Abstract: Objective To observe the effect of betulinic acid ( BA) on receptor activator ofnuclear factor-κb ligand ( RANKL) induced osteoblast differentiation in preosteoblast cell line(MC3T3-E1), and to explore its possible mechanism. Methods MC3T3-E1 cells were cultured invitro with treatment of different concentrations (0, 5, 10, 20 μmol / L) of BA for 48 h. The proliferation of MC3T3-E1 was detected by CCK-8. Cells were divided into 5 groups. Control group, RANKL group, RANKL + BA group, RANKL + BA + 740Y-P (PI3K agonist) group and RANKL + BA + Curcumin (MEK / ERK agonist) group. The level of interleukin-6 (IL-6) in cell supernatant was detected by ELISA. The activity of alkaline phosphatase (ALP) was detected by ALP activity detection kit. The expression levels of osteocalcin (OCN), osteopontin (OPN), Collagen I, p-P65, P65, MEK / ERK and PI3K signaling pathway proteins were detected by Western blotting. Results BA of 5 μmol / L and 10 μmol / L was of no significant toxicity to MC3T3 cells. Thepretreatment with 10 μmol / L BA could significantly reduce the level of IL-6 and the ratio of p-P65 /P65 which were induced by RANKL (P < 0. 05). The down-regulation of OCN, OPN, Collagen I proteins induced by RANKL were significantly reversed by treatment of 10 μmol / L BA (P < 0. 05).10 μmol / L BA could obviously inhibit the phosphorylation of MEK, ERK and PI3K ( P < 0. 05). 10 μmol / L BA could significantly increase the expression levels of osteogenic marker proteins(OCN, OPN, Collagen I), and could increase the positive rate and activity of APL staining. Theabove effects were significantly weakened by the MEK / ERK agonist curcumin and PI3K agonist740Y-P (P< 0. 05). Conclusion BA can improve the inflammatory micro-environment by inhibiting NF-κB activation and IL-6 secretion, and can promote the osteoblast differentiation through inhibition of MEK / ERK and PI3K pathways.

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