Soufeng Sanjie formula and its active ingredient formononetin alleviate osteoarthritis via PPARG-AKT-ERK1/2 promoted cartilage extracellular matrix anabolism

Ethnopharmacological relevance: In Traditional Chinese Medicine (TCM), osteoarthritis (OA) is referred to as "Gu Bi" (bone bi syndrome), which is characterized by chronic joint disease, cartilage damage, and deterioration of the extracellular matrix (ECM). The Soufeng Sanjie formula (SF), comprising Scolopendra, Scorpions, Astragali radix, and Black soybean seed coats, is traditionally employed in the treatment of "bone bi" disease and has been utilized in managing OA. Nonetheless, the effects of SF on ECM deterioration and cartilage destruction in OA, as well as its principal bioactive components, remain inadequately understood.

Aim of the study: This study aims to investigate SF's therapeutic potential against ECM deterioration and cartilage destruction in OA, explore its underlying mechanisms, and identify its primary bioactive components.

Materials and methods: A mouse model of knee OA was established via anterior cruciate ligament transection (ACLT). The analgesic effect of SF on arthritis pain was assessed by measuring cold pain thresholds. Cartilage damage was evaluated using Safranin O-fast green staining and the Osteoarthritis Research Society International (OARSI) scores. Immunohistochemical staining was employed to evaluate the influence of SF on cartilage metabolism, whereas cellular experiments were conducted to examine SF's capacity to enhance chondrocyte anabolism. The expression levels of SRY-box transcription factor 9 (SOX9), Collagen type II alpha 1 (COL2A1), and aggrecan (ACAN) were analyzed using Western blotting and quantitative reverse transcription polymerase chain reaction (qPCR). Alcian blue staining was utilized to identify the bioactive components of SF that affect chondrocyte anabolism. This was further supported by preliminary analyses using network pharmacology and molecular docking techniques.

Results: SF has been shown to effectively reduce cartilage degradation and enhance the expression of SOX9, ACAN, and COL2A1 in the cartilage of OA mice. Furthermore, SF promotes ECM anabolism in chondrocyte cells, as evidenced by the increased production of chondrocyte acidic Polysaccharides and elevated mRNA levels of SOX9, ACAN, and COL2A1. Additionally, Formononetin (FMN) was screened as a key compound of SF with the ability to enhance cartilage ECM anabolism. FMN significantly upregulated the expression of SOX9, ACAN, and COL2A1, alleviated pain symptoms, and reduced cartilage damage in OA mice. Network pharmacology and molecular docking analyses indicated that FMN targets the Peroxisome Proliferator-activated Receptor gamma (PPARG), thereby activating the Akt and ERK1/2 signaling pathways in chondrocyte cells. And the chondroprotective effect of FMN is attenuated by PPARG inhibitors. Additionally, both CETSA and SPR analyses demonstrated direct binding between FMN and PPARG.

Conclusion: Our study demonstrated that SF significantly mitigated cartilage degradation in OA mice by ECM anabolism. FMN was identified as a potential active constituent of SF that supports chondrocyte anabolism and decelerates OA progression through the PPARG-AKT-ERK1/2 signaling pathway. Taken together, this herbal formulation offers new clinical treatment prospects for OA.

AKT-ERK1/2; Cartilage extracellular matrix anabolism; Formononetin; Osteoarthritis; PPARG; Soufeng sanjie formula.