低氧誘導(dǎo)因子在類風(fēng)濕關(guān)節(jié)炎發(fā)病機(jī)制中的作用

于若寒，趙金霞，劉湘源

(北京大學(xué)第三醫(yī)院風(fēng)濕免疫科，北京 100191)

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·綜述·

低氧誘導(dǎo)因子在類風(fēng)濕關(guān)節(jié)炎發(fā)病機(jī)制中的作用

于若寒，趙金霞，劉湘源△

(北京大學(xué)第三醫(yī)院風(fēng)濕免疫科，北京 100191)

低氧誘導(dǎo)因子；關(guān)節(jié)炎，類風(fēng)濕；缺氧

類風(fēng)濕關(guān)節(jié)炎(rheumatoid arthritis，RA)是一種系統(tǒng)性自身免疫性疾病，病理特征為異常的滑膜增生伴血管翳形成、軟骨和骨的侵蝕破壞，最終導(dǎo)致關(guān)節(jié)畸形。目前RA確切發(fā)病機(jī)制未明，環(huán)境和遺傳因素相互作用共同促進(jìn)了RA的發(fā)病。低氧微環(huán)境是RA重要的病理特點(diǎn)，炎癥滑膜組織的高代謝需求和滑膜的快速增生共同導(dǎo)致了RA關(guān)節(jié)內(nèi)低氧狀態(tài)。研究表明,低氧通過低氧誘導(dǎo)因子(hypoxia-inducible factor，HIF)調(diào)控的一系列轉(zhuǎn)錄因子的活化促進(jìn)RA的疾病進(jìn)展[1]。HIF可通過促進(jìn)RA滑膜細(xì)胞(RA fibroblast-like synoviocytes，RA-FLS)增殖和侵襲、調(diào)節(jié)炎性細(xì)胞因子分泌、誘導(dǎo)血管生成及軟骨破壞參與RA的發(fā)病和病情進(jìn)展[2-4]。鑒于低氧在RA發(fā)病機(jī)制中的重要作用，本文對HIF與RA發(fā)病機(jī)制的研究進(jìn)展進(jìn)行綜述。

1 HIF信號通路

1.1 HIF的結(jié)構(gòu)

HIF介導(dǎo)多細(xì)胞生物對低氧應(yīng)激的主要轉(zhuǎn)錄反應(yīng)在1991年首次被發(fā)現(xiàn)[5]。HIF是一種異源二聚體復(fù)合物，由受氧調(diào)節(jié)的α亞單位(HIF-1α、HIF-2α和HIF-3α)和穩(wěn)定的β亞單位(HIF-1β)構(gòu)成，每個亞單位均含一個helix-loop-helix區(qū)域使其識別并結(jié)合到低氧誘導(dǎo)基因調(diào)節(jié)序列中的HIF DNA結(jié)合位點(diǎn)[6-7]。HIF-α的3種亞型中，HIF-1α和HIF-2α在結(jié)構(gòu)和功能上有較大的相似性，而HIF-3α的研究較少，有研究認(rèn)為HIF-3α可能作為抑制元件對HIF-1α和HIF-2α進(jìn)行負(fù)調(diào)控[8]。HIF-α對氧濃度的調(diào)節(jié)非常敏感，在氧濃度低于6.0%時，細(xì)胞HIF-1α水平以指數(shù)的方式快速增長，在氧濃度為0.5%(相當(dāng)于PO210～15 mmHg，1 mmHg=0.133 kPa)時達(dá)到最高值[2]。大量積累的HIF-α與HIF-1β結(jié)合，從胞漿進(jìn)入胞核內(nèi)，并結(jié)合基因組上的低氧應(yīng)答元件(hypoxia response elements，HREs)， 從而開啟相關(guān)靶基因的轉(zhuǎn)錄。

1.2 HIF的調(diào)節(jié)機(jī)制

脯氨酸羥基化酶(prolyl hydroxylase domain proteins，PHDs)在對HIF-α的調(diào)節(jié)中發(fā)揮關(guān)鍵作用。常氧下，PHDs將HIF-α蛋白上的兩個關(guān)鍵位置的脯氨酸進(jìn)行羥基化修飾[9]，被修飾后的HIF-α可被腫瘤抑制因子vHL(von Hippel-Lindau)所識別和結(jié)合，然后進(jìn)行vHL降解復(fù)合物所介導(dǎo)的K48泛素化修飾，最后經(jīng)26S蛋白酶體途徑將HIF-α降解[10-11]。PHDs的活性受其底物O2的直接調(diào)控，低氧下，PHDs酶活力下降，無法完成對HIF-α的羥基化修飾，這時vHL也就無法識別、結(jié)合及降解HIF-α，大量積累的HIF-α與HIF-1β結(jié)合，由胞漿進(jìn)入胞核[12]，與HREs結(jié)合，上調(diào)血管生成、糖酵解、細(xì)胞遷移、生長和凋亡基因等的表達(dá)。此外，PHDs的活性還受其輔因子Fe2+和α-酮戊二酸的影響[8]。

另外，天門冬氨酸羥基化酶(factor inhibiting HIF，F(xiàn)IH)對HIF-α的調(diào)節(jié)也有一定作用，它通過特異性地對HIF-1α的Asn803進(jìn)行羥基化修飾，從而削弱HIF-1α與其轉(zhuǎn)錄輔因子p300/CBP的結(jié)合能力，降低HIF-1α對其靶基因的轉(zhuǎn)錄活性[13]。與PHDs一樣，F(xiàn)IH的酶活性也受O2的直接調(diào)控，只有在常氧下才對HIF-1α有修飾作用。除此之外，生長因子和炎性因子，如白細(xì)胞介素(interleukin，IL)-1β、腫瘤壞死因子-α(tumor necrosis factor-α，TNF-α)均能影響HIF-α的蛋白水平及轉(zhuǎn)錄活性[14-15]。

對不同HIF調(diào)節(jié)酶(PHD-1、PHD-2、PHD-3和FIH-1)的研究發(fā)現(xiàn)，常氧下PHD-2含量最豐富，低氧可使其水平增加約2倍；而PHD-3含量最少，但對低氧最敏感，低氧下其表達(dá)可增加19倍；相反，PHD-1和FIH-1幾乎不受低氧影響。常氧下敲除PHD-2后，HIF-1α和HIF-2α蛋白表達(dá)水平與低氧時水平相當(dāng)；而敲除PHD-3可使HIF-2α與HREs的結(jié)合增加，對HIF-1α蛋白表達(dá)影響甚小，甚至降低HIF-1α表達(dá)水平。以上研究提示，PHD-2是調(diào)控RA-FLS中HIF穩(wěn)定性及其下游靶基因的最重要的酶[16]。

2 低氧對RA發(fā)病機(jī)制的影響

越來越多的證據(jù)表明低氧調(diào)節(jié)RA許多重要的病理生理過程，包括滑膜炎癥、血管生成和軟骨破壞等。

2.1 低氧與滑膜炎癥

滑膜炎是RA最主要的病理特征。低氧下，HIF在細(xì)胞核內(nèi)大量累積。研究表明,HIF是RA滑膜炎癥的重要調(diào)節(jié)因素，HIF-α可上調(diào)細(xì)胞因子[TNF-α、IL-1、IL-6、IL-8、IL-15、IL-17、IL-33、干擾素-γ(interferon，IFN-γ)]、趨化因子[CXCL8(C-X-C motif chemokine ligand 8)、CXCL12、CCL20(CC-chemokine ligand 20)]、血管細(xì)胞粘附分子-1(vascular cell adhesion molecule-1，VCAM-1)、促血管生成因子[血管內(nèi)皮生長因子(vascular endothelial growth factor，VEGF)、血小板反應(yīng)蛋白-1(thrombospondin-1，TSP-1)]、基質(zhì)金屬蛋白酶(matrix metalloproteinase，MMP)(MMP-1、MMP-2、MMP-3、MMP-9、MMP-12、MMP-13)和Toll樣受體的表達(dá)[1]，從而促進(jìn)RA的滑膜炎癥、血管生成、軟骨破壞和骨侵蝕[17]。低氧下RA-FLS的遷移、侵襲能力也顯著增加[18]。HIF-1α基因敲除RA小鼠模型臨床表現(xiàn)和病理特征顯著改善，特別是滑膜炎癥、血管翳形成和軟骨破壞[19],同樣，阻斷HIF-1α信號通路顯著降低RA-FLS的遷移和侵襲能力及MMPs分泌[18, 20-21]。Ryu等[22]分別在DBA/1J小鼠膝關(guān)節(jié)內(nèi)注射Ad-HIF-1α或Ad-Epas1(HIF-2α)腺病毒致局部過表達(dá)HIF-1α或HIF-2α，3周后局部過表達(dá)HIF-2α的關(guān)節(jié)組織顯示典型的RA樣改變，如滑膜過度增生、滑膜炎、軟骨破壞、血管生成和血管翳形成。Epas1(HIF-2α)基因敲除顯著減少小鼠膠原誘導(dǎo)性關(guān)節(jié)炎的發(fā)生率和嚴(yán)重性，致病性細(xì)胞因子IL-1β、IL-6、IL-12、IL-17A、IL-17F、TNF-α和IFN-γ的表達(dá)顯著下調(diào)。但在上述研究中HIF-1α的過表達(dá)并沒有引起小鼠關(guān)節(jié)結(jié)構(gòu)的任何改變，這可能提示HIF-1α、HIF-2α在RA發(fā)病機(jī)制中的作用不同。

盡管與HIF-1α結(jié)構(gòu)有很大的相似性，HIF-2α在RA中的作用與HIF-1α有所不同。首先，HIF-1α和HIF-2α在RA滑膜組織中的表達(dá)位置不同，HIF-2α在滑膜襯里層高表達(dá)，而HIF-1α則在RA滑膜組織的襯里層下層和更深層表達(dá)[22]。其次，HIF-2α在RA發(fā)病機(jī)制中的作用似乎是通過炎癥因子介導(dǎo)的，Ryu等[22]的研究發(fā)現(xiàn)IL-1β和TNF-α可上調(diào)小鼠FLSHIF-2α的表達(dá)，低氧僅可使FLS表達(dá)HIF-2α輕度增加，提示炎癥因子可能是HIF-2α表達(dá)上調(diào)的主要原因。過表達(dá)HIF-2α可增加小鼠FLS的增殖能力，上調(diào)核因子κB(nuclear factor κB, NF-κB)受體激活蛋白配體(receptor activator of NF-κB ligand，RANKL)的表達(dá)和破骨細(xì)胞的產(chǎn)生及基質(zhì)降解酶、趨化因子、炎癥介質(zhì)水平。敲除HIF-2α后，IL-1β誘導(dǎo)的細(xì)胞增殖能力、MMPs、趨化因子和炎癥介質(zhì)被抑制，還有研究發(fā)現(xiàn)HIF-2α通過上調(diào)IL-6的表達(dá)而導(dǎo)致RA的發(fā)病。以上結(jié)果提示HIF-2α在RA的發(fā)病中起重要作用，且是通過IL-6介導(dǎo)的。

滑膜炎癥的一個重要病理改變是新生血管形成。新生血管為擴(kuò)增的炎癥細(xì)胞群提供營養(yǎng)和氧，并促進(jìn)白細(xì)胞的進(jìn)入，因此導(dǎo)致了滑膜炎的持續(xù)。低氧明顯促進(jìn)了血管生成[23]，低氧通過HIF-1α或HIF-2α調(diào)控許多血管生成介質(zhì)的表達(dá)，如一氧化氮合成酶、VEGF、CXCL8、CCL20和基質(zhì)細(xì)胞衍生因子-1(stromal cell-derived factor-1，SDF-1)，從而導(dǎo)致血管擴(kuò)張，增加血管通透性[24]。HIF還可活化血管生成素、Tie-2(tyrosie kinase with Eg and EGF homo-logy domain)、纖維母細(xì)胞生長因子(fibroblast growth factor，F(xiàn)GF)和血小板源生長因子(platelet-derived growth factor，PDGF)， 從而導(dǎo)致內(nèi)皮細(xì)胞增殖、遷移和血管重建[25]。

低氧和炎癥因子具有協(xié)同作用。低氧通過調(diào)節(jié)HIF-1α和HIF-2α的表達(dá)促進(jìn)滑膜細(xì)胞分泌炎癥因子，反過來，炎癥因子，如IL-1、TNF-α、 高遷移族蛋白-1(high mobility group box-1 protein，HMGB1)和IL-33，能誘導(dǎo)RA-FLS表達(dá)HIF-1α和HIF-2α，因此，形成一個調(diào)節(jié)回路，促進(jìn)RA滑膜炎癥的持續(xù)[15, 22, 26]。IL-17A與低氧可協(xié)同促進(jìn)RA-FLS的遷移和侵襲能力，抑制HIF-1α和NF-κB后這種作用顯著減弱，另外，抑制NF-κB使HIF-1α的表達(dá)顯著減弱，提示IL-17A在低氧下通過NF-κB信號通路活化HIF-1α[27]。HMGB1通過促進(jìn)RA-FLS表達(dá)HIF-1α而促進(jìn)血管形成，從而加重滑膜炎癥[28]。

HIF信號通路與其他信號通路之間存在交互作用。信號轉(zhuǎn)導(dǎo)子和轉(zhuǎn)錄活化子3(signal transducer and activator transcription factor 3，STAT3)-siRNA和Janus 激酶2(Janus kinase 2，JAK2)的抑制劑(WP1066)可抑制低氧誘導(dǎo)的HIF-1α表達(dá)，同樣HIF-1α siRNA也可抑制低氧誘導(dǎo)的STAT3的表達(dá)，阻斷STAT3后炎性細(xì)胞因子表達(dá)顯著減少，提示HIF-1α通路和STAT3通路在RA炎癥中存在交互作用[29]。低氧促進(jìn)RA-FLS Notch信號通路組分的表達(dá)，Notch-1 siRNA可抑制低氧誘導(dǎo)的HIF-1α和VEGF表達(dá)，提示Notch-1和HIF-1α之間存在交互作用[30]。絲裂原細(xì)胞外激酶1/2(mitogen extracellular kinase 1/2，MEK1/2)抑制劑PD98059和磷脂酰肌醇三激酶(phosphoinositide 3-kinase，PI3K)抑制劑LY294002能顯著抑制細(xì)胞因子誘導(dǎo)的HIF-1α表達(dá)，說明PI3K和細(xì)胞外信號調(diào)節(jié)激酶通路在炎癥因子誘導(dǎo)的HIF-1α表達(dá)中有一定作用[14]。低氧可上調(diào)Toll樣受體(Toll-like receptor，TLR)配體誘導(dǎo)的RA-FLS炎癥性細(xì)胞因子、MMPs、VEGF等的釋放，過表達(dá)HIF-1α可加強(qiáng)聚肌胞苷酸(polyinosinic-polycytidylic acid，polyIC)誘導(dǎo)的IL-6、IL-8和TNF-α的增加，敲除HIF-1α后這種效應(yīng)可被抑制，提示HIF-1α與TLR刺激的免疫反應(yīng)協(xié)同促進(jìn)RA的滑膜炎癥[31]。另外，過表達(dá)NF-κB可促進(jìn)HIF-1α的表達(dá)，而NF-κB亞單位的敲除使HIF-1α的表達(dá)下降[32]。NF-kB抑制劑Bay能完全抑制細(xì)胞因子誘導(dǎo)的HIF-1α活化[33]。

2.2 低氧與軟骨破壞

關(guān)節(jié)軟骨的破壞和骨的侵蝕是RA病理的重要表現(xiàn)。隨著RA炎癥的進(jìn)展，過度增生的血管翳侵入、破壞關(guān)節(jié)軟骨。低氧下的滑膜細(xì)胞可通過分泌大量MMPs破壞關(guān)節(jié)軟骨。HIF-1α和HIF-2α均能顯著促進(jìn)滑膜細(xì)胞產(chǎn)生多種MMPs和聚蛋白多糖酶-1(a disintegrin and metalloproteinase with thrombospondin motifs-4，ADAMTS4)[2, 34],HIF-2α可能通過誘導(dǎo)IL-6的產(chǎn)生促進(jìn)軟骨細(xì)胞分泌MMP-3和MMP-13，通過誘導(dǎo)TNF-α的產(chǎn)生促進(jìn)滑膜細(xì)胞表達(dá)MMPs和炎癥性介質(zhì)[35]。

低氧還可通過活化破骨細(xì)胞介導(dǎo)骨的破壞。低氧可使破骨細(xì)胞數(shù)量、骨吸收及骨質(zhì)溶解相關(guān)酶活性明顯增加，HIF-1αsiRNA能完全阻斷低氧誘導(dǎo)的這些效應(yīng)[36]。Zhao等[37]的研究發(fā)現(xiàn)低氧可增加破骨細(xì)胞的分化，同時伴有一些特異的自噬功能，抑制自噬可顯著降低低氧狀態(tài)下破骨細(xì)胞的分化，提示自噬對低氧誘導(dǎo)的破骨細(xì)胞分化有重要的作用。他們的研究還發(fā)現(xiàn)，低氧狀態(tài)下自噬的活化是由HIF-1α依賴的BCL2結(jié)合蛋白3(BCL-2 interacting protein 3，BNIP3)的上調(diào)引起的。將HIF-1α或BNIP3敲除能夠顯著降低低氧誘導(dǎo)的自噬的活化和破骨細(xì)胞的生成增加。低氧下HIF介導(dǎo)的通路還可為破骨細(xì)胞提供能量[38]。Hiraga等[39]的研究發(fā)現(xiàn)，低氧和HIF-1α通過抑制成骨細(xì)胞的分化和促進(jìn)破骨細(xì)胞的形成導(dǎo)致乳腺癌的溶骨性骨轉(zhuǎn)移。以上研究提示低氧可促進(jìn)骨破壞的發(fā)生，并且HIF在其中起重要作用。

2.3 低氧對免疫細(xì)胞的影響

T細(xì)胞是在RA發(fā)病機(jī)制中起重要作用的細(xì)胞,低氧在T細(xì)胞的形成分化和效應(yīng)功能中起重要作用。Foxp3是調(diào)節(jié)性T細(xì)胞(regulatory T cells，Treg)特異的標(biāo)志物，低氧可使人Jurkat T細(xì)胞表達(dá)Foxp3增加，這種效應(yīng)可被HIF-1αsiRNA抑制，而HIF-1α過表達(dá)增加Foxp3的表達(dá)[40-41]。同樣，人外周血CD4+T細(xì)胞轉(zhuǎn)染過表達(dá)HIF-1α的慢病毒后Foxp3表達(dá)增加，HIF-1αsiRNA能逆轉(zhuǎn)Foxp3的高表達(dá)[42]。相反，也有研究發(fā)現(xiàn)HIF-1α通過與Foxp3結(jié)合，使Treg被蛋白酶體降解而減少其形成[43-44]。造成研究結(jié)論不一致的原因可能是HIF-1α對Treg的調(diào)節(jié)不是直接作用的，低氧對Treg的調(diào)節(jié)依賴于HIF-1α和轉(zhuǎn)化生長因子β的共同作用及局部微環(huán)境的細(xì)胞因子[41]。HIF-1α可調(diào)節(jié)Th17/Treg細(xì)胞的平衡，HIF-1α的缺失可影響Th17細(xì)胞的分化[43-44]。HIF-1α過表達(dá)還可促進(jìn)RA-FLS介導(dǎo)的Th1和Th17細(xì)胞的增加，從而促進(jìn)IFN-γ和IL-17的產(chǎn)生[31]。相反，HIF-2α對Th17細(xì)胞的分化沒有影響，但HIF-2α可通過上調(diào)IL-6的表達(dá)從而影響Th17細(xì)胞的分化[22]。總之，這些數(shù)據(jù)提示HIF對T細(xì)胞的分化有重要影響，且對Th17細(xì)胞的分化是必要的。

3 潛在的治療作用

越來越多的證據(jù)表明低氧和HIF參與了RA許多重要的病理生理過程，包括滑膜炎癥、血管生成和軟骨破壞，提示HIF可能是RA潛在的治療靶點(diǎn)。以低氧為靶點(diǎn)的治療方法多來自低氧對腫瘤的研究，包括應(yīng)用前體物質(zhì)、特定的HIF抑制劑或基因治療，低氧前體物質(zhì)可在缺氧組織中選擇性活化，從而把活性物質(zhì)運(yùn)送至缺氧細(xì)胞[45]。這些治療方法應(yīng)用低氧為靶向機(jī)制以運(yùn)送治療性物質(zhì)到特定的疾病部位，但由于低氧不僅是一些疾病的特征,還是正常生理情況下的動態(tài)過程，因此，這種治療方法可能會帶來額外的副作用。過去5年中，在腫瘤和HIF相關(guān)疾病中報(bào)道了很多具有抑制活性的HIF抑制劑[46]。盡管在腫瘤和其他HIF相關(guān)疾病中，關(guān)于HIF抑制劑的初步臨床研究取得了令人欣慰的結(jié)果，但由于HIF通路在RA中的復(fù)雜性以及服用HIF抑制劑后的藥代動力學(xué)問題，使得RA中這些抑制劑的研究尚不成熟，還有待于進(jìn)行臨床試驗(yàn)以評估其有效性[47]。有人提出局部應(yīng)用這些物質(zhì)(如關(guān)節(jié)內(nèi)注射)以減少全身應(yīng)用帶來的藥代動力學(xué)問題及副作用，但由于RA通常是多關(guān)節(jié)性的，因此這種方法很難應(yīng)用于臨床。

4 展望

低氧對RA的發(fā)病具有重要作用，但具體調(diào)控機(jī)制甚為復(fù)雜，目前尚不完全清楚。研究認(rèn)為，低氧通過HIF信號通路介導(dǎo)一些靶基因的活化促進(jìn)RA的病理過程，除此之外，低氧還調(diào)節(jié)免疫細(xì)胞的分化，與RA疾病的炎癥狀態(tài)相互促進(jìn)，與其他信號通路也存在交互作用，從而促進(jìn)RA病理的持續(xù)。因此，低氧不是通過某單一方面影響RA發(fā)病，而是可能在基因轉(zhuǎn)錄或蛋白水平對RA發(fā)病有著整體的影響作用。未來仍需要對低氧在RA發(fā)病機(jī)制中的作用進(jìn)行更深入的研究，對低氧調(diào)控機(jī)制的更深入理解將有助于以低氧為新的靶點(diǎn)對RA進(jìn)行治療。

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(2016-08-11收稿)

(本文編輯：趙 波)

SUMMARY Rheumatoid arthritis (RA) is a destructive chronic autoimmune disease characterized by synovium inflammation, cartilage destruction, bone erosion and the presence of autoantibodies. Hypoxia is a prominent micro-environmental feature in a range of disorders including RA. A combination of increased oxygen consumptionby inflamed resident cells and infiltrating immune cells along with a disrupted blood supply due to vascular dysfunction contribute to tissue hypoxia in RA. Hypoxia in turn regulates a number of key signaling pathways that help adaptation. The primary signaling pathway activated by hypoxia is the hypoxia-inducible factor (HIF) pathway. It has been shown that HIFs are highly expressed in the synovium of RA. HIFs mediate the pathogenesis of RA through inducing inflammation, angiogenesis, cell migration, and cartilage destruction, and inhibiting the apoptosis of synovial cells and inflammatory cells. HIF expressed in RA can be regulated in both oxygen-dependent and independent fashions, like inflammatory cytokines, leading to the aggravation of this disease. Considering the vital role of HIF in the pathogenesis of RA, we reviewed the new advances about hypoxia and RA. In this review, we firstly discussed the hypoxia-inducible factor and its regulation, and then, the pathologic role of hypoxia in RA, mainly elucidating the role of hypoxia in synovitis and cartilage destruction and immune cells. Finally, we provided evidence about the potential therapeutic target for treating RA.

Role of hypoxia-inducible factor in the pathogenesis of rheumatoid arthritis

YU Ruo-han, ZHAO Jin-xia, LIU Xiang-yuan△

(Department of Rheumatology and Immunology, Peking University Third Hospital, Beijing 100191, China)

Hypoxia-inducible factor; Arthritis, rheumatoid; Anoxia

國家自然科學(xué)基金(81273293，81471599)資助 Supported by National Natural Science Foundation of China (81273293, 81471599)

時間：2016-11-2 9：01：39

http://www.cnki.net/kcms/detail/11.4691.R.20161102.0901.002.html

R593.22

A

1671-167X(2016)06-1095-05

10.3969/j.issn.1671-167X.2016.06.031

△ Corresponding author’s e-mail, liu-xiangyuan@263.net