仙茅苷调节lncRNA MEG3/miR-181a-5p通路对骨质疏松大鼠成骨细胞自噬的影响
Effect of Xianmao glycoside on the regulation of lncRNA MEG3/miR-181a-5p pathway on autophagy of osteoblasts in osteoporosis rats
  
DOI:10.3969/j.issn.1006-7108.2023.10.002
中文关键词:  长链非编码RNA MEG3  骨质疏松症  微小RNA-181a-5p  成骨细胞自噬
英文关键词:long non-coding RNA MEG3  osteoporosis  micro RNA-181a-5p  osteoblast autophagy
基金项目:河南省高等学校重点科研项目(23B360006);国家中医临床研究基地科研专项课题(2019JDZX055);河南省中医药科学研究专项课题(2016ZY3015)
作者单位
王勤俭 张睿昕* 张攀 李泊泊 河南省中医院 河南中医药大学第二附属医院河南 郑州 450000 
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中文摘要:
      目的 探讨仙茅苷(Curculigoside,CUR)对骨质疏松症(osteoporosis,OP)大鼠成骨细胞自噬的影响以及对lncRNA MEG3/miR-181a-5p信号轴的调节作用。方法 体外培养骨髓间充质干细胞(BM-MSCs)诱导成骨细胞分化,CCK-8法筛选CUR浓度;数据库预测lncRNA MEG3与miR-181a-5p结合位点;利用转染技术将BM-MSCs细胞分为NC组、CUR组、CUR+3-MA组、CUR+pc-NC组和CUR+pc-LncRNA MEG3组,qRT-PCR检测lncRNA MEG3、miR-181a-5p的表达水平,碱性磷酸酶(ALP)染色法检测细胞ALP活性,MDC法检测细胞自噬体数量,免疫荧光检测细胞微管相关蛋白1-轻链3(LC3)、自噬效应蛋白(Beclin1)的表达。肌肉注射地塞米松磷酸钠建立OP大鼠,大鼠分为对照组(CT组)、模型组(OP组)、OP+CUR组(灌胃15 mg/kg CUR),CT检测大鼠胫骨骨形态学,Western blot和qRT-PCR分别检测LC3Ⅱ/Ⅰ、Beclin1与lncRNA MEG3、miR-181a-5的表达。结果 25 μg/mL以上浓度的CUR显著提高BM-MSCs细胞增殖活力(P<0.05);lncRNA MEG3与miR-181a-5p具有靶向结合位点;与NC组相比,CUR组细胞ALP活性、自噬体数量以及LC3、Beclin1表达增加(P<0.05);与CUR组相比,CUR+3-MA组细胞ALP活性、自噬体数量以及LC3、Beclin1表达减少(P<0.05);与CUR+pc-NC组相比,CUR+pc-LncRNA MEG3组细胞ALP活性、自噬体数量以及LC3、Beclin1表达减少(P<0.05)。OP组大鼠骨形态学评价以及LC3Ⅱ/Ⅰ、Beclin1、miR-181a-5p表达较CT组下降,lncRNA MEG3表达增加(P < 0.05);OP+ CUR组大鼠骨形态学评价以及LC3Ⅱ/Ⅰ、Beclin1、miR-181a-5p表达较OP组增加,lncRNA MEG3表达降低(P<0.05)。结论 CUR可能通过调节LncRNA MEG3/miR-181a-5p信号轴促进OP大鼠成骨细胞自噬活性。
英文摘要:
      Objective To investigate the impact of curculigoside(CUR)on autophagy of osteoblasts in rats with osteoporosis(OP)and its regulatory effect on the lncRNA MEG3/miR-181a-5p signal axis. Methods Bone marrow mesenchymal stem cells(BM-MSCs)were cultured in vitro to induce osteoblast differentiation, and the concentration of CUR was screened by CCK-8 method; the database was applied to predict the binding sites of lncRNA MEG3 to miR-181a-5p; BM-MSCs were grouped into NC group, CUR group, CUR+3-MA group, CUR+pc-NC group, and CUR+pc-LncRNA MEG3 group using transfection technology, the expression levels of lncRNA MEG3 and miR-181a-5p were detected by qRT-PCR, alkaline phosphatase (ALP) staining was applied to detect the ALP activity of the cells, the number of autophages was detected by MDC method, immunofluorescence assay was applied to detect the expression of microtubule associated protein 1-light chain 3(LC3)and autophagy effector protein(Beclin1). OP rats were established by intramuscular injection of dexamethasone sodium phosphate, and the rats were grouped into control group(CT group), model group(OP group), and OP+CUR group(15 mg/kg CUR by gavage), CT was applied to detect the morphology of the tibial bone in rats, Western blot and qRT-PCR were applied to detect the expression of LC3 Ⅱ/Ⅰ, Beclin1, and lncRNA MEG3, miR-181a-5, respectively. Results The concentration of CUR above 25 μg/mL obviously increased the proliferation activity of BM-MSCs cells(P<0.05); LncRNA MEG3 and miR-181a-5p had targeted binding sites; compared with NC group, the activity of ALP, the number of autophages, and the expression of LC3 and Beclin1 in CUR group increased(P<0.05); compared with the CUR group, the activity of ALP, the number of autophages, and the expression of LC3 and Beclin1 in the CUR+3-MA group decreased(P<0.05); compared with the CUR+pc-NC group, the activity of ALP, the number of autophages, and the expression of LC3 and Beclin1 in the CUR+pc-LncRNA MEG3 group decreased(P<0.05). The bone morphological evaluation and the expression of LC3 II/I, Beclin1, miR-181a-5p in OP group were lower than those in CT group, while the expression of lncRNA MEG3 was increased(P<0.05); the bone morphological evaluation and the expression of LC3 II/I, Beclin1, miR-181a-5p in the OP+CUR group were higher than those in the OP group, while the expression of lncRNA MEG3 was lower(P<0.05).Conclusion CUR may promote autophagic activity of osteoblasts in OP rats by regulating the LncRNA MEG3/miR-181a-5p signal axis.
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