摘要
目的 观察槲皮素(quercetin, QR)对核因子κB受体活化因子配体(RANKL)诱导RAW264.7细胞产生的破骨细胞NLRP3通路和自噬的影响。方法 采用RANKL诱导RAW264.7细胞分化为破骨细胞,通过CCK8实验筛选出安全范围内浓度的QR;TRAP染色法鉴定并计数破骨细胞;ELISA法检测细胞上清液中炎症因子IL-18、IL-1β的分泌量,扫描电子显微镜(SEM)下观察(0.1、1、5μmol/L)浓度QR作用于破骨细胞产生的骨吸收陷窝;透射电子显微镜(TEM)观察破骨细胞内自噬小体;Western blot检测自噬和NLRP3通路相关蛋白表达。结果 超过10μmol/L浓度的QR会显著抑制RAW264.7细胞活性。QR能明显减少RANKL诱导的TRAP阳性细胞的数量,减少功能性破骨细胞产生的骨吸收陷窝面积占比。5μmol/L QR能明显减少破骨细胞内自噬小体的数量,下调Beclin1,增强p62的蛋白表达,降低LC3II/I的比值。5μmol/L QR能下调破骨细胞上清液中炎症因子IL-18、IL-1β的分泌量,并抑制NLRP3、caspase-1蛋白的表达和及下游效应因子IL-18、IL-1β蛋白的表达。结论 QR可能通过抑制NLRP3信号通路及抑制破骨细胞自噬,影响破骨细胞生成及行使功能。
Objective To observe the effect of quercetin(QR)on the NLRP3 pathway and autophagy of osteoclasts generated from RAW264.7 cells induced by nuclear factorκB receptor activating factor ligand(RANKL).Methods RANKL was utilized to induce RAW264.7 cells to differentiate into osteoclasts.CCK8 assay was conducted to screen for QR at concentrations within the safe range.TRAP staining was used to identify and count osteoclasts.The secretion of inflammatory factors IL-18 and IL-1βin cell supernatants was detected with ELISA Bone resorption pits produced by the action of QR on osteoclasts at concentrations of 0.1,1,and 5μmol/L were observed under scanning electron microscopy(SEM).The autophagic vesicles in osteoclasts were observed with transmission electron microscopy(TEM).The expression of autophagy and NLRP3 pathway-related proteins were detected using Western blotting.Results Concentrations of QR above 10μmol/L significantly inhibited RAW264.7 cell activity.QR significantly reduced the number of RANKL-induced TRAP-positive cells and decreased the percentage of bone resorption pit area generated by functional osteoclasts.QR at 5μmol/L significantly reduced the number of autophagosome in osteoclasts,down-regulated Beclin1,enhanced protein expression of p62,and decreased the LC3II/I ratio.QR at 5μmol/L down-regulated the secretion of inflammatory factors IL-18 and IL-1βin the supernatant of osteoclasts,and inhibited the expression of NLRP3 and caspase-1 proteins and the expression of downstream effector IL-18 and IL-1βproteins.Conclusion QR may affect osteoclastogenesis and function by inhibiting the NLRP3 signaling pathway and by suppressing osteoclast autophagy.
作者
熊玥
黄丞蔚
程余婷
刘官娟
宋娜
石晓玉
廖健
XIONG Yue;HUANG Chengwei;CHENG Yuting;LIU Guanjuan;SONG Na;SHI Xiaoyu;LIAO Jian(Stomatological Hospital of Guizhou Medical University,Guiyang 550003,China;Oshika Dental,Shantou 515000,China;Prince of Songkhla University,Hat Yai 90110,Thailand)
出处
《中国骨质疏松杂志》
北大核心
2025年第5期650-656,共7页
Chinese Journal of Osteoporosis
