Background: Total flavonoids of Rhizoma drynariae (TFRD) is broadly used in the treatment of orthopedic diseases. Nevertheless, the effects and underlying mechanism of TFRD on tendon-bone healing after anterior cruciate ligament reconstruction (ACLR) remain unclear. Methods: The ACLR mouse model was established. Hematoxylin and Eosin (HE) staining was used for histological analysis of tendon-bone healing. Western blot was utilized to detect the levels of osteogenic related factors (ALP, OCN, RUNX2). The viability and alkaline phosphatase (ALP) activity of bone mesenchymal stem cells (BMSCs) were determined by Cell Counting Kit-8 (CCK-8) and ALP assays. The interaction of estrogen related receptor alpha (ESRRA), estrogen related receptor beta (ESRRB), and golgi-localized.-ear containing ADP ribosylation factor-binding protein 1 (Gga1) was detected by luciferase reporter assays. The levels of important proteins on the TGF-ss/MAPK pathway were measured by western blot. Results: TFRD improved tendon-bone healing, restored biomechanics of ACLR mice and activated the TGF-ss/MAPK pathway. TFRD treatment also enhanced the viability and osteogenic differentiation of BMSCs in vitro. Then, we demonstrated that TFRD targeted ESRRA and ESRRB to transcriptionally activate Gga1 expression. Knockdown of ESRRA, ESRRB, or Gga1 suppressed the viability and osteogenic differentiation of TFRD-induced BMSCs, which was revealed to be restored by Gga1 overexpression. The overexpression of ESRRA, ESRRB, or Gga1 was demonstrated to promote the BMSC viability and osteogenic differentiation. TGF-ss 1 treatment can reverse the impact of Gga1 inhibition on osteogenic differentiation in TFRD-induced BMSCs. Conclusion: TFRD improves tendon-bone healing in ACLR mouse models and facilitates the osteogenic differentiation of BMSCs through the ERR1/2-Gga1-TGF ss/MAPK pathway, which might deepen our understanding of the underlying mechanism of TFRD in tendon-bone healing.
