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

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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 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.

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Authors and 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

    Chun-hong Zhou, Hua Yang, Li-fang Zou, Lin-zhong Yu, Hui-hui Cao, Zi-bin Lu &  Jun-shan Liu

  2. Department of Pharmacy, Zhujiang Hospital, Southern Medical University, Guangzhou, 510282, China

    Chun-hong Zhou, Hua Yang, Li-fang Zou, Lin-zhong Yu, Hui-hui Cao, Zi-bin Lu &  Jun-shan Liu

  3. State Key Laboratory of Innovative Natural Medicine and Traditional Chinese Medicine Injections, Jiangxi Qingfeng Pharmaceutical Co. Ltd., Ganzhou, Jiangxi Province, 341000, China

    Di-fa Liu & Zhang-wei Wang

  4. Affiliated Dongguan People’s Hospital, Southern Medical University, Dongguan, Guangdong Province, 523076, China

    Li-e Deng

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  1. Chun-hong Zhou
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  2. Hua Yang
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  3. Li-fang Zou
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  10. Jun-shan Liu
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Contributions

Zhou CH and Yang H performed the experiments and wrote the manuscript. Zhou CH and Zou LF analyzed data in this study. Liu DF and Zou LF assisted in many experiments. Yu LZ, Cao HH, and Deng LE contributed to the reagents. Wang ZW, Lu ZB and Liu JS designed the idea and supervised this study. All authors participated in this work and agreed to publish this manuscript.

Corresponding author

Correspondence to Jun-shan Liu.

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The authors declare no conflicts of interest.

Additional information

Supported by Guangdong Province Universities and Colleges Pearl River Scholar Funded Scheme (No. GDHVPS2018) and Young Elite Scientists Sponsorship Program by the China Association of Chinese Medicine (No. 2019-QNRC2-C14)

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Zhou, Ch., Yang, H., Zou, Lf. et al. Ethyl Lithospermate Reduces Lipopolysaccharide-Induced Inflammation through Inhibiting NF-κB and STAT3 Pathways in RAW 264.7 Cells and Zebrafish. Chin. J. Integr. Med. 29, 1111–1120 (2023). https://doi.org/10.1007/s11655-023-3643-y

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