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  

Salvianolic acid A contributes to cartilage endplate cell restoration by regulating miR-940 and miR-576-5p

作　　者：展嘉文[1,2] 王尚全 陈明[1,2] 孙凯 于杰[2,3] 李玲慧 魏戌 孙武 陈忻[3] 蔡楚豪[1] 张伟业 韩涛[1,2] 尹煜辉[1] 唐彬[3] 朱立国 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(Sport Medicine center,Wangjing Hospital,China Academy of Chinese Meidical Science,Beijing 100102,China;Beijing Key Laboratory of Manipulative Technique Beijing100102,China;The Second Department of Spine,Wangjing Hospital,China Academy of Chinese Medical Science,Beijing 100102,China;Department of Academic Development,Wangjing Hospital,China Academy of Chinese Medical Science,Beijing100102,China)

机构地区：[1]中国中医科学院望京医院运动医学中心,北京100102 [2]中医正骨技术北京市重点实验室,北京100102 [3]中国中医科学院望京医院脊柱二科,北京100102 [4]中国中医科学院附属望京医院学术发展处,北京100102

出　　处：《中国骨伤》2023年第10期982-989,共8页China Journal of Orthopaedics and Traumatology

基　　金：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)。

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、10μM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 typeⅡa1,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:10μM 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。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 10μM)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 10μM SAA treatment for 48 h significant-y 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-1βon 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.

关 键 词：椎间盘 丹酚酸A 终板软骨细胞 细胞外基质 MICRO-RNA 

分 类 号：R336[医药卫生—人体生理学]