The Effect Of3β,19α-dihydroxy-20β-acetate-serrat-14-en-21β-ol On Osteoblast Activity In Vitro

Lycopodium obscurum L. from Lycopodium, as a traditional herbal medicine waswidely used in clinics which has an obvious effect in the treatment of bruises,fractures and bone-repairing. Modern clinical medicine research also confirmed that itdid have a significant effect in the treatment of bone fractures. However, most studiesof the treatment of Lycopodium obscurum L. on bone disease were based on clinicaldata. The effective components and the molecular mechanism of the treatment are notclear yet. In this study, a new huperzia triterpenoid, the compound extracted fromLycopodium obscurum L. named as3β,19α-dihydroxy-20β-acetate-serrat-14-en-21β-ol (DHAS) was studied on its effecton osteoblast proliferation, alkaline phosphatase activity and osteogenic geneexpression, and we tried to elucidate that whether the compound was one of the activeingredient of the Lycopodium obscurum L. in enhancing bone fracture healing and themolecular mechanisms.This study including the following three parts:(1) The effect of DHAS on osteoblast proliferation rate.(2) The effect of DHAS on the ALP activity in osteoblast.(3) The effect of DHAS on the expression of osteoblast gene. The gene conclude：AP-1family（Fos subfamily：c-fos, fra-1, fra-2and Jun subfamily：c-jun, jun-d）and NFATc1, transcription-related factors(runx2, osterix and BMP-2) andbone-related genes(ALP, BSP, COL-I, OCN, OPN).In this study, plenty osteoblasts of high-purity were obtained from newly-bornmouse calvarias. By morphological observation and the detection of ALP activity, thecells were confirmed to be osteoblasts: obtained cells showed a strong osteogenicactivity which can be used as a cell model for further study.MTT assay results showed that the cell proliferation rate could be improved byDHAS. The2μM/L and the8μM/L of DHAS showed strong enhancement to cellproliferation. Hence, they were chosen to be the optimal concentration for furtherstudy.The alkaline phosphatase activity of osteoblasts showed that the activity ofalkaline phosphatase had been increased by every concentration of DHAS and theenhancement could be increased in the later period of treatment.The results of Real-Time PCR showed that DHAS had a complicated affectionon the gene expression of osteoblasts. Generally speaking, DHAS could increase theexpression of the early-expressed gene such as AP-1family, COL-I, and ALP in a long treatment. However, for the genes which expressed in the matrix period (BMP)and in the mineralization period (OCN, OPN, BSP, etc.) had the negative expressionwith the treatment of DHAS in early cultured stage. While in the later cultured stage,the expression could be increased.The results of this study showed that DHAS could promote the proliferation rate,the activity of ALP, the expression the bone-related gene in osteoblasts, and itparticularly beneficial to the expression of early growth and biological activity ofosteoblasts. DHAS could increase the number of osteoblasts, the osteoblast activityand it helped accelerating the speed of recovery. DHAS may be one of the effectivecomponents of Lycopodium obscurum L. on the treatment of bone disease.