BackgroundSnakebites is a neglected disease and in Brazil is considered a serious health problem, with the majority of the snakebites caused by the genus Bothrops. Antivenom therapy and other first-aid treatments do not reverse local myonecrose which is the main sequel caused by the envenomation. Several studies have shown the effectiveness of low level laser (LLL) therapy in reducing local myonecrosis induced by Bothropic venoms, however the mechanism involved in this effect is unknown. In this in vitro study, we aimed to analyze the effect of LLL irradiation against cytotoxicity induced by Bothrops jararacussu venom on myoblast C2C12 cells.MethodologyC2C12 were utilized as a model target and were incubated with B. jararacussu venom (12.5 μg/mL) and immediately irradiated with LLL at wavelength of red 685 nm or infrared 830 nm with energy density of 2.0, 4.6 and 7.0 J/cm2. Effects of LLL on cellular responses of venom-induced cytotoxicity were examined, including cell viability, measurement of cell damage and intra and extracellular ATP levels, expression of myogenic regulatory factors, as well as cellular differentiation.ResultsIn non-irradiated cells, the venom caused a decrease in cell viability and a massive release of LDH and CK levels indicating myonecrosis. Infrared and red laser at all energy densities were able to considerably decrease venom-induced cytotoxicity. Laser irradiation induced myoblasts to differentiate into myotubes and this effect was accompanied by up regulation of MyoD and specially myogenin. Moreover, LLL was able to reduce the extracellular while increased the intracellular ATP content after venom exposure. In addition, no difference in the intensity of cytotoxicity was shown by non-irradiated and irradiated venom.ConclusionLLL irradiation caused a protective effect on C2C12 cells against the cytotoxicity caused by B. jararacussu venom and promotes differentiation of these cells by up regulation of myogenic factors. A modulatory effect of ATP synthesis may be suggested as a possible mechanism mediating cytoprotection observed under laser irradiation.

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Bleeding is a common feature in envenoming caused by Bothrops snake venom due to extensive damage to capillaries and venules, producing alterations in capillary endothelial cell morphology. It has been demonstrated, in vivo, that photobiomodulation (PBM) decreases hemorrhage after venom inoculation; however, the mechanism is unknown. Thus, the objective was to investigate the effects of PBM on a murine endothelial cell line (tEnd) exposed to Bothrops jararaca venom (BjV). Cells were exposed to BjV and irradiated once with either 660- or 780-nm wavelength laser light at energy densities of 4 and 5 J/cm(2), respectively, and irradiation time of 10 s. Cell integrity was analyzed by crystal violet and cell viability/mitochondrial metabolism by MTT assay. The release of lactic dehydrogenase (LDH) was quantified as a measure of cell damage. In addition, cytokine IL1-β levels were measured in the supernatant. PBM at 660 and 780 nm wavelength was able to increase cellular viability and decrease the release of LDH and the loss of cellular integrity. In addition, the concentration of pro-inflammatory cytokine IL1-β was reduced after PBM by both wavelengths. The data reported herein indicates that irradiation with red or near-infrared laser resulted in protection on endothelial cells after exposure to Bothrops venom and could be, at least in part, a reasonable explanation by the beneficial effects of PBM inhibiting the local effects induced by Bothrops venoms, in vivo.

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INTRODUCTIONEnvenoming caused by snakes of the genus Bothrops is often associated with severe and complex local pathological manifestations, with dermonecrosis and myonecrosis being the most severe damage. The specific treatment for Bothrops accident is the use of the antivenom, with the function to neutralize the circulating venom, but its action does not extend the local manifestations. Previous studies in our group have shown that photobiomodulation using low intensity laser (LBI) increases the viability of C2C12 muscle cells when incubated with B. jararacussu venom (BjV) and further promotes the proliferation of these cells.AIMto analyze the effect of LBI on muscle repair and the oxidative stress caused by B. jararacussu venom on C2C12 myoblast cells.METHODOLOGYMyoblasts cells were plated at 1×104 cells/well and incubated for 24 hours for cell adhesion. BjV (12.5ug/ml) was applied and the cells were immediately irradiated with LBI (660 and 780 nm, power of 100 mW, irradiated time 10 sec, energy density of 4 J/cm2) and incubated for 1 h. Then, a wound was created by scratching cells with a sterile pipette tip and the cell migration into the wound surface was analyzed 24 h after the venom incubation. Oxidative stress (NO, H2O2, SOD, Gpx and TBARS) were analyzed 1 h after BjV incubation. The NO concentration was evaluated by the GRIESS and the H2O2 by the phenol red method. The concentration of SOS and Gpx was made by spectrometry. The analysis of TBARS by the Elisa method.RESULTSResults showed that the LBI was able to almost completely close the risk of the wound, similar to the control group. C2C12 cells were able to produce both NO and H2O2 one hour after the addition of the venom being statistically different from the control. LBI irradiation significantly reduced NO release, but did not alter the release of H2O2. No significant difference was observed of anti‐oxidant enzymes in C2C12 cells incubated to BjV. LBI Irradiation induced a significant increase of SOD in comparison to the venom group, but not modify Gpx release. BjV did not cause lipid peroxidation and LBI treatment did not modify this effect.CONCLUSIONPhotobiomodulation caused muscle cell protection and this protection seems to be related to decrease of reactive species of oxygen and nitrogen and increase of anti‐oxidants.Support or Funding InformationComissão de Aperfeiçoamento de Pessoal do Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.