低氧状态下IL-6对骨细胞表达RANKL的影响

Effect of IL-6 on the expression of RANKL in osteocytes under hypoxia

目的探讨低氧状态下炎症因子白介素-6(IL-6)对骨细胞分泌核因子κB受体活化因子配体(RANKL)的影响及相关机制。方法建立大鼠下颌截骨术模型,应用IL-6和氯化钴(CoCl2)建立体外低氧炎症微环境培养MLO-Y4小鼠长骨骨细胞系,采用苏木素-伊红(HE)染色、抗酒石酸酸性磷酸酶(TRAP)染色和免疫荧光染色观察术后大鼠下颌骨破骨细胞活化情况和低氧诱导因子(HIF-1α)、RANKL的表达水平;利用实时荧光定量聚合酶链反应(RT qPCR)、细胞免疫荧光、蛋白质印迹(Western blot)和小干扰RNA(si RNA)转染实验检测低氧状态下IL-6对MLO-Y4小鼠长骨骨细胞系细胞分泌HIF-1α和RANKL的影响。结果术后2周及4周大鼠下颌骨破骨细胞活化水平和HIF-1α、RANKL表达水平均提高。低氧状态下,IL-6可使MLO-Y4细胞HIF-1α、RANKL的表达水平提高。低氧状态下IL-6转染组的HIF-1α和RANKL蛋白表达水平均低于转染对照组和对照组。结论低氧状态下IL-6可以通过上调MLO-Y4细胞的HIF-1α表达水平促进RANKL的分泌。

Objective To investigate the effect and related mechanisms of IL-6 on the expression of RANKL in osteocytes under hypoxia.Methods A rat mandibular osteotomy model was established,and the osteocyte-like cell line MLO-Y4 was cultured in vitro with hypoxic inflammation microenvironment.HE staining,TRAP staining and immunofluorescence staining were used to observe the activation of mandibular osteoclasts and the expression of HIF-1αand RANKL.RT qPCR,cellular immunofluorescence,Western blot and siRNA transfection experiment were used to detect the expression levels of HIF-1αand RANKL in MLO Y4 treated with IL-6 under hypoxia or normoxia.Results The mandibular osteoclast activation level and HIF-1αand RANKL expression levels in rats increased after 2 or 4 weeks.Under hypoxic conditions,IL-6 stimulated the expression of HIF-1αand RANKL in MLO-Y4 cells.The levels of HIF-1αand RANKL protein expression in the IL-6 transfection group were lower than those in the transfection control group and the control group under hypoxia.Conclusions Under hypoxia,IL-6 could facilitate the formation of RANKL through up-regulating the expression of HIF 1αprotein in MLO-Y4 cells.