Description of trunk neural crest migration and peripheral nervous system formation in the Egyptian cobra Naja haje haje.

The neural crest is a stem cell population that forms in the neurectoderm of all vertebrates and gives rise to a diverse set of cells such as sensory neurons, Schwann cells and melanocytes. Neural crest development in snakes is still poorly understood. From the point of view of evolutionary and comparative anatomy is an interesting topic given the unique anatomy of snakes. The aim of the study was to characterize how trunk neural crest cells (TNCC) migrate in the developing elapid snake Naja haje haje and consequently, look at the beginnings of development of neural crest derived sensory ganglia (DRG) and spinal nerves. We found that trunk neural crest and DRG development in Naja haje haje is like what has been described in other vertebrates and the colubrid snake strengthening our knowledge on the conserved mechanisms of neural crest development across species. Here we use the marker HNK1 to follow the migratory behavior of TNCC in the elapid snake Naja haje haje through stages 1-6 (1-9 days postoviposition). We observed that the TNCC of both snake species migrate through the rostral portion of the somite, a pattern also conserved in birds and mammals. The development of cobra peripheral nervous system, using neuronal and glial markers, showed the presence of spectrin in Schwann cell precursors and of axonal plexus along the length of the cobra embryos. In conclusion, cobra embryos show strong conserved patterns in TNCC and PNS development among vertebrates.

Egyptian cobra (Naja haje haje) venom phospholipase A2: a promising antiviral agent with potent virucidal activity against simian rotavirus and bovine coronavirus.

Viral infections are linked to a variety of human diseases. Despite the achievements made in drug and vaccine development, several viruses still lack preventive vaccines and efficient antiviral compounds. Thus, developing novel antiviral agents is of great concern, particularly the natural products that are promising candidates for such discoveries. In this study, we have purified an approximately 15 kDa basic phospholipase A2 (PLA2) enzyme from the Egyptian cobra Naja haje haje venom. The purified N. haje PLA2 showed a specific activity of 22 units/mg protein against 6 units/mg protein for the whole crude venom with 3.67-fold purification. The antiviral activity of purified N. haje PLA2 has been investigated in vitro against bovine coronavirus (BCoV) and simian rotavirus (RV SA-11). Our results showed that the CC50 of PLA2 were 33.6 and 29 µg/ml against MDBK and MA104 cell lines, respectively. Antiviral analysis of N. haje PLA2 showed an inhibition of BCoV and RV SA-11 infections with a therapeutic index equal to 33.6 and 16, respectively. Moreover, N. haje PLA2 decreased the BCoV and RV SA-11 titers by 4.25 log10 TCID50 and 2.5 log10 TCID50, respectively. Thus, this research suggests the potential antiviral activity of purified N. haje PLA2 against BCoV and RV SA-11 infections in vitro.

Concerted hepatoprotective effect of bradykinin potentiating factor and low dose of γ- radiation on Naja haje envenomed rats via Bax/Bcl2 pathway.

This study investigates the concerted hepatoprotective effects for three doses of bradykinin potentiating factor (BPF) and/or followed by exposure to a low dose of γ-radiation (LDR) against Naja haje envenoming in rats. Male rats were injected with three consecutive doses of BPF (1 µg/g i.p. for 3 days), followed by exposure to a low dose of gamma radiation (0.5 Gy), and then rats were injected with a dose of Naja haje venom (250 µg/kg i.p.). Results showed that Naja haje causes liver damage, significant elevation of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), cytochrome c, Nitric oxide (NO), malondialdehyde (MDA) and significant depletion in glutathione peroxidase (GPx) contents. In addition, significant depletion in B-cell lymphoma 2 (Bcl-2) and significant elevation in BcL-2 associated X (Bax protein), nuclear factor kappa B (NF-κB), interleukin-1ß (IL-1ß) in hepatocytes. Bradykinin potentiating factor and/or low dose of γ-radiation caused improvement in liver damage caused by Naja haje venom by a significant decrease in ALT, AST, ALP levels, Bax, cytochrome c, NF-κB, IL-1ß, NO and MDA contents, BPF alone or combined with low dose radiation caused a significant increase in Bcl2 and GPx contents. In conclusion, the concerted impact of BPF and LDR may provide an effective venom detoxification tool that helps to reduce hepatic toxicity and extends the lifespan.

Anti-Cancer Effect of Moroccan Cobra Naja haje Venom and Its Fractions against Hepatocellular Carcinoma in 3D Cell Culture.

Hepatocellular carcinoma (HCC) is the most common primary liver cancer in adults, the fifth most common malignancy worldwide and the third leading cause of cancer related death. An alternative to the surgical treatments and drugs, such as sorafenib, commonly used in medicine is necessary to overcome this public health problem. In this study, we determine the anticancer effect on HCC of Moroccan cobra Naja haje venom and its fraction obtained by gel filtration chromatography against Huh7.5 cancer cell line. Cells were grown together with WI38 human fibroblast cells, LX2 human hepatic stellate cell line, and human endothelial cells (HUVEC) in MCTS (multi-cellular tumor spheroids) models. The hepatotoxicity of venom and its fractions were also evaluated using the normal hepatocytes cell line (Fa2N-4 cells). Our results showed that an anti HCC activity of Moroccan cobra Naja haje venom and, more specifically, the F7 fraction of gel filtration chromatography exhibited the greatest anti-hepatocellular carcinoma effect by decreasing the size of MCTS. This effect is associated with a low toxicity against normal hepatocytes. These results strongly suggest that the F7 fraction of Moroccan cobra Naja haje venom obtained by gel filtration chromatography possesses the ability to inhibit cancer cells proliferation. More research is needed to identify the specific molecule(s) responsible for the anticancer effect and investigate their mechanism of action.

Proteomic analysis of three medically important Nigerian Naja (Naja haje, Naja katiensis and Naja nigricollis) snake venoms.

Proteomics technologies enable a comprehensive study of complex proteins and their functions. The venom proteomes of three medically important Nigerian Elapidae snakes Naja haje, Naja katiensis and Naja nigricollis was studied using HILIC coupled with LC-MS/MS analysis. Results revealed a total of 57, 55, and 46 proteins in the venoms of N. haje, N. katiensis, and N. nigricollis, respectively, with molecular mass ranging between 5 and 185  kDa. These snakes have 38 common proteins in addition to 3 uncommon proteins: actiflagelin, cathelicidin, and cystatin identified in their venoms. The identified proteins belonged to 14 protein families in N. haje and N. katiensis, and 12 protein families in N. nigricollis. Of the total venom proteins, 3FTx was the most abundant protein family, constituting 52% in N. haje and N. katiensis, and 41% in N. nigricollis, followed by PLA2, constituting 37% in N. nigricollis, 26% in N. haje, and 24% in N. katiensis. Other protein families, including LAAO, CRISPs, VEGF, PLB, CVF, SVMP, SVH, AMP, PI, Globin, Actin, and C-type lectins, were also detected, although, at very low abundances. Quantification of the relative abundance of each protein revealed that alpha and beta fibrinogenase and PLA2, which constituted 18-26% of the total proteome, were the most abundant. The 3 uncommon proteins have no known function in snake venom. However, actiflagelin activates sperm motility; cystatin inhibits angiogenesis, while cathelicidin exerts antimicrobial effects. The three Nigerian Naja genus proteomes displayed 70% similarity in composition, which suggests the possibility of formulating antivenom that may cross-neutralise the venoms of cobra species found in Nigeria. These data provide insights into clinically relevant peptides/proteins present in the venoms of these snakes. Data are available via ProteomeXchange with identifier PXD024627.

Molecular modeling and structural analysis of nAChR variants uncovers the mechanism of resistance to snake toxins.

Nicotinic acetylcholine receptors (nAChRs) are neuromuscular proteins responsible for muscle contraction upon binding with chemical stimulant acetylcholine (ACh). The α-neurotoxins of snake mimic the structure of ACh and attacks nAChRs, which block the flow of ACh and leads to numbness and paralysis. The toxin-binding site of alpha subunit in the nAChRs is highly conserved throughout chordate lineages with few exceptions in resistance organisms. In this study, we have analyzed the sequence and structures of toxin-binding/resistant nAChRs and their interaction stability with toxins through molecular docking and molecular dynamics simulation (MDS). We have reported the potential glycosylation residues within the toxin-binding cleft adding sugar moieties through N-linked glycosylation in resistant organisms. Residue variations at key positions alter the secondary structure of binding cleft, which might interfere with toxin binding and it could be one of the possible explanations for the resistance to snake venoms. Analysis of nAChR-α-neurotoxin complexes has confirmed the key interacting residues. In addition, drastic variation in the binding stability of Mongoose nAChR-α-Bungarotoxin (α-BTX) and human nAChR-α-BTX complexes were found at specific phase of MDS. Our findings suggest that specific mutations in the binding site of toxin are potentially preventing the formation of stable complex of receptor-toxin, which might lead to mechanism of resistance. This in silico study on the binding cleft of nAChR and the findings of interacting residues will assist in designing potential inhibitors as therapeutic targets.

Hepatotoxicity and oxidative stress induced byNaja haje crude venom

Background Snake venoms are synthesized and stored in venom glands. Most venoms are complex mixtures of several proteins, peptides, enzymes, toxins and non-protein components. In the present study, we investigated the oxidative stress and apoptosis in rat liver cells provoked by Naja haje crude injection (LD50) after four hours.Methods Wistar rats were randomly divided into two groups, the control group was intraperitoneally injected with saline solution while LD50-dose envenomed group was intraperitoneally injected with venom at a dose of 0.025 μg/kg of body weight. Animals were killed four hours after the injection. Lipid peroxidation, nitric oxide and glutathione levels were measured as oxidative markers in serum and liver homogenate. In addition, liver function parameters and activities of antioxidant enzymes were determined.ResultsN. haje crude venom (0.025 μg/kg of body weight) enhanced lipid peroxidation and nitric oxide production in both serum and liver with concomitant reduction in glutathione, catalase, glutathione reductase and glutathione-S-transferase activities. Superoxide dismutase and glutathione peroxidase activities were significantly increased in liver of envenomed rats. These findings were associated with apoptosis induction in the liver. In addition, N. haje crude venom caused hepatic injury as indicated by histopathological changes in the liver tissue with an elevation in total bilirubin, serum alanine aminotransferase, aspartate aminotransferase, γ-glutamyl transpeptidase, and alkaline phosphatase.Conclusions Based on the present results, it can hypothesized that N. haje crude venom is a potent inducer of toxin-mediated hepatotoxicity associated with apoptosis in the liver.(AU)

Pharmacological characterization of rat paw edema induced by Naja haje arabica venom

This investigation was performed in order to assess the inflammatory response induced by Naja haje arabica venom (NhaV) in rat hind paw. The inflammatory response was estimated by measuring the edema with a Plethysmometer. The venom (0.625-10mug/paw) produced a dose and time-dependent increase in non-hemorrhagic paw edema. The response to NhaV was maximal within 15 min and disappeared in 24 h. Five mug/paw of NhaV was chosen to test the effect of various drugs on the edema induced by this venom. Quinacrine (QNC), a phospholipase A2 (PLA2) inhibitor, and dipyridamole (DPM), an adenosine transport inhibitor, attenuated venom-induced edema in rat paw (P<0.001). Commercially available antivenom was ineffective when administered intravenously, whereas its local administration with NhaV attenuated the edema formation (P<0.001). In conclusion, NhaV-induced edema in rat paw involves PLA2 and adenosine mechanisms. Additionally, the use of polyspecific antivenom, intravenously, was ineffective in preventing NhaV-induced edema.(AU)

Biochemical and morphological analysis of cell death induced by Egyptian cobra (Naja haje) venom on cultured cells

We investigated the in vitro process of cell death caused by Egyptian cobra venom on primary human embryonic kidney (293T) and mouse myoblast (C2C12) cell lines. The aim of these studies was to provide further information about triggering cell death, and suggest methods for eliminating unwanted cells, such as tumour cells. Both cell lines were treated with 10, 20, and 50 m g/ml of Egyptian cobra (Naja haje) venom in serum free media (SFM) and incubated for 8 hours. Total activities of the lactate dehydrogenase (LDH) and creatine kinase (CK) released in the culture during venom incubation were used as an indicator of the venom in vitro cytotoxicity. Cell injury was morphologically recognized and apoptosis determined by a Fluorescing Apoptosis Detection System and confirmed by staining nuclear DNA with DAPI. Our data clearly demonstrated marked cytotoxic effects and acute cell injury for both cell lines. Release of LDH and CK into the culture media induced by the venom correlates well with the morphological changes and extent of cell death. Mostly, these consequences were time and dose-dependent in both cell lines. The results obtained from this study indicated that cobra venom cause cell death by two different mechanisms: necrosis and induction of apoptosis. The apoptotic mechanism, accompanied by cell necrosis, mediated cell destruction of both tested cell lines; however, necrosis was predominant in the C2C12 cell line while apoptosis, in 293T cells. This unusual form of cell death induced by cobra venom may represent a combination of apoptosis and necrosis within the same cell. This is a first-hand investigation showing the apoptotic effects of N. haje venom at the cellular level. However, the contribution of the apoptotic pathway may be dependent on concentration and/or time of exposure to snake venom.(AU)