Unveiling the Potent Fibrino(geno)lytic, Anticoagulant, and Antithrombotic Effects of Papain, a Cysteine Protease from Carica papaya Latex Using κ-Carrageenan Rat Tail Thrombosis Model.

While fibrinolytic enzymes and thrombolytic agents offer assistance in treating cardiovascular diseases, the existing options are associated with a range of adverse effects. In our previous research, we successfully identified ficin, a naturally occurring cysteine protease that possesses unique fibrin and fibrinogenolytic enzymes, making it suitable for both preventing and treating cardiovascular disorders linked to thrombosis. Papain is a prominent cysteine protease derived from the latex of Carica papaya. The potential role of papain in preventing fibrino(geno)lytic, anticoagulant, and antithrombotic activities has not yet been investigated. Therefore, we examined how papain influences fibrinogen and the process of blood coagulation. Papain is highly stable at pH 4-11 and 37-60 °C via azocasein assay. In addition, SDS gel separation electrophoresis, zymography, and fibrin plate assays were used to determine fibrinogen and fibrinolysis activity. Papain has a molecular weight of around 37 kDa, and is highly effective in degrading fibrin, with a molecular weight of over 75 kDa. Furthermore, papain-based hemostatic performance was confirmed in blood coagulation tests, a blood clot lysis assay, and a κ-carrageenan rat tail thrombosis model, highlighting its strong efficacy in blood coagulation. Papain shows dose-dependent blood clot lysis activity, cleaves fibrinogen chains of Aα, Bß, and γ-bands, and significantly extends prothrombin time (PT) and activated partial thromboplastin time (aPTT). Moreover, the mean length of the infarcted regions in the tails of Sprague-Dawley rats with κ-carrageenan was shorter in rats administered 10 U/kg of papain than in streptokinase-treated rats. Thus, papain, a cysteine protease, has distinct fibrin and fibrinogenolytic properties, suggesting its potential for preventing or treating cardiovascular issues and thrombosis-related diseases.

Nemopilema nomurai jellyfish venom attenuates phenotypic modulation of PDGF BB-induced vascular smooth muscle cells and κ-carrageenan-induced rat tail thrombosis.

Jellyfish venoms have long been recognized as a potentially rich source of natural bioactive compounds with pharmacological potential for the creation of innovative drugs. Our previous study demonstrated that Nemopilema nomurai jellyfish venom (NnV) has a chymotrypsin-like serine protease with fibrinolytic activity in vitro. Therefore, the present study aims to investigate the potential effect of NnV on cell migration, proliferation, and differentiation of vascular smooth muscle cells (VSMC; A7r5 cells) involved in the probable mechanism pathways. We also determined its anti-thrombotic effect through κ-carrageenan-induced Sprague-Dawley (SD) rat tail thrombus model. NnV inhibits on Platelet-derived growth factor (PDGF)-BB-stimulated A7r5 cells migration and proliferation by decreasing matrix metalloproteinase 2 (MMP-2) level and phosphorylation of ERK and Akt in a dose-dependent manner, but not p38. Furthermore, NnV regulates the phenotype transition of differentiation in PDGF-BB-stimulated A7r5 cells via ɑ-SMA and calponin in a dose-dependent manner. In an in vivo study, NnV treatment demonstrated clear anti-thrombotic activity in a dose-dependent manner, which was associated with decreased thrombus formation and length in κ-carrageenan-induced SD rat tail. These findings suggested that NnV has a novel fibrinolytic enzyme that can be used to prevent and/or treat thrombosis-related cardiovascular disorders.

Pharmacoinformatic Investigation of Silymarin as a Potential Inhibitor against Nemopilema nomurai Jellyfish Metalloproteinase Toxin-like Protein.

Jellyfish stings pose a major threat to swimmers and fishermen worldwide. These creatures have explosive cells containing one large secretory organelle called a nematocyst in their tentacles, which contains venom used to immobilize prey. Nemopilema nomurai, a venomous jellyfish belonging to the phylum Cnidaria, produces venom (NnV) comprising various toxins known for their lethal effects on many organisms. Of these toxins, metalloproteinases (which belong to the toxic protease family) play a significant role in local symptoms such as dermatitis and anaphylaxis, as well as systemic reactions such as blood coagulation, disseminated intravascular coagulation, tissue injury, and hemorrhage. Hence, a potential metalloproteinase inhibitor (MPI) could be a promising candidate for reducing the effects of venom toxicity. For this study, we retrieved the Nemopilema nomurai venom metalloproteinase sequence (NnV-MPs) from transcriptome data and modeled its three-dimensional structure using AlphaFold2 in a Google Colab notebook. We employed a pharmacoinformatics approach to screen 39 flavonoids and identify the most potent inhibitor against NnV-MP. Previous studies have demonstrated the efficacy of flavonoids against other animal venoms. Based on our analysis, Silymarin emerged as the top inhibitor through ADMET, docking, and molecular dynamics analyses. In silico simulations provide detailed information on the toxin and ligand binding affinity. Our results demonstrate that Silymarin's strong inhibitory effect on NnV-MP is driven by hydrophobic affinity and optimal hydrogen bonding. These findings suggest that Silymarin could serve as an effective inhibitor of NnV-MP, potentially reducing the toxicity associated with jellyfish envenomation.

Identification of cardiorespiratory toxic components of Nemopilema nomurai jellyfish venom using sequential chromatography methods.

Jellyfish stings pose a significant threat to humans in coastal areas worldwide, with venomous jellyfish species stinging millions of individuals annually. Nemopilema nomurai is one of the largest jellyfish species, with numerous tentacles rich in nematocysts. N. nomurai venom (NnV) is a complex mixture of proteins, peptides, and small molecules that serve as both prey-capture and defense mechanisms. Yet, the molecular identity of its cardiorespiratory and neuronal toxic components of NnV has not been clearly identified yet. Here, we isolated a cardiotoxic fraction, NnTP (Nemopilema nomurai toxic peak), from NnV using chromatographic methods. In the zebrafish model, NnTP exhibited strong cardiorespiratory and moderate neurotoxic effects. LC-MS/MS analysis identified 23 toxin homologs, including toxic proteinases, ion channel toxins, and neurotoxins. The toxins demonstrated a synergistic effect on the zebrafish, leading to altered swimming behavior, hemorrhage in the cardiorespiratory region, and histopathological changes in organs such as the heart, gill, and brain. These findings provide valuable insights into the mechanisms underlying the cardiorespiratory and neurotoxic effects of NnV, which could be useful in developing therapeutic strategies for venomous jellyfish stings.

Comparative Study of Toxic Effects and Pathophysiology of Envenomations Induced by Carybdea brevipedalia (Cnidaria: Cubozoa) and Nemopilema nomurai (Cnidaria: Scyphozoa) Jellyfish Venoms.

Jellyfish stings can result in local tissue damage and systemic pathophysiological sequelae. Despite constant occurrences of jellyfish stings in oceans throughout the world, the toxinological assessment of these jellyfish envenomations has not been adequately reported in quantitative as well as in qualitative measurements. Herein, we have examined and compared the in vivo toxic effects and pathophysiologic alterations using experimental animal models for two representative stinging jellyfish classes, i.e., Cubozoa and Scyphozoa. For this study, mice were administered with venom extracts of either Carybdea brevipedalia (Cnidaria: Cubozoa) or Nemopilema nomurai (Cnidaria: Scyphozoa). From the intraperitoneal (IP) administration study, the median lethal doses leading to the deaths of mice 24 h post-treatment after (LD50) for C. brevipedalia venom (CbV) and N. nomurai venom (NnV) were 0.905 and 4.4697 mg/kg, respectively. The acute toxicity (i.e., lethality) of CbV was much higher with a significantly accelerated time to death value compared with those of NnV. The edematogenic activity induced by CbV was considerably (83.57/25 = 3.343-fold) greater than NnV. For the evaluation of their dermal toxicities, the epidermis, dermis, subcutaneous tissues, and skeletal muscles were evaluated toxinologically/histopathologically following the intradermal administration of the venoms. The minimal hemorrhagic doses (MHD) of the venoms were found to be 55.6 and 83.4 µg/mouse for CbV and NnV, respectively. Furthermore, the CbV injection resulted in extensive alterations of mouse dermal tissues, including severe edema, and hemorrhagic/necrotic lesions, with the minimum necrotizing dose (MND) of 95.42 µg/kg body weight. The skin damaging effects of CbV appeared to be considerably greater, compared with those of NnV (MND = 177.99 µg/kg). The present results indicate that the toxicities and pathophysiologic effects of jellyfish venom extracts may vary from species to species. As predicted from the previous reports on these jellyfish envenomations, the crude venom extracts of C. brevipedalia exhibit much more potent toxicity than that of N. nomurai in the present study. These observations may contribute to our understanding of the toxicities of jellyfish venoms, as well as their mode of toxinological actions, which might be helpful for establishing the therapeutic strategies of jellyfish stings.

Exploring the Fibrin(ogen)olytic, Anticoagulant, and Antithrombotic Activities of Natural Cysteine Protease (Ficin) with the κ-Carrageenan-Induced Rat Tail Thrombosis Model.

Although fibrinolytic enzymes and thrombolytic agents help in cardiovascular disease treatment, those currently available have several side effects. This warrants the search for safer alternatives. Several natural cysteine protease preparations are used in traditional medicine to improve platelet aggregation and thrombosis-related diseases. Hence, this study aimed to investigate the effect of ficin, a natural cysteine protease, on fibrin(ogen) and blood coagulation. The optimal pH (pH 7) and temperature (37 °C) for proteolytic activity were determined using the azocasein method. Fibrinogen action and fibrinolytic activity were measured both electrophoretically and by the fibrin plate assay. The effect of ficin on blood coagulation was studied by conventional coagulation tests: prothrombin time (PT), activated partial thromboplastin time (aPTT), blood clot lysis assay, and the κ-carrageenan thrombosis model. The Aα, Bß, and γ bands of fibrinogen are readily cleaved by ficin, and we also observed a significant increase in PT and aPTT. Further, the mean length of the infarcted regions in the tails of Sprague-Dawley rats was shorter in rats administered 10 U/mL of ficin than in control rats. These findings suggest that natural cysteine protease, ficin contains novel fibrin and fibrinogenolytic enzymes and can be used for preventing and/or treating thrombosis-associated cardiovascular disorders.

Cloning of Metalloproteinase 17 Genes from Oriental Giant Jellyfish Nemopilema nomurai (Scyphozoa: Rhizostomeae).

We previously demonstrated that Nemopilema nomurai jellyfish venom metalloproteinases (JVMPs) play a key role in the toxicities induced by N. nomurai venom (NnV), including dermotoxicity, cytotoxicity, and lethality. In this study, we identified two full-length JVMP cDNA and genomic DNA sequences: JVMP17-1 and JVMP17-2. The full-length cDNA of JVMP17-1 and 17-2 contains 1614 and 1578 nucleotides (nt) that encode 536 and 525 amino acids, respectively. Putative peptidoglycan (PG) binding, zinc-dependent metalloproteinase, and hemopexin domains were identified. BLAST analysis of JVMP17-1 showed 42, 41, 37, and 37% identity with Hydra vulgaris, Acropora digitifera, Megachile rotundata, and Apis mellifera venom metalloproteinases, respectively. JVMP17-2 shared 38 and 36% identity with H. vulgaris and A. digitifera, respectively. Alignment results of JVMP17-1 and 17-2 with other metalloproteinases suggest that the PG domain, the tissue inhibitor of metalloproteinase (TIMP)-binding surfaces, active sites, and metal (ion)-binding sites are highly conserved. The present study reports the gene cloning of metalloproteinase enzymes from jellyfish species for the first time. We hope these results can expand our knowledge of metalloproteinase components and their roles in the pathogenesis of jellyfish envenomation.

Rosa davurica Pall. improves DNCB-induced atopic dermatitis in mice and regulated TNF-Alpa/IFN-gamma-induced skin inflammatory responses in HaCaT cells.

PURPOSE: Rosa davurica Pall., is mainly distributed in Korea, Japan, northeastern China, southeastern Siberia, and eastern Asia. It has been extensively used to treat various kinds of diseases by reason of the significant antioxidant, antiviral and anti-inflammatory activities. However, the pharmacological mechanism of Rosa davurica Pall. in atopic dermatitis (AD) is still ill defined and poorly understood. This study was to examine the anti-inflammatory effects and its mechanism on AD of Rosa davurica Pall. leaves (RDL). METHODS: To evaluate the therapeutic potential of RDL against AD, we have investigated the effects of RDL on the inflammatory reactions and the productions of inflammatory chemokines and cytokines that were induced by tumor necrosis factor-α (TNF-α)/interferon-γ (IFN-γ) in HaCaT cells. Futhermore, we examined the effects of RDL on the signaling pathways of mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB). For the in-vivo studies, RDL extract was topically applied to the dinitrochlorobenzene (DNCB)-induced AD mice, then its therapeutic effect was evaluated physiologically and morphologically. RESULTS: After the stimulation of HaCaT cells with TNF-α/IFN-γ, RDL considerably reduced the release of inflammatory mediators such as nitric oxide (NO), PEG2 and other cytokines. RDL also reduced the phosphorylations of MAPK and NF-κB in TNF-α/IFN-γ-stimulated HaCaT cells. In vivo topical application of RDL to DNCB-induced AD mice significantly reduced the dorsal skin and ear thickness, clinical dermatitis severity, and mast cells. Treatment with RDL also markedly decreased the levels of serum IgE, IL-6 and the number of WBCs in the blood. CONCLUSION: Our studies indicate that RDL inhibits the AD-like skin lesions by modulating skin inflammation. Consequently, these results suggest that RDL may be served as a possible alternative therapeutic treatment for skin disorder such as AD.

Proteomic Changes during the Dermal Toxicity Induced by Nemopilema nomurai Jellyfish Venom in HaCaT Human Keratinocyte.

Jellyfish venom is well known for its local skin toxicities and various lethal accidents. The main symptoms of local jellyfish envenomation include skin lesions, burning, prickling, stinging pain, red, brown, or purplish tracks on the skin, itching, and swelling, leading to dermonecrosis and scar formation. However, the molecular mechanism behind the action of jellyfish venom on human skin cells is rarely understood. In the present study, we have treated the human HaCaT keratinocyte with Nemopilema nomurai jellyfish venom (NnV) to study detailed mechanisms of actions behind the skin symptoms after jellyfish envenomation. Using two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF/MS), cellular changes at proteome level were examined. The treatment of NnV resulted in the decrease of HaCaT cell viability in a concentration-dependent manner. Using NnV (at IC50), the proteome level alterations were determined at 12 h and 24 h after the venom treatment. Briefly, 70 protein spots with significant quantitative changes were picked from the gels for MALDI-TOF/MS. In total, 44 differentially abundant proteins were successfully identified, among which 19 proteins were increased, whereas 25 proteins were decreased in the abundance levels comparing with their respective control spots. DAPs involved in cell survival and development (e.g., Plasminogen, Vinculin, EMILIN-1, Basonuclin2, Focal adhesion kinase 1, FAM83B, Peroxisome proliferator-activated receptor-gamma co-activator 1-alpha) decreased their expression, whereas stress or immune response-related proteins (e.g., Toll-like receptor 4, Aminopeptidase N, MKL/Myocardin-like protein 1, hypoxia up-regulated protein 1, Heat shock protein 105 kDa, Ephrin type-A receptor 1, with some protease (or peptidase) enzymes) were up-regulated. In conclusion, the present findings may exhibit some possible key players during skin damage and suggest therapeutic strategies for preventing jellyfish envenomation.

Dataset of Swimming behavioral alterations in Danio rerio by Nemopilema nomurai jellyfish venom.

This article reports data associated with Prakash et al. [1]. Nemopilema nomurai jellyfish venom (NnV) can lead to neurotoxicity in zebrafish (Danio rerio) model. In the present study, zebrafish were treated with NnV by intraperitoneal injection and the swimming behavior of each fish was evaluated using a score scale. The dose of NnV in each treatment group was based on the protein concentration of NnV. Swimming is the main locomotory movements in the fishes. NnV modulated the swimming behavior of Danio rerio in a dose-dependent manner. In this article provided data are directly related to the previously published research article - "Danio rerio as an alternative vertebrate model for jellyfish venom study: the toxinological aspects of Nemopilema nomurai venom" [1] where the downregulation of acetylcholinesterase activity as well as histopathological alterations were observed from the brain of Danio rerio treated with NnV. Here we provide datasets, including mortality rate table, swimming behavior graph, and videos of zebrafish after NnV envenomation.

Danio rerio as an alternative vertebrate model for jellyfish venom study: The toxinological aspects of Nemopilema nomurai venom.

Nemopilema nomurai venom (NnV) is severely toxic to many organisms. However, the mechanism of its poisoning has not been properly understood yet. The present work demonstrates that zebrafish (Danio rerio) is an alternative vertebrate model for studying NnV jellyfish venom for the first time. In this model, NnV appears to cause severe hemorrhage and inflammation in cardiopulmonary regions of zebrafish. NnV also altered the swimming behavior of zebrafish accompanied by a significant downregulation of acetylcholinesterase (AChE) activity in brain tissues. Histopathological changes observed for various organs of D. rerio caused by NnV corresponded to an increase in lactate dehydrogenase (LDH) activity in tissues. NnV also significantly altered glutathione S-transferase (GST) activity in cardiopulmonary and brain tissues of D. rerio. SDS-PAGE revealed many protein bands of NnV of various sizes after silver staining. Taken together, these results indicate that Danio rerio can be a useful alternative animal model for jellyfish venom toxicology studies. Findings of the present study also suggest that Danio rerio could be used to develop an effective treatment strategy and discover the mechanism of action of jellyfish venom envenomation.

Protective effect of epigallocatechin-3-gallate (EGCG) on toxic metalloproteinases-mediated skin damage induced by Scyphozoan jellyfish envenomation.

Jellyfish stingings are currently raising serious public health concerns around the world. Hence, the search for an effective first aid reagent for the envenomation has been the goal of many investigators in the field. There have been a few previous reports of in vivo as well as in vivo studies suggesting the metalloproteinase activity of scyphozoan jellyfish venom, such as N. nomurai venom (NnV), plays a major role in the pathogenesis. These results have inspired us to develop a metalloproteinase inhibitor as a candidate for the treatment of Scyphozoan jellyfish envenomation. It has been previously demonstrated that the major polyphenol component in green tea, epigallocatechin-3-gallate (EGCG), can inhibit metalloproteinase activity of snake venoms. In fact, plant polyphenols as potential therapeutics have been shown to exert positive effects on neutralizing snake venoms and toxins. In the present study, we found that EGCG significantly inhibits the toxic proteases of NnV in a concentration-dependent manner. Human keratinocyte (HaCaT) and Human dermal fibroblast (HDF) cell culture studies showed that EGCG treatment can protect the cells from NnV-induced cytotoxicity which has been accompanied by the down-regulation of human matrix metalloproteinase (MMP)-2 and -9. Simulated rat NnV envenomation study disclosed that topical treatments with EGCG considerably ameliorated the progression of the dermonecrotic lesions caused by NnV. EGCG also reduced the activitions of tissue MMP-2 and MMP-9, which seem to be crucial players in the dermal toxic responses induced by NnV. Therefore, we propose that EGCG might be an effective therapeutic agent for the treatment of cutaneoous jellyfish symptoms.

Rosa davurica Pall. Improves Propionibacterium acnes-Induced Inflammatory Responses in Mouse Ear Edema Model and Suppresses Pro-Inflammatory Chemokine Production via MAPK and NF-κB Pathways in HaCaT Cells.

Acne, also known as acne vulgaris, is a common disorder of human skin involving the sebaceous gland and Propionibacterium acnes (P. acnes). Although there are a number of treatments suggested for acne, many of them have limitations in their safety and have efficacy issues. Therefore, there is a high demand to develop safe and effective novel acne treatments. In the present study, we demonstrate the protective effects of Rosa davurica Pall. leaves (RDL) extract against P. acnes-induced inflammatory responses in vitro and in vivo. The results showed that RDL dose-dependently inhibited the growth of skin bacteria, including P. acnes (KCTC3314) and aerobic Staphylococcus aureus (KCTC1621) or Staphylococcus epidermidis (KCTC1917). The downregulation of proinflammatory cytokines by RDL appears to be mediated by blocking the phosphorylations of mitogen-activated protein kinase (MAPK) and subsequent nuclear factor-kappa B (NF-κB) pathways in P. acnes-stimulated HaCaT cells. In a mouse model of acne vulgaris, histopathological changes were examined in the P. acnes-induced mouse ear edema. The concomitant intradermal injection of RDL resulted in the reduction of ear swelling in mice along with microabscess but exerted no cytotoxic effects for skin cells. Instrumental analysis demonstrated there were seven major components in the RDL extract, and they seemed to have important roles in the anti-inflammatory and antimicrobial effects of RDL. Conclusively, our present work showed for the first time that RDL has anti-inflammatory and antimicrobial effects against P. acnes, suggesting RDL as a promising novel strategy for the treatment of acne, including natural additives in anti-acne cosmetics or pharmaceutical products.

Proteomic Analysis of Novel Components of Nemopilema nomurai Jellyfish Venom: Deciphering the Mode of Action.

Nowadays, proliferation of jellyfish has become a severe matter in many coastal areas around the world. Jellyfish Nemopilema nomurai is one of the most perilous organisms and leads to significant deleterious outcomes such as harm to the fishery, damage the coastal equipment, and moreover, its envenomation can be hazardous to the victims. Till now, the components of Nemopilema nomurai venom (NnV) are unknown owing to scant transcriptomics and genomic data. In the current research, we have explored a proteomic approach to identify NnV components and their interrelation with pathological effects caused by the jellyfish sting. Altogether, 150 proteins were identified, comprising toxins and other distinct proteins that are substantial in nematocyst genesis and nematocyte growth by employing two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI/TOF/MS). The identified toxins are phospholipase A2, phospholipase D Li Sic Tox beta IDI, a serine protease, putative Kunitz-type serine protease inhibitor, disintegrin and metalloproteinase, hemolysin, leukotoxin, three finger toxin MALT0044C, allergens, venom prothrombin activator trocarin D, tripeptide Gsp 9.1, and along with other toxin proteins. These toxins are relatively well characterized in the venoms of other poisonous species to induce pathogenesis, hemolysis, inflammation, proteolysis, blood coagulation, cytolysis, hemorrhagic activity, and type 1 hypersensitivity, suggesting that these toxins in NnV can also cause similar deleterious consequences. Our proteomic works indicate that NnV protein profile represents valuable source which leads to better understanding the clinical features of the jellyfish stings. As one of the largest jellyfish in the world, Nemopilema nomurai sting is considered to be harmful to humans due to its potent toxicity. The identification and functional characterization of its venom components have been poorly described and are beyond our knowledge. Here is the first report demonstrating the methodical overview of NnV proteomics research, providing significant information to understand the mechanism of NnV envenomation. Our proteomics findings can provide a platform for novel protein discovery and development of practical ways to deal with jellyfish stings on human beings.

Therapeutic Effect of Rumex japonicus Houtt. on DNCB-Induced Atopic Dermatitis-Like Skin Lesions in Balb/c Mice and Human Keratinocyte HaCaT Cells.

Rumex japonicus Houtt. (RJ) is traditionally used in folk medicines to treat patients suffering from skin disease in Korea and other parts of East Asia. However, the beneficial effect of RJ extract on atopic dermatitis (AD) has not been thoroughly examined. Therefore, this study aimed to investigate the anti-inflammatory effects of RJ on AD in vitro and in vivo. Treatment with RJ inhibited the phosphorylation of mitogen-activated protein kinase (MAPK) as well as the activation of nuclear factor-kappa B (NF-κB) in tumor necrosis factor-α (TNF-α) stimulated in HaCaT cells. The five-week-old Balb/c mice were used as an AD-like mouse model by treating them with 1-chloro-2, 4-dinitrobenzene (DNCB). Topical administration of RJ to DNCB-treated mice significantly reduced clinical dermatitis severity, epidermal thickness, and decreased mast cell and eosinophil infiltration into skin and ear tissue. These results suggest that RJ inhibits the development of AD-like skin lesions by regulating the skin inflammation responses in HaCaT cells and Balb/c mice. Thus, RJ may be a potential therapeutic agent for AD.

A comparative study on the equine and camelid antivenoms upon cardiovascular changes induced with Hemiscorpius lepturus venom in rats.

OBJECTIVES: In this study, the neutralizing abilities of the equine and the recently introduced camelid antivenoms on the hemodynamic parameters (inotropism, chronotropism, and arrhythmogenicity) were assessed following envenomation by Hemiscorpius lepturus venom in rats. MATERIALS AND METHODS: At first, the electrophoretic profiles of both products were obtained by using the SDS-PAGE method (12.5%) and stained with Coomassie blue and silver nitrate. Secondly, different doses of the camelid antivenom (10, 50, and 100 µl) were given intravenously in 10 min before venom injection (400 µg/rat). The neutralizing potencies of camelid and equine antivenoms were measured by preincubation (100 µl) with H. lepturus venom for 30 min at room temperature. Finally, equal amounts of the antivenoms were injected intravenously to observe the hemodynamic changes. RESULTS: Based on the electrophoretic profile, it was evident that undesired proteins significantly decreased in equine antivenom, owing to impurities. Pretreatment with the camelid antivenom (100 µl), neutralized the elevation of the mean arterial pressure evoked with scorpion venom injection (88.15±4.56 versus 10.2±1.23 percent at the 8th min). The Incubation of the venom and the camelid antivenom counteracted the hemodynamic changes, but the equine product had no effect. The intravascular injection of the equine antivenom transiently increased the mean arterial pressure as compared to the control (108.67±8.63 mmHg versus 52.67±1.93 mmHg at the 10th min). CONCLUSION: The most obvious finding emerging from this study was that the camelid antivenom neutralized the hemodynamic changes in rats significantly, but in comparison, the equine antivenom had just a minor ability.

Hemodynamic Changes in Experimentally Envenomed Anaesthetized Rats by Intravenous Injection of Hemiscorpius lepturus Venom.

BACKGROUND: We investigated the hemodynamic changes (Inotropic, chronotropic and arrhythmogenic) in intravenously envenomed anesthetized rats with Hemiscorpius lepturus venom. The neutralizing potencies of different drugs and commercial antivenom were assessed simultaneously. METHODS: Different doses of the crude venom (100, 200 and 400µg/rat) were injected during five minutes via the femoral vein and cardiovascular changes were recorded in rats in Razi Institute Corporation, Karaj, Iran in 2017. The drugs (Atropine, lidocaine, propranolol and prazosin) were injected before the venom for determination of the counteracting effects. Different volumes (100, 500 and 1000µl) of the antivenom were pre envenomed to neutralize cardiovascular changes. RESULTS: Temporary hypertension and bradycardia with no arrhythmogenic effects were depicted within twenty minutes. There was a difference in arterial pressure between the venom (400µg/rat) and the vehicle at 8 minutes (114.68±5.1mmHg versus 70.2±4.3mmHg). Elevation of the mean arterial pressure was inhibited by propranolol (2 mg/kg) and neutralized by prazosin (1mg/kg) while lidocaine (4mg/kg) and atropine (1mg/kg) had no effects. Premedication with Iranian commercial antivenom (1000µl) produced surprisingly temporary hypertension compared to the vehicle (140.84±4.5 versus 84.3±3.2). It had no neutralizing properties on blood pressure variation before the venom injection. Volume-expanded hypertension phenomenon was ruled out in a parallel study. CONCLUSION: This venom has vasoconstrictive effects in rats probably due to the presence of norepinephrine like materials in its content or liberated from adrenal gland inhibited by prazosin premedication. The neutralizing effects of antivenom on venom-induced hypertension are questionable.

Proteomic Investigation to Identify Anticancer Targets of Nemopilema nomurai Jellyfish Venom in Human Hepatocarcinoma HepG2 Cells.

Nemopilema nomurai is a giant jellyfish that blooms in East Asian seas. Recently, N. nomurai venom (NnV) was characterized from a toxicological and pharmacological point of view. A mild dose of NnV inhibits the growth of various kinds of cancer cells, mainly hepatic cancer cells. The present study aims to identify the potential therapeutic targets and mechanism of NnV in the growth inhibition of cancer cells. Human hepatocellular carcinoma (HepG2) cells were treated with NnV, and its proteome was analyzed using two-dimensional gel electrophoresis, followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF/MS). The quantity of twenty four proteins in NnV-treated HepG2 cells varied compared to non-treated control cells. Among them, the amounts of fourteen proteins decreased and ten proteins showed elevated levels. We also found that the amounts of several cancer biomarkers and oncoproteins, which usually increase in various types of cancer cells, decreased after NnV treatment. The representative proteins included proliferating cell nuclear antigen (PCNA), glucose-regulated protein 78 (GRP78), glucose-6-phosphate dehydrogenase (G6PD), elongation factor 1γ (EF1γ), nucleolar and spindle-associated protein (NuSAP), and activator of 90 kDa heat shock protein ATPase homolog 1 (AHSA1). Western blotting also confirmed altered levels of PCNA, GRP78, and G6PD in NnV-treated HepG2 cells. In summary, the proteomic approach explains the mode of action of NnV as an anticancer agent. Further characterization of NnV may help to unveil novel therapeutic agents in cancer treatment.

Nemopilema nomurai jellyfish venom exerts an anti-metastatic effect by inhibiting Smad- and NF-κB-mediated epithelial-mesenchymal transition in HepG2 cells.

Epithelial-mesenchymal transition (EMT) is a key initial step in metastasis for malignant cancer cells to obtain invasive and motile properties. Inhibiting EMT has become a new strategy for cancer therapy. In our previous in vivo study, Nemopilema nomurai jellyfish venom (NnV) -treated HepG2 xenograft mice group showed that E-cadherin expression was strongly detected compared with non-treated groups. Therefore, this study aimed to determine whether NnV could inhibit the invasive and migratory abilities of HepG2 human hepatocellular carcinoma cells and to examine its effect on EMT. Our results revealed that transforming growth factor (TGF)-ß1 induced cell morphological changes and downregulated E-cadherin and ß-catenin expression, but upregulated N-cadherin and vimentin expression through the Smad and NF-κB pathways in HepG2 cells. Treatment of TGF-ß1-stimulated HepG2 cells with NnV reversed the EMT-related marker expression, thereby inhibiting cell migration and invasion. NnV also significantly suppressed the activation of p-Smad3, Smad4, and p-NF-κB in a dose-dependent manner. These data indicated that NnV can significantly suppress cell migration and invasion by inhibiting EMT in HepG2 cells, and therefore might be a promising target for hepatocellular carcinoma therapeutics.