强力霉素介导的蛇毒磷脂酶和金属蛋白酶抑制作用

IF 1.2 4区 医学 Q2 MEDICINE, GENERAL & INTERNAL Military Medicine Pub Date : 2024-11-05 DOI:10.1093/milmed/usae184
Daniel K Arens, Meaghan A Rose, Emelyn M Salazar, Merideth A Harvey, Eun Y Huh, April A Ford, Daniel W Thompson, Elda E Sanchez, Yoon Y Hwang
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引用次数: 0

摘要

简介:根据《联合创伤系统军事临床实践指南--全球毒蛇啮咬管理》,蛇咬伤在所有战区都是一种令人担忧的威胁。蛇毒是一种复杂的毒素混合物,包括磷脂酶 A2 (PLA2) 和蛇毒金属蛋白酶 (SVMP),可产生肌毒性、血液毒性和细胞毒性损伤。以抗体为基础的抗蛇毒血清是标准的治疗方法,但包括小分子抑制剂在内的新方法近年来也备受关注。强力霉素是人类金属蛋白酶和 PLA2 的有效抑制剂。3 种不同系统发育的蛇的酶活性:在多西环素的抑制条件下测试了 Agkistrodon piscivorus、Naja kaouthia 和 Daboia russelii 的酶活性:使用 EnzChek 磷脂酶 A2 和明胶酶检测试剂盒测定了 N. kaouthia、D. russelii 和 A. piscivorus 毒液中单独和与强力霉素一起使用时的 PLA2 和 SVMP 酶活性。采用单因素方差分析和Tukey事后检验进行比较分析。在巴格白化(BALB/c)成年小鼠模型中,采用肌肉注射法测定了毒液的中位致死剂量、强力霉素的有效剂量和肌酸激酶(CK)抑制水平。使用 Spearman-Karber 方法确定了中位致死剂量和有效剂量,并使用单因素方差分析和 Tukey 后检验比较了肌酸激酶抑制水平:结果:与仅使用毒液的对照组相比,使用 0.32、0.16 和 0.08 毫克/毫升多西环素可将所有 3 种毒液中的磷脂酶 A2 活性降低 1.5% 至 44.0%,且其降低呈剂量依赖性(P 结论:多西环素可降低磷脂酶 A2 活性,但其降低幅度与毒液对照组无关):多西环素降低了PLA2-和SVMP相关的致死率,尤其是在A. piscivorus蛇毒中毒中,而且在有限的D. russelii蛇毒中毒中,这揭示了多西环素作为蛇咬伤治疗药物的前景。此外,接受过多西环素处理毒液的小鼠肌肉损伤的常见指标--CK活性也受到了抑制。ED50 研究中确定的多西环素浓度相当于 70 公斤体重的人服用 1,456 至 5,061 毫克的剂量。毒液产量和蛇的种类等因素会影响实际所需剂量。需要对大剂量强力霉素的安全性及其对多种蛇类的有效性进行研究,以便将其完全应用于人类。在这项工作的基础上,多西环素可作为一种治疗方法,用于更高级别的治疗,保护肌肉免受损伤,并降低不同蛇类的致死率。
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Doxycycline-Mediated Inhibition of Snake Venom Phospholipase and Metalloproteinase.

Introduction: Warfighters are exposed to life-threatening injuries daily and according to the Joint Trauma System Military Clinical Practice Guideline-Global Snake Envenomation Management snakebites are a concerning threat in all theaters of operation. Snake venom is a complex mixture of toxins including phospholipases A2 (PLA2) and snake venom metalloproteinases (SVMP) that produce myotoxic, hemotoxic, and cytotoxic injuries. Antibody-based antivenom is the standard of care but new approaches including small-molecule inhibitors have gained attention in recent years. Doxycycline is an effective inhibitor of human metalloproteinases and PLA2. The enzymatic activities of 3 phylogenetically distinct snakes: Agkistrodon piscivorus, Naja kaouthia, and Daboia russelii were tested under inhibitory conditions using doxycycline.

Materials and methods: Enzymatic activity of PLA2 and SVMP was measured in N. kaouthia, D. russelii, and A. piscivorus venom alone and with doxycycline using EnzChek Phospholipase A2 and Gelatinase Assay Kits. A 1-way ANOVA with Tukey's post-hoc test was used to conduct comparative analysis. The median lethal dose of the venoms, the effective dose of doxycycline, and creatine kinase (CK) inhibition levels were measured in a murine model with adult Bagg Albino (BALB/c) mice using intramuscular injections. Median lethal and effective doses were determined using Spearman-Karber's method and a 1-way ANOVA with Tukey's post-hoc test was used to compare CK inhibition levels.

Results: Phospholipases A2 activity was reduced to 1.5% to 44.0% in all 3 venoms in a dose-dependent manner using 0.32, 0.16, and 0.08 mg/mL doxycycline when compared to venom-only controls (P < .0001) (Fig. 1A). Snake venom metalloproteinases activity was reduced to 4% to 62% in all 3 venoms in a dose-dependent manner using 0.32, 0.16, and 0.08 mg/mL doxycycline (P < .0001) (Fig. 1B). The lethal dose (LD50) values of the venoms in the murine model were calculated as follows: A. piscivorus = 20.29 mg/kg (Fig. 2A), N. kaouthia = 0.38 mg/kg (Fig. 2B), and D. russelii = 7.92 mg/kg (Fig. 2C). The effective dose (ED50) of doxycycline in A. piscivorus was calculated to be 20.82 mg/kg and 72.07 mg/kg when treating D. russelii venom. No ED50 could be calculated when treating N. kaouthia venom (Fig. 3). Creatine kinase activity was significantly decreased in all 3 venoms treated with doxycycline (P < .0001) (Fig. 4).

Conclusion: Doxycycline reduced PLA2- and SVMP-related lethality, particularly in A. piscivorus envenomings and in a limited capacity with D. russelii revealing its promise as a treatment for snakebites. In addition, CK activity, a common indicator of muscle damage was inhibited in mice that received doxycycline-treated venom. The doxycycline concentrations identified in the ED50 studies correspond to 1,456 to 5,061 mg dosages for a 70 kg human. Factors including venom yield and snake species would affect the actual dosage needed. Studies into high-dose doxycycline safety and its effectiveness against several snake species is needed to fully translate its use into humans. Based on this work, doxycycline could be used as a treatment en route to higher echelons of care, providing protection from muscle damage and reducing lethality in different snake species.

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来源期刊
Military Medicine
Military Medicine MEDICINE, GENERAL & INTERNAL-
CiteScore
2.20
自引率
8.30%
发文量
393
审稿时长
4-8 weeks
期刊介绍: Military Medicine is the official international journal of AMSUS. Articles published in the journal are peer-reviewed scientific papers, case reports, and editorials. The journal also publishes letters to the editor. The objective of the journal is to promote awareness of federal medicine by providing a forum for responsible discussion of common ideas and problems relevant to federal healthcare. Its mission is: To increase healthcare education by providing scientific and other information to its readers; to facilitate communication; and to offer a prestige publication for members’ writings.
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