Huo Xue Tong Luo capsule epigenetically activates β-catenin, promotes Prx1-lineage cells proliferation and endochondral bone formation during fracture healing.

BACKGROUND

In recent years, traditional Chinese medicine (TCM) has been noted to possess unique advantages in fracture treatment. In our previous studies, we have demonstrated that Huo Xue Tong Luo capsule (HXTL) promotes osteogenesis and inhibits osteoporosis. However, the effect of the HXTL on fracture healing has not been studied.

METHODS

Mouse bone marrow mesenchymal stem cells (MSCs) were isolated and cultured. Western blotting, quantitative RT-PCR, immunohistochemical staining, immunofluorescence staining, and RNA-seq analysis were applied to detect the levels of genes or proteins. The mouse femoral fracture model was used to investigate the effect of HXTL on fracture healing.

RESULTS

In this study, our in vitro experiments confirmed that HXTL could enhance the proliferation and osteogenic differentiation of MSCs in a dose-dependent manner. Then, we established femur fracture healing model using the Prx1-CreERT2; tdTomato lineage tracing mice, and revealed that HXTL accelerated the proliferation and osteogenic differentiation of Prx1 + MSCs at the fracture site during the early and middle stages of fracture repair, thereby accelerating the process of healing and allowing the fracture callus to enter into the mineralization and remodeling phases earlier. Mechanistically, our RNA-seq analysis data revealed that HXTL activated Wnt/β-catenin signaling pathway, and the chromatin immunoprecipitation (ChIP)-PCR result demonstrated that HXTL transcriptionally activated β-catenin expression by modulating H3K4me3 status at its promoter region.

CONCLUSION

Taken together, our results showed that HXTL epigenetically activated β-catenin, promoted Prx1-lineage cells proliferation and endochondral bone formation during fracture healing, which provides insights into the potential therapeutic application of HXTL in bone regeneration and fracture healing.