Network pharmacology combined with experimental validation to investigate the effect of Rongjin Niantong Fang on chondrocyte apoptosis in knee osteoarthritis

Abstract

Knee osteoarthritis (KOA) is a chronic degenerative disease that affects the quality of life of middle-aged and elderly individuals, and is one of the major factors leading to disability. Rongjin Niantong Fang (RJNTF) can alleviate the clinical symptoms of patients with KOA, but the molecular mechanism underlying its beneficial effects on KOA remains unknown. Using pharmacological analysis and in vitro experiments, the active components of RJNTF were analyzed to explore their potential therapeutic targets and mechanisms in KOA. The potential targets and core signaling pathways by which RJNTF exerts its effects on KOA were obtained from databases such as Gene Expression Omnibus, Traditional Chinese Medicine Systems Pharmacology and Analysis Platform. Subsequently, chondrocyte apoptosis was modeled using hydrogen peroxide (H 2 O 2 ). Cell Counting Kit-8 assay involving a poly [ADP-ribose] polymerase-1 (PARP1) inhibitor, DAPI staining, reverse transcription-quantitative PCR, Annexin V-FITC/PI staining and flow cytometry, western blotting and co-immunoprecipitation analysis were used to determine the therapeutic efficacy of RJNTF on KOA and to uncover the molecular mechanism. It was found that PARP1-knockdown lentivirus, incubation with PARP1 inhibitor PJ34, medium and high doses of RJNTF significantly reduced H 2 O 2 -induced chondrocyte apoptosis. Medium and high doses of RJNTF downregulated the expression of cleaved caspase-3, cleaved PARP1 and PAR total proteins, as well as nucleus proteins of apoptosis-inducing factor (AIF) and migration inhibitory factor (MIF), and upregulated the expression of caspase-3, PARP1 total protein, as well as the cytoplasmic expression of AIF and MIF, suggesting that RJNTF may inhibit chondrocyte apoptosis through the PARP1/AIF signaling pathway.