力學改變激活軟骨細胞介導創傷性骨關節炎的研究進展

張興宇(綜述)　向　萌(審校)

(1復旦大學基礎醫學院生理與病理生理學系　上海　200032; 2復旦大學上海醫學院2013級臨床醫學八年制　上海　200032)

作為一種慢性、進行性的退行性關節病,創傷性骨關節炎(post-traumatic osteoarthritis,PTOA)主要繼發于關節創傷、運動外傷[1-3]。瑞典的一項回顧性研究發現手術會提高PTOA發病率[4]。近年來研究顯示[5-6],關節外傷遷延為骨關節炎的風險約為20%,有時甚至超過50%。對于嚴重關節外傷,多采用關節鏡下清理術、重建手術或者關節置換術,但手術并不能降低罹患PTOA的風險[7]。

軟骨細胞合成膠原纖維和蛋白多糖等基質成分,是形成和維持軟骨的重要細胞。關節軟骨在層次上分為淺表區、過渡區、深區及鈣化軟骨區(圖1)。Grogan等[8]發現,軟骨細胞生物學功能多樣,參與蛋白翻譯、受體對話、信號轉導等。高強度外力瞬間作用于關節可造成關節軟骨嚴重損傷[9],激活軟骨細胞引起局部炎癥反應[10],產生多種病理變化[11-13](圖2),患者關節反復疼痛,可伴有關節腫脹、僵硬、負重能力下降及活動障礙等表現,嚴重者可致畸形甚至殘疾。PTOA發病機制主要涉及:(1) 創傷后關節部分區域的生物力學改變[14];(2) 軟骨細胞介導的炎性介質的釋放[10,15]。因此,軟骨細胞的活化和損傷是骨關節炎的重要發病機制之一,明確PTOA的發病機制有助于開展PTOA的預防和治療。

(1) Superficial zone;(2) Transitional zone;(3) Deep zone;(4) Tidemark;(5) Subchondral bone.

圖1關節軟骨的基本結構
Fig1Basicstructureofarticularcartilage

力傳導通路激活軟骨細胞的研究進展研究表明,軟骨細胞的初級纖毛可視為機械性刺激感受器,接受力學刺激信號[16-17]。若初級纖毛缺失,關節軟骨則相應增厚且剛度降低,深區和軟骨下骨的壓縮模量顯著減小[18]。創傷后關節物理形態改變造成軟骨細胞表面的初級纖毛彎曲或拉伸[19],激活了軟骨細胞膜表面的受體[20],例如整合素、G蛋白、離子通道等通過信號轉導,參與調控PTOA的病程進展[21]。軟骨細胞介導骨關節炎信號通路的最新研究進展主要如下(圖3)。

(1) Swelling joint;(2) Damaged surface;(3) Infiltration;(4) Osteophyte;(5) Thickening and stiffening of subchondral bone.

圖2創傷后關節軟骨的病理變化
Fig2Pathologicalchangesofarticularcartilageafterinjury

Ca2+信號相關通路Ca2+信號參與多種信號轉導通路,例如G蛋白耦聯受體、電壓門控Ca2+通道等。軟骨細胞力傳導通路也聚焦于Ca2+信號通路[22]。軟骨細胞初級纖毛接受力傳導信號后,鞭毛內運輸蛋白由質膜向胞內傳遞信號[23],而蛋白裝卸與運輸離不開Ca2+依賴的激酶活化[24]。整合素(integrin)、嘌呤受體2 (purine receptor 2,P2)、瞬時受體電位蛋白V4型(transient receptor potential vanilloid 4,TRPV4)和Piezo受體參與活化Ca2+信號。

整合素是連接基質和細胞蛋白骨架的跨膜受體,創傷后膠原纖維、黏附分子、纖連蛋白等胞外片段與整合素結合,誘發整合素構象改變,進而活化胞內的黏附斑激酶FAK,激活下游Ca2+依賴的蛋白激酶,從而介導胞外信號向胞內轉導[25]。Garciadiego-cazares等[26]在大鼠PTOA模型中發現,PTOA早期整合素α1、α5亞單位有較高表達,推測可能與軟骨細胞增殖和軟骨基質的維持有關,并可對抗PTOA早期病變[27];而在PTOA中后期αV亞單位有較高表達,并且此時軟骨組織中α5亞單位表達量顯著降低,這提示αV亞單位可能介導PTOA發病過程中軟骨細胞的肥大和功能亢進。但是,也有研究指出α5亞單位促進了骨關節炎的典型病變[28-29]。以上研究均說明PTOA中存在異常力傳導信號,通過整合素通路調控軟骨細胞功能[30]。

P2可介導應變誘發的新式信號轉導通路[31]。研究證實,軟骨細胞P2X受體和P2Y受體與經Connexin43通道外流的ATP結合后,介導胞內Ca2+信號調節激酶ERK1/2的磷酸化過程[32],轉活轉錄輔助因子CITED2與轉錄因子Sp1和HIF1α結合后,促使軟骨細胞下調基質金屬蛋白酶(matrix metalloproteinase,MMP)家族中MMP1/13的表達。但是該通路在輕度周期性單軸應變(5%,1 Hz)才能起作用。在較強應變條件下P2通路如何調控軟骨細胞MMPs表達尚未見報道。

圖3　創傷導致的力學改變對軟骨細胞力傳導通路的影響Fig 3　Influences of mechanical alterations induced by injury on the chondrocyte mechanotransduction

TRPV4為一種Ca2+通道,在軟骨細胞初級纖毛和質膜上均有表達[33]。正常狀態下,TRPV4介導的Ca2+信號通路對維持基質和關節穩態有重要作用,TRPV4介導的軟骨基質低滲透壓信號通過升高胞質內的Ca2+濃度調控細胞基因表達[34-35]。TRPV4突變或功能被抑制時,則影響基質的合成及關節的力學特性[35],致使關節退變。O’conor等[35]提出利用TRPV4激動劑GSK1016790A增強基質合成,從而治療PTOA的思路。

Piezo1和Piezo2受體是軟骨細胞膜上的跨膜蛋白,屬于機械門控性Ca2+通道。Lee等[36]發現其在豬原代軟骨細胞上有豐富表達,同時證實Piezo1和Piezo2受體參與維持Ca2+內流。高應變力信號破壞軟骨穩態,利用GsMTx4阻斷Piezo1/2通道或特異性小干擾RNA迫使Piezo1/2通道失活,均可抑制創傷后軟骨組織中軟骨細胞死亡,由此提出降低Piezo1/2介導的關節損傷性力傳導信號來治療PTOA的新思路。

Hedgehog信號通路Hedgehog信號通路在骨關節炎發病中也起到重要作用。碎片蛋白1(patched homologue 1,Ptch1)受體與基質蛋白成分結合后,活化初級纖毛上的潤滑蛋白(smoothened,Smo),從而促進Gli家族蛋白活化并入核作用于Wnt和Ptc基因。Thompson等[21]研究發現,10%周期性張應變作用于牛軟骨細胞后,通過該通路促進軟骨基質中蛋白多糖酶ADAMTS-5的表達,加速基質退變。

經典Wnt信號通路經典Wnt信號通路以卷曲蛋白Frizzled和低密度脂蛋白LRP5/6為主要復合受體,當配體Wnt蛋白與受體結合后,胞內β-連環蛋白(β-catenin)含量升高,與核內轉錄因子T細胞因子/淋巴增強因子(TCF/LEF)結合后,啟動靶基因轉錄,從而促進軟骨細胞的增殖[37]、Ⅱ型膠原的分泌[38]。然而,Wnt通路與骨關節炎的發生具有爭議。Yuasa等[39]指出該通路促進兔軟骨細胞MMP-3/13以及ADAMTS-5表達,損傷關節軟骨;而Nalesso等[40]和Ma等[41]研究發現人軟骨細胞β-連環蛋白與NF-κB相互作用可抑制MMP1/3/13表達,保護軟骨。

TGF-β/BMP信號通路轉化生長因子(transforming growth factor,TGF)超家族中的TGF-β和骨形成蛋白(bone morphogenetic protein,BMP)等配體與細胞膜受體胞外區結合后,使胞內信號蛋白Smads磷酸化,轉移到細胞核內與特異性靶基因序列結合,或與DNA結合蛋白相互作用調控靶基因的表達[42]。研究證實,PTOA患者關節中TGF-β水平過低或者過高均誘導軟骨喪失正常力學特性和功能,促進MMPs過表達[43-44],引起軟骨退變、基質降解、骨贅形成等PTOA特征性改變[45-46]。

上述4種信號通路雖是獨立的,但也存在相互作用。在PTOA發病中,軟骨細胞中整合素除與Ca2+通路相關,還與TGF-β/BMP信號通路中的GDF-5和BMP-7等相關,BMP-7的上調可增加整合素αV亞單位表達,這與中后期PTOA的發病相關,而GDF-5與α5亞單位表達相關,PTOA中可能被抑制。而在另一研究中,Wnt信號通路誘導的WISP1蛋白作用于TGF-β信號通路可能促進骨關節炎中典型病變的發生[47]。多種信號通路相互作用說明力傳導信號介導PTOA發病機制的復雜性。

力傳導通路改變軟骨細胞生物學功能創傷作用于軟骨可直接導致軟骨細胞死亡[48],或者通過Fas/FasL[49]、一氧化氮[9]、核酸表達[50-51]等途徑間接誘導軟骨細胞凋亡,并促進軟骨細胞分泌基質分解酶。創傷后軟骨細胞大量分泌此類酶,加速惡化軟骨原有力學結構,從而導致更嚴重的力學失衡,加重軟骨基質損毀。

非免疫系統細胞(軟骨細胞、成纖維細胞及滑膜細胞等)分泌促炎因子[52]進一步加強軟骨細胞的凋亡[51],破壞基質,加重軟骨力學負荷的失衡,甚至導致軟骨發生損毀而直接暴露軟骨下骨[1]。大量研究表明PTOA中存在抗炎因子[53-54]。

軟骨基質分解酶類帶有血小板凝血酶敏感蛋白樣模體的解聚素和蛋白金屬蛋白酶(a disintegrin and metalloproteinase with thrombospondin-like motifs,ADAMTS-4/5)是降解軟骨基質蛋白聚糖的主要酶類。TNF-α和IL-1可促進軟骨細胞ADAMTS-4的表達量上調[55],這可能是由其基因啟動子中的特異性CpG島去甲基化介導的[56]。ADAMTS-5的表達不僅受前述多種信號通路的調控[21,39],多種miRNA也具有調控ADAMTS-5基因表達的作用,例如miR-148a[57]、miR-15a[58]、miR-105[59]具有下調作用。

MMPs降解膠原的蛋白成分促進關節損傷。骨關節炎患者關節中的MMPs活性比正常關節顯著增高,不僅降解已有膠原纖維,還會抑制新生基質形成,造成創傷所致的損傷難以愈合。中后期骨關節炎軟骨細胞中MMP3/9/13基因啟動子的CpG島去甲基化,有助于相關蛋白酶量的增加[60-61]。與ADAMTS-5相似,MMPs的表達也受到miRNA的調控,例如miR-127-5p[62]、miR-148a[57]、miR-320[63]下調MMP-13的表達;miR-140[64]、miR- 411[65]卻上調MMP-13的表達。近年來,類似miRNA不斷被發現,miRNA正成為PTOA的研究熱點。Saito等[66]給予人軟骨細胞單軸周期性拉應變(10%,0.5 Hz),發現組蛋白去乙酰化酶的拮抗劑可抑制MMP3/13的表達。

促炎因子與抗炎因子損傷相關分子模式(damage-associated molecular pattern,DAMP)是組織或細胞受到損傷、缺氧、應激等因素刺激后釋放到細胞外的一類物質。軟骨在創傷損傷后,通過模式識別受體(pattern recognition receptor,PRR)識別的DAMP,包括透明質酸[67]、核酸片段、ATP等。透明質酸、核酸片段通過Toll樣受體活化轉錄因子NF-κB增強促炎因子TNF-α、IL-1β和IL-6的表達。研究顯示,在小鼠膝關節關節面骨折后急性期反應,關節內主要是IL-1β起促炎作用[68]。另外,滑膜液和軟骨內存在大量的TNF-α、IL-1β和IL-6等因子[69],同時基質分解酶類的表達增加[44,64,70]。提示DAMP與PRR相互作用,通過炎癥加快基質降解,促進軟骨細胞的凋亡,加劇PTOA病變。

機體為維持關節穩態,需要保證炎癥與抗炎反應的動態平衡。抗炎因子在PTOA中也扮演重要角色,包括IL-1受體拮抗劑(IL-1 receptor antagonist,IL-1Ra)、IL-4、IL-10、IL-13、TGF-β1等[71-73]。Zhang等[54]在成年兔中轉染IL-1Ra和TGF-β1,發現單一抗炎因子就能促進損傷軟骨的修復,兩因子共表達則有協同效應。Agarwal等[74]研發的納米級IL-1Ra-poly-pyridine顆粒通過阻斷NF-κB途徑抑制IL-1β合成,進而抑制炎癥。另外,IL-10能促進軟骨細胞合成蛋白多糖[72],有研究提示鍛煉也可促進滑膜內和軟骨中IL-10的合成[75-76]。

結語由于關節軟骨內并不存在免疫細胞,因此轉變為成纖維細胞樣的軟骨細胞可視為軟骨細胞生物學和分子生物學應答的中心,其中力學因素和炎癥因素的協同作用,對PTOA的發病和進展有重要效應。然而,對關節軟骨不同區域內軟骨細胞的力學信號通路和炎癥反應通路的機制研究還很有限。這些通路中分子作用的靶點將是預防、治療PTOA和良好預后的研究方向。

[1]RACINE J,AARON RK.Post-traumatic osteoarthritis after ACL injury[J].RIMedJ(2013),2014,97(11):25-28.

[2]DRIBAN JB,HOOTMAN JM,SITLER MR,etal.Is participation in certain sports associated with knee osteoarthritis? A systematic review[J].JAthlTrain,2017,52(6):497-506.

[3]SHOWERY JE,KUSNEZOV NA,DUNN JC,etal.The rising incidence of degenerative and posttraumatic osteoarthritis of the knee in the united states military[J].JArthroplasty,2016,31(10):2108-2114.

[4]NORDENVALL R,BAHMANYAR S,ADAMI J,etal.Cruciate ligament reconstruction and risk of knee osteoarthritis:the association between cruciate ligament injury and post-traumatic osteoarthritis.a population based nationwide study in Sweden,1987-2009[J].PLoSOne,2014,9(8):e104681.

[5]ANDERSON DD,CHUBINSKAYA S,GUILAK F,etal.Post-traumatic osteoarthritis:improved understanding and opportunities for early intervention[J].JOrthopRes,2011,29(6):802-809.

[6]AJUIED A,WONG F,SMITH C,etal.Anterior cruciate ligament injury and radiologic progression of knee osteoarthritis:a systematic review and meta-analysis[J].AmJSportsMed,2014,42(9):2242-2252.

[7]SAMAAN MA,RINGLEB SI,BAWAB SY,etal.Anterior cruciate ligament (ACL) loading in a collegiate athlete during sidestep cutting after ACL reconstruction:a case study[J].Knee,2016,23(4):744-752.

[8]GROGAN SP,DUFFY SF,PAULI C,etal.Zone-specific gene expression patterns in articular cartilage[J].ArthritisRheum,2013,65(2):418-428.

[9]PRINCE DE,GREISBERG JK.Nitric oxide-associated chondrocyte apoptosis in trauma patients after high-energy lower extremity intra-articular fractures[J].JOrthopTraumatol,2015,16(4):335-341.

[10]LEWIS JS,HEMBREE WC,FURMAN BD,etal.Acute joint pathology and synovial inflammation is associated with increased intra-articular fracture severity in the mouse knee[J].OsteoarthritisCartilage,2011,19(7):864-873.

[11]LOESER RF,GOLDRING SR,SCANZELLO CR,etal.Osteoarthritis:a disease of the joint as an organ[J].ArthritisRheum,2012,64(6):1697-1707.

[12]THOMAS CM,FULLER CJ,WHITTLES CE,etal.Chondrocyte death by apoptosis is associated with cartilage matrix degradation[J].OsteoarthritisCartilage,2007,15(1):27-34.

[13]BONNANS C,CHOU J,WERB Z.Remodelling the extracellular matrix in development and disease[J].NatRevMolCellBiol,2014,15(12):786-801.

[14]WELLSANDT E,GARDINIER ES,MANAL K,etal.Decreased knee joint loading associated with early knee osteoarthritis after anterior cruciate ligament injury[J].AmJSportsMed,2016,44(1):143-151.

[15]BOSCHETTI F,PERETTI GM.Tensile and compressive properties of healthy and osteoarthritic human articular cartilage[J].Biorheology,2008,45(3-4):337-344.

[16]MCGLASHAN SR,CLUETT EC,JENSEN CG,etal.Primary cilia in osteoarthritic chondrocytes:from chondrons to clusters[J].DevDyn,2008,237(8):2013-2020.

[17]RUHLEN R,MARBERRY K.The chondrocyte primary cilium[J].OsteoarthritisCartilage,2014,22(8):1071-1076.

[18]IRIANTO J,RAMASWAMY G,SERRA R,etal.Depletion of chondrocyte primary cilia reduces the compressive modulus of articular cartilage[J].JBiomech,2014,47(2):579-582.

[19]JENSEN CG,POOLE CA,MCGLASHAN SR,etal.Ultrastructural,tomographic and confocal imaging of the chondrocyte primary cilium in situ[J].CellBiolInt,2004,28(2):101-110.

[20]MCGLASHAN SR,JENSEN CG,POOLE CA.Localization of extracellular matrix receptors on the chondrocyte primary cilium[J].JHistochemCytochem,2006,54(9):1005-1014.

[21]THOMPSON CL,CHAPPLE JP,KNIGHT MM.Primary cilia disassembly down-regulates mechanosensitive hedgehog signalling:a feedback mechanism controlling ADAMTS-5 expression in chondrocytes[J].OsteoarthritisCartilage,2014,22(3):490-498.

[22]MATTA C,ZAKANY R,MOBASHERI A.Voltage-dependent calcium channels in chondrocytes:roles in health and disease[J].CurrRheumatolRep,2015,17(7):43.

[23]DEREN ME,YANG X,GUAN Y,etal.Biological and chemical removal of primary cilia affects mechanical activation of chondrogenesis markers in chondroprogenitors and hypertrophic chondrocytes[J].IntJMolSci,2016,17(2):188.

[24]LIANG Y,PANG Y,WU Q,etal.FLA8/KIF3B phosphorylation regulates kinesin-Ⅱ interaction with IFT-B to control IFT entry and turnaround[J].DevCell,2014,30(5):585-597.

[25]KONG F,LI Z,PARKS WM,etal.Cyclic mechanical reinforcement of integrin-ligand interactions[J].MolCell,2013,49(6):1060-1068.

[26]GARCIADIEGO-CAZARES D,AGUIRRE-SANCHEZ HI,ABARCA-BUIS RF,etal.Regulation of alpha5 and alphaV integrin expression by GDF-5 and BMP-7 in chondrocyte differentiation and osteoarthritis[J].PLoSOne,2015,10(5):e127166.

[27]SHIN SY,POZZI A,BOYD SK,etal.Integrin alpha1beta1 protects against signs of post-traumatic osteoarthritis in the female murine knee partially via regulation of epidermal growth factor receptor signalling[J].OsteoarthritisCartilage,2016,24(10):1795-1806.

[28]ALMONTE-BECERRIL M,COSTELL M,KOURI JB.Changes in the integrins expression are related with the osteoarthritis severity in an experimental animal model in rats[J].JOrthopRes,2014,32(9):1161-1166.

[29]CANDELA ME,WANG C,GUNAWARDENA AT,etal.Alpha 5 integrin mediates osteoarthritic changes in mouse knee joints[J].PLoSOne,2016,11(6):e156783.

[30]JANG KW,BUCKWALTER JA,MARTIN JA.Inhibition of cell-matrix adhesions prevents cartilage chondrocyte death following impact injury[J].JOrthopRes,2014,32(3):448-454.

[31]HE Z,LEONG DJ,ZHUO Z,etal.Strain-induced mechanotransduction through primary cilia,extracellular ATP,purinergic calcium signaling,and ERK1/2 transactivates CITED2 and downregulates MMP-1 and MMP-13 gene expression in chondrocytes[J].OsteoarthritisCartilage,2016,24(5):892-901.

[32]KNIGHT MM,MCGLASHAN SR,GARCIA M,etal.Articular chondrocytes express connexin 43 hemichannels and P2 receptors- a putative mechanoreceptor complex involving the primary cilium?[J].JAnat,2009,214(2):275-283.

[33]PHAN MN,LEDDY HA,VOTTA BJ,etal.Functional characterization of TRPV4 as an osmotically sensitive ion channel in porcine articular chondrocytes[J].ArthritisRheum,2009,60(10):3028-3037.

[34]WALTER BA,PURMESSUR D,MOON A,etal.Reduced tissue osmolarity increases TRPV4 expression and pro-inflammatory cytokines in intervertebral disc cells[J].EurCellMater,2016,32:123-136.

[35]O′CONOR CJ,LEDDY HA,BENEFIELD HC,etal.TRPV4-mediated mechanotransduction regulates the metabolic response of chondrocytes to dynamic loading[J].ProcNatlAcadSciUSA,2014,111(4):1316-1321.

[36]LEE W,LEDDY HA,CHEN Y,etal.Synergy between Piezo1 and Piezo2 channels confers high-strain mechanosensitivity to articular cartilage[J].ProcNatlAcadSciUSA,2014,111(47):E5114-E5122.

[37]QU F,WANG J,XU N,etal.WNT3A modulates chondrogenesis via canonical and non-canonical Wnt pathways in MSCs[J].FrontBiosci(LandmarkEd),2013,18:493-503.

[38]NIU Q,LI F,ZHANG L,etal.Role of the Wnt/beta-catenin signaling pathway in the response of chondrocytes to mechanical loading[J].IntJMolMed,2016,37(3):755-762.

[39]YUASA T,OTANI T,KOIKE T,etal.Wnt/beta-catenin signaling stimulates matrix catabolic genes and activity in articular chondrocytes:its possible role in joint degeneration[J].LabInvest,2008,88(3):264-274.

[40]NALESSO G,SHERWOOD J,BERTRAND J,etal.WNT-3A modulates articular chondrocyte phenotype by activating both canonical and noncanonical pathways[J].JCellBiol,2011,193(3):551-564.

[41]MA B,VAN BLITTERSWIJK CA,KARPERIEN M.A Wnt/beta-catenin negative feedback loop inhibits interleukin-1-induced matrix metalloproteinase expression in human articular chondrocytes[J].ArthritisRheum,2012,64(8):2589-2600.

[42]TEKARI A,LUGINBUEHL R,HOFSTETTER W,etal.Transforming growth factor beta signaling is essential for the autonomous formation of cartilage-like tissue by expanded chondrocytes[J].PLoSOne,2015,10(3):e120857.

[43]AREF-ESHGHI E,LIU M,HARPER PE,etal.Overexpression of MMP13 in human osteoarthritic cartilage is associated with the SMAD-independent TGF-beta signalling pathway[J].ArthritisResTher,2015,17:264.

[44]KUNISCH E,KINNE RW,ALSALAMEH RJ,etal.Pro-inflammatory IL-1beta and/or TNF-alpha up-regulate matrix metalloproteases-1 and -3 mRNA in chondrocyte subpopulations potentially pathogenic in osteoarthritis:in situ hybridization studies on a single cell level[J].IntJRheumDis,2016,19(6):557-566.

[45]SCHARSTUHL A,GLANSBEEK HL,VAN BEUNINGEN HM,etal.Inhibition of endogenous TGF-beta during experimental osteoarthritis prevents osteophyte formation and impairs cartilage repair[J].JImmunol,2002,169(1):507-514.

[46]ZHEN G,WEN C,JIA X,etal.Inhibition of TGF-beta signaling in mesenchymal stem cells of subchondral bone attenuates osteoarthritis[J].NatMed,2013,19(6):704-712.

[47]VAN DEN BOSCH MH,BLOM AB,VAN LENT PL,etal.Canonical Wnt signaling skews TGF-beta signaling in chondrocytes towards signaling via ALK1 and Smad 1/5/8[J].CellSignal,2014,26(5):951-958.

[48]STOLBERG-STOLBERG JA,FURMAN BD,GARRIGUES NW,etal.Effects of cartilage impact with and without fracture on chondrocyte viability and the release of inflammatory markers[J].JOrthopRes,2013,31(8):1283-1292.

[49]CAMEJO FA,ALMEIDA LE,DOETZER AD,etal.FasL expression in articular discs of human temporomandibular joint and association with osteoarthrosis[J].JOralPatholMed,2014,43(1):69-75.

[50]JIN L,ZHAO J,JING W,etal.Role of miR-146a in human chondrocyte apoptosis in response to mechanical pressure injury in vitro[J].IntJMolMed,2014,34(2):451-463.

[51]LI J,HUANG J,DAI L,etal.miR-146a,an IL-1beta responsive miRNA,induces vascular endothelial growth factor and chondrocyte apoptosis by targeting Smad4[J].ArthritisResTher,2012,14(2):R75.

[52]APPLETON CT,PITELKA V,HENRY J,etal.Global analyses of gene expression in early experimental osteoarthritis[J].ArthritisRheum,2007,56(6):1854-1868.

[53]BEHRENDT P,PREUSSE-PRANGE A,KLUTER T,etal.IL-10 reduces apoptosis and extracellular matrix degradation after injurious compression of mature articular cartilage[J].OsteoarthritisCartilage,2016,24(11):1981-1988.

[54]ZHANG P,ZHONG ZH,YU HT,etal.Exogenous expression of IL-1Ra and TGF-beta1 promotes in vivo repair in experimental rabbit osteoarthritis[J].ScandJRheumatol,2015,44(5):404-411.

[55]XUE J,WANG J,LIU Q,etal.Tumor necrosis factor-alpha induces ADAMTS-4 expression in human osteoarthritis chondrocytes[J].MolMedRep,2013,8(6):1755-1760.

[56]CHEUNG KS,HASHIMOTO K,YAMADA N,etal.Expression of ADAMTS-4 by chondrocytes in the surface zone of human osteoarthritic cartilage is regulated by epigenetic DNA de-methylation[J].RheumatolInt,2009,29(5):525-534.

[57]VONK LA,KRAGTEN AH,DHERT WJ,etal.Overexpression of hsa-miR-148a promotes cartilage production and inhibits cartilage degradation by osteoarthritic chondrocytes[J].OsteoarthritisCartilage,2014,22(1):145-153.

[58]LU X,LIN J,JIN J,etal.Hsa-miR-15a exerts protective effects against osteoarthritis by targeting aggrecanase-2 (ADAMTS5) in human chondrocytes[J].IntJMolMed,2016,37(2):509-516.

[59]JI Q,XU X,XU Y,etal.miR-105/Runx2 axis mediates FGF2-induced ADAMTS expression in osteoarthritis cartilage[J].JMolMed(Berl),2016,94(6):681-694.

[60]ROACH HI,YAMADA N,CHEUNG KS,etal.Association between the abnormal expression of matrix-degrading enzymes by human osteoarthritic chondrocytes and demethylation of specific CpG sites in the promoter regions[J].ArthritisRheum,2005,52(10):3110-3124.

[61]HARPER PE,LIU M,AREF-ESHGHI E,etal.Gene expression analysis and DNA methylation in the promoter region of matrix metalloproteinase-13 (MMP-13) and osteoarthritis[J].OsteoarthritisCartilage,2015,23(2):A305.

[62]PARK SJ,CHEON EJ,LEE MH,etal.MicroRNA-127-5p regulates matrix metalloproteinase 13 expression and interleukin-1beta-induced catabolic effects in human chondrocytes[J].ArthritisRheum,2013,65(12):3141-3152.

[63]MENG F,ZHANG Z,CHEN W,etal.MicroRNA-320 regulates matrix metalloproteinase-13 expression in chondrogenesis and interleukin-1beta-induced chondrocyte responses[J].OsteoarthritisCartilage,2016,24(5):932-941.

[64]LIANG ZJ,ZHUANG H,WANG GX,etal.MiRNA-140 is a negative feedback regulator of MMP-13 in IL-1beta-stimulated human articular chondrocyte C28/I2 cells[J].InflammRes,2012,61(5):503-509.

[65]WANG G,ZHANG Y,ZHAO X,etal.MicroRNA-411 inhibited matrix metalloproteinase 13 expression in human chondrocytes[J].AmJTranslRes,2015,7(10):2000-2006.

[66]SAITO T,NISHIDA K,FURUMATSU T,etal.Histone deacetylase inhibitors suppress mechanical stress-induced expression of RUNX-2 and ADAMTS-5 through the inhibition of the MAPK signaling pathway in cultured human chondrocytes[J].OsteoarthritisCartilage,2013,21(1):165-174.

[67]SCHEIBNER KA,LUTZ MA,BOODOO S,etal.Hyaluronan fragments act as an endogenous danger signal by engaging TLR2[J].JImmunol,2006,177(2):1272-1281.

[68]FURMAN BD,MANGIAPANI DS,ZEITLER E,etal.Targeting pro-inflammatory cytokines following joint injury:acute intra-articular inhibition of interleukin-1 following knee injury prevents post-traumatic arthritis[J].ArthritisResTher,2014,16(3):R134.

[69]TSUCHIDA AI,BEEKHUIZEN M,T HART MC,etal.Cytokine profiles in the joint depend on pathology,but are different between synovial fluid,cartilage tissue and cultured chondrocytes[J].ArthritisResTher,2014,16(5):441.

[70]YAMANISHI Y,BOYLE DL,CLARK M,etal.Expression and regulation of aggrecanase in arthritis:the role of TGF-beta[J].JImmunol,2002,168(3):1405-1412.

[71]LANG A,NEUHAUS J,PFEIFFENBERGER M,etal.Optimization of a nonviral transfection system to evaluate Cox-2 controlled interleukin-4 expression for osteoarthritis gene therapyinvitro[J].JGeneMed,2014,16(11-12):352-363.

[72]WOJDASIEWICZ P,PONIATOWSKI L A,SZUKIEWICZ D.The role of inflammatory and anti-inflammatory cytokines in the pathogenesis of osteoarthritis[J].MediatorsInflamm,2014,2014:561459.

[73]JOVANOVIC D,PELLETIER JP,ALAAEDDINE N,etal.Effect of IL-13 on cytokines,cytokine receptors and inhibitors on human osteoarthritis synovium and synovial fibroblasts[J].OsteoarthritisCartilage,1998,6(1):40-49.

[74]AGARWAL R,VOLKMER TM,WANG P,etal.Synthesis of self-assembled IL-1Ra-presenting nanoparticles for the treatment of osteoarthritis[J].JBiomedMaterResA,2016,104(3):595-599.

[75]ROJAS-ORTEGA M,CRUZ R,VEGA-LOPEZ MA,etal.Exercise modulates the expression of IL-1beta and IL-10 in the articular cartilage of normal and osteoarthritis-induced rats[J].PatholResPract,2015,211(6):435-443.

[76]HELMARK IC,MIKKELSEN UR,BORGLUM J,etal.Exercise increases interleukin-10 levels both intraarticularly and peri-synovially in patients with knee osteoarthritis:a randomized controlled trial[J].ArthritisResTher,2010,12(4):R126.