Background. Benzoylmesaconine (BMA), the most abundant monoester alkaloid in Aconitum plants, has some biological activities and is a potential therapeutic agent for inflammation-related diseases. However, the potential anti-inflammatory mechanisms of BMA have not been clarified. Purpose. This study aimed to investigate the underlying molecular mechanisms of the anti-inflammatory action of this compound using lipopolysaccharide (LPS)-activated RAW264.7 macrophages. Methods. The release of pro-inflammatory cytokines and mediators were detected by nitric oxide (NO) assays, reactive oxygen species (ROS) assays, and enzyme-linked immunosorbent assays (ELISA) in LPS-activated RAW264.7 macrophage cells. Quantitative real-time PCR was used to measure the gene expression of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-6, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2). Cell viability was determined using a cell counting kit-8 (CCK-8) assay. The expression of iNOS, COX-2, mitogen-activated protein kinase (MAPK), and nuclear factor-κB (NF-κB)-related proteins were detected by western blot, and nuclear translocation of p65 was observed by immunofluorescence. Results. BMA significantly decreased the production of IL-1β, IL-6, TNF-α, PGE₂, NO, and ROS and inhibited the protein and mRNA levels of COX-2 and iNOS in LPS-activated RAW264.7 macrophages. Moreover, LPS-induced phosphorylation of IκBα, JNK, p38, and ERK; degradation of IκBα; and nuclear translocation of p65 were significantly suppressed by BMA treatment. Conclusion. These findings demonstrate that the anti-inflammatory effect of BMA was through the suppression of the NF-κB and MAPK signaling pathways and that it may be a therapeutic agent targeting specific signal transduction events required for inflammation-related diseases.

中文翻译：

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苯甲酰美沙康在脂多糖刺激的RAW264.7细胞中的抗炎作用机制

背景。苯甲酰美沙乌碱 (BMA) 是乌头植物中含量最丰富的单酯生物碱，具有一定的生物活性，是炎症相关疾病的潜在治疗剂。然而，BMA 的潜在抗炎机制尚未阐明。目的。本研究旨在利用脂多糖 (LPS) 激活的 RAW264.7 巨噬细胞研究该化合物抗炎作用的潜在分子机制。方法。通过一氧化氮 (NO) 测定、活性氧 (ROS) 测定和酶联免疫吸附测定 (ELISA) 在 LPS 激活的 RAW264.7 巨噬细胞中检测促炎细胞因子和介质的释放。定量实时 PCR 用于测量白细胞介素 (IL)-1 β、肿瘤坏死因子 (TNF) -α、IL-6、诱导型一氧化氮合酶 (iNOS) 和环氧合酶-2 (COX-2 ) 的基因表达）。使用细胞计数试剂盒 8 (CCK-8) 测定法测定细胞活力。Western blot检测iNOS、COX-2、丝裂原活化蛋白激酶(MAPK)和核因子-κB (NF-κB )相关蛋白的表达，免疫荧光观察p65的核转位。结果。BMA 显着降低了 LPS 激活的 RAW264.7 巨噬细胞中 IL-1 β、IL-6、TNF- α、PGE ₂、NO 和 ROS 的产生，并抑制了 COX-2 和 iNOS 的蛋白质和 mRNA 水平。此外，LPS 诱导的IκBα 、 JNK 、p38 和 ERK的磷酸化；I κ B α的降解；BMA处理显着抑制了p65的核转位和核转位。结论。这些发现表明，BMA 的抗炎作用是通过抑制 NF- κB 和 MAPK 信号通路，它可能是针对炎症相关疾病所需的特定信号转导事件的治疗剂。