## 2 Results and Discussion
### Molecular Docking and Molecular Dynamics Simulation Analysis  
To elucidate the biomolecular interactions between spilanthol and pivotal proteins involved in the TLR4 signaling pathway, molecular docking and MD simulations were undertaken. The Protein Data Bank (PDB) serves as a vital source, containing three-dimensional structural data on numerous biologically important macromolecules, including proteins and ligands. This extensive resource serves as a cornerstone for researchers seeking profound insights into molecular biology. In our study, we focused on specific macromolecular constituents involved in the TLR pathway. Accordingly, three-dimensional structural representations of the target proteins were retrieved from the PDB. Subsequently, we conducted docking experiments and assessed the interaction energy using the GOLD program. The relevant PDB identifiers together with the GOLD fitness score for spilanthol and specific inhibitors are tabulated (Table 1). Figure 3: The distribution of genes in the TLR signaling pathway. \(\longrightarrow\) represents the activation effect, T arrows represent the inhibition effect, and dashed lines represent activation or inhibition effects. The intersection genes are highlighted in red. Interestingly, spilanthol demonstrated moderate binding affinity to multiple targets, and notably, spilanthol exhibited a substantial affinity for the active sites of IKK\(\alpha\) and NF-kB1 proteins, as indicated by GOLD fitness scores of 59.70 and 49.73, respectively. Through analysis of the superimposed three-dimensional interactions, we observed that spilanthol fit favorably within the active site that is comparable to the inhibitors CID16048085 and CID5280961 (Figure 4A). To delve deeper into the matter, MD simulation of the protein-ligand association complexes was conducted for 100 ns. MD simulations of the complexes formed between spilanthol-IKK\(\alpha\) and NF-kB1 proteins were all located within the active sites. We calculated the root-mean-square displacement (RMSD) throughout the simulation by tracking the geometric coordinates of all atoms in the complex (Supplementary Figure S3). In the IKK\(\alpha\)-CID16048085 and IKK\(\alpha\)-spilanthol, the RMSD values exhibited an initial increase to approximately 4.00-4.50 A, persisting until the end of the simulation. In the same way as IKK\(\alpha\)-spilanthol, the RMSD values rose to 5.00-5.50 A throughout the entire 100 ns simulation. In the case of NF-kB1-CID5280961 and NF-kB1-spilanthol complex, the RMSD values initially increased, followed by a subsequent rise to 6.00-6.50 A throughout the entire 100 ns simulation. Given that the active site of NF-kB1 resides within the loop region, the RMSD values of genistein and spilanthol displayed some fluctuations; however, during the final 20 ns, the RMSD values remained stable until the completion of the simulation. The binding affinity values of the protein-ligand complexes were determined using the SIE method, with data extracted from the analysis of 1000 snapshots obtained during the final 20 ns of the simulations (Table 2). The average binding free energy values (AGbind) for different complexes involving IKK\(\alpha\) and various ligands are as follows: IKK\(\alpha\)-CID5280961 and IKK\(\alpha\)-spilanthol exhibit values of \(-3.81\pm 1.74\) and \(-6.65\pm 0.37\) kcal/mol, respectively. Similarly, for NF-kB1 complexes, the values for NF-kB1-CID16048085 and NF-kB1-spilanthol are \(-3.05\pm 0.62\) and \(-5.06\pm 1.79\) kcal/mol, respectively. The SIE method exposes a remarkable and robust binding affinity between spilanthol and both IKK\(\alpha\) and NF-kB1. This affinity is notably stronger than that observed with their inhibitors. The map of protein-ligand interactions shows the frequency of interactions over the last 20 ns, as determined by the average structure. (Figure 4B) Molecular docking and dynamics collectively demonstrate the inherent capability of spilanthol to bind with NF-kB1 and IKK\(\alpha\), thereby reinforcing the findings of the network pharmacology analysis.