成骨细胞来源的外泌体介导微小RNA-494影响骨质疏松症大鼠骨代谢与骨重建平衡
Osteoblast-derived exosome mediating the effect of microRNA-494 on bone metabolism and bone remodeling balance in osteoporotic rats
林维 1李超艺 1唐捷 1张丕军1
作者信息
- 1. 海南医学院第二附属医院关节外科,海口 570311
- 折叠
摘要
目的 探讨成骨细胞来源的外泌体(Exo)中微小RNA(miR)-494 对骨质疏松症(OP)大鼠骨代谢与骨重建平衡的影响及其机制.方法 从MC3T3-E1 成骨细胞系中分离鉴定Exo,并将miR-494 电转至Exo,Real-time PCR测定miR-494 表达水平.40 只SD大鼠随机分为对照组、模型组、miR-494 模拟剂(miR-494)组和miR-494 抑制剂组,每组 10 只.除对照组外,其余 3 组大鼠切除卵巢构建OP模型.模型制作成功后,miR-494 组与miR-494抑制剂组分别接受含相应miRNA的Exo尾静脉注射,剂量为 3×109 个微粒.4 周后,Micro-CT检测各组大鼠的骨参数,ELISA测定血清骨标志物,HE染色观察骨组织病理变化,抗酒石酸酸性磷酸酶(TRACP)染色检测破骨细胞数量,Western blotting测定骨重建相关蛋白及Toll样受体4(TLR4)相关通路蛋白表达水平.结果 成功从MC3T3-E1 细胞中分离得到典型杯状或圆形,直径约 100 nm的Exo,其含有CD63、CD9、肿瘤易感性基因 101(TSG101)、热休克蛋白 70(HSP70)蛋白,且能被MC3T3-E1 细胞摄取.经电转处理后,miR-494 模拟剂和miR-494 抑制剂的Exo中miR-494 的相对表达量明显升高和降低(P＜0.05).与模型组相比,miR-494 组大鼠骨参数降低,血清骨标志物骨钙蛋白(BGP)、TRACP、Ⅰ型胶原C端肽(CTX-Ⅰ)升高,骨保护蛋白(OPG)、Ⅰ型前胶原N端肽(PⅠNP)降低,骨小梁结构紊乱,破骨细胞增加,骨组织内骨形态发生蛋白 2(BMP-2)、Runt相关转录因子 2(RUNX2)下调,核因子κB受体激活剂配体(RANKL)上调,TLR4、核因子κB(NF-κB)p65 及髓样分化主要响应蛋白 88(MyD88)上调(P＜0.05).而miR-494 抑制剂组情况则相反,骨参数和OPG、PⅠNP升高,BGP、TRACP、CTX-Ⅰ降低,骨结构恢复正常,破骨细胞减少,骨组织内BMP-2、RUNX2 上调,RANKL下调,TLR4、NF-κB p65 及MyD88 下调(均P＜0.05).结论 Exo传递miR-494 可加重OP大鼠骨代谢异常,并抑制骨重建平衡,推测其作用机制可能与调控TLR4 通路有关.
Abstract
Objective To investigate the effect of osteoblast-derived exosome(Exo)mediating microRNA(miR)-494 on bone metabolism and bone remodeling balance in osteoporosis(OP)rats and its mechanism.Methods Exosomes was isolated and identified from MC3T3-E1 osteoblast cell line and transferred to Exo by electrical transfer.Forty SD rats were randomly divided into control,model,miR-494 mimic(miR-494),and miR-494 inhibitor group,10 rats in each group.The ovaries were removed to construct the OP model except the control group.After modeling,the miR-494 group and miR-494 inhibitor group received tail vein injections of exosomes containing the corresponding miRNA,at a dose of 3×109 particles.Four weeks later,bone parameters were detected in each group of rats by Micro-CT,serum bone markers were measured by ELISA,pathological changes in bone tissue were observed by HE staining,osteoclast numbers were detected by tartrate-resistant acid phosphatase(TRACP)staining,and the expression levels of bone remodeling-related proteins and toll-like receptor 4(TLR4)pathway-related proteins were determined by Western blotting.Results Typical cup-shaped or round exosomes were successfully isolated with a diameter of about 100 nm from MC3T3-E1 cells,which contained CD63,CD9,tumor susceptibility gene 101(TSG101),heat shock protein 70(HSP70)proteins and can be taken up by MC3T3-E1 cells.Compared with the model group,the bone parameters of the rats in the miR-494 mimic group decreased,serum bone markers bone Gla protein(BGP),TRACP,C-terminal telopeptide of type Ⅰ collagen(CTX-Ⅰ)increased,osteoprotegerin(OPG),procollagen type Ⅰ N-terminal propeptide(PⅠNP)decreased,bone trabeculae structure was disordered,osteoclasts increased,bone morphogenetic protein 2(BMP-2),Runt related transcription factor 2(RUNX2)in bone tissue downregulated,receptor activator of nuclear factor kappa-B ligand(RANKL)upregulated,TLR4,nuclear factor kappa-B p65(NF-κB p65)and myeloid differentiation primary response 88(MyD88)upregulated(all P＜0.05).In contrast,the situation of the miR-494 inhibitor group was opposite,bone parameters and OPG,PⅠNP increased,BGP,TRACP,CTX-Ⅰdecreased,bone structure returned to normal,osteoclasts decreased,BMP-2,RUNX2 in bone tissue upregulated,RANKL downregulated,TLR4,NF-κB p65 and MyD88 downregulated(all P＜0.05).Conclusion The transfer of miRNA-494 by Exo aggravates abnormal bone metabolism in OP rats and inhibits bone remodeling balance,suggesting that the mechanism of action may be related to the regulation of TLR4 pathway.
关键词
骨质疏松症/微小RNA-494/外泌体/骨代谢/骨重建平衡/实时定量聚合酶链反应/免疫印迹法/大鼠Key words
Osteoporosis/MicroRNA-494/Exosome/Bone metabolism/Bone remodeling balance/Real-time PCR/Western blotting/Rat引用本文复制引用
基金项目
海南省卫生健康行业科研项目(琼卫科教[2022]3号22A200067)
出版年
2024
NETL
NSTL
万方数据