[Inhibition of M1 polarization and inflammatory response in RAW264.7 macrophages by harpagide through PGC-1α-mediated...

This study aimed to investigate whether harpagide can alleviate lipopolysaccharide(LPS) and interferon-gamma(IFN-γ)-induced M1 polarization and inflammatory response in RAW264.7 macrophages by regulating peroxisome proliferator-activated receptor gamma coactivator-1 alpha(PGC-1α) and improving mitochondrial function. The GEO dataset GSE183077 was used for bioinformatics analysis to identify differentially expressed genes(DEGs) of M1 and M0 macrophages, which was followed by Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment analyses. The M1 macrophage model was established in RAW264.7 macrophages by stimulation with LPS and IFN-γ. Cell counting kit-8(CCK-8) assays were used to evaluate cell viability and determine the optimal concentration of harpagide for treatment. The Griess assay was performed to measure nitric oxide(NO) levels. The mRNA expression levels of interleukin-6(IL-6), interleukin-1β(IL-1β), and tumor necrosis factor-α(TNF-α) were determined by real-time quantitative polymerase chain reaction(qRT-PCR). JC-1, Mito-Tracker, and Mito-SOX fluorescent probes were used to assess mitochondrial membrane potential, mitochondrial function, and mitochondrial reactive oxygen species(ROS) levels, respectively. Western blot analysis was conducted to detect the protein expression levels of PGC-1α, NOD-like receptor family pyrin domain containing 3(NLRP3), dynamin-related protein 1(DRP1), inducible nitric oxide synthase(iNOS), and TNF-α. Transcriptomic analysis revealed that inflammatory responses and mitochondrial fission-related biological processes were significantly activated during M1 macrophage polarization. In vitro experiments confirmed that, compared with the control group, the model group exhibited an increased NO level in cell culture supernatant, elevated mRNA expression of IL-6, IL-1β, and TNF-α, impaired mitochondrial function, decreased mitochondrial membrane potential, and elevated mitochondrial ROS levels. Meanwhile, PGC-1α protein expression was reduced, whereas NLRP3, DRP1, iNOS, and TNF-α protein levels were upregulated. Following harpagide treatment, these changes were reversed in a dose-dependent manner, namely decreased NO production and mRNA expression of IL-6, IL-1β, and TNF-α, improved mitochondrial function, restored mitochondrial membrane potential and mitochondrial ROS levels, significantly upregulated PGC-1α expression, and decreased expression of NLRP3, DRP1, iNOS, and TNF-α. In conclusion, harpagide alleviates inflammatory responses probably by modulating mitochondrial fission in macrophages and inhibiting NLRP3 inflammasome activation via activating PGC-1α.