Menezes Ramon R P P B de, Mello Clarissa P, Lima Dânya B, Tessarolo Louise D, Sampaio Tiago Lima, Paes Lívia C F, Alves Natacha T Q, Assis Junior Eudmar M, Lima Junior Roberto C P, Toyama Marcos H, Martins Alice M C
Department of Physiology and Pharmacology, School of Medicine, Federal University of Ceará, Fortaleza, Ceará, Brazil.
Department of Clinical and Toxicological Analysis, Federal University of Ceará, Fortaleza, Ceará, Brazil.
PLoS One. 2016 Mar 14;11(3):e0151029. doi: 10.1371/journal.pone.0151029. eCollection 2016.
Viperidae venom has several local and systemic effects, such as pain, edema, inflammation, kidney failure and coagulopathy. Additionally, bothropic venom and its isolated components directly interfere on cellular metabolism, causing alterations such as cell death and proliferation. Inflammatory cells are particularly involved in pathological envenomation mechanisms due to their capacity of releasing many mediators, such as nitric oxide (NO). NO has many effects on cell viability and it is associated to the development of inflammation and tissue damage caused by Bothrops and Bothropoides venom. Bothropoides insularis is a snake found only in Queimada Grande Island, which has markedly toxic venom. Thus, the aim of this work was to evaluate the biological effects of Bothropoides insularis venom (BiV) on RAW 264.7 cells and assess NO involvement. The venom was submitted to colorimetric assays to identify the presence of some enzymatic components. We observed that BiV induced H2O2 production and showed proteolytic and phospholipasic activities. RAW 264.7 murine macrophages were incubated with different concentrations of BiV and then cell viability was assessed by MTT reduction assay after 2, 6, 12 and 24 hours of incubation. A time- and concentration-dependent effect was observed, with a tendency to cell proliferation at lower BiV concentrations and cell death at higher concentrations. The cytotoxic effect was confirmed after lactate dehydrogenase (LDH) measurement in the supernatant from the experimental groups. Flow cytometry analyses revealed that necrosis is the main cell death pathway caused by BiV. Also, BiV induced NO release. The inhibition of both proliferative and cytotoxic effects with L-NAME were demonstrated, indicating that NO is important for these effects. Finally, BiV induced an increase in iNOS expression. Altogether, these results demonstrate that B. insularis venom have proliferative and cytotoxic effects on macrophages, with necrosis participation. We also suggest that BiV acts by inducing iNOS expression and causing NO release.
蝰蛇科毒液具有多种局部和全身作用，如疼痛、水肿、炎症、肾衰竭和凝血病。此外，具窍蝮蛇毒液及其分离成分直接干扰细胞代谢，导致细胞死亡和增殖等变化。炎症细胞因其能够释放许多介质，如一氧化氮（NO），而特别参与病理性蛇咬中毒机制。NO对细胞活力有多种影响，并且与具窍蝮蛇属和岛蝮属毒液引起的炎症和组织损伤的发展有关。岛蝮是一种仅在大凯马达岛发现的蛇，其毒液具有明显的毒性。因此，本研究的目的是评估岛蝮毒液（BiV）对RAW 264.7细胞的生物学作用并评估NO的参与情况。对该毒液进行比色测定以鉴定某些酶成分的存在。我们观察到BiV诱导H2O2产生，并表现出蛋白水解和磷脂酶活性。将RAW 264.7小鼠巨噬细胞与不同浓度的BiV孵育，然后在孵育2、6、12和24小时后通过MTT还原试验评估细胞活力。观察到时间和浓度依赖性效应，在较低BiV浓度下有细胞增殖趋势，在较高浓度下有细胞死亡趋势。通过测量实验组上清液中的乳酸脱氢酶（LDH）证实了细胞毒性作用。流式细胞术分析表明坏死是BiV引起的主要细胞死亡途径。此外，BiV诱导NO释放。L-NAME对增殖和细胞毒性作用的抑制作用得到证实，表明NO对这些作用很重要。最后，BiV诱导诱导型一氧化氮合酶（iNOS）表达增加。总之，这些结果表明岛蝮毒液对巨噬细胞具有增殖和细胞毒性作用，并伴有坏死参与。我们还认为BiV通过诱导iNOS表达和导致NO释放而起作用。
