Korean Medicine (KM)-Application Center, Korea Institute of Oriental Medicine, 70, Cheomdanro, Dong-gu, Daegu 41062, Republic of Korea.
Nutrients. 2024 Sep 27;16(19):3266. doi: 10.3390/nu16193266.
This study aimed to analyze the immunostimulatory activity of gamisoyosan (GSS) on the activation of macrophages in RAW 264.7 cells and its underlying mechanisms.
The effects of GSS on the secretion of nitric oxide (NO), immunomodulatory mediators, cytokines and mRNAs, and related proteins were assessed using the Griess assay, Western blotting, quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, and HDCFDA, respectively. The level of phagocytosis was determined by the neutral red method while the immune function of GSS was determined using adhesion and wound-healing assays.
GSS-treated macrophages significantly increased the production of NO, immunomodulatory enzymes, cytokines, and intracellular reactive oxygen species without causing cytotoxicity. GSS effectively improved macrophage immune function by increasing their phagocytic level, adhesion function, and migration activity. Mechanistic studies via Western blotting revealed that GSS notably induced the activation of the Toll-like receptor (TLR) 4-mediated mitogen-activated protein kinase, nuclear factor-κB, and protein kinase B signaling pathways.
Overall, our results indicated that GSS could activate macrophages through the secretion of immune-mediated transporters via TLR4-dependent signaling pathways. Thus, GSS has potential value as an immunity-enhancing agent.
本研究旨在分析加味四君子汤（GSS）对 RAW 264.7 细胞中巨噬细胞活化的免疫刺激活性及其作用机制。
采用 Griess 法、Western blot 法、实时定量聚合酶链反应（qPCR）、酶联免疫吸附试验（ELISA）和 HDCFDA 分别检测 GSS 对一氧化氮（NO）、免疫调节介质、细胞因子和 mRNAs 及相关蛋白分泌的影响。中性红法测定吞噬作用水平，黏附及划痕愈合试验测定 GSS 的免疫功能。
GSS 处理的巨噬细胞可显著增加 NO、免疫调节酶、细胞因子和细胞内活性氧的产生，而无细胞毒性。GSS 可通过提高吞噬水平、黏附功能和迁移活性，有效改善巨噬细胞免疫功能。Western blot 结果显示，GSS 可显著诱导 Toll 样受体 4（TLR4）介导的丝裂原活化蛋白激酶、核因子-κB 和蛋白激酶 B 信号通路的激活。
总之，本研究结果表明，GSS 可通过 TLR4 依赖的信号通路，通过分泌免疫调节转运蛋白来激活巨噬细胞。因此，GSS 具有作为免疫增强剂的潜在价值。
