Kidney Sensitivity to the Non-Toxic Fraction F1 in Physiological and Non-Physiological State

Background: Scorpion venom contains several bioactive components that could be useful in therapeutic. Methods: In this study, the effects of the repeated administration of the nontoxic fraction of Androctonus australis hector (Aah) venom after carcinogenesis induced by N-Nitrosodiethylamine (DEN) were investigated on rats. Metabolic parameters and oxidative stress markers were evaluated in sera and kidney. Results: The obtained result revealed that DEN had disturbed the metabolic activities (an increase of transaminases and LDH activities) and had led to oxidative stress (a significant increase of NO and MDA levels and depletion of GSH rate) compared to the control. However, repeated injection of F1 shows a kind of modulatory effect on metabolism and oxidative stress. It shows a variable effect on metabolic markers (turn ASAT activity to normal level, recovered ALAT, and activities). A decrease of MDA level in sera and kidney and noticed an increase of GSH level in the kidney were observed. Conclusion: F1 fraction purified from Aah venom contains many bioactive components. These biomolecules lead to enzymatic activity changes and to modulate oxidative stress in the kidney in normal and carcinogenic model. These changes seem to be beneficial in the pathological case

Exploration of the antithrombotic effect of a C type lectin purified from Cerastes cerastes venom by protein-protein docking

Background: Thromboembolic diseases are a major clinical problem due to their high prevalence and their often fatal consequences. In the present study, an anticoagulant galactoside binding C type lectin “Cc-Lec” was purified and characterized. Methods: Cc-Lec was purified by affinity chromatography on a column of Sepharose 4B coupled with D-lactose. Its homogeneity was verified by SDS-PAGE and ESI-MS. Cc-Lec 3D structure modelization was achieved by homology to Convulxin a snake venom C type lectin. Cc-Lec anticoagulant effect was explored in vivo by i.p. administration to mice, in vitro by native PAGE analysis and in silico by protein-protein docking approach. Results: Cc-Lec is a 34 271,59 Da protein, composed of 160 residues of amino acids for each subunit. Its 3D structure is organized into a homodimer cross-linked with a disulfide bridge and composed of three alpha helices and seven beta strands for each monomer. Cc-Lec functional characterization revealed a durable anticoagulant effect in vivo after 6 and 48h of i.p. administration to mice. This anticoagulant effect is mediated by interaction with FXa and FIXa as showed by native PAGE analysis. Moreover, protein-protein docking results reinforced this data and showed that Cc-Lec interacts with coagulation factors X and IX through their -carboxyglutamic domains. The interaction with factors X and IX requires calcium or calcium and magnesium ions respectively. Conclusion: The anticoagulant effect of Cc-Lec makes it a promising pharmacological target for the diagnosis and/or the therapy of the thromboembolic diseases

New approach in the improvement of antiscorpionic immunotherapy: Input of TMG use as adjuvant for irradiated venom.

Background: Severe envenomation is a major public health problem in certain parts of the world such as North Africa, Central and South America, the Middle East, and South Asia. The toxic effects of scorpion envenomation are due to massive release of sympathetic and parasympathetic neurotransmitters; the severity is related to cardiac and hemodynamic changes, with cardiogenic shock and pulmonary edema contributing to the main causes of death. Due to this severity, intra-venous immunotherapy should be given to patients stung by scorpions as soon as possible. The production of specific antibodies of immunotherapy requires the use of highly immunogenic preparation to immunize sera producing animals, thus this preparation has to meet strict bioethics rules. The aim of this study was to determine the immunostimulating effect of TiterMax Gold adjuvant (TMG) associated to irradiated Androctonus australis hector venom in comparison to commonly used Freund’s complete adjuvant preparation Methods: Two groups of mice were immunized twice at one month intervals with TMG of FCA adjuvants associated to irradiated venom. The induced inflammatory and adaptive immune responses were evaluated. One month after the last booster, animals were challenged to ascending doses of native venom to evaluate their immunoprotectivness. Results: TMG preparation use induced inflammation characterized by low activation of polynuclear cell levels and MPO and EPO seric activities compared to FCA group. Evaluation of the titers of specific antibodies showed that TMG preparation was more effective in inducing higher titers of neutralizing antibodies. Results also showed that animals immunized with TMG preparation were highly protected up to 6 LD50 of native venom. Conclusion: These results allow to suggest the irradiated venom-TMG preparation as a more effective and secure preparation that could replace the FCA preparation in immunizing animal producers of antiscorpionic immune sera

Assessment of Inflammatory response of developed vaccine against scorpion envenomation using attenuated venom

Background: In Algeria Scorpion envenoming is very common, vaccine therapy based on irradiated venom antigens could be an attracted approach to prevent the lethal and pathophysiological effects induced after scorpion envenomation. The study aimed to evaluate the inflammatory response of detoxified Androctonus australis hector (Aah) venom used in vaccine approach against scorpion stings. Methods: This approach was undertaken with groups of three New-Zealand Oryctolagus cuniculus rabbits. The animals were inoculated three times at one-month interval. During the immunization schedule, blood samples were collected weekly after each injection. Cell count, serum peroxidase activities (MPO, EPO) and antibody titer (IgG) were evaluated. Six months after immunization, a protective effect of immunized rabbits with detoxified venom was evaluated by injection of different lethal doses of native Aah venom and mortality was recorded. Results: During the immunization schedule, low levels of peripheral neutrophil, eosinophil cell count and peroxidase activities were observed in sera of immunized animals with detoxified venom. Furthermore, immunological response showed high level of lymphocyte titers accompanied with high IgG titer at one month after immunization followed by gradual decrease that persisted at six months. More interestingly, detoxified venom can induce an immunoprotective effect six months after immunization against challenge with lethal doses until 6 DL50 of native venom. Conclusion: It seems that detoxified venom associated to alum adjuvant triggers a specific immune response with low inflammation. Despite antibody titer decreased with time, the protection remained higher. These results allowed this vaccine as a possible alternative to immunotherapy

Involvement of the AT1 Receptor in the Pathophysiology of the Respiratory Failure Induced by Scorpion Venom

Background: Renin-Angiotensin System (RAS) is related to cardiovascular diseases. It is also involved in the pathogenesis of pulmonary dysfunction, through the induction of proinflammatory mediators in lungs. Respiratory failure is a life-threatening complication in scorpion envenomation, a public health problem in Algeria. Our aim was to explore the role of angiotensin type I receptors (AT1R) in the respiratory dysfunction induced in an envenomed model with Androctonus australis hector (Aah) venom, in the presence or absence of an AT1R antagonist. Methods: Respiratory failure is assessed by the arterial blood gas analysis (pO2 and pCO2, sO2, hemoglobin and HCO3 concentration and pH), by neutrophil infiltration (myeloperoxidase activity) and oxidative stress markers (Nitrites, MDA, H2O2, catalase and GSH), in lungs and the pulmonary artery. Results: A severe respiratory dysfunction, including a marked perturbation of the oxygenation parameters and the acid base balance, reflecting a state of hypoxia and acidosis were induced by the venom. An important infiltration of neutrophils into the tissues and the generation of ROS with the alteration of the antioxidant system were found. Inhibition of the AT1R before envenomation revealed the recovery of the respiratory parameters in sera. The reduction of inflammatory cells infiltration and the prevention of the redox status imbalance were also recorded. Conclusion: These results indicate the involvement of Ang II throught the AT1R in the respiratory failure induced by Aah’s venom. However, further studies are still required to understand the RAS role in the pulmonary injury developed in the envenoming syndrome, a serious public health surgery that must be immediately treated

CcMP-II Metalloproteinase from Cerastes Cerastes Snake Venom Acts both on Blood Vessel ECM and Platelets

Background: Snake venoms contain various metalloproteinases that are highly toxic, inducing in severe bleeding by interfering with the blood coagulation and degrading the basement membrane or extracellular matrix (ECM) components. It has been suggested that hemorrhagic metalloproteinases interact in a specific way with platelet surface proteins resulting in an alteration of platelet function.  Hemorrhage and tissue necrosis are common manifestations of viperid envenomations in humans, largely due to the actions of prominent metalloproteinases. CcMP-II a weakly hemorrhagic metalloproteinase purified from Cerastes cerastes venom, as already described it belongs to the class P-II snake venom metalloproteinase. Methods: In this study, we characterized the biological effects of CcMP-II, including the ability of the enzyme to hydrolyze extracellular matrix components and plasma proteins, as well as its histopathological effects induced in capillaries. Then a study of the anti-aggregating activity was carried out and controlled for 10 min using a Chronolog aggregometer (ServiBIO) (USTHB, Faculty of Biological Sciences, BP 32, El-Alia Bab Ezzouar, 16111, Algiers, Algeria in 2019). The histopathological changes induced by CcMP-II were also analyzed and the 1 µm semi-thin sections are then stained with Toluidine Blue and the observation of these sections was realized using a photomicroscope. Toluidine Blue, magnification: 100x. Results: Obtained results showed that CcMP-II metalloproteinase hydrolyzed selectively the A α-chain of fibrinogen. This enzyme hydrolyzed also laminin and type IV collagen in time- and dose-dependent manner. CcMP-II inhibits collagen-induced platelet aggregation of human PRP with an IC50 value of 0.11 nM.  Pathological changes induced by CcMP-II after intramuscular injection in mice gastrocnemius were also studied. Histological study showed that the main morphological alterations in capillary vessels are characterized by a separation of endothelial cells from the surrounding basement membrane and reduction in the width of these cells, together with loss of basement membrane in some areas, through which erythrocytes and plasma are extravasated. Conclusion: In conclusion, CcMP-II, as a member of the P-II class of SVMPs, presents a selective specificity toward fibrinogen, laminin and type IV collagen. It was an α- fibrinogenase metalloproteinase, which inhibits collagen-stimulated platelet aggregation. This study will contribute to understanding better the functional mechanisms of the metalloproteinases that may be useful in the development of therapeutic agents related to thrombotic disorders

Isolated biomolecules of pharmacological interest in hemostasis from Cerastes cerastes venom

Biomolecules from Cerastes cerastes venom have been purified and characterized. Two phospholipases isolated from Cerastes cerastes venom share 51% of homology. CC2-PLA2 exhibits antiplatelet activity that blocks coagulation. CCSV-MPase, a non-hemorrhagic Zn2+-metalloproteinase, significantly reduced the plasmatic fibrinogen level and hydrolyzes only its Bβ chain. Serine proteinases such as RP34, afaâcytin and CC3-SPase hydrolyze the fibrinogen and are respectively α, αβ and αβ fibrinogenases. In deficient human plasma, afaâcytin replaces the missing factors VIII and IX, and activates purified human factor X into factor Xa. It releases serotonin from platelets and directly aggregates human (but not rabbit) blood platelets. RP34 proteinase also had no effect on both human and rabbit blood platelet aggregation. CC3-SPase revealed a pro-coagulant activity. However, the insolubility of the obtained clot indicates that CC3-SPase does not activate factor XIII. In addition, CC3-SPase clotting activity was carried out with human plasmas from volunteer patients deficient in clotting factors. Results showed that CC3-SPase shortens clotting time of plasma deficient in factors II and VII but with weaker clotting than normal plasma. The clotting time of plasma deficient in factor II is similar to that obtained with normal plasma; suggesting that CC3-SPase is able to replace both factors IIa and VII in the coagulation cascade and thus could be involved in the blood clotting process via an extrinsic pathway. These results imply that CC3-SPase and afaâcytin could repair hemostatic abnormalities and may replace some factors missing in pathological deficiency. Afaâcytin also exhibits α fibrinase property similar to a plasmin-like proteinase. Despite its thrombin-like characteristics, afaâcytin is not inhibited by plasmatic thrombin inhibitors. The procoagulant properties of afaâcytin might have potential clinical applications

TNF-α Involvement in Insulin Resistance Induced by Experimental Scorpion Envenomation

<div><h3>Background</h3><p>Scorpion venom induces systemic inflammation characterized by an increase in cytokine release and chemokine production. There have been few experimental studies assessing the effects of scorpion venom on adipose tissue function <em>in vivo</em>.</p> <h3>Methodology/Principal Findings</h3><p>To study the adipose tissue inflammation (ATI) induced by <em>Androctonus australis hector</em> (<em>Aah</em>) venom and to assess possible mechanisms of ATI, mice (n = 6, aged 1 month) were injected with <em>Aah</em> (0.45 mg/kg), toxic fraction of <em>Aah</em> (FTox-G50; 0.2 mg/kg) or saline solution (control). Inflammatory responses were evaluated by ELISA and cell sorting analyses in adipose tissue 45 minutes and 24 hours after injection. Quantitative real-time PCR was used to assess the regulation of genes implicated in glucose uptake. The titers of selected inflammatory cytokines (IL-1β, IL-6 and TNF-α) were also determined in sera and in insulin target tissues. The serum concentration of IL-1β rose 45 minutes after envenomation and returned to basal level after 24 hours. The pathophysiological effects of the venom after 24 hours mainly involved M1-proinflammatory macrophage infiltration in adipose tissue combined with high titers of IL-1β, IL-6 and TNF-α. Indeed, TNF-α was strongly induced in both adipose tissue and skeletal muscle. We studied the effects of <em>Aah</em> venom on genes implicated in insulin-stimulated glucose uptake. Insulin induced a significant increase in the expression of the mRNAs for hexokinase 2 and phosphatidylinositol 3-kinase in both skeletal muscle and adipose tissue in control mice; this upregulation was completely abolished after 24 hours in mice envenomed with <em>Aah</em> or FTox-G50.</p> <h3>Conclusions/Significance</h3><p>Our findings suggest that <em>Aah</em> venom induces insulin resistance by mechanisms involving TNF-α-dependent Map4k4 kinase activation in the adipose tissue.</p> </div

Anti-TNF-α treatment effect on insulin sensitivity in adipose tissue from <i>FTox-G50</i>-treated mice.

<p>Four-week-old mice were injected with etanercept (anti-TNFα, 1 mg/kg/injection) one hour before injection of FTox-G50. Twenty-four hours later, adipose explants were harvested and stimulated with insulin (100 nmol/L) <i>ex vivo</i> for 1 hour as shown in the scheme (top). (A) Insulin-induced changes in Hk 2 mRNA levels in adipose explants were determined by quantitative RT-PCR. (B) Insulin-induced changes in Map4k4 mRNA levels in adipose explants were determined by quantitative RT-PCR. Ratio of Hk2 and Map4k4 mRNA expression in response to insulin over basal level (non-insulin stimulated) in adipose tissue for each individual mouse is represented. Data were analyzed by unpaired t test*, P<0.05. n = 6. (C) Cellular mechanisms of decreased glucose uptake after <i>Aah</i> injection. <i>Aah</i> venom causes pronounced upregulation of TNF-α, IL1-β and IL-6 expression in the adipose tissue, exacerbating the inflammatory state. As the inflammatory state intensifies 24 hours after envenomation, TNF-α and other factors are upregulated causing activation of Map4k4 expression and blunting the insulin response in adipocytes by decreasing of Hk2 expression. Up and down arrows indicate increased and decreased expression, respectively.</p

<i>Ex vivo</i> assessment of adipose tissue insulin sensitivity in FTox-G50-injected mice.

<p>Adipose explants from controls and mice treated 24 hours earlier with <i>Aah</i> venom or FTox-G50 were harvested and stimulated with or without insulin (100 nmol/L) <i>ex vivo</i> for 1 hour, as shown in the scheme (top). The control graph represents the ability of insulin to promote glucose uptake into adipose tissue. Insulin-induced changes in <i>Hk2</i>. (A) and <i>Pik3r2</i> expression (B) mRNA levels were determined by quantitative RT-PCR in adipose explants from controls and mice injected with native and toxic fraction of venom. Ratio of Hk2 and Pik3r2 mRNA expression in response to insulin over basal level (non-insulin stimulated) in adipose tissue for each individual mouse is represented. Data were analyzed by unpaired t test; *P<0.05, **P<0.01..n = 6.</p