Department of Studies in Biochemistry, University of Mysore, Manasagangotri, Mysore, Karnataka, India.
The Ohio State University Comprehensive Cancer Center, Columbus, Ohio, United States of America.
PLoS Negl Trop Dis. 2021 Feb 2;15(2):e0008596. doi: 10.1371/journal.pntd.0008596. eCollection 2021 Feb.
Echis carinatus (EC) is known as saw-scaled viper and it is endemic to the Indian subcontinent. Envenoming by EC represents a major cause of snakebite mortality and morbidity in the Indian subcontinent. Zinc (Zn++) dependent snake venom metalloproteases (SVMPs) present in Echis carinatus venom (ECV) is well known to cause systemic hemorrhage and coagulopathy in experimental animals. An earlier report has shown that ECV activates neutrophils and releases neutrophil extracellular traps (NETs) that blocks blood vessels leading to severe tissue necrosis. However, the direct involvement of SVMPs in the release of NETs is not clear. Here, we investigated the direct involvement of EC SVMPs in observed pathological symptoms in a preclinical setup using specific Zn++ metal chelator, Tetraethyl thiuram disulfide (TTD)/disulfiram. TTD potently antagonizes the activity of SVMPs-mediated ECM protein degradation in vitro and skin hemorrhage in mice. In addition, TTD protected mice from ECV-induced footpad tissue necrosis by reduced expression of citrullinated H3 (citH3) and myeloperoxidase (MPO) in footpad tissue. TTD also neutralized ECV-induced systemic hemorrhage and conferred protection against lethality in mice. Moreover, TTD inhibited ECV-induced NETosis in human neutrophils and decreased the expression of peptidyl arginine deiminase (PAD) 4, citH3, MPO, and p-ERK. Further, we demonstrated that ECV-induced NETosis and tissue necrosis are mediated via PAR-1-ERK axis. Overall, our results provide an insight into SVMPs-induced toxicities and the promising protective efficacy of TTD can be extrapolated to treat severe tissue necrosis complementing anti-snake venom (ASV).
圆斑蝰（EC）又称锯鳞蝰蛇，是印度次大陆的特有种。EC 蛇毒中的锌（Zn++）依赖性蛇毒金属蛋白酶（SVMPs）已知会导致印度次大陆的蛇伤死亡率和发病率升高。圆斑蝰蛇毒（ECV）中存在的锌（Zn++）依赖性蛇毒金属蛋白酶（SVMPs）已被证明会在实验动物中引起全身性出血和凝血功能障碍。之前的一项研究表明，ECV 会激活中性粒细胞并释放中性粒细胞胞外诱捕网（NETs），从而阻塞血管，导致严重的组织坏死。然而，SVMPs 是否直接参与 NETs 的释放尚不清楚。在这里，我们使用特定的 Zn++金属螯合剂四乙基秋兰姆二硫代磷酸酯（TTD）/戒酒硫，在临床前模型中研究了 EC SVMPs 在观察到的病理症状中的直接参与。TTD 可有效拮抗 SVMPs 介导的细胞外基质蛋白降解和小鼠皮肤出血。此外，TTD 通过减少足垫组织中瓜氨酸化 H3（citH3）和髓过氧化物酶（MPO）的表达，保护小鼠免受 ECV 诱导的足垫组织坏死。TTD 还中和了 ECV 诱导的全身性出血，并在小鼠中提供了针对致死性的保护。此外，TTD 抑制了 ECV 诱导的人中性粒细胞 NETosis，并降低了肽基精氨酸脱亚氨酶（PAD）4、citH3、MPO 和 p-ERK 的表达。进一步，我们证明了 ECV 诱导的 NETosis 和组织坏死是通过 PAR-1-ERK 轴介导的。总的来说，我们的研究结果提供了对 SVMPs 诱导的毒性的深入了解，并表明 TTD 的有希望的保护效果可以推断为治疗严重组织坏死，补充抗蛇毒血清（ASV）。
