论文部分内容阅读
目的观察淫羊藿甙对体外培养大鼠成骨细胞丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)信号通路的影响以及MAPK信号通路在淫羊藿甙促成骨细胞核心结合因子α1(core bindingfactor-1,Cbfa1)蛋白表达中的作用,以探讨淫羊藿甙对成骨细胞作用的信号传导机制。方法用酶消化法分离24h内新生SD大鼠颅盖骨成骨细胞,进行原代培养。在培养液中加入淫羊藿甙(10ng/mL),作用于成骨细胞5min、10min、30min、60min,抽提总蛋白,用Western blot法检测细胞中ERK、p-ERK、P38和p-P38蛋白的表达。用雌二醇(10-8mol/L)作用于成骨细胞5min、10min、30min,同上法检测细胞中ERK、p-ERK、P38和p-P38蛋白的表达。用淫羊藿甙(10ng/mL)、雌二醇(10-8mol/L)和u0126或SB203580单独或共同干预成骨细胞24h,抽提核蛋白,用Western blot法检测Cbfa1蛋白的表达。结果淫羊藿甙作用于成骨细胞,30min时可促进ERK蛋白的磷酸化,并可持续至60min,和空白组比较,差异有显著性(P<0.05);淫羊藿甙作用于成骨细胞,5min时即可促进P38蛋白的磷酸化,在30min时达高峰,并可持续至60min,和空白组比较,差异有显著性(P<0.05)。②雌二醇作用于成骨细胞,在30min时可促进ERK蛋白的磷酸化,和空白组比较,差异有显著性(P<0.05);雌二醇作用于成骨细胞,在10min时可促进P38蛋白的磷酸化,并可持续至30min,和空白组比较,差异有显著性(P<0.05)。③淫羊藿甙和雌二醇均能促进成骨细胞中Cbfa1蛋白的表达,和空白组相比,差异有显著性(P<0.05)。u0126和SB203580可以抑制淫羊藿甙和雌二醇促进Cbfa1蛋白表达的作用。结论淫羊藿甙和雌二醇均可以激活成骨细胞中ERK/MAPK和P38/MAPK信号通路;②淫羊藿甙和雌二醇均能促进成骨细胞中核转录因子Cbfa1的表达,并且ERK/MAPK和P38/MAPK信号通路参与了此过程。
Objective To observe the effects of Epimedium on mitogen-activated protein kinase (MAPK) signaling pathway in rat osteoblasts and MAPK signaling pathway in epimedium-promoted osteoblast core binding factor α1 ( The role of core binding factor-1, Cbfa1) in protein expression, to investigate the signal transduction mechanism of epimedium on osteoblasts. Methods Osteoblasts of neonatal Sprague-Dawley rats were isolated by enzyme digestion and cultured for 24 hours. Epimedium (10 ng/mL) was added to the culture medium and osteoblasts were treated for 5 min, 10 min, 30 min, and 60 min to extract total protein. ERK, p-ERK, P38, and p-cells were detected by Western blot. P38 protein expression. Osteoblasts were treated with estradiol (10-8 mol/L) for 5 min, 10 min, and 30 min. The expressions of ERK, p-ERK, P38, and p-P38 were detected by the same method as above. Osteoblasts were treated with Epimedium (10 ng/mL), Estradiol (10-8 mol/L), and u0126 or SB203580, either alone or together, for 24 hours to extract nucleoprotein. The expression of Cbfa1 protein was detected by Western blot. Results Epimedium acted on osteoblasts, which promoted the phosphorylation of ERK protein at 30 min and lasted for 60 min. Compared with the blank group, the difference was statistically significant (P<0.05); Epimedium acted on osteogenesis Phosphorylation of P38 protein was promoted by cells at 5 min, peaked at 30 min, and continued to 60 min, compared with the blank group, the difference was statistically significant (P<0.05). 2 Estradiol acts on osteoblasts and promotes the phosphorylation of ERK at 30 min. Compared with the blank group, the difference is significant (P<0.05); estradiol acts on osteoblasts and can be promoted at 10 min. Phosphorylation of P38 protein lasted for 30 min, compared with the blank group, the difference was significant (P<0.05). 3 Epimedium and estradiol can promote the expression of Cbfa1 protein in osteoblasts, compared with the blank group, the difference was significant (P<0.05). U0126 and SB203580 can inhibit the expression of Cbfa1 protein by epimedium and estradiol. Conclusion Epimedium and estradiol can activate the ERK/MAPK and P38/MAPK signaling pathways in osteoblasts; 2 Epimedium and estradiol can promote the expression of nuclear transcription factor Cbfa1 in osteoblasts, and ERK The /MAPK and P38/MAPK signaling pathways are involved in this process.