补肾活血方含药血清对退变型人原代软骨细胞microRNA表达谱的影响
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摘要
目的:探究补肾活血方含药血清对白细胞介素-1β(interleukin-1β,IL-1β)刺激的人原代软骨细胞microRNA芯片表达的影响。方法:采用免疫组化和甲苯胺蓝染色鉴定了原代培养的人软骨细胞,然后采用IL-1β(10 ng/m L)刺激人原代软骨细胞,并且加入补肾活血方含药血清处理24 h。抽提细胞RNA采取反转录荧光标记microRNA,标记完成之后取样品与microRNA表达谱芯片进行杂交,并用激光扫描仪检测其杂交信号,再用相应软件分析所得数据。结果:与对照组比较,补肾活血方含药血清导致9个已知的microRNA表达呈明显上升(诱导倍数> 2)以及4个已知的microRNA表达明显下降(抑制倍数> 2)。RT-qPCR结果表明补肾活血方含药血清对miR-140-5p的表达具有明显诱导作用。结论:补肾活血方可能通过调节miR-140-5p靶向ADAMTS-5和MMP-13等基因在维持软骨细胞外基质稳态中发挥作用。
Objective: To investigate the effect of Bushen Huoxue Recipe on gene expression profile of interleukin-1β( IL-1β)-stimulated human chondrocytes. Methods: For in vitro study,cultured human chondrocytes were treated with IL-1β( 10 ng·m L~(-1)) in the presence or absence of Bushen Huoxue Recipe-contained serum for 24 h. Cell RNA was extracted and reverse-transcripted into fluorescently-labeled microRNA which was hybridized with microRNA expression profile chip. Furthermore,laser scanner was used to detect the hybridization signal. Finally,the data was analyzed by related software. Results: Compared with normal control group,Bushen Huoxue Recipe obviously increased expression of 9 known microRNAs( fold > 2),and markedly reduced expression of 4 known microRNAs( fold > 2). In addition,RT-q PCR detection demonstrated that Bushen Huoxue Recipe significantly elevated miR-140-5p expression in IL-1β-stimulated human chondrocytes( P < 0. 05). Conclusion: These results demonstrated that the Bushen Huoxue Recipe could maintain chondrocyte extracellular matrix homeostasis by regulation of miR-140-5p and its targets of ADAMTS-5 and MMP-13 genes.
Objective: To investigate the effect of Bushen Huoxue Recipe on gene expression profile of interleukin-1β( IL-1β)-stimulated human chondrocytes. Methods: For in vitro study,cultured human chondrocytes were treated with IL-1β( 10 ng·m L~(-1)) in the presence or absence of Bushen Huoxue Recipe-contained serum for 24 h. Cell RNA was extracted and reverse-transcripted into fluorescently-labeled microRNA which was hybridized with microRNA expression profile chip. Furthermore,laser scanner was used to detect the hybridization signal. Finally,the data was analyzed by related software. Results: Compared with normal control group,Bushen Huoxue Recipe obviously increased expression of 9 known microRNAs( fold > 2),and markedly reduced expression of 4 known microRNAs( fold > 2). In addition,RT-q PCR detection demonstrated that Bushen Huoxue Recipe significantly elevated miR-140-5p expression in IL-1β-stimulated human chondrocytes( P < 0. 05). Conclusion: These results demonstrated that the Bushen Huoxue Recipe could maintain chondrocyte extracellular matrix homeostasis by regulation of miR-140-5p and its targets of ADAMTS-5 and MMP-13 genes.
引文
[1]Johnson VL,Hunter DJ.The epidemiology of osteoarthritis[J].Best Pract Res Clin Rheumatol,2014,28(1):5-15.
[2]Hopham LT,Lai TQ,Mai LD,et al.Prevalence of Radiographic Osteoarthritis of the Knee and Its Relationship to Self-Reported Pain[J].Plos One,2014,9(4):e94563.
[3]杨锦华,曹惠英,冯仲锴,等.原发性膝关节骨关节炎中医证候流行病学调查[J].中医正骨,2005,17(7):19-22.
[4]徐传毅,樊粤光,宁显明.肾虚血瘀与膝骨性关节炎关系初探[J].新中医,2002,34(3):7-9.
[5]李钊.冯新送教授从肾虚血瘀治疗骨性关节炎经验介绍[J].新中医,2011(11):152-153.
[6]钟树玉,林石明.肾虚血瘀型膝骨性关节炎的中医药治疗概况[J].北方药学,2015,12(1):98-100.
[7]曾意荣,樊粤光,刘少军,等.补肾活血中药治疗肾虚血瘀型膝骨性关节炎的临床研究[J].广州中医药大学学报,2007,24(4):276-278.
[8]王羽丰,许学猛,邓晋丰.补肾活血胶囊影响兔膝关节退变性疾病组织形态学的实验研究[J].中国中医骨伤科杂志,1999,7(4):9-12.
[9]许学猛,王羽丰,邓晋丰,等.补肾活血胶囊影响兔膝关节退变性疾病骨内压变化的实验研究[J].中国中医骨伤科杂志,2001,9(4):24-27.
[10]赵传喜,吴淮,曹金梅,等.补肾活血中药干预对去卵巢膝骨关节炎大鼠E2及CTX-Ⅱ的影响[J].风湿病与关节炎,2012,1(3):43-45.
[11]赵传喜,刘欣,吴淮,等.关节镜清理术配合补肾强筋胶囊治疗退行性膝关节炎远期疗效观察[J].现代中西医结合杂志,2012,21(33):3713-3714.
[12]胡零三,许学猛,李鹏,等.白酒送服补肾强筋胶囊治疗膝骨性关节疗效观察[J].按摩与康复医学,2013,4(10):37-38.
[13]姚楠,陈能,许学猛,等.补肾强筋胶囊含药血清对IL-1β刺激的人原代软骨细胞的影响[J].临床医学工程,2017,24(3):327-329.
[14]刘文刚,陈能,许学猛,等.补肾强筋胶囊治疗围绝经期及绝经后期膝骨性关节炎临床研究[J].河南中医,2017,37(5):911-913.
[15]Lagosquintana M,Rauhut R,Lendeckel W,et al.Identification of novel genes coding for small expressed RNAs[J].Science,2001,294(5543):853-858.
[16]Bartel DP.MicroRNAs:genomics,biogenesis,mechanism,and function[J].Cell,2004,116(2):281-297.
[17]Min Z,Zhang R,Yao J,et al.MicroRNAs associated with osteoarthritis differently expressed in bone matrix gelatin(BMG)rat model[J].Int J Clin Exp Med,2015,8(1):1009-1017.
[18]Iliopoulos D,Malizos KN,Oikonomou P,et al.Integrative microRNAand proteomic approaches identify novel osteoarthritis genes and their collaborative metabolic and inflammatory networks[J].Plos One,2008,3(11):e3740.
[19]Akhtar N,Rasheed Z,Ramamurthy S,et al.MicroRNA-27b regulates the expression of matrix metalloproteinase 13 in human osteoarthritis chondrocytes[J].Arthritis Rheum,2010,62(5):1361-1371.
[20]赵勇,王钢.MicroRNA基因调控在膝骨关节炎发生发展作用中的研究进展[J].中国骨伤,2016,29(4):383-385.
[21]Si HB,Zeng Y,Liu SY,et al.Intra-articular injection of microRNA-140(miRNA-140)alleviates osteoarthritis(OA)progression by modulating extracellular matrix(ECM)homeostasis in rats[J].Osteoarthritis Cartilage,2017,25(10):1698-1707.
[22]Wojdasiewicz P,Poniatowski LA,Szukiewicz D.The Role of inflammatory and anti-inflammatory cytokines in the pathogenesis of osteoarthritis[J].Mediators Inflamm,2014:561459.doi:10.1155/2014/561459.
[23]Le LT,Swingler TE,Clark IM.Review:the role of microRNAs in osteoarthritis and chondrogenesis[J].Arthritis Rheumatol,2013,65(8):1963-1974.
[24]Liang Z,Zhuang H,Wang G,et al.MiRNA-140 is a negative feedback regulator of MMP-13 in IL-1β-stimulated human articular chondrocyte C28/I2 cells[J].Inflamm Res,2012,61(5):503-509.
[25]Miyaki S,Sato T,Inoue A,et al.MicroRNA-140 plays dual roles in both cartilage development and homeostasis[J].Genes Dev,2010,24(11):1173-1185.
[26]Tardif G,Hum D,Pelletier JP,et al.Regulation of the IGFBP-5 and MMP-13 genes by the microRNAs miR-140 and miR-27a in human osteoarthritic chondrocytes[J].BMC Musculoskelet Disord,2009,10:148-158.
[27]Liu M,Zhong S,Kong R,et al.Paeonol alleviates interleukin-1β-induced inflammatory responses in chondrocytes during osteoarthritis[J].Biomed Pharmacother,2017,95:914-921.
[28]Yang X,Zhang Q,Gao Z,et al.Baicalin alleviates IL-1β-induced inflammatory injury via down-regulating miR-126 in chondrocytes[J].Biomed Pharmacother,2018,99:184-190.
[29]Yang Y,Wang Y,Wang Y,et al.Tormentic Acid Inhibits IL-1β-Induced Inflammatory Response in Human Osteoarthritic Chondrocytes[J].Inflammation,2016,39(3):1151-1159.
[30]Xu XX,Zhang XH,Diao Y,et al.Achyranthes bidentate saponins protect rat articular chondrocytes against interleukin-1β-induced inflammation and apoptosis in vitro[J].Kaohsauig J Med Sci,2017,33(2):62-68.
[2]Hopham LT,Lai TQ,Mai LD,et al.Prevalence of Radiographic Osteoarthritis of the Knee and Its Relationship to Self-Reported Pain[J].Plos One,2014,9(4):e94563.
[3]杨锦华,曹惠英,冯仲锴,等.原发性膝关节骨关节炎中医证候流行病学调查[J].中医正骨,2005,17(7):19-22.
[4]徐传毅,樊粤光,宁显明.肾虚血瘀与膝骨性关节炎关系初探[J].新中医,2002,34(3):7-9.
[5]李钊.冯新送教授从肾虚血瘀治疗骨性关节炎经验介绍[J].新中医,2011(11):152-153.
[6]钟树玉,林石明.肾虚血瘀型膝骨性关节炎的中医药治疗概况[J].北方药学,2015,12(1):98-100.
[7]曾意荣,樊粤光,刘少军,等.补肾活血中药治疗肾虚血瘀型膝骨性关节炎的临床研究[J].广州中医药大学学报,2007,24(4):276-278.
[8]王羽丰,许学猛,邓晋丰.补肾活血胶囊影响兔膝关节退变性疾病组织形态学的实验研究[J].中国中医骨伤科杂志,1999,7(4):9-12.
[9]许学猛,王羽丰,邓晋丰,等.补肾活血胶囊影响兔膝关节退变性疾病骨内压变化的实验研究[J].中国中医骨伤科杂志,2001,9(4):24-27.
[10]赵传喜,吴淮,曹金梅,等.补肾活血中药干预对去卵巢膝骨关节炎大鼠E2及CTX-Ⅱ的影响[J].风湿病与关节炎,2012,1(3):43-45.
[11]赵传喜,刘欣,吴淮,等.关节镜清理术配合补肾强筋胶囊治疗退行性膝关节炎远期疗效观察[J].现代中西医结合杂志,2012,21(33):3713-3714.
[12]胡零三,许学猛,李鹏,等.白酒送服补肾强筋胶囊治疗膝骨性关节疗效观察[J].按摩与康复医学,2013,4(10):37-38.
[13]姚楠,陈能,许学猛,等.补肾强筋胶囊含药血清对IL-1β刺激的人原代软骨细胞的影响[J].临床医学工程,2017,24(3):327-329.
[14]刘文刚,陈能,许学猛,等.补肾强筋胶囊治疗围绝经期及绝经后期膝骨性关节炎临床研究[J].河南中医,2017,37(5):911-913.
[15]Lagosquintana M,Rauhut R,Lendeckel W,et al.Identification of novel genes coding for small expressed RNAs[J].Science,2001,294(5543):853-858.
[16]Bartel DP.MicroRNAs:genomics,biogenesis,mechanism,and function[J].Cell,2004,116(2):281-297.
[17]Min Z,Zhang R,Yao J,et al.MicroRNAs associated with osteoarthritis differently expressed in bone matrix gelatin(BMG)rat model[J].Int J Clin Exp Med,2015,8(1):1009-1017.
[18]Iliopoulos D,Malizos KN,Oikonomou P,et al.Integrative microRNAand proteomic approaches identify novel osteoarthritis genes and their collaborative metabolic and inflammatory networks[J].Plos One,2008,3(11):e3740.
[19]Akhtar N,Rasheed Z,Ramamurthy S,et al.MicroRNA-27b regulates the expression of matrix metalloproteinase 13 in human osteoarthritis chondrocytes[J].Arthritis Rheum,2010,62(5):1361-1371.
[20]赵勇,王钢.MicroRNA基因调控在膝骨关节炎发生发展作用中的研究进展[J].中国骨伤,2016,29(4):383-385.
[21]Si HB,Zeng Y,Liu SY,et al.Intra-articular injection of microRNA-140(miRNA-140)alleviates osteoarthritis(OA)progression by modulating extracellular matrix(ECM)homeostasis in rats[J].Osteoarthritis Cartilage,2017,25(10):1698-1707.
[22]Wojdasiewicz P,Poniatowski LA,Szukiewicz D.The Role of inflammatory and anti-inflammatory cytokines in the pathogenesis of osteoarthritis[J].Mediators Inflamm,2014:561459.doi:10.1155/2014/561459.
[23]Le LT,Swingler TE,Clark IM.Review:the role of microRNAs in osteoarthritis and chondrogenesis[J].Arthritis Rheumatol,2013,65(8):1963-1974.
[24]Liang Z,Zhuang H,Wang G,et al.MiRNA-140 is a negative feedback regulator of MMP-13 in IL-1β-stimulated human articular chondrocyte C28/I2 cells[J].Inflamm Res,2012,61(5):503-509.
[25]Miyaki S,Sato T,Inoue A,et al.MicroRNA-140 plays dual roles in both cartilage development and homeostasis[J].Genes Dev,2010,24(11):1173-1185.
[26]Tardif G,Hum D,Pelletier JP,et al.Regulation of the IGFBP-5 and MMP-13 genes by the microRNAs miR-140 and miR-27a in human osteoarthritic chondrocytes[J].BMC Musculoskelet Disord,2009,10:148-158.
[27]Liu M,Zhong S,Kong R,et al.Paeonol alleviates interleukin-1β-induced inflammatory responses in chondrocytes during osteoarthritis[J].Biomed Pharmacother,2017,95:914-921.
[28]Yang X,Zhang Q,Gao Z,et al.Baicalin alleviates IL-1β-induced inflammatory injury via down-regulating miR-126 in chondrocytes[J].Biomed Pharmacother,2018,99:184-190.
[29]Yang Y,Wang Y,Wang Y,et al.Tormentic Acid Inhibits IL-1β-Induced Inflammatory Response in Human Osteoarthritic Chondrocytes[J].Inflammation,2016,39(3):1151-1159.
[30]Xu XX,Zhang XH,Diao Y,et al.Achyranthes bidentate saponins protect rat articular chondrocytes against interleukin-1β-induced inflammation and apoptosis in vitro[J].Kaohsauig J Med Sci,2017,33(2):62-68.