Cistanoside A promotes osteogenesis of primary osteoblasts by alleviating apoptosis and activating autophagy through involvement of the Wnt/β-catenin signal pathway
Tongying Chen, Feng-He Gao, Dan Luo, Shihao Wang, Yu Zhao, Shuhua Liu, Jiachun Huang, Yanping Lin, Zhihai Zhang, Hongxing Huang, Lei Wan
Abstract
Background: As a phenylethanoid glycoside extracted from Cistanche deserticola, cistanoside A has been shown to have antioxidative effects. In recent years, it has been found to play an important role in osteoporosis. Methods: Primary osteoblasts were randomly divided into a cistanoside A (Cis A)-1 group (5 µM), a Cis A-2 group (10 µM), and a Cis A-3 group (20 µM) to screen the optimal dose. Then, cells were treated with Rapamycin (Rapa), 3-MA, Dickkopf-1 (DKK-1), 3MA + Cis A (10 µM), and DKK-1 + Cis A (10 µM). After a certain period of routine culture, Alkaline Phosphatase (ALP) and Alizarin Red S Staining were performed again and the cells were collected for subsequent experiments including immunofluorescence staining, western blotting, transmission electron microscopy, mitochondrial membrane measurement, and Annexin-V-Fluorescein isothiocyanate (Annexin-V-FITC). Results: The optimal Cis A dose that preserved osteoblast viability and activated osteogenesis was 10 µM. It appeared that Cis A (10 µM) decreased apoptosis and augmented autophagy via increasing microtubule-associated protein light chain 3 (LC3)-I/II expressions as well as raising Wnt/β-catenin signal pathway activity. The addition of 3-MA further inhibited osteogenic differentiation and suppressed Wnt/β-catenin signal pathway activity to increase apoptosis while reducing autophagy levels. A combination of Cis A and DKK-1 resulted in higher levels of apoptosis but lower levels of autophagy. Conclusions: Cis A appears to be a potent inducer of autophagy and inhibitor of apoptosis in primary osteoblasts by working through the Wnt/β-catenin signal pathway, thereby resulting in enhanced osteogenic differentiation.