Amity Institute of Biotechnology, Amity University Rajasthan, Amity Education Valley, Kant Kalwar, NH-11C, Jaipur-Delhi Highway, Jaipur, India.
Department of Biotechnology, IIS (deemed to be University), Gurukul Marg, SFS, Mansarovar, Jaipur, India.
Clin Chim Acta. 2021 Feb;513:43-49. doi: 10.1016/j.cca.2020.12.004. Epub 2020 Dec 10.
Recent reports from this lab have demonstrated a higher incidence of NETs, nitrosative, as well as oxidative stress, and have a direct correlation with the severity of sepsis and organ damage. However, the mechanistic perspective of NETs induced organ damage has not been understood at the cellular and molecular level. Interaction of inducible nitric oxide synthase (iNOS) with Rac2 in regulating reactive oxygen species (ROS) and reactive nitrogen species (RNS) generation and its implications in microbial killing has been reported. This study was, therefore, undertaken in neutrophils of sepsis patients to investigate the functional importance of iNOS-Rac2 interaction in ROS/ RNS, peroxynitrite generation, NETs generation, and NETs mediated cell death.
The study was conducted on 100 patients with sepsis and 50 healthy volunteers. Interaction between iNOS and Rac2 was performed using co-immunoprecipitation and co-immunolabeling assay. Free radicals involving ROS and RNS were evaluated using cytochrome c reduction assay. NETs formation was evaluated by fluorescence microscopy. The cytotoxic effect of NETs was assessed on lung carcinoma cell line (A549) using colorimetric Alamar blue assay.
Enhanced interaction between iNOS and Rac2 was found in sepsis neutrophils in comparison with control. This was accompanied by an increased level of superoxide (O), nitric oxide (NO), and peroxynitrite (ONOO) which were decreased in the presence of NAC, DPI, and 1400 W, signifying the role of iNOS-Rac2 interaction. Enhanced NETs release from activated sepsis neutrophils were abrogated in the presence of DPI. NETs from sepsis neutrophils exert a cytotoxic effect on lung epithelial cells (A549) in a concentration-dependent manner.
Our findings exhibit the functional role of iNOS-Rac2 interaction in ROS/RNS, peroxynitrite generation, NETs generation, and NETs mediated cell death.
本实验室最近的报告表明，中性粒细胞胞外诱捕网（NETs）、硝化和氧化应激的发生率更高，并且与脓毒症的严重程度和器官损伤直接相关。然而，NETs 诱导的器官损伤的机制尚未在细胞和分子水平上得到理解。诱导型一氧化氮合酶（iNOS）与 Rac2 的相互作用在调节活性氧（ROS）和活性氮（RNS）的产生及其在微生物杀伤中的作用已被报道。因此，本研究在脓毒症患者的中性粒细胞中进行，以研究 iNOS-Rac2 相互作用在 ROS/RNS、过氧亚硝酸盐生成、NETs 生成和 NETs 介导的细胞死亡中的功能重要性。
该研究在 100 名脓毒症患者和 50 名健康志愿者中进行。使用共免疫沉淀和共免疫标记测定法检测 iNOS 和 Rac2 之间的相互作用。使用细胞色素 c 还原测定法评估涉及 ROS 和 RNS 的自由基。通过荧光显微镜评估 NETs 的形成。使用比色法 Alamar 蓝测定法评估 NETs 对肺癌细胞系（A549）的细胞毒性作用。
与对照组相比，脓毒症中性粒细胞中发现 iNOS 和 Rac2 之间的相互作用增强。这伴随着超氧化物（O）、一氧化氮（NO）和过氧亚硝酸盐（ONOO）水平的增加，而在 NAC、DPI 和 1400W 的存在下，这些水平降低，表明 iNOS-Rac2 相互作用的作用。在 DPI 的存在下，从激活的脓毒症中性粒细胞中释放的增强的 NETs 被阻断。脓毒症中性粒细胞来源的 NETs 以浓度依赖的方式对肺上皮细胞（A549）产生细胞毒性作用。
我们的研究结果表明，iNOS-Rac2 相互作用在 ROS/RNS、过氧亚硝酸盐生成、NETs 生成和 NETs 介导的细胞死亡中发挥功能作用。
