Exploration of Pharmacological Mechanism of Kaempferol in Treating Rheumatoid Arthritis based on Network Pharmacology, Molecular Modelling, and Experimental Validation

Background: The autoimmune inflammatory disease known as rheumatoid arthritis (RA) has a complicated and poorly understood etiology. Fibroblast-like synoviocytes (FLSs) have tumor-like characteristics in RA, including aggressive growth and heightened activation that leads to the release of proinflammatory factors. These processes are essential for the gradual deterioration of joint tissues. Kaempferol, with the chemical formula 3,5,7-trihydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one, is found in many different types of plants and plant families. The pharmacological effects of this substance have been welldocumented. The benefits of this substance encompass protection for the heart and brain, as well as fighting inflammation, bacteria, cancer, osteoporosis, and allergies. It also has properties that can help with anxiety, pain relief, and hormonal balance. However, its precise function in the management of RA is still unclear. Objective: To investigate the effect of kaempferol on apoptosis in RA FLSs and elucidate the underlying mechanisms. Methods: We used the CCK-8 assay to assess the effects of different kaempferol concentrations on RA FLSs. We also used flow cytometry with Annexin V-FITC/PI staining to analyse cell cycle distribution and quantify apoptotic cells. To verify apoptosis, the TUNEL test was employed. Important proteins associated with apoptosis were verified to be expressed using western blotting. Finally, network pharmacology analysis was used to identify potential kaempferol targets, and their interactions with AKT1, PIK3R1, and HSP90AA1 proteins were studied using molecular docking and molecular dynamics simulations. Results: Kaempferol treatment significantly increased apoptosis in RA FLSs, up-regulating the pro-apoptotic protein Bax and down-regulating the anti-apoptotic protein Bcl-2. Specifically, kaempferol at 100 and 200 μM increased the apoptosis index to 29.77 ± 6.02% and 55.63 ± 11.05%, respectively, compared to the control. The induction of caspase-9 and caspase-3 cleavage was observed, indicating the activation of the mitochondrial pathway. Kaempferol also inhibited the phosphorylation of PI3K and Akt, with a significant reduction in their activation. Molecular docking studies demonstrated that kaempferol interacted with AKT1, PIK3R1, and HSP90AA1 proteins, with binding energies of -6.51, -4.26, and -6.51 kcal/mol, respectively, suggesting a strong affinity and potential direct impact on these proteins. Conclusion: Kaempferol induces apoptosis in RA FLSs by inhibiting phosphorylation of the PI3K/Akt signaling pathway, increasing levels of pro-apoptotic proteins, and decreasing levels of anti-apoptotic proteins. Thus, kaempferol, a naturally occurring flavonoid, has great promise in the management of RA.