Abstract: BACKGROUND: Studies have indicated that asiaticoside possesses multiple pharmacological functions, including anti-inflammatory and antioxidant properties, and exerts a positive effect on osteogenic differentiation. It may serve as a potential therapeutic agent for osteoporosis.
OBJECTIVE: To investigate the effects of asiaticoside on osteogenic differentiation in osteoporotic rats. 
METHODS: (1) Animal models of osteoporosis were established by removing both ovaries in 52 female Sprague-Dawley rats. Simultaneously, equivalent volumes of adipose tissue near the ovaries were removed bilaterally from 10 female Sprague-Dawley rats as the sham operation group. At 8 weeks after modeling, 50 model rats were randomly divided into five intervention groups: model group (n=10) was administered saline via oral gavage; low-dose asiaticoside group (n=10) was administered 16 mg/(kg·d) asiaticoside via oral gavage; high-dose asiaticoside group (n=10) was administered 32 mg/(kg·d) asiaticoside via oral gavage; positive control group (n=10) was subjected to oral administration of sodium alendronate tablets at 7.35 mg/(kg·d); and high-dose asiaticoside + activator group (n=10) received concurrent oral administration of 32 mg/(kg·d) asiaticoside + 300 mg/(kg·d) NLRP3 activator, once daily for 6 consecutive weeks. After drug administration, tissue samples were collected. Serum levels of interleukin-1β and interleukin-18 were measured. Micro-CT scans were performed on the distal femur. Hematoxylin-eosin staining was used to observe histological changes in the femur. Western blot assay was conducted to detect the expression of tripartite motif-containing protein 24 (TRIM24), NLRP3, and cleaved caspase-1 in the femur. (2) Rat bone marrow mesenchymal stem cells at the logarithmic growth stage were divided into four groups: blank group with no treatment, asiaticoside group treated with 20 µmol/L asiaticoside, asiaticoside+empty carrier group treated with 20 µmol/L asiaticoside for 48 hours after transfection with the empty vector plasmid for 48 hours, and asiaticoside+NLRP3 overexpression group treated with 20 µmol/L asiaticoside for 48 hours after transfection with the NLRP3 overexpression plasmid for 48 hours. After 7 days of inducing osteogenic differentiation, the activity of alkaline phosphatase and mRNA expression of osteopontin, osteocalcin, TRIM24, and NLRP3 were detected. 
RESULTS AND CONCLUSION: (1) Animal experiment: The levels of interleukin-1β and interleukin-18 in the low-dose asiaticoside group, high-dose asiaticoside group, and positive control group were all lower than those in the model group (P < 0.05). The levels of interleukin-1β and interleukin-18 in the high-dose asiaticoside + activator group were higher than those in the high-dose asiaticoside group (P < 0.05). Micro-CT scans and hematoxylin-eosin staining revealed that compared with the model group, the microstructure and histomorphology of the femur were significantly improved in the low-dose and high-dose asiaticoside groups as well as in the positive control group. However, NLRP3 activator partially inhibited the effects of high-dose asiaticoside. Compared with the model group, the low-dose asiaticoside group, high-dose asiaticoside group, and positive control group showed increased expression of TRIM24 protein (P < 0.05) and decreased expression of NLRP3 and cleaved caspase-1 protein (P < 0.05). Compared with the high-dose asiaticoside group, the high-dose asiaticoside + activator group showed lower protein expression of TRIM24 (P < 0.05), but higher protein expression of NLRP3 and cleaved caspase-1 (P < 0.05). (2) Cell experiment: The alkaline phosphatase activity in the asiaticoside group was higher than that in the blank group and the asiaticoside + NLRP3 overexpression group (P < 0.05). RT-PCR revealed that the asiaticoside group showed higher mRNA expression levels of osteopontin, osteocalcin, and TRIM24 but lower NLRP3 mRNA expression compared with the control group and the asiaticoside + NLRP3 overexpression group (all P < 0.05). To conclude, asiaticoside may promote osteogenic differentiation of bone marrow mesenchymal stem cells and delay osteoporosis progression in osteoporotic rats by upregulating TRIM24 and inhibiting NLRP3 expression.

Key words: asiaticoside, osteoporosis, osteogenic differentiation, tripartite motif-containing protein 24, NLRP3, bone marrow mesenchymal stem cells, femur, tissue construction