[Effect of osthole on proliferation of neonatal rat osteoblast and the relative mechanism research].
OBJECTIVE
To investigate the effects of osthole on proliferation of neonatal rat osteoblast and the mechanism.


METHODS
Ten 24 hours old SD rats were executed by dislocating. The cranium of rats were removed and cut into blocks of 1 mm x 1 mm size. After digested by trypsin for 15 min, the cranium were digested by type I collagenase for one hour two times. The mixed cells were cultured in thermostat incubator with 5% CO2 under the condition of 37 degrees C. To identify the cells, ALP staining and alizarin red staining were performed after cultured 48 h and 28 d. The osteoblasts were randomly divided into five groups. Cells were treated with osthole at concentrations of 100, 50, 25, 12.5, 0 micromol/L. CCK-8 method was used to evaluate the proliferation after 24 h,48 h and 72 h. The expression of PCNA and beta-catenin protein were detected through the method of Western Blot after one week.


RESULTS
The cells had irregular shapes and showed typical features of osteoblast. The results of ALP staining and alizarin red staining were both positive. CCK-8 detection showed that the osthole with final concentration of 100 micromol/L inhibited the proliferation of osteoblast after 24 h, while the osthole with final concentrations of 50 micromol/L and 25 micromol/L displayed the inhibition effect after 48 h. The osthole of 12.5 micromol/L had no obvious influence on the proliferation of osteoblast. The result of Western Blot showed that osthole reduced the expression of PCNA and beta-catenin protein in a dose-dependent manner.


CONCLUSIONS
The osthole with final concentrations of 100, 50, 25 micromol/L inhibited the proliferation of osteoblast (P < 0.05). The osthole with final concentrations of 12.5 micromol/L had no obvious influence on the proliferation of osteoblast (P > 0.05). These findings demonstrate that osthole may inhibit the proliferation of osteoblast by regulating the Wnt/beta-catenin signaling in osteoblast.
Shi's Center of Orthopaedics and Traumatology, Shuguang Hospital Affiliated to Shanghai University of TCM, Shanghai 201203, China.
Ling-Hui
LH
Li
Dao-Fang
DF
Ding
Guo-Qing
GQ
Du
Hao
H
Gong
Hui-Hao
HH
Wang
Hong-Sheng
HS
Zhan
D000818:Animals; D000831:Animals, Newborn; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D003374:Coumarins / Q000494:pharmacology*; D005260:Female; D008297:Male; D010006:Osteoblasts / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects; D051176:beta Catenin / Q000378:metabolism
D003374:Coumarins; D051176:beta Catenin; C046627:osthol