Melittin-induced metabolic changes on the Madin-Darby Bovine Kidney cell line

ABSTRACT In this study, the toxic effects of melittin on Madin-Darby Bovine Kidney cells (MDBK) were analyzed with respect to mitochondrial functionality by reduction of MTT and flow cytometry, apoptosis potential, necrosis, oxygen reactive species (ROS) production, lipid peroxidation, and DNA fragmentation using flow cytometry and cell membrane destabilization by confocal microscopy. The toxicity presented dose-dependent characteristics and mitochondrial activity was inhibited by up to 78.24 ±3.59% (P<0.01, n = 6) in MDBK cells exposed to melittin (10μg/mL). Flow cytometry analysis revealed that melittin at 2μg/mL had the highest necrosis rate (P<0.05) for the cells. The lipoperoxidation of the membranes was also higher at 2μg/mL of melittin (P<0.05), which was further confirmed by the microphotographs obtained by confocal microscopy. The highest ROS production occurred when the cells were exposed to 2.5μg/mL melittin (P<0.05), and this concentration also increased DNA fragmentation (P<0.05). There was a significative and positive correlation between the lipoperoxidation of membranes with ROS (R=0.4158), mitochondrial functionality (R=0.4149), and apoptosis (R=0.4978). Thus, the oxidative stress generated by melittin culminates in the elevation of intracellular ROS that initiates a cascade of toxic events in MDBK cells.

摘要 本研究针对蜂毒肽（melittin）对马-达二氏牛肾细胞（Madin-Darby Bovine Kidney cells, MDBK）的毒性效应展开分析，通过MTT还原法与流式细胞术检测线粒体功能、细胞凋亡潜能、细胞坏死、活性氧（ROS）生成、脂质过氧化及DNA断裂情况，并借助共聚焦显微镜观察细胞膜稳定性变化。研究结果显示，蜂毒肽的毒性呈现剂量依赖性特征；当MDBK细胞暴露于10μg/mL蜂毒肽时，线粒体活性被抑制达78.24±3.59%（P<0.01, n = 6）。流式细胞术分析表明，2μg/mL蜂毒肽处理组的细胞坏死率最高（P<0.05）。该浓度下的细胞膜脂质过氧化水平同样显著升高（P<0.05），这一结果通过共聚焦显微镜获取的显微照片得到进一步验证。当细胞暴露于2.5μg/mL蜂毒肽时，ROS生成量达到峰值（P<0.05），且该浓度同样会加剧DNA断裂（P<0.05）。细胞膜脂质过氧化与ROS、线粒体功能及细胞凋亡分别存在显著正相关关系，相关系数依次为R=0.4158、R=0.4149及R=0.4978。综上，蜂毒肽诱导的氧化应激会引发胞内ROS水平升高，进而启动MDBK细胞内一系列毒性级联反应。