Effects of nitric oxide on C2C12 cell inflammatory responses and notexin-induced muscle injury / Efectos del óxido nítrico en las respuestas inflamatorias de las células C2C12 y la lesión muscular inducida por notexina

SUMMARY: Skeletal muscle injury is an acute inflammatory condition caused by an inflammatory response. To reduce inflammatory cell infiltration and relieve skeletal muscle injury, efficient treatment is urgently needed. Nitric oxide is a free radical molecule reported to have anti-inflammatory effects. In this study, we showed that NO could inhibit the inflammatory response of C2C12 cells in vitro and protect rat skeletal muscle injury from notexin in vivo. NO synthase inhibitor (L-NG-Nitroarginine Methyl Este?L-NAME) and NO donor (sodium nitroprusside dehydrate ?SNP) were used to explore the vital role of lipopolysaccharides (LPSs) in LPS-stimulated C2C12 myoblasts.The expression of IL-18 and IL-1b was upregulated by L-NAME and downregulated by SNP, as indicated by the ELISA results. NO can reduce ASC, Caspase-1, and NLRP3 mRNA and protein levels. Furthermore, NO was detected in the rat model. The results of immunohistochemical staining showed that the production of DMD decreased. We conducted qRT-PCR and western blotting to detect the expression of Jo-1, Mi-2, TLR2, and TLR4 on day 6 post injury following treatment with L-NAME and SNP. The expression of Jo-1, Mi-2, TLR2, and TLR4 was upregulated by L-NAME and significantly reversed by SNP. NO can alleviate C2C12 cell inflammatory responses and protect rat skeletal muscle injury from notexin.

RESUMEN: La lesión del músculo esquelético es una afección inflamatoria aguda causada por una respuesta inflamatoria. Para reducir la infiltración de células inflamatorias y aliviar la lesión del músculo esquelético es necesario un tratamiento eficaz. El óxido nítrico es una molécula de radicales libres que tiene efectos antiinflamatorios. En este estudio, demostramos que el ON podría inhibir la respuesta inflamatoria de las células C2C12 in vitro y proteger la lesión del músculo esquelético de rata de la notexina in vivo. El inhibidor de ON sintasa (L-NG-nitroarginina metil este, L-NAME) y el donante de ON (nitroprusiato de sodio deshidratado, SNP) se utilizaron para explorar el papel vital de los lipopolisacáridos (LPS) en los mioblastos C2C12 estimulados por LPS. La expresión de IL- 18 e IL-1b fue regulada positivamente por L-NAME y regulada negativamente por SNP, como indican los resultados de ELISA. El ON puede reducir los niveles de proteína y ARNm de ASC, Caspasa-1 y NLRP3. Además, se detectó ON en el modelo de rata. Los resultados de la tinción inmunohistoquímica mostraron que disminuyó la producción de DMD. Realizamos qRT-PCR y transferencia Western para detectar la expresión de Jo-1, Mi-2, TLR2 y TLR4 el día 6 después de la lesión después del tratamiento con L-NAME y SNP. La expresión de Jo-1, Mi-2, TLR2 y TLR4 fue regulada positivamente por L- NAME y significativamente revertida por SNP. El ON puede aliviar las respuestas inflamatorias de las células C2C12 en ratas, y proteger la lesión del músculo esquelético de la notexina.

Kinetic and toxicological effects of synthesized palladium(II) complex on snake venom (Bungarus sindanus) acetylcholinesterase

The venom of the krait (Bungarus sindanus), an Elapidae snake, is highly toxic to humans and contains a great amount of acetylcholinesterase (AChE). The enzyme AChE provokes the hydrolysis of substrate acetylcholine (ACh) in the nervous system and terminates nerve impulse. Different inhibitors inactivate AChE and lead to ACh accumulation and disrupted neurotransmission. Methods: The present study was designed to evaluate the effect of palladium(II) complex as antivenom against krait venom AChE using kinetics methods. Results: Statistical analysis showed that krait venom AChE inhibition decreases with the increase of Pd(II) complex (0.025-0.05 µM) and exerted 61% inhibition against the AChE at a fixed concentration (0.5 mM) of ACh. Kinetic analysis using the Lineweaver Burk plot showed that Pd(II) caused a competitive inhibition. The compound Pd(II) complex binds at the active site of the enzyme. It was observed that K m (Michaelis-Menten constant of AChE-ACh into AChE and product) increased from 0.108 to 0.310 mM (45.74 to 318.35%) and V max remained constant with an increase of Pd(II) complex concentrations. In AChE K Iapp was found to increase from 0.0912 to 0.025 µM (29.82-72.58%) and did not affect the V maxapp with an increase of ACh from (0.05-1 mM). K i (inhibitory constant) was estimated to be 0.029µM for snake venom; while the K m was estimated to be 0.4 mM. The calculated IC50 for Pd(II) complex was found to be 0.043 µM at constant ACh concentration (0.5 mM). Conclusions: The results show that the Pd(II) complex can be deliberated as an inhibitor of AChE.(AU)

Non-neurotoxic activity of Malayan krait (Bungarus candidus) venom from Thailand

Envenoming by kraits (genus Bungarus) is a medically significant issue in South Asia and Southeast Asia. Malayan krait (Bungarus candidus) venom is known to contain highly potent neurotoxins. In recent years, there have been reports on the non-neurotoxic activities of krait venom that include myotoxicity and nephrotoxicity. However, research on such non-neurotoxicity activities of Malayan krait venom is extremely limited. Thus, the aim of the present study was to determine the myotoxic, cytotoxic and nephrotoxic activities of B. candidus venoms from northeastern (BC-NE) and southern (BC-S) Thailand in experimentally envenomed rats. Methods: Rats were administered Malayan krait (BC-NE or BC-S) venom (50 µg/kg, i.m.) or 0.9% NaCl solution (50 µL, i.m.) into the right hind limb. The animals were sacrificed 3, 6 and 24 h after venom administration. The right gastrocnemius muscle and both kidneys were collected for histopathological analysis. Blood samples were also taken for determination of creatine kinase (CK) and lactate dehydrogenase (LDH) levels. The human embryonic kidney cell line (HEK-293) was used in a cell proliferation assay to determine cytotoxic activity. Results: Administration of BC-NE or BC-S venom (50 µg/kg, i.m.) caused time-dependent myotoxicity, characterized by an elevation of CK and LDH levels. Histopathological examination of skeletal muscle displayed marked muscle necrosis and myofiber disintegration 24 h following venom administration. Both Malayan krait venoms also induced extensive renal tubular injury with glomerular and interstitial congestion in rats. BC-NE and BC-S venoms (100­0.2 µg/ mL) caused concentration-dependent cytotoxicity on the HEK-293 cell line. However, BC-NE venom (IC50 =8 ± 1 µg/mL; at 24 h incubation; n = 4) was found to be significantly more cytotoxic than BC-S venom (IC50 =15 ± 2 µg/mL; at 24 h incubation; n = 4). In addition, the PLA2 activity of BC-NE venom was significantly higher than that of BC-S venom. Conclusions: This study found that Malayan krait venoms from both populations possess myotoxic, cytotoxic and nephrotoxic activities. These findings may aid in clinical diagnosis and treatment of envenomed patients in the future.(AU)

Prophylactic effect of Mucuna pruriens Linn (velvet bean) seed extract against experimental Naja sputatrix envenomation: Gene expression studies.

Mucuna pruriens is widely used in traditional medicine for treatments of various diseases. In certain region of Nigeria, the seed is used as oral prophylactics for snakebite. Rats pretreated with the aqueous extract from M. pruriens seed (MPE) were protected against the lethal effects of Naja sputatrix (Javan spitting cobra) venom [Tan et al., J Ethnopharmacol, 123 (2009) 356]. The pretreatment also protected against venom-induced histopathological changes in rat heart. To contribute to the understanding of the mechanism of cardio-protective action, the present study examined the effects of MPE-pretreatment on gene expression profile of rat heart as well as effect of MPE-pretreatment on N. sputatrix venom-induced gene expression alterations in rat heart. The gene expression profiles were examined by microarray analysis and verified by real time PCR. The results showed that pretreatment with MPE caused 50 genes in the rat heart substantially up-regulated of which 19 were related to immune responses, 7 were related to energy production and metabolism. The up-regulation of genes related to energy metabolism probably plays a role in maintaining the viability of the heart. Four other genes that were up-regulated (alpha synuclein, natriuretic peptide precursor, calsequestrin and triadin) were involved in the maintenance of homeostasis of the heart or maintaining its viability, thereby contributing to the direct protective action. The results demonstrated that protective effect of MPE pretreatment against snake venom poisoning may involve a direct action on the heart.

Use of antivenoms for the treatment of envenomation by Elapidae snakes in Guinea, Sub-Saharan Africa

Background In Guinea Elapids are responsible for 20% of envenomations. The associated case fatality rate (CFR) ranged 15-27%, irrespective of treatment. Results We studied 77 neurotoxic envenomations divided in 3 groups: a set of patients that received only traditional or symptomatic treatments, and two other groups that received either 2 or 4 initial vials of Antivipmyn® Africa renewed as necessary. CFR was 27.3%, 15.4% and 17.6%, respectively. Although antivenom treatment was likely to reduce CFR, it didn’t seem to have an obvious clinical benefit for the patients, suggesting a low treatment efficacy. Mean delay to treatment or clinical stages were not significantly different between the patients who recovered and the patients who died, or between groups. Interpretation of these results is complicated by the lack of systematic studies under comparable conditions. Of particular importance is the absence of assisted ventilation, available to patients in all the other clinical studies of neurotoxic envenomation. Conclusion The apparent lack of clinical benefit may have several causes. The hypothesis of a limited therapeutic window, i.e. an insufficient formation of antigen-antibody complexes once toxins are bound to their targets and/or distributed beyond the reach of antivenom, should be explored. .

What is the most dangerous snake?

Letter to Editor of Journal of Venomous Animals and Toxins.

Pattern of renal pathology in fatal envenomation by indian cobra (naja naja).

Deaths due to poisonous snakebite are a significant health related problem especially the rural heartland of in tropical countries. Renal involvement in snakebite is well documented especially so in bites by the Viperidae group. The Elapidae family consisting of cobra and kraits among other varieties are mainly considered neurotoxic. The venom of neurotoxic variety predominantly has direct depressing action on the respiratory center and neuromuscular junction. We investigated the renal changes at autopsy and histology of fatal cobra bites. This series included autopsy examination of 14 cases of fatal cobra bite in our hospital-based study. Dissected kidneys were sectioned, stained with hematoxylin & eosin stain and histological examination was done under light microscope. Five cases from head injury subject were used as control. The study reveals renal involvement in 64.28 %of fatal bites by Indian cobra (Naja naja) primarily considered neurotoxic. The major renal changes were tubular necrosis 1(7.14%), cortical necrosis 3 (21.42%) and interstitial nephritis 3(21.42%). This fact is worth giving due consideration during management and monitoring of cases of envenomation by cobra.

In vitro hemolytic activity of Bothrops lanceolatus (fer-de-lance) venom

Bothrops lanceolatus venom contains a variety of enzymatic and biological activities. The present work investigated the hemolytic activity of this venom and its phospholipase A2 (PLA2). Bothrops lanceolatus venom (6.7 µg/mL) caused indirect hemolysis of cow, horse, rat and sheep erythrocytes, with horse erythrocytes being the most sensitive; no direct hemolysis was observed. Hemolysis in sheep erythrocytes was concentration-dependent (5-11.7 µg/mL) and markedly attenuated by heating the venom for 30 minutes at ≥ 40°C and by the PLA2 inhibitor p-bromophenacyl bromide. An acidic PLA2 (5 µg/mL) purified from B. lanceolatus venom also caused hemolysis. This PLA2 showed immunoprecipitin lines with antivenom against B. lanceolatus, which suggests that the enzymatic and hemolytic activities of this enzyme may be neutralized during antivenom therapy. These results indicate that B. lanceolatus venom and its PLA2 can cause hemolysis in vitro.(AU)

Antisnake venom activity of ethanolic seed extract of Strychnos nux vomica Linn.

The whole seed extract of S. nux vomica (in low doses) effectively neutralized Daboia russelii venom induced lethal, haemorrhage, defibrinogenating, PLA2 enzyme activity and Naja kaouthia venom induced lethal, cardiotoxic, neurotoxic, PLA2 enzyme activity. The seed extract potentiated polyvalent snake venom antiserum action in experimental animals. An active compound (SNVNF) was isolated and purified by thin layer chromatography and silica gel column chromatography, which effectively antagonised D. russelii venom induced lethal, haemorrhagic, defibrinogenating, oedema, PLA2 enzyme activity and N. kaouthia induced lethal, cardiotoxic, neurotoxic, PLA, enzyme activity. Polyvalent snake venom antiserum action was significantly potentiated by the active compound. Spectral studies revealed it to be a small, straight chain compound containing methyl and amide radicals. Detailed structure elucidation of the compound (SNVNF) is warranted before its clinical trials as a snake venom antagonist.

Electrophysiological and ultrastructural analysis of the neuromuscular blockade and miotoxicity induced by the Micrurus nigrocinctus snake venom

Micrurus nigrocinctus is the most abundant coral snake in Central America. The venom of this specie induced a concentration-dependent (10-20 mug/ml) depolarization in the isolated mouse phrenic nerve-diaphragm preparations incubated ate 37 degree. D-Tubocurrarine (10 mug/ml) and alpha betaungarotoxin (3-5 mug/ml) were able to partially protect against the depolarization induced by the venom (10 mug/ml), suggesting the involvement of subsynaptic cholinergic receptors. This venom (10 mug/ml) also increased the frequency and amplitude of miniature end-plate potentials (mepps) during the first 10-20 min of incubation. Subsequently, the mepps progressively decreased and disappeared after 60 min. These responses were accompanied by ultrastructural changes involving the nerve terminals, the subsynaptic junctional folds and the muscle mitochondria. The synaptic gutter was shallow and, very often, "shrunken" terminal with omega-shaped axolemmal identations and a decreased number of synaptic vecicles were present. A common finding was the presence of numerous finger-like, membrane-bounded bodies interposed between the terminal and the Schwann cells or postsynaptic sarcolemma. The preincubation of the venom with specific antivenom or the incubation of the preparations at room temperature (24-26 degree) reduced the number and intensity of the ultrastructural alterations. The last finding suggests the involvement of a enzymatic process, probably a phospholipase A2, present in the venom. There was a good correlation between the electrophysiological and ultrastructural effects induced by the venom which allow us to conclude that M. nigrocinctus venom has a presynaptic action in the initial stages of intoxication followed by sub- and postsynaptic effects, the last being the most important cause of neuromuscular blockade. A direct action of the venom on muscle fibers may also contributes to the irreversible blockade.

Toxicities, LD50 prediction and in vivo neutralisation of some elapid and viperid venoms.

Toxicity levels of elapid (Naja naja and Naja oxiana) viperid (Vipera lebetina and Vipera russelli) venoms for mice and rat for intraperitoneal intravenous and intramuscular routes have been determined. The data have been analysed using a mathematical expression to calculate lethal venom concentrations in human snake bite cases. Further, in vivo neutralisation of snake venom potency (after experimental injection) using high voltage-low current electric shock treatment has been attempted. This treatment postponed the death further by 60-90 min in mice in case of elapid envenomation. In case of viperid envenomation such a postponement of death time was not noticed. The death postponement induced by the shock treatment probably refers to structural impairments that occur at molecular level in venom components and their consequent altered interactions with the target tissue or system.

Venenos animales / Animals poisons

Esta colaboración trata de explicar lo relativo de los conceptos manejados en relación a los venenos animales y su acción terapéutica homeopática a través de la dinamización hahnemanniana. Las diferencias entre su acción tóxica y patogenética; su justificación a través de los parágrafos 19, 20 y 110 del Organón de Hahnemann. El planteo que otros conceptos son posibles en ciencia y la sugerencia de tender a una epistemología homeopática

Neutralization of coral snake Micrurus nigrocinctus venom by a monovalent antivenom

The neutralizing ability of a monovalent anti-Micrurus nigrocinctus (coral snake) antivenom produced in Costa Rica was tested against the letal, myotoxic and phospholipase A, activities of homologous venom. In addition, immunodiffusion and Western blot analyses were performed. in experiments where venom and antivenom were incubated prior to the test, antivenom was effective in neutralizing lethal, myotoxic and phospholipase A2, activities, with Effective Doses 50% of 2700 *l antivenom/mg venom, 1840 *l antivenom/mg venom, and 3630 *l antivenom, respectively. When coral snake antivenom was administered different times after coral snake venom injection, neutralization of lethality was achieved ehen antivenom was injected iv immediately and 15 min after venom. In contrast, lethaly was not reduced when antivenom was administered by the route. Only partial neutralization of myotoxixity was observed even when antivenom was injected iv immediately after envenomation. Immunodiffusion and immunoblot analyses demonstrated the presence of antibodies in antivenom against several, but not all, venom components

Is direct cardiotoxicity the primary cause of death following i. v. injection of the basic phospholipase A2 from Naja Nigricollis venom?

Con objeto de elucidar la función del Ca intracelular en la transmisión neuromuscular investigamos en preparaciones de músculo de rana los efectos del ácido1,2-bis(o-aminofenoxi)etano-N,N,N',N'-tetraacético (BAPTA) sobre el aumento-potenciación por frecuencia (anteriormente llamado facilitación por frecuencia) el que ha sido de utilidad para identificar los sitios de acción de varios agentes colinérgicos. La disminución de los iones Ca del espacio intracelular por BAPTA sólo suprimió el componente dependiente de Ca del fenómeno (ma) sin modificar el factor de estimulación dependiente de frecuencia (K). La depresión causada por BAPTA en la facilitación de corto plazo del potencial de placa (EPP) fue la misma tanto en reposo como en la estimulación. El efecto del BAPTA fue parcialmente antagonizado, por el ionóforo de Ca A23187. Esto sugiere que la capacidad de "buffer" de Ca del BAPTA se mantiene durante la estimulación repetitiva de baja frecuencia. BAPTA no modificó la potenciación post-tetánica de los EPP miniatura en medio libre de Ca. Estos resultados indican que los iones Ca son esenciales para la liberación de transmisor y para la facilitación de corto plazo, pero no son responsables de todos los cambios en la liberación de transmisor