Vitisin B 經由雌激素受體調節Src 激酶及下游MAPK 之非基因型訊息路徑而刺激骨質新生

Vitisin B stimulates osteoblastogenesis via estrogen receptor-mediated activation of non-genomic Src and MAPKs pathway

Vitisin B 是小本山葡萄主要成分之一，民間常使用該本土植物於發炎性骨疾，宣稱可強壯筋骨。我們先前發現餵食富含vitisin B 的提取物可明顯改善卵巢切除鼠的骨質流失現象，現在進一步利用MC3T3-E1 造骨細胞，研究 vitisin B 對骨質新生的影響。結果發現與骨分化/ 成熟有關的鹼性磷解酶活性及礦化程度，均因處理vitisin B 而明顯提高。vitisin B 也使骨基質以及特有轉錄因子的表現量增加，且上述促進作用皆會被雌激素受體（ER）拮抗劑所抑制。不像典型的雌激素作用劑17-estradiol，vitisin B 不會刺激ER 或ER 調控的轉錄活性，但反而可快速磷酸化ER 受體及Src 激酶、繼而磷酸化p38 及ERK 這二個MAPK，這些作用也都會被ER 拮抗劑所抑制。進一步發現p38 及ERK 抑制劑會干擾vitisin B 誘導的造骨細胞分化，而 Src 抑制劑也會阻斷vitisin B 誘導的MAPK 活化與礦化。綜上，本研究發現vitisin B 具改善骨質流失的功效，其機轉可能是經由雌激素受體去活化細胞內非基因路徑的Src 激酶及下游的MAPK 訊息分子，進一步啟動造骨細胞特有的轉錄因子活化，繼而促進骨基質生成及礦化。

 

Background/Purpose: Vitisin B is a major component existed in Vitis thunbergii, a herbal medicine used in Taiwan for treatment of inflammatory bone diseases. We recently reported that vitisin B stimulated differentiation in primary cultured osteoblasts and treatment with vitisin B-enriched preparation obviously ameliorated ovariectomy-induced bone loss in mice. This study further delineated the action mechanism(s) that how vitisin B stimulates osteoblastogenesis by using MC3T3-E1 osteoblasts. Methods: Cell differentiation and mineralization were identified by alkaline phosphatase (ALP) activity and Alizarin red S staining, respectively. RT-PCR and western blot were used to analyze the expression of osteoblast-associated genes and signal molecules. The transcriptional activity of estrogen receptor (ER) was also assessed. Results: Vitisin B significantly increased ALP activity, bone mineralization, mRNA expression of osteoids (type 1 collagen, bone sialprotein and osteocalcin) and bone-characteristic transcription factors (runt-related transcription factor-2 and osterix) through ER since such responsiveness were obviously repressed by ER antagonist ICI182,780. Unlike 17β-estradiol (E2), vitisin B failed to stimulate either ERα- or ERβ-mediated transcriptional activity. Nevertheless, vitisin B rapidly induced ERα and Src phosphorylations within 5 min and evoked late phosphorylations of p38 and ERK after 15-30 min stimulation through ER. Furthermore, p38 inhibitor SB203580 and MEK inhibitor PD98059 significantly inhibited vitisin B induced differentiation. Src inhibitor PP2 significantly repressed vitisin B-induced activation of MAPK and finalmineralization suggesting that Src might play an important role. Conclusions: Vitisin B might act through ER-mediated activation of Src and downstream MAPK to stimulate osteoblastogenesis which contributed to its beneficial effect in prevent bone loss.