蜕皮甾酮对糖皮质激素诱导成骨细胞增殖及分化的影响

The effects of ecdysterone on proliferation and differentiation of glucocorticoid-induced osteoblasts

目的观察β-蜕皮甾酮(β-Ecd)对糖皮质激素诱导的成骨细胞增殖及分化的影响及机制.方法取Wistar雄性大鼠,处死后分离培养BMSCs,消化传代.采用地塞米松(Dex)诱导培养成骨细胞,并分为空白对照组、Dex组、Dex+阿仑膦酸钠组、Dex+β-Ecd组.成骨细胞在96孔板上培养,在加入Dex基础上,分别加入阿仑膦酸钠、β-Ecd,作用处理24h、48h、72h,采用CCK-8细胞计数法检测细胞增殖;碱性磷酸酶活性检测观察β-Ecd对成骨细胞分化的影响.采用转录酶-聚合酶链反应(RT-PCR)检测相关基因mRNA表达.结果诱导14d后,成骨细胞外基质呈强紫色,表明成骨细胞诱导成功.与空白对照组比较,Dex在较低浓度下能明显刺激碱性磷酸酶活性,在较高浓度时抑制碱性磷酸酶活性,且两者均呈剂量依赖性.不同浓度的β-Ecd对碱性磷酸酶活性的刺激作用与单用Dex和阿仑膦酸钠相似.Dex能显著降低Runx2和OCN的表达,而RANKL在mRNA和蛋白质水平上则均表现为显著增加.不同浓度的β-Ecd对Dex诱导的成骨细胞Runx2、OCN和RANKL表达的变化呈剂量依赖性的逆转作用.与空白对照组比较,低浓度Dex处理24h后细胞增殖明显增强,而高浓度则抑制细胞增殖.不同浓度的β-Ecd作用24h对细胞增殖无明显促进作用,但作用48h、72h与单用Dex处理比较,差异有统计学意义(P<0.05).结论β-Ecd能以剂量依赖性的方式逆转Dex对成骨细胞分化和增殖的抑制.该结果将为β-Ecd治疗糖皮质激素性骨质疏松症提供强有力的理论支持.

Objective To observe the effect of ecdysterone on proliferation and difFerentiarion of osteoblasts induced by glucocorticoid and its mechanism.Methods Male Wistar male rats were sacrificed and cultured for BMSCs digestion and passage.Osteoblasts were induced by dexamethasone and divided into blank control group,dexamethasone group,dexamethasone+alendronate group,dexamethasone+β—Ecdysone group.Osteoblasts were cultured on 96-well plate and treated with alendronate and beta—ecdysterone for 24h,48 h and 72h respectively.The proliferation of osteoblasts was detected by CCK—8 counting method.Alkaline phosphatase activity was detected to observe the effect of β-Ecdysone on the differentiation of osteoblasts and the effects of cell differentiation.The mRNA expression o f related genes was RT-PCR detected.Results After induction of 14 days,the extracellular matrix of osteoblasts showed strong purple,indicating that that osteoblasts were successfully induced.Compared with the blank control group,dexamethasone significantly stimulated the activity of alkaline phosphatase at lower concentrations and inhibited the activity of alkaline phosphatase at higher concentrations in a dose—dependent manner.The stimulating effect of different concentrations of β-Ecdysone on alkaline phosphatase activity was similar to that of dexamethasone and alendronate.Dexamethasone significantly decreased the expression of Runx2 and OCN,while RANKL increased significantly at both mRNA and protein levek.Different concentrations of Ecdysterone reversed the expression of Runx2,OCN and RANKL in osteoblasts induced by dexamethasone.Compared with the blank control group,the cell proliferation wasignificantly increased after 24h treatment with low concentration of dexamethasone,but inhibited by high concentration of dexamethasone.Different concentrations of β—Ecdysone had no obvious effect on cell proliferation after 24 hours,but there was significant difference between 48 hours and 72 hours of β—Ecdysone treatment and dexamethasone treatment alone(P<0.05).Conclusion β—Ecdysone can reverse the differentiation and proliferation of osteoblasts induced by dexamethasone in a dose—dependent manner.The results will provide strong theoretical support for the treatment of glucocorticoid osteoporosis with β—Ecdysone.