Duhuo Jisheng Decoction Mitigates Osteochondral Degeneration in Knee Osteoarthritis through Modulation of Complement C3a/C3AR1-IKKβ-NF-κB Signaling

Knee osteoarthritis (KOA) is characterized by progressive osteochondral destruction driven by inflammatory and catabolic signaling, yet no approved structure-modifying pharmacotherapy currently exists. Duhuo Jisheng Decoction (DHJSD), a classic traditional Chinese medicine (TCM) formula widely used for KOA, requires a clearer understanding of its mechanism of action. To address this, we integrated multiple approaches: a destabilization of the medial meniscus (DMM) mouse model for initial therapeutic efficacy ( n = 3 per group), bulk articular cartilage RNA-sequencing (RNA-seq) (Gene Expression Omnibus (GEO): GSE319157), network pharmacology, focused in vivo dose validation, in vitro dose-bridging assays, primary chondrocyte phospho-signaling assays, ex vivo complement activation, ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) serum metabolomics, molecular docking and dynamics simulations, cellular target engagement assays cellular thermal shift assay (CETSA), isothermal dose–response fingerprint CETSA (ITDRF-CETSA), and drug affinity responsive target stability (DARTS), and an in vivo C3a challenge-rescue experiment to interrogate the complement–IκB kinase beta (IKKβ)–nuclear factor kappa-B (NF-κB) axis. DHJSD treatment was associated with preservation of osteochondral integrity and cartilage matrix homeostasis in DMM mice. The initial dose cohort (2, 4, and 8 g/kg/day) showed a non-monotonic efficacy pattern; an independent focused validation cohort centered on 4 g/kg/day confirmed this dose as the most consistent for mechanistic interrogation within the refined range tested. RNA-seq and network pharmacology identified a complement–NF-κB program as a DHJSD-responsive network. Bulk-cartilage complement C3a receptor 1 (C3AR1) transcript behavior including anchor-cohort reverse transcription quantitative PCR (RT-qPCR) confirmation of the RNA-seq direction served as tissue-level contextual information, whereas mechanistic inference regarding C3AR1 was based on chondrocyte-level RT-qPCR, cartilage-localized protein expression, and downstream IKKβ–NF-κB signaling readouts. In primary chondrocytes, interleukin-1 beta (IL-1β) induced C3 and C3AR1 transcripts and activated the IKKβ–inhibitor of nuclear factor kappa B alpha (IκBα)–v-rel reticuloendotheliosis viral oncogene homolog A (RelA, P65) cascade; DHJSD-medicated serum derived from the 4 g/kg dose most consistently attenuated these responses. Intra-articular C3a challenge partially blunted DHJSD-mediated protection, whereas IKKβ inhibition with BMS-345541 partially restored structural and signaling endpoints. UHPLC-Q-TOF-MS prioritized geniposidic acid (GA) as a blood-exposed candidate constituent, and CETSA, ITDRF-CETSA, and DARTS provided convergent cellular evidence supporting GA–IKKβ interaction, although direct biochemical affinity remains to be determined. Collectively, these data support a restrained mechanistic model in which DHJSD attenuates a complement-amplified IKKβ–NF-κB inflammatory circuit in experimental KOA. GA emerges as a prioritized candidate requiring pharmacokinetic (PK) and biochemical validation, and adequately powered dose-optimization studies are still warranted.