CAJ | 학술논문

目的：探讨淫羊藿甙调节 MC3T3-E1成骨细胞增殖分化过程中雌激素受体途径及与之关联的 MAPK 信号的作用。方法用不同浓度淫羊藿甙处理 MC3T3-E1成骨细胞不同时间,观察细胞活性、细胞周期相关蛋白表达及 ERK、P38和 JNK 蛋白磷酸化的改变,然后用 MAPK 通路的抑制剂与淫羊藿甙联合或单独处理细胞,观察 MAPK 通路在淫羊藿甙对成骨细胞增殖中的作用。用碱性磷酸酶活性和茜素红矿化结节染色评价淫羊藿甙对 MC3T3-E1成骨细胞成熟和矿化的影响,并用 ICI182780和 Nilutamide 与淫羊藿甙共同处理 MC3T3-E1成骨细胞,实时 PCR 检测碱性磷酸酶、Ⅰ型胶原和骨钙素(osteocalcin)基因的表达。结果淫羊藿甙浓度依赖性促进 MC3T3-E1成骨细胞的活性,可上调 cyclin E 和 PCNA 并下调 Cdkn2b的表达。 U0126和 SP600125可减弱淫羊藿甙对成骨细胞活性和基因表达的调节。淫羊藿甙可快速激活ERK 和 JNK 信号(5 min 内),但不改变 P38信号的磷酸化,ICI182780可部分抑制淫羊藿甙对 ERK 和 JNK信号的激活。淫羊藿甙能上调成骨细胞中碱性磷酸酶、Ⅰ型胶原和骨钙素基因的表达,并促进矿化结节的形成,ICI182780可以部分阻断淫羊藿甙的促分化和矿化作用。结论淫羊藿甙可快速激活成骨细胞中ERK 和 JNK 信号而影响细胞周期相关蛋白基因的表达,进而促进成骨细胞的增殖,且可部分通过雌激素受体信号促进成骨细胞的分化和矿化。
목적：탐토음양곽대조절 MC3T3-E1성골세포증식분화과정중자격소수체도경급여지관련적 MAPK 신호적작용。방법용불동농도음양곽대처리 MC3T3-E1성골세포불동시간,관찰세포활성、세포주기상관단백표체급 ERK、P38화 JNK 단백린산화적개변,연후용 MAPK 통로적억제제여음양곽대연합혹단독처리세포,관찰 MAPK 통로재음양곽대대성골세포증식중적작용。용감성린산매활성화천소홍광화결절염색평개음양곽대대 MC3T3-E1성골세포성숙화광화적영향,병용 ICI182780화 Nilutamide 여음양곽대공동처리 MC3T3-E1성골세포,실시 PCR 검측감성린산매、Ⅰ형효원화골개소(osteocalcin)기인적표체。결과음양곽대농도의뢰성촉진 MC3T3-E1성골세포적활성,가상조 cyclin E 화 PCNA 병하조 Cdkn2b적표체。 U0126화 SP600125가감약음양곽대대성골세포활성화기인표체적조절。음양곽대가쾌속격활ERK 화 JNK 신호(5 min 내),단불개변 P38신호적린산화,ICI182780가부분억제음양곽대대 ERK 화 JNK신호적격활。음양곽대능상조성골세포중감성린산매、Ⅰ형효원화골개소기인적표체,병촉진광화결절적형성,ICI182780가이부분조단음양곽대적촉분화화광화작용。결론음양곽대가쾌속격활성골세포중ERK 화 JNK 신호이영향세포주기상관단백기인적표체,진이촉진성골세포적증식,차가부분통과자격소수체신호촉진성골세포적분화화광화。
Objective To explore the detailed underlying molecular and signaling mechanisms in the effects of icariin on bone formation by an in vitro cell model. Methods The proliferation of MC3T3-E1 osteoblast-like cells was evaluated by MTT, and gene expression of cell cycle related proteins in MC3T3-E1 cells after icariin treatment was detected by real-time PCR. The phosphorylation of MAPK signals, including ERK, P38, and JNK was determined by Western blot, and then the inhibitors of MAPK signals were used to treat cells with icariin alone or together to determine the role of MAPKs in the process of icariin treatment on MC3T3-E1 cell proliferation. Alkaline phosphatase and Alizarin red staining were used to detect the formation of mineralization nodules, and gene expressions of alkaline phosphatase, type Ⅰ collagen, and osteocalcin in osteoblasts after being treated by icariin were evaluated by real-time PCR. ICI182780, and nilutamide was used to decide the participation of estrogen and androgen receptor signals in the process of icariin treatment on the differentiation and mineralization of MC3T3-E1 cells. Results Treatment with icariin promoted MC3T3-E1 cell growth in a time- and dose-dependent manner. This treatment also revealed that icariin increased the expression of mRNAs encoding both cyclin E and PCNA, positive regulators of cell growth, but decreased levels of mRNAs encoding Cdkn2b, a negative regulator of cell cycle progression. When MC3T3-E1 cells were cultured in a differentiated condition, icariin enhanced mineralized nodule formation and increased the expression of mRNAs encoding alkaline phosphatase, type Ⅰ collagen, and osteocalcin. Treatment with icariin significantly induced phosphorylation of both ERK and JNK and this phosphorylated effect occurred very rapidly within 5 minutes and reached peak at 15 minutes. Furthermore, the stimulated effects of icariin on proliferation and gene expression of cyclin E, PCNA, and Cdkn2b in MC3T3-E1 cells were dramatically attenuated by treatment with both U0126 and SP600125, inhibitors of MAPKs. Interestingly, such stimulating effects of icariin were at least partly reduced by treatment with ICI182780, an inhibitor of estrogen receptor. Icariin induced mineralized nodule formation and gene expression of alkaline phosphatase, type Ⅰ collagen, and osteocalcin in MC3T3-E1 cells were also partly reduced when the cells were treated with ICI182780. Conclusions Our findings indicate that the anabolic effect of icariin on bone formation is, at least partly, mediated through the MAPK signaling pathway in order to modulate osteoblast proliferation and differentiation.