丹酚酸A调控miR-940与miR-576-5p促进退变终板软骨细胞修复的作用研究
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作者Author单位AddressE-Mail
展嘉文 ZHAN Jia-wen 中国中医科学院望京医院运动医学中心, 北京 100102
中医正骨技术北京市重点实验室, 北京 100102
Sport Medicine center, Wangjing Hospital, China Academy of Chinese Meidical Science, Beijing 100102, China
Beijing Key Laboratory of Manipulative Technique Beijing 100102, Chiese
 
王尚全 WANG Shang-quan 中国中医科学院望京医院运动医学中心, 北京 100102
中医正骨技术北京市重点实验室, 北京 100102
Sport Medicine center, Wangjing Hospital, China Academy of Chinese Meidical Science, Beijing 100102, China
Beijing Key Laboratory of Manipulative Technique Beijing 100102, Chiese
 
陈明 CHEN Ming 中国中医科学院望京医院运动医学中心, 北京 100102
中医正骨技术北京市重点实验室, 北京 100102
Sport Medicine center, Wangjing Hospital, China Academy of Chinese Meidical Science, Beijing 100102, China
Beijing Key Laboratory of Manipulative Technique Beijing 100102, Chiese
 
孙凯 SUN Kai 中医正骨技术北京市重点实验室, 北京 100102
中国中医科学院望京医院脊柱二科, 北京 100102
Beijing Key Laboratory of Manipulative Technique Beijing 100102, Chiese
The Second Department of Spine, Wangjing Hospital, China Academy of Chinese Medical Science, Beijing 100102, China
 
于杰 YU Jie 中医正骨技术北京市重点实验室, 北京 100102
中国中医科学院望京医院脊柱二科, 北京 100102
Beijing Key Laboratory of Manipulative Technique Beijing 100102, Chiese
The Second Department of Spine, Wangjing Hospital, China Academy of Chinese Medical Science, Beijing 100102, China
 
李玲慧 LI Ling-hui 中国中医科学院望京医院运动医学中心, 北京 100102
中医正骨技术北京市重点实验室, 北京 100102
Sport Medicine center, Wangjing Hospital, China Academy of Chinese Meidical Science, Beijing 100102, China
Beijing Key Laboratory of Manipulative Technique Beijing 100102, Chiese
 
魏戌 中国中医科学院附属望京医院学术发展处, 北京 100102 Department of Academic Development, Wangjing Hospital, China Academy of Chinese Medical Science, Beijing 100102, China  
孙武 SUN Wu 中国中医科学院望京医院脊柱二科, 北京 100102 The Second Department of Spine, Wangjing Hospital, China Academy of Chinese Medical Science, Beijing 100102, China  
陈忻 CHEN Xin 中国中医科学院望京医院脊柱二科, 北京 100102 The Second Department of Spine, Wangjing Hospital, China Academy of Chinese Medical Science, Beijing 100102, China  
蔡楚豪 CAI Chu-hao 中国中医科学院望京医院运动医学中心, 北京 100102 Sport Medicine center, Wangjing Hospital, China Academy of Chinese Meidical Science, Beijing 100102, China  
张伟业 ZHANG Wei-ye 中国中医科学院望京医院运动医学中心, 北京 100102 Sport Medicine center, Wangjing Hospital, China Academy of Chinese Meidical Science, Beijing 100102, China  
韩涛 HAN Tao 中国中医科学院望京医院运动医学中心, 北京 100102
中医正骨技术北京市重点实验室, 北京 100102
Sport Medicine center, Wangjing Hospital, China Academy of Chinese Meidical Science, Beijing 100102, China
Beijing Key Laboratory of Manipulative Technique Beijing 100102, Chiese
 
尹煜辉 YIN Yu-hui 中国中医科学院望京医院运动医学中心, 北京 100102 Sport Medicine center, Wangjing Hospital, China Academy of Chinese Meidical Science, Beijing 100102, China  
唐彬 TANG Bin 中国中医科学院望京医院脊柱二科, 北京 100102 The Second Department of Spine, Wangjing Hospital, China Academy of Chinese Medical Science, Beijing 100102, China  
朱立国 ZHU Li-guo 中医正骨技术北京市重点实验室, 北京 100102
中国中医科学院望京医院脊柱二科, 北京 100102
Beijing Key Laboratory of Manipulative Technique Beijing 100102, Chiese
The Second Department of Spine, Wangjing Hospital, China Academy of Chinese Medical Science, Beijing 100102, China
 
期刊信息:《中国骨伤》2023年,第36卷,第10期,第982-989页
DOI:10.12200/j.issn.1003-0034.2023.10.014
基金项目:国家自然科学基金项目(编号:81930118,81804120,81774330);国家中医药管理局中医药创新团队及人才支持计划项(编号:ZYYCXTD-C-202003);中国中医科学院基本科研业务费优秀青年科技人才(创新类)培养专项(编号:ZZ13-YQ-038)
中文摘要:

目的:观察丹酚酸A(salvianolic acid A,SAA)对退变终板软骨细胞(cartilaginous endplates cells,CEPCs)的干预作用,及其调控的潜在非编码RNA(micro-RNA,miRNA)作用靶点。

方法:从腰椎间盘手术标本中分离CEPCs,用不同浓度SAA(2、5、10 μM处理24、48、72 h,利用CCK-8检测细胞活性确定SAA的最适剂量和干预时间。通过阿利新蓝染色和对血小板反应蛋白解整合素金属肽酶-5(A disintegrin and metalloproteinase with thrombospondin-5,ADAMTS-5)、基质金属肽酶3(matrix metallopepridase 3,MMP-3)、Ⅱ型胶原a1(clollagen typeⅡ a1,Col2a1)的蛋白表达检测,分析SAA对IL-1β诱导的退变CEPCs的干预作用。进一步结合生信分析,以及实时荧光定量PCR(quantitative real-time,qRT-PCR)与Western blot检测,并应用miRNA模拟物(miR-mimics) 与抑制剂(miR-inhibitor),验证SAA对潜在靶miRNAs的调控作用。

结果:10 μM SAA处理48 h显著提高了CEPCs活性,增加了白细胞介素(interlenkin-1β,IL-1β)所抑制的糖胺聚糖积累与Col2a1表达,并降低了细胞基质中ADAMTS-5与MMP3表达(P<0.05)。通过生物信息分析筛选与差异基因表达检测,确定了SAA的潜在靶miRNAs为miR-940和miR-576-5p。进一步在SAA处理组中加入miR-940-mimic或miR-576-5p-mimic,与SAA处理组相比过表达miR-940或miR-576-5p后CEPCs基质中ADAMTS-5与MMP3表达显著升高,Col2a1表达显著降低。

结论:SAA能够提高CEPCs活性,改善退变CEPCs的基质成分表达,而SAA的调控作用与抑制miR-940和miR-576-5p表达有关,两者可能是SAA调节CEPCs退变的作用靶点。
【关键词】椎间盘  丹酚酸A  终板软骨细胞  细胞外基质  micro-RNA
 
Salvianolic acid A contributes to cartilage endplate cell restoration by regulating miR-940 and miR-576-5p
ABSTRACT  

Objective To investigate whether Salvianolic acid A (SAA) can restore cartilage endplate cell degeneration of intervertebral discs and to identify the mechanism via regulation of micro-RNA.

Methods Cartilage endplate cells were isolated from lumbar intervertebral disc surgical samples and were treated with serum containing a series of concentrations of SAA (2,5,and 10 μM) for 24,48,and 72 h to identify a proper dose and treatment time of SAA. The effect SAA on interlenkin-1β (IL-1β)-induced extracellular matrix degradation of cartilage endplate cells were analyzed by Alcian blue staining and assessment of the expression levels of ADAMTS-5,MMP3 and Col2a1. Further,the potential target miRNAs were preliminarily screened by micro-RNA sequencing combining qRT-PCR and Western blot,and then,the miRNAs mimics and inhibitors were used to verify the regulatory effect of SAA on potential target miRNAs.

Results The 10 μM SAA treatment for 48 h significantly enhanced the viability of cartilage endplate cells,and increased Col2a1 expression and glycosaminoglycan accumulation that were repressed by IL-1β,and reduced the effect of IL-1β on ADAMTS-5,and MMP3. Screening analysis based on micro-RNA sequencing and Venny analysis identified the downstream micro-RNAs,including miR-940 and miR-576-5p. Then,the miR-940-mimic or miR-576-5p-mimic were transfected into CEPCs. Compared with the SAA group,the expression of ADAMTS-5 and MMP3 increased significantly and the expression of COL2A1 obviously decreased after overexpression of miR-940 or miR-576-5p in CEPCs.

Conclusion Salvianolic acid A attenuated the IL-1β-induced extracellular matrix degradation of cartilage endplate cells by targeting regulate the miR-940 and the miR-576-5p.
KEY WORDS  Intervertebral disc  Salvianolic acid A  Cartilaginous endplates cells  Extracellular matrix  Micro-RNA
 
引用本文,请按以下格式著录参考文献:
中文格式:展嘉文,王尚全,陈明,孙凯,于杰,李玲慧,魏戌,孙武,陈忻,蔡楚豪,张伟业,韩涛,尹煜辉,唐彬,朱立国.丹酚酸A调控miR-940与miR-576-5p促进退变终板软骨细胞修复的作用研究[J].中国骨伤,2023,36(10):982~989
英文格式:ZHAN Jia-wen,WANG Shang-quan,CHEN Ming,SUN Kai,YU Jie,LI Ling-hui,SUN Wu,CHEN Xin,CAI Chu-hao,ZHANG Wei-ye,HAN Tao,YIN Yu-hui,TANG Bin,ZHU Li-guo.Salvianolic acid A contributes to cartilage endplate cell restoration by regulating miR-940 and miR-576-5p[J].zhongguo gu shang / China J Orthop Trauma ,2023,36(10):982~989
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