Coelho Vanessa Rodrigues, Viau Cassiana Macagnan, Staub Renata Bartolomeu, De Souza Marcele Silva, Pflüger Pricila, Regner Gabriela Gregory, Pereira Patrícia, Saffi Jenifer
Laboratory of Neuropharmacology and Preclinical Toxicology, Federal University of Rio Grande do Sul, Porto Alegre, Brazil.
Neuroimmunomodulation. 2017;24(3):171-181. doi: 10.1159/000481095. Epub 2017 Nov 7.
The present study evaluated the ability of rosmarinic acid (RA) to inhibit microglia activation induced by lipopolysaccharide (LPS) in the N9 murine microglial cell line, and investigated the putative mechanisms involved in this process.
In all tests, N9 murine microglial cells were pretreated with RA (0.1, 1.0, and 10 μM) for 20 h and exposed to LPS (1 μM/mL) for 4 h. Cell viability was measured by Trypan blue exclusion assay. Flow cytometry was used to detect reactive oxygen species (ROS), quantify cleaved caspase-3, and analyze the mitochondrial electrochemical potential. iNOS, Arg-1, TNF-α, IL-1β, and IL-6 proteins were analyzed by Western blotting, and their antigens were detected using the chemiluminescence technique. The effect of RA on DNA was evaluated by the Comet assay.
RA attenuated the expression of the M1 marker iNOS and the levels of proinflammatory factors, including TNF-α, IL-1β, and IL-6; it increased the expression of the M2 marker Arg-1, and inhibited, at least in part, ROS generation and loss of mitochondrial outer membrane permeabilization through the inhibition of cleaved caspase-3 activation. RA also inhibited DNA damage, reassuring cell protection.
The results suggested a protective effect of RA through downregulation of inflammatory cytokines and cleaved caspase-3.
本研究评估了迷迭香酸（RA）抑制N9小鼠小胶质细胞系中脂多糖（LPS）诱导的小胶质细胞活化的能力，并研究了该过程中涉及的潜在机制。
在所有试验中，N9小鼠小胶质细胞先用RA（0.1、1.0和10 μM）预处理20小时，然后暴露于LPS（1 μM/mL）4小时。通过台盼蓝排斥试验测量细胞活力。流式细胞术用于检测活性氧（ROS）、定量裂解的半胱天冬酶-3并分析线粒体电化学电位。通过蛋白质印迹分析诱导型一氧化氮合酶（iNOS）、精氨酸酶-1（Arg-1）、肿瘤坏死因子-α（TNF-α）、白细胞介素-1β（IL-1β）和白细胞介素-6（IL-6）蛋白，并使用化学发光技术检测其抗原。通过彗星试验评估RA对DNA的影响。
RA减弱了M1标志物iNOS的表达以及包括TNF-α、IL-1β和IL-6在内的促炎因子水平；它增加了M2标志物Arg-1的表达，并至少部分抑制了ROS的产生以及通过抑制裂解的半胱天冬酶-3活化导致的线粒体外膜通透性丧失。RA还抑制了DNA损伤，从而证实了细胞保护作用。
结果表明RA通过下调炎性细胞因子和裂解的半胱天冬酶-3发挥保护作用。
