Background: Senile osteoporosis (SOP), which is caused by unbalanced bone remodeling, leads to significant economic and societal burdens globally. The combination of Epimedii Folium (EF) and Ligustri Lucidi Fructus (LLF) serves as a commonly-used prescription for SOP in Traditional Chinese Medicine (TCM). This study aimed to evaluate the osteoprotective effects of EF and LLF in combination on SOP rats based on the constructed multilayer perception (MLP)-artificial neural network (ANN) model. Methods: 15 month old male Sprague-Dawley rats were administrated with EF, LLF or the combination of EF and LLF (EF&LLF) for 2 months, while 17 month old rats were used as the aging control group. All the rats were anesthetized with 25% ethyl carbamate, then their serum liver and bone tissues were taken. We detected bone mass, bone mineral density (BMD), biomechanics and the microstructure of bone trabecula by micro-CT and H&E staining to evaluate the degree of osteoporosis. Blood lipids and serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and γ-glutamyl transferase (GGT) and liver pathology were use to assess the side effects of drugs. Levels of alkaline phosphatase (ALP) and Tartrate-resistant acid phosphatase (TRACP) and the ratio of ALP to TRACP both in serum and bone were measured for the evaluation of bone turnover rate. The bone mRNA and protein expression of osteoprotegerin (OPG), nuclear factor-kappa B ligand (RANKL), macrophage colony-stimulating factor (M-CSF), d2 isoform of vacuolar (H +) ATPase (ATP6V0d2), insulin-like growth factor (IGF-1), bone morphogenetic protein-2 (BMP2), M-CSF, Wnt5a, transforming growth factor-β1 (TGF-β1) were detected for evaluating bone metabolism. Results: The results showed that EF&LLF improved bone mass and bone quality by preventing bone loss, increasing maximal load as well as protecting the micro-structural retrogressive change of trabecular bone in SOP rats; ameliorated the steatosis in the liver and decreased blood lipids and serum ALT, AST and GGT; enhanced bone remodeling by stimulating the expression of ALP and TRACP. At the molecular levels, EF&LLF stimulated the osteoclastogenesis by upregulating the protein and mRNA expression of OPG, RANKL, M-CSF and ATP6V0d2; meanwhile, EF&LLF stimulated
Osteoblast (OB) and osteoclast (OC) play important roles in bone formation and bone resorption, which can communicate with each other through cytokine paracrine. Previous studies have confirmed that Epimedii Folium (EF) and Ligustri Lucidi Fructus (LLF) used alone or in combination can treat osteoporosis (OP) through regulating bone remodeling, but the effects of EF and LLF on osteoblastogenesis, osteoclastogenesis, and OB-OC communication are unclear. In this study, we investigated the direct and indirect effects of EF and LLF on OBs and OCs via monoculture and coculture (transwell) models of OBs and OCs. We found that the combination of EF and LLF (EF&LLF) could promote osteoblastogenesis and inhibit osteoclastogenesis directly and indirectly. In order to study the mechanisms of EF&LLF on indirectly regulating osteoblastogenesis and osteoclastogenesis, we detected the expression of cytokines by which OBs and OCs could communicate with each other. We found that EF&LLF could downregulate the expression of RANKL and M-CSF and the protein ratio of RANKL/OPG of OBs and Atp6v0d2 expression of OCs and upregulate the expression of OPG and TGF-β1 of OBs and the expression of TGF-β1, BMP-2, and IGF-1 of OCs, indicating that EF&LLF could regulate cytokine expressions of OBs/OCs to affect OB-OC communication. In addition, EF&LLF had a better effect on regulating cytokines of OBs and OCs than EF or LLF in single use. This study suggested that EF&LLF exhibited the effects of promoting osteoblastogenesis and inhibiting osteoclastogenesis via acting on OB-OC communication and provided some scientific evidences for EF&LLF against OP.
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