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Synergism is a significant phenomenon present in snake venoms that may be an evolving strategy to potentiate toxicities. Synergism exists between different toxins or toxin complexes in various snake venoms, with phospholipaseA 2 s (PLA 2 s) (toxins or subunits) the main enablers. The predominant toxins, snake venom PLA 2 s, metalloproteases (SVMPs), serine proteases (SVSPs) and three-finger toxins (3FTxs), play essential roles in synergistic processes. The hypothetical mechanisms of synergistic effect can be generalized under the effects of amplification and chaperoning. The Toxicity Score is among the few quantitative methods to assess synergism. Selection of toxins involved in synergistically enhanced toxicity as the targets are important for development of novel antivenoms or inhibitors. [ABSTRACT FROM AUTHOR]

A novel phospholipaseA 2 (PLA 2 ) inhibitory protein (PLI) was purified from the serum of Macropisthodon rudis , a non-venomous snake mainly found in southern China. The molecular mass of the purified PLI was 160 kDa as determined by Superdex 200HR; however, the PLI protein had only one subunit of 25.4 kDa as determined by 12% SDS-PAGE, indicating an oligomeric protein. PLI cDNA obtained by PCR from the liver of Macropisthodon rudis, revealed 549 bps coding for a mature protein of 183 amino acid residues. Based on an amino acid sequence alignment with venomous and non-venomous snakes, this inhibitor was determined to be in the γ type family of PLI. In vitro experiments showed that PLIγ inhibited enzymatic, inflammatory, and antibacterial activities of snake venom PLA 2 isolated from Agkistrodon acutus. [ABSTRACT FROM AUTHOR]

Snake envenomation is well known to cause grievous pathological signs, including haemorrhagic discharge, necrosis, and respiratory distress. However, inflammatory reactions are also common envenoming manifestations that lead to successive damage, such as oedema, ulceration, lymphadenectasis, systemic inflammatory response syndrome (SIRS) and even multiple organ dysfunction syndrome (MODS). Interference with the inflammatory burst is hence important in the clinical treatment of snake envenomation. Here, we summarize the typical snake toxins (or venoms) that cause inflammatory reactions and the underlying signaling pathways. In brief, inflammatory reactions are usually triggered by snake venom phospholipase A2 (svPLA2), snake venom metalloprotease (SVMP), snake venom serine protease (SVSP) and C-type lectin/snaclec (CTL) as well as disintegrin (DIS) via multiple signaling pathways. They are nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing 3 (NLRP3), nuclear factor kappa-B (NF-κB), mitogen-activated protein kinase (MAPK), janus kinase/signal transducer and activator of transcription (JAK-STAT) and phosphoinositide 3-Kinase/protein kinase B (PI3K/PKB also called PI3K-AKT) signaling pathways. Activation of these pathways promotes the expression of pro-inflammatory molecules such as cytokines, especially interleukin-1β (IL-1β) which causes further inflammatory cascades and manifestations, such as swelling, fever, pain, and severe complications. Remarkably, almost half of introduced snake toxins (or venoms) have anti-inflammatory effects through blocking these pathways and suppressing the expression of pro-inflammatory molecules. Investigation of affected inflammation-related signaling pathways is meaningful to achieve better clinical treatment. [Display omitted] • Inflammatory reactions after snakebite are usually triggered by snake toxins via multiple mechanisms. • Multiple mechanisms mainly refer to five signalling pathways, which finally point to cytokines, especially interleukin-1β. • This review introduced 14 snake venoms (or toxins) in total, almost half of which exhibit anti-inflammatory effects. • Due to the limitation of antivenom, more treatments are explored based on mechanisms affected by snake venoms (or toxins). [ABSTRACT FROM AUTHOR]

Abstract The gamma-type inhibitor of snake venom phospholipase A 2 (PLIγ) is expressed extensively in livers of both venomous and non-venomous snakes. It is not clear why PLIγs from different snake species possess diverse activities. To obtain high activity PLIγs and interpret the sequence-function relationships, we used DNA shuffling to hybridize the PLIγs of Sinonatrix annularis (saPLIγ) and Elaphe carinata (ecPLIγ). Chimera PLIγs (cPLIγ) of ∼550 bp were obtained by a series of gene manipulations including DNase I digestion, primer-free PCR, and PCR amplification with PLIγs primer pair. After successful insertion of cPLIγs into pCANTAB5e phage vector, the transformed TG1 strain of Esherichia coli was achieved. The cPLIγ phage library was produced and panned in a five-pace snake venom-coated immune tube. Three high affinity cPLIγ isoforms survived two rounds of panning. Prokaryote expression by the pET28c vector was employed for production of the three cPLIγs and the two parental PLIγs. These all showed anti-hemorrhage activity with cPLIγ 2 demonstrating superior inhibition to the parent PLIγs. Sequence alignment showed that the three kinds of cPLIγ were produced by gene splicing of S. annularis and E. carinata at different sites. Primary sequence changes brought regional changes in secondary and tertiary structure, which may explain the differences in PLIγ activity. Highlights • PLIγ can neutralize snake venom PLA2s, and is regarded as a promising antivenom candidate. • DNA shuffling and phage surface display is an effective way for PLIγ directed evolution. • The chimera PLIγ 2 showed even superior anti-hemorrhage activity than the parent PLIγs. • The α-helix of 150–155aa was believed to be correlative to the activity of PLIγ. [ABSTRACT FROM AUTHOR]

Phospholipase A2s (PLA2) is a major component of snake venom with diverse pathologic toxicities and, therefore, a potential target for antivenom therapy. Varespladib was initially designed as an inhibitor of mammal PLA2s, and was recently repurposed to a broad-spectrum inhibitor of PLA2 in snake venom. To evaluate the protective abilities of varespladib to hemorrhage, myonecrosis, and systemic toxicities that are inflicted by different crude snake venoms, subcutaneous ecchymosis, muscle damage, and biochemical variation in serum enzymes derived from the envenomed mice were determined, respectively. Varespladib treatment showed a significant inhibitory effect to snake venom PLA2, which was estimated by IC50 in vitro and ED50 in vivo. In animal models, the severely hemorrhagic toxicity of D. acutus and A. halys venom was almost fully inhibited after administration of varespladib. Moreover, signs of edema in gastrocnemius muscle were remarkably attenuated by administration of varespladib, with a reduced loss of myonecrosis and desmin. Serum levels of creatine kinase, lactate dehydrogenase isoenzyme 1, aspartate transaminase, and alanine transaminase were down-regulated after treatment with varespladib, which indicated the protection to viscera injury. In conclusion, varespladib may be a potential first-line drug candidate in snakebite envenomation first aid or clinical therapy. [ABSTRACT FROM AUTHOR]

SaPLIγ is a novel gamma phospholipase A 2 inhibitor (PLI) recently isolated from Sinonatrix annularis , a Chinese endemic non-venomous snake. To explore the neutralization effects of saPLIγ in snakebite envenomation, a dose equivalent to LD 50 of Deinagkistrodon acutus , Agkistrodon halys and Naja atra venom with/without saPLIγ was inoculated into the gastrocnemius muscle of female Kunming mice. The ability of saPLIγ to inhibit myonecrosis and systemic toxicity were evaluated through investigations of muscle histopathology, and determination of the serum levels of creatine kinase (CK), lactate dehydrogenase isoenzyme1 (LDH1) and aspartate transferase (AST). Edema of the gastrocnemius muscle was evaluated by calculating the width difference between the inoculated limb and the contralateral leg. Desmin loss in the gastrocnemius muscle was determined by Western blot analysis. Co-immunoprecipitation and shotgun LC-MS/MS analyses were performed to identify venom proteins that interact with saPLIγ. All the envenomed mice had significantly elevated serum CK, LDH1 and AST levels, whereas the levels were decreased significantly in the presence of saPLIγ. Histopathological evaluation of gastrocnemius muscle sections showed severe snake venom-induced damage, characterized by leukocyte infiltration and erythrocyte leakage, leading to local edema. Myonecrosis, hemorrhage and desmin loss were significantly attenuated by saPLIγ. SaPLIγ interacted with a wide range of venom proteins, including PLA 2 s, metalloproteinases and C type lectins, which may contribute to broad anti-venom effects. [ABSTRACT FROM AUTHOR]

Phospholipase A 2 (PLA 2 ) is a calcium-dependent enzyme that is involved in inflammatory processes such as the liberation of free arachidonic acid from the membrane pool for the biosynthesis of eicosanoids. Snake venom are known containing PLA 2 s (svPLA 2 s) which exhibit a wide variety of pharmacological effects including neurotoxicity, cardiotoxicity, myotoxicity and hemorrhage. Therefore, inhibition of svPLA 2 would be advantageous to successful envenomation treatment. A gamma type PLI (PLA 2 inhibitor) has been extracted from the serum of Sinonatrix annularis , a non-venomous snake indigenous to China. This showed strong inhibition of Deinagkistrrodon acutus PLA 2 , however, the PLIγ level in the serum and snake resource are not sufficiently sustainable for further research. To overcome these limitations, we constructed a His6-PLIγ pET28 fusion expression vector and transformed Escherichia coli BL21. To improve the expression of PLIγ, an orthogonal experiment [L 16 (4) 5 ] was performed to optimize induction parameters. The optimized condition was determined to be: induction by 0.4 mM isopropyl-β-d-thiogalactoside (IPTG) for 6 h to the recombinant BL21 after its OD 600 was 0.8, with continuous shaking cultivation at 190 rpm and 35 °C. Under these conditions, the amount of expressed protein could reach 57 mg/L. The His6-PLIγ was purified by nickel affinity chromatography and renatured by On-column refolding. The resulting PLIγ showed a good inhibitory effect of enzymatic activities to venom PLA 2 isolated from D. acutus . Moreover, the PLIγ had a wide anti-hemorrhage activities to D. acutus , Naja atra and Agkistrodon halys venom. [ABSTRACT FROM AUTHOR]