Progressive Hemorrhage and Myotoxicity Induced by Echis carinatus Venom in Murine Model: Neutralization by Inhibitor Cocktail of N,N,N',N'-Tetrakis (2-Pyridylmethyl) Ethane-1,2-Diamine and Silymarin

Abstract

Viperbite is often associated with severe local toxicity, including progressive hemorrhage and myotoxicity, persistent even after the administration of anti-snake venom (ASV). In the recent past, investigations have revealed the orchestrated actions of Zn2+ metalloproteases (Zn2+MPs), phospholipase A2s (PLA2s) and hyaluronidases (HYs) in the onset and progression of local toxicity from the bitten site. As a consequence, venom researchers and medical practitioners are in deliberate quest of potent molecules alongside ASV to tackle the brutal local manifestations induced by aforesaid venom toxins. Based on these facts, we have demonstrated the protective efficacy of inhibitor cocktail containing equal ratios of N,N,N',N'-tetrakis (2-pyridylmethyl) ethane-1,2-diamine (TPEN) and silymarin (SLN) against progressive local toxicity induced by Echis carinatus venom (ECV). In our previous study we have shown the inhibitory potentials of TPEN towards Zn2+MPs of ECV (IC50: 6.7 μM). In this study we have evaluated in vitro inhibitory potentials of SLN towards PLA2s (IC50: 12.5 μM) and HYs (IC50: 8 μM) of ECV in addition to docking studies. Further, we have demonstrated the protection of ECV induced local toxicity with 10 mM inhibitor cocktail following 15, 30 min (for hemorrhage and myotoxicity); 60 min (for hemorrhage alone) of ECV injection in murine model. The histological examination of skin and thigh muscle sections taken out from the site of ECV injection substantiated the overall protection offered by inhibitor cocktail. In conclusion, the protective efficacy of inhibitor cocktail is of high interest and can be administered locally alongside ASV to treat severe local toxicity.

Fig 1. Phospholipases A₂ inhibitors.

Aristolochic acid (8-methoay-6-nitrophenanthro (3, 4-d) 1, 3-dioxole-S-carboxylic acid); ascorbyl palmitate; oleanolic acid; and ursolic acid (3β-hydroxy-urs-12-en-28-oic acid).

Fig 2. Multiple sequence alignment of D. russelii and E. carinatus venom sPLA₂s.

(a) Structure superposition shown as ribbon structure; (b) sequence alignment of sPLA₂s. The sequences shares identical domain architecture with 57% identity, 70% similarity with His48, Asp49, Trp31, and Lys69 as conserved active site residues

Fig 3. Energetically favorable binding modes of AA, AP, OA, and UA calculated using IFD method.

Glide score (a) and glide energy (b) (calculated in kcal/mol) associated with best binding modes of AA, AP, OA, and UA with the active site of ECVPLA₂. The hydrogen bonding and hydrophobic interactions between the enzyme and AA (c), AP (d), OA (e), and UA (f) respectively are depicted using the LigPlot software. AA, AP, OA, and UA are labeled using three letter codes “Ara”, “Apa”, “Ola”, and “Ura” respectively with a common residue number 999(Z).

Fig 4. Hyaluronidase inhibitors.

Cromolyn sodium salt, sodium aurothiomalate hydrate, and silymarin.

Fig 5. Predicted structure of ECVHY.

(a) Target—template sequence alignment, (b) model validation, (c) target-template structure superposition and (d) conserved active site residues. The template structures—bee venom hyaluronidase (PDB ID: 1FCQ-template 1) and human hyaluronidase (PDB ID: 2PE4- template 2) showed 33.3% and 42% sequence identity and 92% and 70% query coverage with the target sequence—Echis ocellatus venom hyaluronidase (UniProt ID: A3QVN2).

Fig 6. Energetically favorable binding modes of CSS, SAH, and SLN calculated using IFD method.

Glide score (a) and glide energy (b) (calculated in kcal/mol) associated with best binding modes of CSS, SAH, and SLN with the active site of modeled ECVHY. The hydrogen bonding and hydrophobic interactions between the enzyme and CSS (c), SAH (d), and SLN (e) respectively are depicted using the LigPlot software. CSS, SAH, and SLN are labeled using respective three letter codes with a common residue number 999(Z).

Fig 7. Energetically favorable binding modes of AP and SLN calculated using Induced fit docking method.

Glide score (calculated in kcal/mol) associated with best binding modes of AP and SLN with the active site of ECVPLA₂ (a) and modeled ECVHY (a1). The hydrogen bonding and hydrophobic interaction of AP and SLN with ECVPLA₂ (b, c) and modeled ECVHY (b1, c1) respectively are depicted using the LigPlot software.

Fig 8

(a) Inhibition of phospholipase A2 activity of ECV by AP and SLN. The reaction mixture (350 μl) contained enzyme in 100 mM Tris HCl pH 7.4, 5 mM CaCl₂ and various concentrations of AP and SLN (0.1 nM-10 mM). The reactions were initiated by adding 30 μl substrate and incubated at 37°C for 45 min. (b) Inhibition of edema-inducing activity of ECV by AP and SLN: Mouse intra plantar surface (footpad) was injected with constant 3 μg ECV + various concentrations of AP and SLN following 15 min of ECV injection. After 45 min, the mice were euthanized and both hind limbs were removed at the ankle joint and weighed individually to calculate the edema ratio. *, ^# p < 0.05, ^## p < 0.01, and ***, ^### p < 0.001 compared to ECV induced edema ratio.

Fig 9. Inhibition of hyaluronidase activity of ECV by AP and SLN.

Reaction mixture 300 μl contained 100 μg ECV in 100 mM acetate buffer pH 5.5, 150 mM NaCl and various concentrations of AP and SLN (0.1 nM-10 mM). The reactions were initiated by adding 50 μl substrate (hyaluronic acid) and incubated at 37°C for 2.5 h. After terminating the reaction, the contents were processed for color development.

Fig 10. Hemorrhagic activity of ECV and its protection by inhibitor cocktail of TPEN and SLN upon independent injections.

(i) Dorsal surface of mouse skin showing hemorrhagic spots. (ii) Area of hemorrhagic spots measured using graph paper. Mice were injected intradermally with constant 3 μg ECV (3 MHD dose) and various doses of inhibitor cocktail of TPEN and SLN (0.3 to 10 mM) at different time points (15 to 60 min) after venom injection. After 3 h, mice were sacrificed and hemorrhagic spots on the inner surface were examined for protection of ECV induced hemorrhage; a. negative control (30μl Saline); b. positive control (hemorrhagic spot appeared after 3 h of 3 μg ECV injection); c. hemorrhagic spot appeared after 15 min of 3 μg ECV injection; d, e, and f: 0.3, 3, and 10 mM inhibitor cocktail injected after 15 min of ECV injection; g. hemorrhagic spot appeared after 30 min of 3 μg ECV injection; h and i: 3 and 10 mM inhibitor cocktail injected after 30 min of ECV injection; j. hemorrhagic spot appeared after 60 min of 3 μg ECV injection; k and l: 3 and 10 mM inhibitor cocktail injected after 60 min of ECV injection; m, n and o: 0.3, 3 and 10 mM inhibitor cocktail alone (cocktail control). *p < 0.05, **p < 0.01, and ***, ^### p < 0.001 compared to ECV induced hemorrhage.

Fig 11. Photomicrographs of mice skin transverse sections observed at 100 X magnification showing protection against ECV induced hemorrhage by inhibitor cocktail.

(a) Saline-injected control section showed intact dermal layer (D), basement membrane (BM) and surrounding blood vessels (BV). 3μg ECV injected sections dissected at different time points—15 min (c); 30 min (g); 60 min (j); and 180 min (b) showed disorganized dermis, basement membrane and disruption of blood vessels in time dependent fashion. On independent injection (following 15, 30, and 60 min of ECV administration) inhibitor cocktail showed dose-dependent protection against venom-induced hemorrhage—(d), (e), (f): 0.3, 3, and 10 mM inhibitor cocktail injected after 15 min of ECV injection; (h) and (i): 3 and 10 mM inhibitor cocktail injected after 30 min of ECV injection; (k) and (l): 3 and 10 mM inhibitor cocktail injected after 60 min of ECV injection. Cocktail control—(m), (n) and (o): 0.3, 3, and 10 mM inhibitor cocktail alone injected sections showed intact ECM and the basement membrane surrounding the blood vessels. The dark arrow represents the degraded portions of tissue sections.

Fig 12. (i) Serum creatine kinase (CK) and lactate dehydrogenase (LDH) levels and (ii) histopathology of mice injected (i.m.) with ECV and its protection by inhibitor cocktail.

Mice were injected with 5 μg ECV + different doses of inhibitor cocktail (independently after 30 min of ECV injection). After 3 h, mice were sacrificed and serum CK and LDH levels were assayed using AGAPPE kit. **, ^## p < 0.01 and ***, ^### p < 0.001 compared to ECV induced CK and LDH values. Further, dissected thigh muscles from the site of ECV injection were processed for hematoxylin and eosin staining and were observed at 200 X magnification. (a) Saline control showed characteristic muscular striations and intact myocytes. Five μg ECV injected sections dissected at different time points—30 min (c); and 180 min (b) showed disorganization in muscular striations and myocytes in time dependent fashion as evidenced by proportionate elevation of serum CK and LDH activities compared to control. On independent injection, inhibitor cocktail—(d), (e): 3 and 10 mM showed dose-dependent protection against ECV induced myotoxicity. 10 mM inhibitor cocktail alone-injected section (f) showed characteristic muscular striations and intact myocytes. The dark arrows show the damaged portion of muscle sections.