## 4 Discussion
The ethanol extract of Cinnamorum amphora leaves showed anti-inflammatory properties through the suppression of NF-\(\kappa\)B pathway expression [36]. Isoflavones daidzin and daidzein can inhibit the secretion of inflammatory factors andinhibit LPS-induced inflammation by regulating the NF-kB/MAPK pathway in RAW264.7 cells [37]. In our study, the activation of key proteins in LPS-induced macrophage-related signaling pathways by COLFs was examined. Protein expression analysis showed that COLFs could significantly decrease the ratio of p-p65/p65 and IxB\(\alpha\), indicating that COLFs inhibited the degradation of IxB\(\alpha\) through the NF-kB pathway, thereby promoting the binding of NF-kB and P65 in the cytoplasm, reducing the translocation of NF-kB, and finally inhibiting the production of cytokines. The MAPK pathway, which includes regulatory proteins such as JNK, ERK, and p38, is a classical inflammation-related signaling pathway that plays a crucial role in regulating the NF-kB pathway [38,39]. It primarily functions by phosphorylating various molecular signals, thereby amplifying and integrating signals from diverse stimuli and regulating gene expression [40]. Consequently, inhibiting the expression or phosphorylation levels of molecules involved in the MAPK pathway may serve as a target for the development of anti-inflammatory drugs. Hostaflavone A, derived from Hosta plantaginea, exhibits anti-inflammatory effects in macrophages by suppressing the activation of multiple signaling pathways, including the MAPK pathway [41]. Studies have demonstrated that COLFs significantly inhibit the phosphorylation of key proteins such as JNK, ERK, and p38 within the MAPK signaling pathway, indicating that COLFs exert anti-inflammatory effects by regulating the MAPK pathway. The presence of quercetin and kaempfer in COLFs may contribute to their potent anti-inflammatory activity. Previous research has shown that quercetin and kaempfer inhibit NF-kB activation by preventing the nuclear translocation of the p50 and p65 subunits of NF-kB, as well as suppressing the phosphorylation of IxB\(\alpha\) protein, which in turn reduces the expression of pro-inflammatory cytokines [42]. Additionally, quercetin and kaempfer mitigate inflammatory responses by exerting distinct regulatory effects on the MAPK pathway. [PERSON] et al. demonstrated that quercetin inhibits ERK, JNK, and their phosphorylated forms, thereby regulating the MAPK pathway and ultimately producing anti-inflammatory effects [43]. Kaempfer, on the other hand, impedes the activation of the MAPK signaling pathway by modulating the phosphorylation of P38 and JNK [44]. Therefore, quercetin and kaempfer may function by downregulating the NF-kB/MAPK pathway in COLFs. TLRs are a family of cell surface receptors that play a critical role in signal transduction from the cell surface to the interior of the cell [45]. Increasing evidence suggests that TLR4 is a key receptor for lipopolysaccharide-LPS recognition and mediates inflammatory responses via the NF-kB/MAPK pathway [46]. Protein expression analysis demonstrated that COLFs significantly inhibited the expression of the TLR4 receptor protein. These findings indicate that COLFs influence the activity of the NF-kB/MAPK signaling pathway through the TLR4 receptor, thereby modulating its anti-inflammatory function. Despite the progress made in this study, there are certain limitations. The research was conducted using a single mouse macrophage cell line (RAW264.7) for in vitro experiments. Although RAW264.7 is a widely used model for studying inflammatory responses, it may not fully capture the biological complexity of human macrophages. Therefore, our findings will be further validated using other cell lines, particularly human cell lines, in future studies. Additionally, in vivo experiments will be conducted to assess the anti-inflammatory effects of COLFs in animal models, with results to be published at a later date.