佛手苷内酯对磷酸三钙磨损颗粒诱导骨细胞损伤的影响及机制

Effects of bergapten on damages of osteocytes MLO-Y4 induced by TCP wear particles and its mechanism

目的:研究佛手苷内酯(BP)对磷酸三钙(TCP)磨损颗粒诱导骨细胞损伤的影响,并阐明其可能作用机制。方法:将TCP磨损颗粒与小鼠骨细胞MLO-Y4细胞共孵育48 h建立骨细胞体外损伤模型,随机分为正常对照(Control)组、TCP磨损颗粒(TCP,0.1 mg/ml)组、佛手苷内酯(1μmol/L)组、佛手苷内酯(5μmol/L)组和佛手苷内酯(20μmol/L)组。MTT法和Calcein-AM染色检测各组骨细胞活性和形态改变;Hoechst 33342染色和流式细胞术分析各组骨细胞凋亡情况;实时荧光定量PCR检测各组骨细胞特征蛋白牙本质基质蛋白-1(DMP-1)、骨硬化蛋白(SOST)、成纤维细胞生长因子23(FGF23)的mRNA水平;Western blot法检测各组骨细胞中内质网应激标志蛋白葡萄糖调节蛋白78(GRP78)、蛋白激酶R样内质网激酶(PERK)、磷酸化PERK(p-PERK)、真核细胞翻译起始因子2α(eIF2α)、磷酸化eIF2α(p-eIF2α)、活性转录因子(ATF4)和C/EBP同源蛋白(CHOP)等的表达及caspase-3的活化变化。结果:与Control组比较,TCP组骨细胞的活性和DMP-1的mRNA水平显著降低(P<0.05),骨细胞凋亡率及SOST、FGF23的mRNA水平显著增加(P<0.05),GRP78、ATF4和CHOP等蛋白质表达、p-PERK/PERK值和p-eIF2α/eIF2α值显著升高;与TCP组比较,佛手苷内酯组骨细胞损伤明显减轻,骨细胞凋亡率显著减少(P<0.05),GRP78、ATF4和CHOP等蛋白质表达、p-PERK/PERK值和p-eIF2α/PERK值也明显下降(P<0.05)。结论:佛手苷内酯可明显抑制TCP磨损颗粒所致的骨细胞损伤,其机制可能与减弱TCP磨损颗粒诱导的内质网应激反应及PERK通路的活化密切相关。

Objective:To study the effects of bergapten(BP)on damages of osteocytes MLO-Y4 induced by tricalcium phosphate(TCP)wear particles and its mechanism.Methods:MLO-Y4 cells were treated with TCP wear particles for 48 h to establish the model of osteocytes injuries in vitro.The MLO-Y4 cells were divided into the following five groups:control group,TCP wear particles treated(0.1 mg/ml)group,bergapten(1,5 and 20μmol/L)treated groups.MTT assay and Calcein-AM staining were used to determine the viability of MLO-Y4 cells;Hoechst 33342 staining and the flow cytometry were applied to detect the apoptosis of MLO-Y4;real-time PCR was performed to examine the mRNA levels of dentin matrix protein1(DMP-1),sclerostin(SOST)and fibroblast growth factor23(FGF23);Western blot was performed to examine protein expressions of glucose-regulated protein 78(GRP78),protein kinase R-like ER kinase(PERK)phospho-PERK(p-PERK),eukaryotic initiation factor 2α(eIF2α),phospho-eIF2α(p-eIF2α),activating transcription factor 4(AFT4),C/EBP homologous protein(CHOP)and caspase-3 in MLO-Y4 cells.Results:Compared with control group,the MLO-Y4 viability and DMP-1 mRNA level in TCP group were decreased significantly(P<0.05),while the percentage of apoptosis and mRNA levels of SOST and FGF23 were obviously increased(P<0.05),and protein expressions of GRP78,AFT4,CHOP,p-PERK/PERK and p-eIF2α/eIF2αwere up-regulated significantly in MLO-Y4 cells(P<0.05).Compared with TCP group,the damages of MLO-Y4 and cell apoptosis in bergapten treated groups were decrease obviously(P<0.05),the expressions of GRP78,AFT4,CHOP,p-PERK/PERK and p-eIF2α/eIF2αwere down-regulated remarkably(P<0.05).Conclusion:Bergapten can inhibit osteocytes damages induced by TCP wear particles,which may be related to reducing ER stress and PERK pathway activation.