The giant jellyfish Nemopilema nomurai sting can cause local and systemic reactions; however, comparative analysis of the tentacle extract (TE) and nematocyst venom extract (NV), and its toxicity, mechanism, and potential intervention are still limited. This study compared venom from TE and NV for their composition, toxicity, and efficacy in vitro and in vivo used RAW264.7 cells and ICR mice. A total of 239 and 225 toxin proteins were identified in TE and NV by proteomics, respectively. Pathological analysis revealed that TE and NV caused heart and liver damage through apoptosis, necrosis, and inflammation, while TE exhibited higher toxicity ex vivo and in vivo. Biochemical markers indicated TE and NV elevated creatine kinase, lactatedehydrogenase, and aspartate aminotransferase, with the TE group showing a more significant increase. Transcriptomics and Western blotting indicated both venoms increased cytokines expression and MAPK signaling pathways. Additionally, 1 mg/kg PACOCF3 (the phospholipase A2 inhibitor) improved survival from 16.7% to 75% in mice. Our results indicate that different extraction methods impact venom activities, tentacle autolysis preserves toxin proteins and their toxicity, and PACOCF3 is a potential antidote, which establishes a good extraction method of jellyfish venom, expands our understanding of jellyfish toxicity, mechanism, and provides a promising intervention.

OBJECTIVE: To determine whether moxibustion had an anti-inflammatory effect on rheumatoid arthritis (RA) by regulating Annexin 1 expression and interfering with the phospholipaseA2 signaling pathway.
METHODS: Thirty male Sprague-Dawley rats were randomly categorized into five groups (six rats per group): blank control (CON) group, RA model (RA) group, moxibustion (MOX) group, Annexin 1 lentiviral intervention (RNAi-Anxa1) group, and Annexin 1 lentiviral intervention + moxibustion (RNAi-Anxa1 + MOX) group. The rats in the RNAi-Anxa1 and the RNAi-Anxa1 + MOX groups were injected with the lentiviral vector-mediated RNAi-Anxa1 into the rat foot pad. An experimental RA rat model was established by injecting Freund\'s complete adjuvant (FCA) into the RA, MOX, RNAi-Anxa1, and RNAi-Anxa1 + MOX groups. Rats in the MOX and RNAi-Anxa1 + MOX groups received moxibustion treatment. After modeling, using moxibustion \"Shenshu (BL23)\" and \"Zusanli (ST36)\", each point is 5 times, bilateral alternating, once a day, 6 times for a course of treatment, between the courses of rest for a one day. A total of three treatment courses were conducted. Both bilateral pad thicknesses were measured using Vernier calipers on experimental days 1, 7, 14, 21, and 28. The expression of cPLA2α signaling in the synovium of diseased joints was observed using Western blot. The pathology of the rat ankle synovium was observed using hematoxylin-eosin (HE) staining. Interleukin (IL)-1β, IL-10, prostaglandin E2 (PGE2), and leukotriene B4 (LTB4) were detected using enzyme-linked immunosorbent assay.
RESULTS: Moxibustion increased the levels of Annexin 1 and decreased the inflammatory response in rats with RA. After increasing the expression of Annexin 1, the phosphorylated expression of cPLA2α was inhibited, the serum levels of IL-1β, PGE2, and LTB4 decreased, and the level of IL-10 increased. In moxibustion treated RA rats after the Annexin 1 lentiviral intervention, the serum levels of IL-1β, PGE2, LTB4, and IL-10 were almost unchanged.
CONCLUSIONS: Moxibustion enhanced the negative regulation of the cPLA2α signaling pathway, increased the synovial Annexin 1 expression, inhibited the cPLA2α signaling pathway, indirectly inhibited the expression of downstream inflammatory factors, and played a role in reducing inflammation.

Phospholipase A2 (PLA2) is an enzyme present in appreciable quantity in snake venoms which catalyze the hydrolysis of glycerophospholipids at sn-2 position and promote the release of lysophospholipids and fatty acids. 5-methylcoumarin-4-β-glucoside (5MC4BG) and lupeol were previously isolated from the leaves of V. glaberrima. The aim of this research was to evaluate effect of these compounds as potential inhibitors of snake venom toxins of Naja nigricollis using an in vitro and in silico studies. Antisnake venom studies was conducted using acidimetry while the molecular docking analysis against PLA2 enzyme from N. nigricollis was performed using Auto Dock Vina and ADME-Tox analysis was evaluated using swissADME and ProTox-II online servers. The two compounds (5MC4BG and Lupeol) were able to inhibit the hydrolytic actions of PLA2 enzyme with percentage inhibition ranging from 23.99 to 72.36 % and 21.97-24.82 % at 0.0625-1.00 mg/mL respectively while the standard ASV had 82.63 % at 1.00 mg/mL after 10 min incubation at 37 °C. Similar effects were observed after 30 min incubation, although there was significant increase in percentage inhibition of 5MC4BG and lupeol ranging from 66.51 to 83.73 % and 54.87-59.60 % at similar concentrations. Furthermore, the compounds were able to bind to the active site of PLA2 enzyme with high affinity (-7.7 to -6.3 kcal/mol); the standard ligand, Varespladib had a docking score of -6.9 kcal/mol and they exhibited favorable drug-likeness and pharmacokinetic properties and according to toxicity predictions, the two compounds are toxic. In conclusion, the leaf of V. glaberrima contains phytoconstituents with antisnake activity and thus, validates the hypothesis that, the phytoconstituents of V. glaberrima leaves has antisnake venom activity against N. nigricollis venom and thus, should be studied further for the development as antisnake venom agents.

Snakebite envenomation often induces acute kidney injury (AKI) and acute liver injury (ALI), leading to augmented injuries and poor rehabilitation. Phospholipase A2 (PLA2) and metalloproteinase (SVMP) present in venom are responsible for the envenomation-associated events. In this study, mice envenomed with Deinagkistrodon acutus, Naja atra, or Agkistrodon halys pallas venom exhibited typical AKI and ALI symptoms, including significantly increased plasma levels of myoglobin, free hemoglobin, uric acid, aspartate aminotransferase, and alanine aminotransferase and upregulated expression of kidney NGAL and KIM-1. These effects were significantly inhibited when the mice were pretreated with natural inhibitors of PLA2 and SVMP isolated from Sinonatrix annularis (SaPLIγ and SaMPI). The inhibitors protected the physiological structural integrity of the renal tubules and glomeruli, alleviating inflammatory infiltration and diffuse hemorrhage in the liver. Furthermore, the dual therapy alleviated oxidative stress and apoptosis in the kidneys and liver by mitigating mitochondrial damage, thereby effectively reducing the lethal effect of snake venom in the inhibitor-treated mouse model. This study showed that dual therapy with inhibitors of metalloproteinase and phospholipase can effectively prevent ALI and AKI caused by snake bites. Our findings suggest that intrinsic inhibitors present in snakes are prospective therapeutic agents for multi-organ injuries caused by snake envenoming.

BACKGROUND: Snakebite envenomation (SBE) is the world\'s most lethal neglected tropical disease. Bothrops jararaca is the species that causes the greatest number of SBEs in the South and Southeastern of Brazil. The main symptoms are local (inflammation, edema, hemorrhage, and myonecrosis) and systemic (hemorrhage, hemostatic alterations with consumptive coagulopathy, and death) effects. Species of the genus Siparuna, Siparunaceae, are used in folk and traditional medicine to treat SBE. However, limited information is available concerning Brazilian Siparuna species against SBE.
OBJECTIVE: To investigate the correlation between the compounds present in the extracts of five Siparuna species as potential agents against proteolytic activity, plasma coagulation, and phospholipase A2 (PLA2) activity caused by B. jararaca venom, using data obtained by UHPLC-MS/MS, biological activity, and multivariate statistics.
METHODS: The ethanol extracts from leaves of S. ficoides, S. decipiens, S. glycycarpa, S. reginae, and S. cymosa were fractionated by liquid-liquid extraction using different solvents of increasing polarity (hexane, dichloromethane, ethyl acetate, and n-butanol), affording their respective extracts, totaling 25 samples that were assayed through in vitro plasma coagulation and proteolytic activity assays. Moreover, the extracts were analyzed by UHPLC-MS/MS, using electrospray ionization (ESI) and atmospheric-pressure chemical ionization (APCI) in negative and positive ionization modes. The data was processed in MZmine v. 2.53 and evaluated by multivariate statistical tests (PLS) using the software UnscramblerX v. 10.4. These data were also used to build molecular networks (GNPS), and some ions of interest could be annotated using the library of molecules on the GNPS platform.
RESULTS: A total of 19 extracts inhibited B. jararaca-induced plasma coagulation, with emphasis on S. cymosa and S. reginae (800 s). The inhibition of the proteolytic activity was also promising, ranging from 16% (S. glycycarpa) to 99% (S. cymosa, S. decipiens, and S. reginae). In addition, most extracts from S. cymosa and S. reginae inhibited 70-90% of PLA2 activity. Based on data from positive mode APCI analyses, it was possible to obtain a statistic model with reliable predictive capacity which exhibited an average R2 of 0.95 and a Q2 of 0.88, indicating a robust fit. This process revealed five ions, identified as the alkaloids: coclaurine (1), stepholidine (2) O-methylisopiline (3), nornantenine (4) and laurolitsine (5). This is the first study to evidence the potential antivenom of alkaloids from Siparuna species.
CONCLUSIONS: Altogether, our results give support to the popular use of Siparuna extracts in SBE accidents, suggesting their potential as an alternative or complementary strategy against envenoming by B. jararaca venom. The predicted ions in the chemometric analysis for the assayed activities can also be correlated with the blocking activity and encourage the continuation of this study for possible isolation and testing of individual compounds on the used models.

Catuneragam nilotica has been used in ethnomedicine to treat snakebite, inflammation, and diarrhea among others. The aim of this research is to isolate, and characterize potential potential phospholipase A2 (PLA2) inhibitors from the roots of C. nilotica. The plant material was collected, authenticated, and sequentially extracted using solvents of increasing polarity starting from n-hexane, ethyl acetate, and methanol. The extracts as reported in our previous work, were screened in vitro for their inhibitory activity against PLA2 enzyme from N. nigricollis venom using acidimetric assay. In line with the bio-activity guided isolation, methanol extract (being the most active) was subjected to chromatographic separation using silica gel and sephadex LH-20 which resulted in the isolation and characterization of scopoletin, and scopolin; the compounds were able to inhibit the hydrolytic actions of PLA2 enzyme with percentage inhibition ranging from 67.82 to 100.00 % and 65.76-93.15 %, respectively while the standard Antisnake Venom (ASV) had 74.96-85.04 % after 10 min incubation at 37 °C. The molecular docking of the compounds against PLA2 enzyme was performed using Auto Dock Vina while ADME-Tox analysis was evaluated using swissADME and ProTox-II online servers; The findings indicated that both compounds were able to bind to the active site of PLA2 enzyme with high affinity (-6.5 to -6.2 kcal/mol) and they exhibited favorable drug-likeness and pharmacokinetic properties, and according to toxicity predictions, scopolin was found to be non-toxic (LD50 of 5000 mg/kg) while scopoletin has a slight chance of being toxic (LD50 of 3800 mg/kg). In conclusion, the findings of the research revealed that the roots of C. nilotica contains phytoconstituents with anti-PLA2 enzyme activity and thus, validates the ethnomedicinal claim of the use of the plant as herbal therapy against N. nigricollis envenomation.

Crotalus neutralizing factor (CNF) is an endogenous glycoprotein from Crotalus durissus terrificus snake blood that inhibits secretory phospholipases A2 (sPLA2) from the Viperid but not from Elapid venoms (subgroups IA and IIA, respectively). In the present study, we demonstrated that CNF can inhibit group III-PLA2 from bee venom by forming a stable enzyme-inhibitor complex. This finding opens up new possibilities for the potential use of CNF and/or CNF-based derivatives in the therapeutics of bee stings.

In Brazil, the genus Bothrops is responsible for most ophidian accidents. Snake venoms have a wide variety of proteins and peptides exhibiting a broad repertoire of pharmacological and toxic effects that elicit systemic injury and characteristic local effects. The snakes\' natural resistance to envenomation caused by the presence of inhibitory compounds on their plasma have been extensively studied. However, the presence of these inhibitors in different developmental stages is yet to be further discussed. The aim of this study was to evaluate the ontogeny of Bothrops jararaca plasma inhibitor composition and, to this end, plasma samples of B. jararaca were obtained from different developmental stages (neonates, youngs, and adults) and sexes (female and male). SDS-PAGE, Western blotting, affinity chromatography, and mass spectrometry were performed to analyze the protein profile and interaction between B. jararaca plasma and venom proteins. In addition, the presence of γBjPLI, a PLA2 inhibitor previously identified and characterized in B. jararaca serum, was confirmed by Western blotting. According to our results, 9-17% of plasma proteins were capable of binding to venom proteins in the three developmental stages. The presence of different endogenous inhibitors and, more specifically, different PLA2 inhibitor (PLI) classes and antihemorrhagic factors were confirmed in specimens of B. jararaca from newborn by mass spectrometry. For the first time, the αPLI and βPLI were detected in B. jararaca plasma, although low or no ontogenetic and sexual correlation were found. The γPLI were more abundant in adult female, than in neonate and young female, but similar to neonate, young and adult male according to the results of mass spectrometry analysis. Our results suggest that there are proteins in the plasma of these animals that can help counteract the effects of self-envenomation from birth.

Antivenom is currently the standard-of-care treatment for snakebite envenoming, but its efficacy is limited by treatment delays, availability, and in many cases, species specificity. Many of the rapidly lethal effects of envenoming are caused by venom-derived toxins, such as phospholipase A2 (sPLA2); therefore, small molecule direct toxin inhibitors targeting these toxins may have utility as initial and adjunct therapies after envenoming. Varespladib (intravenous, IV) and varespladib-methyl (oral) have been shown to potently inhibit sPLA2s from snake venoms in murine and porcine models, thus supporting their further study as potential treatments for snakebite envenoming. In this pilot study, we tested the ability of these compounds to reverse neurotoxic effects of venom from the Australian and Papuan taipan (Oxyuranus scutellatus) subspecies in juvenile pigs (Sus domesticus). The mean survival time for control animals receiving Australian taipan venom (0.03 mg/kg, n = 3) was 331 min ± 15 min; for those receiving Papuan taipan venom (0.15 mg/kg, n = 3) it was 178 ± 31 min. Thirteen pigs received Australian taipan venom and treatment with either IV or oral varespladib (or with IV to oral transition) and all 13 survived the duration of the study (≥96 h). Eight pigs received Papuan taipan venom followed by treatment: Briefly: Two animals received antivenom immediately and survived to the end of the study. Two animals received antivenom treatment delayed 45 min from envenoming and died within 4 h. Two animals received similarly delayed antivenom treatment and were rescued by varespladib. Two animals were treated with varespladib alone after a 45-min delay. Treatment with varespladib only was effective but required repeat dosing over the course of the study. Findings highlight both the importance of early treatment and, as well, a half-life for the investigational inhibitors now in Phase II clinical trials for snakebite. Varespladib rapidly reversed weakness even when administered many hours post-envenoming and, overall, our results suggest that varespladib and varespladib-methyl could be efficacious tools in the treatment of sPLA2-induced weakness from Oxyuranus envenoming. Further clinical study as initial therapy and as potential method of rescue from some types of antivenom-resistant envenomings are supported by these data.

Snake venom PLA2, a member of the group of hydrolase enzymes, has been recognized as a promising drug target for snake envenomation. In the present study, an attempt was made to identify potential inhibitors of snake venom PLA2 by employing a pharmacophore-based virtual screening, docking, and dynamics approach. A receptor-based pharmacophore model was generated based on the features of the established and bound co-crystal ligand (2-carbamoylmethyl-5-propyl-octahydro-indol-7-yl)-acetic acid in the PLA2 complex. The best pharmacophore model (ADDH) derived, consisted of four features, namely one hydrogen bond acceptor, two hydrogen bond donors, and one hydrophobic region. This common pharmacophore was then used to perform virtual screening against a drug-like diverse database, with due consideration to the Lipinski \'rule of five\', so as to obtain a pool of lead molecules. The short-listed lead molecules were then subjected to docking analysis with that of the Daboia russelli viper venom PLA2 followed by a molecular simulation study for a duration of 100 ns. CAP04815700 was chosen as the best compound based on the simulation parameters, which were then taken for MM/PBSA calculation, and it was revealed that it has a similar effective inhibitory potential as that of the crystal ligand. Further, the cluster analysis also revealed the structural significance of the backbone protein after the interaction with CAP04815700. This study will continue to explore its bioactivity in vitro and in vivo.Communicated by Ramaswamy H. Sarma.