Liensinine Promotes Apoptosis in Rheumatoid Arthritis Fibroblast-Like Synoviocytes via p38 MAPK-Mediated...

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by synovial inflammation and joint destruction. Oxidative stress has been implicated in its pathogenesis. This in vitro study aimed to investigate the effects of liensinine (LIE) on the apoptosis of fibroblast-like synoviocytes (FLSs) and its underlying mechanisms. RA-FLSs were isolated from patients and cultured in low-glucose Dulbecco's Modified Eagle Medium (DMEM). Following LIE treatment, cell viability was assessed using the cell-counting kit-8 assay, and lactate dehydrogenase (LDH) release was measured to evaluate cytotoxicity. Apoptosis was assessed by 4',6-diamidino-2-phenylindole (DAPI) staining and flow cytometry. Oxidative stress was evaluated by reactive oxygen species (ROS) production and mitochondrial membrane potential using 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) staining. Caspase-3/7 activity was measured using the Caspase-Glo^® 3/7 luminescent assay (Promega) following the manufacturer's instructions. RNA sequencing and network pharmacology analysis were performed to identify differentially expressed genes and signaling pathways affected by LIE. Western blotting was used to analyze key proteins in the mitogen-activated protein kinase (MAPK) and apoptosis pathways. Molecular docking and dynamics simulations evaluated LIE binding to P38 and Caspase9. LIE significantly reduced cell viability and increased LDH release in RA-FLSs in a dose-dependent manner. LIE induced apoptosis and elevated oxidative stress, evidenced by increased ROS and altered mitochondrial membrane potential. RNA sequencing and network pharmacology revealed significant changes in genes related to MAPK and apoptosis signaling. Western blot confirmed activation of p38 MAPK and caspase-3/9, suggesting LIE-induced apoptosis correlates with oxidative stress and MAPK pathway activation. LIE exhibited strong binding affinities to P38 and Caspase9, with favorable stability in dynamics simulations. These in vitro findings suggest that LIE has therapeutic potential for RA by targeting MAPK and apoptosis pathways in RA-FLSs, warranting further in vivo validation.