AN & experimentation. WHO Chron, 39 (1995) 51. Halestrap AP, CD147 is tightly associated with lactate 12 Chomczynski P & Saachi N, Single-step method of RNA transporters MCT1 and MCT4 and facilitates their cell isolation by acid guanidinium thiocyanate-phenol-chloroform surface expression, EMBO J, 19 (2000) 3896. extraction, Anal Biochem, 162 (1987) 156. 30 Deschamps AM & Spinale FG, Pathways of matrix 13 Chomczynski P, A reagent for the single-step simultaneous metalloproteinase induction in heart failure: Bioactive isolation of RNA, DNA and proteins from cell and tissue molecules and transcriptional regulation, Cardiovasc Res, samples, BioTechniques, 15 (1993) 532. 69 (2006) 666. 14 Liu G, Loraine AE, Shigeta R, Cline M, Cheng J, 31 Lau YK, Gobin AM & West JL, Overexpression of lysyl Valmeekam V, Sun S, Kulp D & Siani-Rose MA, NetAffx: oxidase to increase matrix crosslinking and improve tissue Affymetrix probe sets and annotations. Nucleic Acids Res, 31 strength in dermal wound healing, Ann Biomed Eng, 34 (2003) 82. (2006) 1239. 15 Sturn A, Quackenbush J & Trajanoski Z, Genesis: Cluster 32 Ho-Chen JK, Bustamante JJ & Soares MJ, Prolactin-like analysis of microarray data, Bioinformatics, 18 (2002) 207. protein-f subfamily of placental hormones/cytokines: 16 Babu MM, Introduction to microarray data analysis, responsiveness to maternal hypoxia, Endocrinology, in Computational genomics: Theory and application, edited 148 (2007) 559. by Grant RP (Horizon Press, Norwich) 2004. 33 Echtay KS, Murphy MP, Smith RAJ, Talbot DA & Brand 17 Dallas PB, Gottardo NG, Firth MJ, Beesley AH, Hoffman K, MD, Superoxide activates mitochondrial uncoupling protein Terry PA, Freitas JR, Boag JM, Cummings AJ & Kees UR, 2 from the matrix side. Studies using targeted antioxidants, Gene expression levels assessed by oligonucleotide J Biol Chem, 277 (2002) 47129. microarray analysis and quantitative real time RT-PCR – 34 Garbers DL & Lowe DG, Guanylyl cyclase receptors, J Biol how well do they correlate, BMC Genomics, 6 (2005) 59. Chem, 269 (1994) 30741. 18 Cher CDN, Armugam A, Zhu YZ & Jeyaseelan K, Molecular 35 Sagnella GA & MacGregor GA, in Textbook of basis of cardiotoxicity upon cobra envenomation. Cell Mol Hypertension, edited by Swales JD (Blackwell Scientific, Life Sci, 62 (2005) 105. Oxford) 1994. 19 Fung SY, Tan NH, Sim SM, Marinello E, Guerranti R & 36 Espiner EA, Richards AM, Yandle TG & Nicholles MG, Aguiyi JC, Mucuna pruriens Linn. Seed extract pretreatment Natriuretic hormones, Endocrinol Metab Clin North Am, protects against cardiorespiratory and neuromuscular 24 (1995) 481. depressant effects of Naja sputatrix (Javan spitting cobra) 37 Oliver PM, Fox JE, Kim R, Rockman HA, Kim HS, Reddick venom in rats, Indian J Exp Biol, 49 (2011) 254. RL, Pandey KN, Milgram SL, Smithies O & Maeda N, 20 Adamczyk A & Strosznajder JB, Alpha-synuclein potentiates Hypertension, cardiac hypertrophy and sudden death in mice Ca2+ influx through voltage-dependant Ca2+ channels, lacking natriuretic peptide receptor A, Proc Natl Acad Sci Neuroreport, 17 (2006) 1883. USA, 4 (1997) 14730. 21 Mair J, Role of cardiac natriuretic peptide testing in heart 38 Phaneuf S & LeeuWenburgh C, Cytochrome c release from failure, Clinical Chemistry, 48 (2002) 977. mitochondria in the aging heart: a possible mechanism for 22 Marty I, Triadin: a multi-protein family, Cell Mol Life Sci, 61 apoptosis with age, Am J Physiol Regul Integr Comp Physiol, (2004) 1850-1853. 282 (2002) R423. 23 Zhang L, Kelley J, Schmeisser G, Kobayashi YM & Jones LR, 39 Wang J, Wei Q, Wang CY, Hill WD, Hess DC, & Dong Z, Complex formation between junctin, triadin, calsequestrin, Minocycline upregulates Bcl-2 and protects against cell and the ryanodine receptor: Proteins of the cardiac death in mitochondria, J Biol Chem, 279 (2004) 19948. junctional sarcoplasmic reticulum membrane, J Biol Chem, 40 Jinno S, Suto K, Nagata A, Igarashi M, Kanaoka Y, 272 (1997) 23389. Nojima H & Okayama H, Cdc25A is a novel FUNG et al.: MUCUNA PRURIENS SEED EXTRACT AGAINST NAJA SPUTATRIX ENVENOMATION 859

phosphatase functioning early in the cell cycle, EMBO J, 44 Smith CD, Carney JM, Starke RP, Oliver CN, Stadtman ER, 13 (1994) 1549. Floyd RA & Markesbery WR, Excess brain protein oxidation 41 Voisin L, Foisy S, Giasson E, Lambert C, Moreau P & and enzyme dysfunction in normal aging and in Alzheimer Meloche S, EGF receptor transactivation is obligatory for disease, Proc Natl Acad Sci USA, 88 (1991) 10540. protein synthesis stimulation by G protein-coupled receptors, 45 Mitchell BM, Dorrance AM & Webb RC, GTP Am J Physiol Cell Physiol, 283 (2002) 446. cyclohydrolase 1 inhibition attenuates vasodilation and 42 Liu B, Chen H, Johns TG & Neufeld AH, Epidermal growth increases blood pressure in rats, Am J Physiol - Heart C, factor receptor activation: an upstream signal for transition of 285 (2003) H2165. quiescent astrocytes into reactive astrocytes after neural 46 Farah CA, Liazoghli D, Perreault S, Desjardins M, Guimont injury, J Neurosci, 26 (2006) 7532. A, Anton A, Lauzon M, Kreibich G, Paiement J & Leclerc N, 43 Xiang YY, Dong H, Wan Y, Li J, Yee A, Yang BB & Lu Interaction of microtubule-associated protein-2 and p63, WY, Versican G3 domain regulates neurite growth J Biol Chem, 280 (2005) 9439. and synaptic transmission of hippocampal neurons by 47 Brand IA & Heinickel A, Key enzymes of carbohydrate activation of epidermal growth factor receptor, J Biol Chem, metabolism as targets of the 11.5 kDa Zn2+ binding protein 281 (2006) 19358. (Parathymosin), JBiol Chem, 266 (1991) 20984.