Abstract: BACKGROUND: Chlorogenic acid is the most abundant polyphenol in dried plums, which, as a food-derived drug, is expected to be used in the treatment of osteoporosis.
OBJECTIVE: To investigate the effect of chlorogenic acid on osteogenic differentiation of osteogenic precursor cells and its mechanism and to verify its effect on bone loss in a mouse model of osteoporosis.
METHODS: (1) In vitro test: MC3T3-E1 cells were cultured in vitro with different concentrations of chlorogenic acid (0, 0.1, 1, 10, 100 mg/L) and cell proliferation was detected using cell counting kit-8. MC3T3-E1 cells were cultured in osteogenic induction media containing 0, 10, 20 mg/L chlorogenic acid, followed by alkaline phosphatase staining, alizarin red staining, and detection of osteogenic genes and BMP-2/RUNX2/SMAD4 signaling pathway-related proteins. (2) In vivo test: Thirty-two female C57BL/6 mice were randomly divided into four groups, sham operation group, ovariectomized group, and low- and high-dose chlorogenic acid groups, with 8 mice in each group. An ovariectomized mouse model was established, and chlorogenic acid was then administered at high (50 mg/kg/d) and low (25 mg/kg/d) concentrations in the latter two groups for 8 continuous weeks, respectively. Micro-CT scan and histomorphology analysis were performed on the femur and tibia of the mice 8 weeks later. 
RESULTS AND CONCLUSION: (1) In vitro test: Chlorogenic acid at a concentration of ≤ 10 mg/L had no obvious effects on the proliferation of MC3T3-E1 cells. With the increased concentration of chlorogenic acid, the alkaline phosphatase activity, calcium deposition and calcium nodule formation ability of MC3T3-E1 cells increased; the mRNA expression levels of osteogenesis-related genes, alkaline phosphatase, osteopontin, osteocalcin, bone morphogenetic protein 2, RUNX2 and SMAD4 increased; and the protein expression levels of bone morphogenetic protein 2, RUNX2, and SMAD4 increased. (2) In vivo test: Micro-CT scan results showed that compared with the sham operation group, the bone mineral density, bone volume fraction, thickness and number of trabecular bone were significantly lower in the ovariectomized group (P < 0.05) and trabecular separation degree was significantly higher in the ovariectomized group (P < 0.05). Treatment with different concentrations of chlorogenic acid could certainly improve the above-mentioned indicators and the high-dose chlorogenic acid group showed significant changes in these indicators (P < 0.05). Hematoxylin-eosin staining showed less bone mass in the ovariectomized group than the sham operation group, and the bone mass increased after chlorogenic acid intervention. (3) To conclude, chlorogenic acid may promote osteogenic differentiation through the BMP-2/RUNX2/SMAD4 signaling pathway and prevent bone loss in ovariectomized mice.

Key words: chlorogenic acid, osteogenic gene, osteogenic differentiation, cell proliferation, osteogenic induction, osteogenic mineralization, signaling pathway, bone loss