骨碎补总黄酮对高糖作用下成骨细胞分化及ERK_(1/2)和p38蛋白激酶表达的影响

Effects of Total Flavonoids in Drynaria fortunei on Osteoblasts Differentiation and the Expression of ERK_(1/2) and p38 MAPK after Treatment by High Glucose in vitro

目的:研究骨碎补总黄酮(TFDF)对高浓度葡萄糖作用下成骨细胞(OB)分化活性及对细胞外信号调节蛋白激酶(ERK1/2)和p38丝裂原活化蛋白激酶(p38MAPK)信号蛋白的影响,为TFDF能否用来防治糖尿病性骨质疏松提供细胞分子学依据。方法:二次酶消化法分离培养新生SD大鼠颅骨OB及活性观察;MTT法检测TFDF对OB的细胞毒性,确定实验药物在培养基中的添加剂量;pNPP、ELISA、茜素红染色法分别检测不同浓度葡萄糖对OB碱性磷酸酶活性(ALP)、Ⅰ型胶原(ColⅠ)、骨钙素(BGP)以及矿化能力的影响,并观察TFDF对高糖作用下OB分化活性的干预作用;Western-blot检测TFDF对高糖作用下OB ERK1/2和p38蛋白磷酸化的情况。结果:原代分离培养新生SD大鼠OB,具有典型的OB分化特性;根据TFDF对OB的毒性试验,选择TFDF的浓度25、50、100 mg/L为实验梯度浓度;葡萄糖(25、50 mmol/L)可以降低OB表达ALP、ColⅠ、BGP,降低其矿化能力;TFDF能剂量依赖性的提高高糖(25 mmol/L)作用下OB表达ALP、ColⅠ、BGP和矿化能力;TFDF(50 mg/L)能提高高糖(25 mmol/L)作用下p38和ERK1/2的蛋白磷酸化。结论:高糖可以降低OB分化和矿化能力,TFDF可提高高糖作用下OB的分化和矿化能力,其作用机理可能和提高p38和ERK1/2信号蛋白的磷酸化有关。

Objective：To observe the effects of Total Flavonoids in Drynaria fortunei（TFDF） on osteoblasts differentiation activity after treatment by high glucose and observe the effects on p38MAPK and ERK1/2 signaling protein in osteoblasts.Methods：Primary osteoblasts of newborn SD rats was extracted and cultured and its biological characteristics was observed.MTT method was used to observe osteoblasts′ cytotoxicity,and to choose a suitable concentration of TFDF in the culture medium.pNPP,ELISA,Alizarin dyeing were used to test ALP,TypeⅠcollagen,osteocalcin and mineralization of osteoblasts after treatment by different concentration of glucose respectively and after treatment by TFDF and high glucose.Western-blot was used to detect p38MAPK and ERK1/2 protein phosphorylation after treatment by TFDF and high glucose.Results：Primary osteoblasts of newborn SD rats could be used well in this experiment.According to the toxicity of TFDF on OB,25,50,100 mg/L of TFDF were selected for the experimental concentration gradient.ALP,TypeⅠcollagen,osteocalcin and mineralization of osteoblasts after treatment with glucose（25,50 mmol/L） were less than those of control group respectively.TFDF could increase ALP,TypeⅠcollagen,osteocalcin activity and mineralization of osteoblasts in a dose-dependent manner after treatment by high glucose（25 mmol/L）.TFDF（50 mg/L） could increase protein phosphorylation of p38MAPK and ERK1/2 of osteoblasts after treatment by high glucose（25 mmol/L）.Conclusion：High glucose can decrease differentiation and mineralization of osteoblasts.TFDF can increase differentiation and mineralization of osteoblasts in a dose-dependent manner after treatment by high glucose.The role of TFDF in the promotion of osteoblasts differentiation is related to protein phosphorylation of p38MAPK and ERK1/2.