Aqueous Fraction of Huogu Formula Promotes Osteogenic Differentiation of Bone Marrow Stromal Cells Through the BMP and Wnt Signaling Pathways

Our recent studies have shown that Huogu (HG) formula was effective both in clinic experience and in experimental osteonecrosis of the femoral head (ONFH). Given that defective of bone marrow stromal cells (MSCs) contribute to the development of osteonecrosis and MSCs show enormous potential in the treatment of ONFH, especially to aging people. How HG impacts the differentiation of MSCs and what is the underlying cellular and molecular mechanism remains largely unknown. Here, we found that an aqueous fraction of HG (HGA) significantly increased the alkaline phosphatase (ALP) activity, mineralized nodules, and migration of MSCs in a dose-dependent manner. Meanwhile, HGA could enhance the mRNA and protein expression of Runt-related transcription factor 2 (Runx2), Alp, Bmp2, osteocalcin (Ocn), and Osterix (Osx). Further investigation of the molecular mechanisms revealed that HGA treatment obviously increased expression, secretion, and activation of bone morphogenetic protein (BMP) 2 and β-catenin, two key regulators of the BMP or Wnt signaling pathway. Furthermore, osteogenic differentiation of MSCs could be blocked by using pharmacological inhibitors for these signaling pathways such as Noggin and Dkk-1. Besides, HGA could inhibit adipogenic differentiation of MSCs. Our study reveals that HGA promotes the osteogenesis of MSCs via the BMP and Wnt signaling pathways. Our findings provide mechanistic insights into the role of HG in treating ONFH.