白皮醇赤芍甲素通过抑制 RAW264.7 细胞和斑马鱼中的 NF-κB 和 STAT3 通路减轻脂多糖诱导的炎症反应。
Ethyl Lithospermate Reduces Lipopolysaccharide-Induced Inflammation through Inhibiting NF-κB and STAT3 Pathways in RAW 264.7 Cells and Zebrafish.
机构信息
Third Level Research Laboratory of State Administration of Traditional Chinese Medicine, Guangdong Provincial Key Laboratory of Chinese Medicine Pharmaceutics, School of Traditional Chinese Medicine, Southern Medical University, Guangzhou, 510515, China.
Department of Pharmacy, Zhujiang Hospital, Southern Medical University, Guangzhou, 510282, China.
出版信息
Chin J Integr Med. 2023 Dec;29(12):1111-1120. doi: 10.1007/s11655-023-3643-y. Epub 2023 Aug 23.
OBJECTIVE
To explore the anti-inflammatory effects of ethyl lithospermate in lipopolysaccharide (LPS)-stimulated RAW 264.7 murine-derived macrophages and zebrafish, and its underlying mechanisms.
METHODS
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide (MTT) assays were performed to investigate the toxicity of ethyl lithospermate at different concentrations (12.5-100 µ mol/L) in RAW 264.7 cells. The cells were stimulated with LPS (100 ng/mL) for 12 h to establish an inflammation model in vitro, the production of pro-inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor α (TNF-α) were assessed by enzyme linked immunosorbent assay (ELISA). Western blot was used to ascertain the protein expressions of signal transducer and activator of transcription 3 (STAT3), nuclear factor kappa B (NF-κB) p65, phospho-STAT3 (p-STAT3, Tyr705), inhibitor of NF-κB (IκB) α, and phospho-I κB α (p-IκB α, Ser32), and confocal imaging was used to identify the nuclear translocation of NF-κB p65 and p-STAT3 (Tyr705). Additionally, the yolk sacs of zebrafish (3 days post fertilization) were injected with 2 nL LPS (0.5 mg/mL) to induce an inflammation model in vivo. Survival analysis, hematoxylin-eosin (HE) staining, observation of neutrophil migration, and quantitative real-time polymerase chain reaction (qRT-PCR) were used to further study the anti-inflammatory effects of ethyl lithospermate and its probable mechanisms in vivo.
RESULTS
The non-toxic concentrations of ethyl lithospermate have been found to range from 12.5 to 100 µ mol/L. Ethyl lithospermate inhibited the release of IL-6 and TNF-α(P<0.05 or P<0.01), decreased IκBα degradation and phosphorylation (P<0.05) as well as the nuclear translocation of NF-κB p65 and p-STAT3 (Tyr705) in LPS-induced RAW 264.7 cells (P<0.01). Ethyl lithospermate also decreased inflammatory cells infiltration and neutrophil migration while increasing the survival rate of LPS-stimulated zebrafish (P<0.05 or P<0.01). In addition, ethyl lithospermate also inhibited the mRNA expression levels of of IL-6, TNF-α, IκBα, STAT3, and NF-κB in LPS-stimulated zebrafish (P<0.01).
CONCLUSION
Ethyl lithospermate exerts anti-Inflammatory effected by inhibiting the NF-κB and STAT3 signal pathways in RAW 264.7 macrophages and zebrafish.
目的
探讨脂多糖（LPS）刺激的 RAW 264.7 鼠源巨噬细胞和斑马鱼中，甲基丹参醌的抗炎作用及其机制。
方法
用 3-[4,5-二甲基噻唑-2-基]-2,5-二苯基四氮唑溴盐（MTT）法测定不同浓度（12.5-100 μmol/L）甲基丹参醌对 RAW 264.7 细胞的毒性。用 LPS（100ng/mL）刺激细胞 12 h 建立体外炎症模型，酶联免疫吸附试验（ELISA）测定促炎细胞因子白细胞介素（IL）-6和肿瘤坏死因子α（TNF-α）的产生。用 Western blot 测定信号转导和转录激活因子 3（STAT3）、核因子κB（NF-κB）p65、磷酸化 STAT3（p-STAT3，Tyr705）、NF-κB 抑制剂（IκB）α和磷酸化 IκBα（p-IκBα，Ser32）的蛋白表达，并用共聚焦成像鉴定 NF-κB p65 和 p-STAT3（Tyr705）的核转位。此外，用 2 nL LPS（0.5mg/mL）注射斑马鱼（受精后 3 天）的卵黄囊，建立体内炎症模型。生存分析、苏木精-伊红（HE）染色、中性粒细胞迁移观察和定量实时聚合酶链反应（qRT-PCR）用于进一步研究甲基丹参醌的抗炎作用及其在体内的可能机制。
结果
发现甲基丹参醌的无毒浓度范围为 12.5-100 μmol/L。甲基丹参醌抑制 LPS 诱导的 RAW 264.7 细胞中 IL-6 和 TNF-α 的释放（P<0.05 或 P<0.01），降低 IκBα 的降解和磷酸化（P<0.05）以及 NF-κB p65 和 p-STAT3（Tyr705）的核转位（P<0.01）。甲基丹参醌还可减少 LPS 刺激的斑马鱼中炎症细胞浸润和中性粒细胞迁移，同时提高其存活率（P<0.05 或 P<0.01）。此外，甲基丹参醌还抑制 LPS 刺激的斑马鱼中 IL-6、TNF-α、IκBα、STAT3 和 NF-κB 的 mRNA 表达水平（P<0.01）。
结论
甲基丹参醌通过抑制 RAW 264.7 巨噬细胞和斑马鱼中的 NF-κB 和 STAT3 信号通路发挥抗炎作用。
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