Malaria Research Group, Department of Biotechnology, Indian Institute of Technology, Guwahati, Assam 781039, India.
Toxicol In Vitro. 2013 Feb;27(1):16-23. doi: 10.1016/j.tiv.2012.09.016. Epub 2012 Oct 3.
Macrophages are an integral part of the immune system, required to produce a robust immune response against an infectious organism. Presence of methemoglobin in body fluids such as blood, cerebrospinal fluid and urine is associated with tissue damage. We tested cytotoxic effects of MetHb and underlying molecular events in mouse macrophage cell line J774A.1. MetHb exposure dose dependently reduced macrophage viability in an MTT assay. Light microscopy and scanning electron microscopic (SEM) observation of MetHb treated macrophage indicated death (less number of cells per field), severe damage to membrane structure and accumulation of particulate matter in the cytosol. The macrophage death during MetHb exposure was due to induction of apoptosis as indicated by annexin-V/FITC staining and DNA fragmentation analysis. MetHb treatment generated a periodic ROS spikes with time in the macrophage cytosol to develop oxidative stress. Scavenging ROS spikes with NAC, mannitol or PBN dose dependently protected macrophages against MetHb induced toxicity, apoptosis and cellular membrane damage. Our work highlighted the contributions of MetHb mediated toxicity toward macrophage and its potential role in tissue damage and immune depression during malaria and other hemolytic disorders.
巨噬细胞是免疫系统的重要组成部分，需要产生强大的免疫反应来对抗感染性生物体。血液、脑脊液和尿液等体液中高铁血红蛋白的存在与组织损伤有关。我们测试了 MetHb 对小鼠巨噬细胞系 J774A.1 的细胞毒性作用及其潜在的分子事件。MTT 检测表明，MetHb 暴露剂量依赖性地降低了巨噬细胞的活力。MetHb 处理后的巨噬细胞的光镜和扫描电子显微镜（SEM）观察表明细胞死亡（视野中细胞数量减少）、细胞膜结构严重受损以及细胞质中颗粒物质的积累。MetHb 暴露导致的巨噬细胞死亡是由于凋亡的诱导，这可以通过 Annexin-V/FITC 染色和 DNA 片段化分析来指示。MetHb 处理会在巨噬细胞质中随时间产生周期性的 ROS 爆发，从而引发氧化应激。用 NAC、甘露醇或 PBN 清除 ROS 爆发可剂量依赖性地保护巨噬细胞免受 MetHb 诱导的毒性、凋亡和细胞膜损伤。我们的工作强调了 MetHb 介导的毒性对巨噬细胞的贡献，以及它在疟疾和其他溶血性疾病期间对组织损伤和免疫抑制的潜在作用。
