## 4 Discussion  
Previous studies have demonstrated that macrophages secrete a significant number of inflammatory mediators and cytokines during inflammation, which further contribute to the progression of the inflammatory response [24]. Therefore, inhibiting the production of these inflammatory mediators and cytokines may represent an effective anti-inflammatory strategy [25]. [PERSON] et al. reported that polyphenols isolated from selenium-enriched green tea exert therapeutic effects on inflammation by inhibiting the production of NO, TNF-\(\alpha\), IL-1\(\beta\), and IL-6 [26]. These findings align with the current study, which demonstrates that COLFs possess strong anti-inflammatory effects, potentially by reducing the production of various inflammatory markers. There are numerous methods to analyze cell phenotype and function, with RNA sequencing (RNA-seq) being a prominent technique for examining gene expression and regulatory mechanisms at the RNA level. RNA-seq is an effective approach for identifying differentially expressed genes and measuring mRNA expression levels within the transcriptome [27]. Reports indicate that RNA-seq technology has been utilized to identify therapeutic targets and molecular markers in various inflammatory diseases [28,29]. One study revealed the interaction between mung bean polysaccharide (MBP) and macrophages, highlighting the alterations in the entire signaling pathway following MBP treatment. RNA-seq, along with GO analysis and KEGG analysis, suggested significant enrichment of the TLR4 and NF-\(\kappa\)B signaling pathways [30]. Hence, RNA-seq technology was used to detect changes in the transcriptome spectrum of each group, and bioinformatics analysis was used to compare the DEGs between groups. In our study, we found that a large number of gene expressions were changed in macrophages after LPS stimulation and similar results were found for previous studies [31]. However, a total of 627 DEGs were produced between the COLF group and LPS group after COLF intervention, including 286 upregulated genes and 341 downregulated genes. GO and KEGG enrichment analyses were performed on the screened DEGs to comprehensively integrate the genomic, chemical, and systemic function information, and to explore the anti-inflammatory mechanism of COLFs on RAW264.7 cells. The results of the GO enrichment analysis indicated that genes involved in biological processes in the LPS and COLF groups were significantly regulated, among which the functional sets of innate immune response, inflammatory response, immune system process, and immune response were significantly enriched. To further investigate the impact of COLFs on signaling pathways, KEGG enrichment analysis was employed. The findings demonstrated that both the LPS and COLF groups exhibited significant enrichment in the regulation of the NF-\(\kappa\)B signaling pathway. COLFs play a major inhibitory function in genes associated with the LPS-NF-\(\kappa\)B signaling pathway, resulting in the reduction in the expression of downstream pro-inflammatory mediators, thereby inhibiting LPS-induced inflammation. NF-\(\kappa\)B serves as a crucial transcription factor that regulates various genes involved in immune and inflammatory responses, modulating both innate and adaptive immune responses [32,33]. NF-\(\kappa\)B is composed of two proteins, p50 and p65 [34]. In resting cells, NF-\(\kappa\)B typically remains inactive in the cytoplasm by binding to \(\kappa\)B. Once activated by extracellular signals, \(\kappa\)B is dissociated from NF-\(\kappa\)B through ubiquitination and phosphorylation [8]. The released NF-\(\kappa\)B translocates to the nucleus, where it is activated by phosphorylation, binds to target DNA, and induces the transcription of inflammatory marker genes [35]. Many studies show that different natural products can affect biological processes by blocking the NF-\(\kappa\)B signaling pathway, which helps lower the release of pro-inflammatory molecules.