New‐QiangGuYin‐Containing Serum Inhibits Osteoclast‐Derived Exosome Secretion and Down‐Regulates Notum to Promote Osteoblast Differentiation

Abstract New‐QiangGuYin (N‐QGY), the addition of sea buckthorn on the basis of QGY formula, is herbal formula widely used clinically in China for the treatment of osteoporosis (OP), but its mechanism warrants further exploration. The mechanisms of QGY and N‐QGY in the treatment of OP are probed from the perspective of osteoclast‐osteoblast balance. Thirty Sprague‐Dawley rats are randomly divided into N‐QGY group, QGY group, and Control group. Beyond control rats that orally took normal saline, other rats are orally administered with isometric N‐QGY or QGY twice every day for 3 days. The drug‐containing serum and control serum are prepared and their effects on osteoclast‐derived exosome secretion are determined by bicinchoninic acid assay (BCA), nanoparticle tracking analysis, and Western blot. GW4869 and Interleukin‐1β (IL‐1β) are adopted as the exosome inhibitor and inducer, respectively. Exosome uptake, cell counting kit‐8, alkaline phosphatase (ALP) staining, alizarin red staining, enzyme‐linked immunosorbent assay, quantitative real‐time polymerase chain reaction, and Western blot are performed to examine the effects of altered osteoclast exosome content on osteogenic differentiation of mesenchymal stem cells (MSCs). N‐QGY, QGY, and GW4869 inhibit osteoclast‐derived exosome secretion and exosome uptake by MSCs, whereas IL‐1β exerted the opposite effects ( p < 0.05). Different from IL‐1β, N‐QGY, QGY, and GW4869 partially elevated MSC viability, osteocalcin secretion, ALP, RUNX Family Transcription Factor 2 (RUNX2) and Osteopontin (OPN) expressions, and calcium deposition in the osteoclast‐MSCs coculture system ( p < 0.05). Mechanically, osteoclasts increased Notum protein level but decreased β‐catenin level, which is enhanced by IL‐1β but is reversed by GW4869, QGY, and N‐QGY ( p < 0.05). And the effect of N‐QGY is more conspicuous than that of QGY ( P <0.05). N‐QGY‐containing serum inhibits exosome levels in osteoclasts, thereby enhancing osteogenic differentiation of MSCs via inhibition of Notum protein and promotion of β‐catenin protein.