The Effect Of 26-nor-8-hydroxy-α-onocerin On Osteoblast Activity In Vitro

Lycopodium obscurum L. from Lycopodium, is a traditional Chinese herbal medicine which is used for treatments of bone fracture, traumatic injury and contusion. Modern clinical medical research has demonstrated that it can enhance osteogenesis. However, we don't know what are the effective components and how do they work, There is no answers to these questions. Therefore, in this thesis, 26-nor-8-hydroxy-α-onocerin(26HO), a novel tetraeyclic triterpenoid extracted from Lycopodium obscurum L. was studied on its effects on osteoblast proliferation, alkaline phosphatase activity and osteogenic gene expression, in order to elucidate the molecular mechanism of its osteogenic activity.In this study, the effect of 26HO on osteoblast proliferation, osteoblast proliferation, alkaline phosphatase activity and osteogenic gene expression(BSP, ON, Col I, OC, Osterix, OP, ALP and Runx2) in vitro was studied.Primary cultured osteoblasts was isolated and purified from calvaria of 2-3 days old mice with a modified tissue culture method. Result showed that purified primary osteoblasts were observed to be in proper morphology and their alkaline phosphatase activity was maintained, which demonstrated that the cells obtained was osteoblasts.MTT assay result showed that osteoblast proliferation could be significantly stimulated if osteoblasts were treated with relatively high concentration (20μM/L, 40μM/L) of 26HO in a short period (1 day). However, the treatment (0-40μM/L) of 26HO would not have a significant effect on osteoblast proliferation after a longer period (3 days).The result of the alkaline phosphatase activity test showed that 26HO could affect alkaline phosphatase activity in osteoblasts. Generally speaking, as incubation time went on, the effect that 26HO of low concentration (5μM/L) had on alkaline phosphatase activity in osteoblasts changed gradually from stimulation (3 days) to inhibition (14 days); while high concentration of 26HO showed the opposite: the effect that high concentration (20-40μM/L) 26HO had on alkaline phosphatase activity in osteoblasts changed gradually from inhibition (3 days) to stimulation (14 days).The result of real-time PCR showed that short-term treatment of osteoblasts with 26HO significantly inhibited the expression of osterix and Runx2 mRNA. Generally speaking, 26HO does not favor the expression of BMP-2 and BSP. The effect of 26HO on the expression of other osteogenic gene showed complexity due to different length of treatment and different concentration of 26HO.To sum up, 26HO could modulate osteoblast proliferation and differentiation, thus is one of the effective components for bone fracture treatment. This study may provide experimental evidence for the clinical application of Lycopodium obscurum L. as osteogenic medicine.