Transcriptomics revealed potential anti-inflammatory mechanisms of ethanol extracts of propolis and tree gum in lipoteichoic acid and interferon-γ-stimulated RAW264.7 macrophages

Abstract

Introduction

Poplar propolis, a traditional Chinese medicine abundant in phenolic acids and flavonoid compounds, demonstrates remarkable anti-inflammatory activity. It is currently widely applied in the pharmaceutical and health product industries for the prevention and adjuvant treatment of various diseases. However, due to the limited availability of natural propolis, its primary plant source—tree gum—is often used as an adulterant, posing significant challenges for the authenticity and quality control of propolis products. Therefore, this study aimed to compare the anti-inflammatory effects of ethanol extracts of poplar propolis (EEP) and tree gum (EEG) on RAW264.7 macrophages co-stimulated with lipoteichoic acid (LTA) and interferon-gamma (IFN-γ) using in vitro cellular assays. Furthermore, transcriptomic analysis was employed to elucidate the molecular mechanisms underlying the anti-inflammatory activities of EEP and EEG.

Methods

Widely targeted metabolomics was used to analyze metabolites of EEP and EEG. A RAW264.7 macrophage inflammation model was established using LTA and IFN-γ co-stimulation, and optimal concentrations of EEP and EEG were determined. ELISA and RT-qPCR were used to assess inflammatory cytokines, inflammation-related genes, and antioxidant genes. Reactive oxygen species (ROS) levels were measured with a DCFH-DA probe. Transcriptomics, RT-qPCR, and Western blotting were employed to evaluate signaling pathway-related markers.

Results

The results showed that 1635 metabolites were identified in EEP and 1545 metabolites were identified in EEG by widely targeted metabolomics. Compared to EEG, EEP more effectively inhibited the release of inflammatory factors, regulated inflammatory gene expression, reduced ROS production, and elevated antioxidant gene expression. RT-qPCR and Western blot analyses revealed that EEP and EEG suppressed the inflammatory response in LTA/IFN-γ-stimulated RAW264.7 macrophages by downregulating cell adhesion molecule expression, inhibiting the MAPK signaling pathway, and activating the autophagy pathway.

Discussion

Propolis exhibited superior efficacy over tree gum in suppressing inflammatory responses and enhancing antioxidant defenses. These findings offer a theoretical foundation for the quality control and further development of propolis as a potential anti-inflammatory agent.