Ethyl Lithospermate Reduces Lipopolysaccharide-Induced Inflammation through Inhibiting NF-κB and STAT3 Pathways in RAW 264.7 Cells and Zebrafish

ZHOU Chun-hong; ZHOU Chun-hong; YANG Hua; YANG Hua; ZOU Li-fang; ZOU Li-fang; LIU Di-fa; LIU Di-fa; YU Lin-zhong; YU Lin-zhong; CAO Hui-hui; CAO Hui-hui; DENG Li-e; DENG Li-e; WANG Zhang-wei; WANG Zhang-wei; LU Zi-bin; LU Zi-bin; LIU Jun-shan

doi:10.1007/s11655-023-3643-y

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Ethyl Lithospermate Reduces Lipopolysaccharide-Induced Inflammation through Inhibiting NF-κB and STAT3 Pathways in RAW 264.7 Cells and Zebrafish

Original Article | Updated：2023-11-21

- Ethyl Lithospermate Reduces Lipopolysaccharide-Induced Inflammation through Inhibiting NF-κB and STAT3 Pathways in RAW 264.7 Cells and Zebrafish
- Chinese Journal of Integrative Medicine Vol. 29, Issue 12, Pages: 1111-1120(2023)
- Affiliations：
  
  1.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
  2.Department of Pharmacy, Zhujiang Hospital, Southern Medical University, Guangzhou(510282), China
  3.State Key Laboratory of Innovative Natural Medicine and Traditional Chinese Medicine Injections, Jiangxi Qingfeng Pharmaceutical Co. Ltd., Ganzhou, Jiangxi Province(341000), China
  4.Affiliated Dongguan People's Hospital, Southern Medical University, Dongguan, Guangdong Province(523076), China
- Author bio：
  
  Associate Prof. LIU Jun-shan, E-mail: liujunshan@smu.edu.cn
- Funds：
- DOI：10.1007/s11655-023-3643-y
  CLC：
- Published：2023-12，
  
  Published Online：23 August 2023，
  
  Accepted：12 May 2023
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ZHOU Chun-hong, ZHOU Chun-hong, YANG Hua, et al. Ethyl Lithospermate Reduces Lipopolysaccharide-Induced Inflammation through Inhibiting NF-κB and STAT3 Pathways in RAW 264.7 Cells and Zebrafish. [J]. Chinese Journal of Integrative Medicine 29(12):1111-1120(2023)
DOI：

ZHOU Chun-hong, ZHOU Chun-hong, YANG Hua, et al. Ethyl Lithospermate Reduces Lipopolysaccharide-Induced Inflammation through Inhibiting NF-κB and STAT3 Pathways in RAW 264.7 Cells and Zebrafish. [J]. Chinese Journal of Integrative Medicine 29(12):1111-1120(2023) DOI： 10.1007/s11655-023-3643-y.

摘要

目的:

探讨Ethyl Lithospermate在脂多糖 (LPS) 刺激的RAW 264.7鼠源巨噬细胞和斑马鱼中的抗炎作用及其内在机制.

方法:

采用3-[4

5-二甲基噻唑-2-基]-2

5-二苯基溴化四氮唑 (MTT) 试验来检测不同浓度 (12.5-100 μ mol/ L) 的Ethyl Lithospermate对RAW 264.7细胞的毒性. 用酶联免疫吸附试验 (ELISA) 检测RAW 264.7细胞中白细胞介素 (IL) -6和肿瘤坏死因子α (TNF-α) 的产生. 用免疫印迹法检测信号转导和转录激活因子 3 (STAT3)、核因子κB (NF-κB) p65和磷酸化STAT3 (p-STAT3

Tyr705)、NF-κB (IκB) α抑制剂和磷酸化IκBα (p-IκBα

Ser32)蛋白的表达

并使用共聚焦成像来鉴定NF-κB p65和p-STAT3 (Tyr705)的核转位. 此外

通过在斑马鱼卵黄囊 (受精后3天) 注射2 nL LPS (5 mg/mL) 以诱导体内炎症模型. 通过生存分析、苏木精-伊红 (HE) 染色、观察中性粒细胞迁移和实时定量聚合酶链反应 (qRT-PCR) 进一步研究Ethyl Lithospermate在体内的抗炎作用及其可能的机制.

结果:

研究发现Ethyl Lithospermate的无毒浓度范围为12.5-100 μmol/L. Ethyl Lithospermate抑制了RAW 264.7 细胞中IL-6和TNF-α的释放 (

0.05或

0.01)

降低了IκBα降解和磷酸化 (

0.05)

以及p-STAT3 (Tyr705)和NF-κB p65的表达和核转位 (

0.01) . 此外

Ethyl Lithospermate还能减少斑马鱼炎症模型中炎症细胞的浸润和中性粒细胞的迁移

提高斑马鱼的存活率 (

0.05

0.01)

抑制IL-6、TNF-α、Iκ Bα、STAT3和NF-κ B的mRNA的表达 (

0.01) .

结论:

Ethyl Lithospermate通过抑制NF-κ B和STAT3信号通路

从而对RAW 264.7巨噬细胞和斑马鱼产生抗炎作用.

Abstract

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 μmol/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-IκBα (p-IκBα

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 μmol/L. Ethyl lithospermate inhibited the release of IL-6 and TNF-α (

0.05 or

0.01)

decreased IκBα degradation and phosphorylation (

0.05) as well as the nuclear translocation of NF-κB p65 and p-STAT3 (Tyr705) in LPS-induced RAW 264.7 cells (

0.01). Ethyl lithospermate also decreased inflammatory cells infiltration and neutrophil migration while increasing the survival rate of LPS-stimulated zebrafish (

0.05 or

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 (

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.

关键词

Ethyl Lithospermate抗炎核因子κB信号转导和转录激活因子3脂多糖

Keywords

ethyl lithospermateanti-inflammationnuclear factor kappa Bsignal transducer and activator of transcription 3lipopolysaccharidezebrafish

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