淫羊藿苷对RAW264.7细胞破骨分化中MAPK信号通路的影响探究

发布时间：2018-11-28 09:57

【摘要】：目的探讨淫羊藿苷(icariin,ICA)抑制破骨分化的量效和时效关系,探究淫羊藿苷在单核细胞向破骨细胞分化的过程中对丝裂原活化蛋白激酶(mitogen-activated protein kinases,MAPKs)信号通路的影响,以了解淫羊藿苷对破骨细胞分化抑制过程的分子生物学机制,为淫羊藿苷进一步应用于临床提供理论基础。方法本研究分为两个部分进行。一淫羊藿苷抑制破骨细胞分化的量效关系研究:按照不同的ICA药物浓度将RAW264.7细胞分为空白对照组(等量培养基对照)、模型对照组(50ng/ml RANKL)、ICA四个浓度梯度组(10-7/10-6/10-5/10-4mol/L ICA+50ng/ml RANKL)。TRAP染色观察破骨细胞形态数目。同时通过western blot检测关键蛋白的表达和磷酸化水平。二淫羊藿苷抑制破骨细胞分化的时效关系研究:设置对照溶剂处理组与淫羊藿苷处理组(ICA浓度为10-5mol/L),每组分别在诱导12、24、36、48、60、72小时后进行TRAP染色观察破骨细胞形态数目,并通过western blot检测关键蛋白的表达和磷酸化水平。结果一RAW264.7诱导培养48小时后可见含3个以上细胞核的多核细胞。Trap染色显示随着ICA浓度的升高,破骨细胞数量减少,破骨细胞形态小,胞核数目较少。模型对照组与空白对照组相比,p38、JNK、ERK磷酸化水平升高,而P38、JNK、ERK表达水平无统计学差异。与不给于ICA的模型对照组相比,各ICA处理组的P38、JNK、ERK磷酸化水平随浓度的升高而降低,其抑制作用呈现浓度依赖性,而P38、JNK、ERK表达水平无统计学差异。当ICA浓度达到10-5mol/L时,对单核细胞破骨分化的抑制作用达到最佳。二溶剂对照组在诱导12小时后即可见TRAP阳性的破骨细胞,且随着诱导时间的增长,破骨细胞数量逐渐增多。与相同诱导时间的溶剂对照组相比,ICA组破骨细胞数量极少,且细胞形态较小。相同诱导时间的溶剂对照组和ICA处理组之间P38、JNK、ERK的磷酸化水平均有差异,P38、JNK、ERK表达水平无统计学差异。溶剂对照组的P38、JNK、ERK的磷酸化水平随诱导时间的增加而逐渐升高,而P38、JNK、ERK表达无统计学差异。ICA处理组的各诱导时间点间两两比较,P38、JNK、ERK的磷酸化水平和蛋白表达水平均无统计学差异。结论淫羊藿苷对单核细胞破骨分化具有稳定的抑制作用,且抑制作用效果随淫羊藿苷浓度的升高而增强。淫羊藿苷对单核细胞破骨分化时MAPK信号通路中关键蛋白p38、ERK和JNK的表达没有影响,但是可以显著抑制其磷酸化水平,且抑制作用与浓度正相关。淫羊藿苷体外抑制破骨分化过程中MAPK通路的最佳浓度为10-5mol/L。淫羊藿苷对破骨分化的抑制作用自12小时起已经达到稳定水平。
[Abstract]:Objective to investigate the dose-effect and time-effect relationship of icariin (icariin,ICA) in inhibiting osteoclast differentiation, and to explore the effect of icariin on mitogen-activated protein kinase (mitogen-activated protein kinases,MAPKs) signaling pathway during monocyte differentiation into osteoclasts. In order to understand the molecular biological mechanism of icariin inhibiting osteoclast differentiation, it provides a theoretical basis for the further application of icariin in clinical practice. Methods the study was divided into two parts. Study on the dose-effect relationship of icariin in inhibiting osteoclast differentiation: RAW264.7 cells were divided into blank control group (equal medium control) and model control group (50ng/ml RANKL),) according to different concentrations of ICA. The number of osteoclasts was observed by 10-7/10-6/10-5/10-4mol/L ICA 50ng/ml RANKL). TRAP staining in four concentration gradient groups of ICA. At the same time, the expression and phosphorylation of key proteins were detected by western blot. The effect of icariin on osteoclast differentiation: the control group and icariin treated group (ICA concentration was 10-5mol/L) were used to observe the number of osteoclasts by TRAP staining. The expression and phosphorylation of key proteins were detected by western blot. Results Mononuclear cells containing more than 3 nuclei were found in a RAW264.7 induction culture for 48 hours. Trap staining showed that with the increase of ICA concentration, the number of osteoclasts decreased, the morphology of osteoclasts was small, and the number of nuclei was less. Compared with the control group, the phosphorylation level of p38 JNKK ERK in the model control group was higher than that in the control group, but there was no significant difference in the expression level of P38 + JNK-ERK between the model control group and the control group. Compared with the model control group without ICA, the phosphorylation level of P38 JNKERK decreased with the increase of ICA concentration, and the inhibitory effect was concentration-dependent, but there was no significant difference in the expression level of P38 JNKERK. When the concentration of ICA reached 10-5mol/L, the inhibitory effect on osteoclast differentiation of monocyte reached the best. TRAP positive osteoclasts were found in the solvent control group 12 hours after induction, and the number of osteoclasts increased with the increase of induction time. Compared with the solvent control group for the same induction time, the number of osteoclasts in ICA group was very small and the morphology of osteoclasts was smaller. The phosphorylation level of P38 JNK-ERK was different between the solvent control group and the ICA treatment group, but there was no significant difference in the expression level of P38 JNK-ERK between the same induction time group and the ICA treatment group. The phosphorylation level of P38 JNK-ERK in the solvent control group increased gradually with the increase of induction time, but there was no significant difference in the expression of P38 JNK-ERK. There was no significant difference in phosphorylation level and protein expression level of ERK. Conclusion Icariin has a stable inhibitory effect on osteoclast differentiation of monocytes, and the inhibitory effect is enhanced with the increase of icariin concentration. Icariin had no effect on the expression of p38 ERK and JNK in the MAPK signaling pathway during osteoclast differentiation, but could significantly inhibit its phosphorylation level, and the inhibitory effect was positively correlated with the concentration. The best concentration of Icariin in inhibiting osteoclast differentiation in vitro was 10-5 mol / L 路L ~ (-1) 路L ~ (-1). The inhibitory effect of icariin on osteoclast differentiation has reached a stable level since 12 hours.
【学位授予单位】：首都医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R783.5

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