Mechanistic Insights into the Anti-Fibrotic Effects of Estrogen via the PI3K-Akt Pathway in Frozen Shoulder

The development of frozen shoulder (FS) is primarily characterized by pathological fibrosis, yet clinical treatment options remain limited. Recent studies have identified estrogen depletion during perimenopause as a significant contributor to the onset of FS and fibrosis. This study investigates the role of estradiol (E2) and the estrogen-related receptor (GPER) in fibrotic processes associated with FS to elucidate the underlying mechanisms. The functional relationship between E2, GPER, and FS progression was examined using a rat immobilization model and synovial-derived fibroblasts (SFs) from FS patients. E2’s effects on GPER expression, fibroblast activation, and tissue fibrosis were evaluated through Western blotting, immunofluorescence staining, collagen contraction assays, wound healing assays, and histological staining. RNA sequencing identified signaling pathways and key regulators involved in E2 treatment. Both E2 and the GPER activator G1 exhibited antifibrotic effects, improving shoulder mobility, reducing extracellular matrix (ECM) deposition in the periarticular capsule, and decreasing the expression of fibrosis-related genes, including fibronectin, α-SMA, and COL3. In contrast, the GPER inhibitor G15 reversed these effects, suggesting that E2 mediates its antifibrotic action through GPER activation. Mechanistically, KEGG pathway analysis revealed that E2 suppresses the PI3K/AKT signaling pathway by inhibiting PI3K and AKT phosphorylation, thereby preventing fibroblast activation and reversing FS-associated fibrosis. These findings provide mechanistic insights into the previously unrecognized role of GPER in FS progression and may open new avenues for research to optimize future clinical therapies.