黃 芳，胡雅琳，姜程今，劉婧芳，何韶衡，李志剛

·論著·

白介素18在顳下頜關(guān)節(jié)炎中的作用研究

黃 芳1，胡雅琳1，姜程今1，劉婧芳1，何韶衡2*，李志剛3*

目的 探究白介素(IL)-18在顳下頜關(guān)節(jié)炎中的作用。方法 2015年11月—2016年4月，30只SPF級(jí)雄性BALB/c小鼠適應(yīng)性喂養(yǎng)1周后，采用隨機(jī)數(shù)字表法分為A、B、C、D、E組，每組6只。B組～E組在第1、21天時(shí)分別注射Ⅱ型膠原酶；從第1天開(kāi)始，A組僅注射0.9%氯化鈉溶液，B組注射0.9%氯化鈉溶液，C組注射IL-18，D組注射IL-18結(jié)合蛋白(IL-18BP)，E組注射IL-18、IL-18BP。第25天，采集各組小鼠外周血、組織灌洗液，采用流式細(xì)胞術(shù)檢測(cè)外周血單核細(xì)胞/顳下頜關(guān)節(jié)組織灌洗液巨噬細(xì)胞中白介素18受體(IL-18R)陽(yáng)性表達(dá)率，ELISA法檢測(cè)外周血血漿/顳下頜關(guān)節(jié)組織灌洗液上清液中腫瘤壞死因子(TNF)-α、IL-1β水平。結(jié)果 B組、C組小鼠外周血單核細(xì)胞中IL-18R陽(yáng)性表達(dá)率大于A組(P<0.05)；C組小鼠外周血單核細(xì)胞中IL-18R陽(yáng)性表達(dá)率大于B組、D組、E組(P<0.05)。B組～E組小鼠顳下頜關(guān)節(jié)組織灌洗液巨噬細(xì)胞中IL-18R陽(yáng)性表達(dá)率大于A組(P<0.05)；C組小鼠顳下頜關(guān)節(jié)組織灌洗液巨噬細(xì)胞中IL-18R陽(yáng)性表達(dá)率大于B組、D組、E組(P<0.05)。B組～E組小鼠外周血血漿中TNF-α、IL-1β水平高于A組(P<0.05)；C組小鼠外周血血漿中TNF-α、IL-1β水平高于B組、D組、E組(P<0.05)。B組～E組小鼠顳下頜關(guān)節(jié)組織灌洗液上清液中TNF-α、IL-1β水平高于A組(P<0.05)；C組小鼠顳下頜關(guān)節(jié)組織灌洗液上清液中TNF-α、IL-1β水平高于B組、D組、E組(P<0.05)；D組小鼠顳下頜關(guān)節(jié)組織灌洗液上清液中TNF-α水平低于B組(P<0.05)；E組小鼠顳下頜關(guān)節(jié)組織灌洗液上清液中TNF-α水平高于D組(P<0.05)。結(jié)論 IL-18能通過(guò)其受體IL-18R刺激單核細(xì)胞/巨噬細(xì)胞產(chǎn)生TNF-α與IL-1β等炎性因子，從而參與顳下頜關(guān)節(jié)炎的發(fā)生發(fā)展，推測(cè)IL-18R可能成為顳下頜關(guān)節(jié)炎的治療靶點(diǎn)。

顳下頜關(guān)節(jié)障礙；關(guān)節(jié)炎；白細(xì)胞介素18

黃芳,胡雅琳,姜程今,等.白介素18在顳下頜關(guān)節(jié)炎中的作用研究[J].中國(guó)全科醫(yī)學(xué)，2017，20(12)：1457-1462.[www.chinagp.net]

HUANG F,HU Y L,JIANG C J,et al.Role for IL-18 in temporomandibular joint arthritis[J].Chinese General Practice，2017，20(12)：1457-1462.

1.JinzhouMedicalUniversity,Jinzhou121000,China

2.AllergyandClinicalImmunologyResearchCentre,theFirstAffiliatedHospitalofJinzhouMedicalUniversity,Jinzhou121000,China

3.DepartmentofProsthodontics,theSecondAffiliatedHospitalofJinzhouMedicalUniversity,Jinzhou121000,China

*Correspondingauthor:HEShao-heng,Professor;E-mail:shoahenghe@126.com

LIZhi-gang,Professor;E-mail:ZGLi700103@163.com

骨關(guān)節(jié)炎是臨床常見(jiàn)病，其能導(dǎo)致各個(gè)關(guān)節(jié)(包括顳下頜關(guān)節(jié))劇痛和功能障礙。顳下頜關(guān)節(jié)炎是顳下頜關(guān)節(jié)紊亂綜合征的主要亞型之一，多發(fā)于20～30歲青壯年，93%的青少年自發(fā)性關(guān)節(jié)炎的早期癥狀可見(jiàn)于顳下頜關(guān)節(jié)[1]。研究發(fā)現(xiàn)，顳下頜關(guān)節(jié)炎與功能性運(yùn)動(dòng)綜合征如張口受限、顳下頜關(guān)節(jié)移動(dòng)彈響和下頜非對(duì)稱移動(dòng)存在著某種聯(lián)系[2-5]。因此早期診斷和及時(shí)治療顳下頜關(guān)節(jié)炎是至關(guān)重要的。

白介素(IL)-18是IL-1超因子家族的一員，是一種促炎性細(xì)胞因子，可由巨噬細(xì)胞、上皮細(xì)胞、T淋巴細(xì)胞、中性粒細(xì)胞、自然殺傷(NK)細(xì)胞和B淋巴細(xì)胞分泌，而單核細(xì)胞和巨噬細(xì)胞為其主要來(lái)源細(xì)胞[6-8]。IL-18在炎癥發(fā)生和免疫應(yīng)答中有促進(jìn)作用[6-7]。研究表明，IL-18與多種炎性疾病，包括缺血性再灌注損傷、移植抑制和自體免疫性疾病等有關(guān)[9-11]。WANNER等[12]對(duì)90例肩部關(guān)節(jié)炎患者進(jìn)行研究發(fā)現(xiàn)，IL-18水平在骨關(guān)節(jié)炎的早晚期均升高；李勇等[13]對(duì)30例膝關(guān)節(jié)炎患者的研究發(fā)現(xiàn)，IL-18水平的升高促進(jìn)了炎性因子前列腺素E2(PGE2)水平的升高，參與了骨關(guān)節(jié)炎的發(fā)生發(fā)展；GOUDA等[14]提取44例類風(fēng)濕關(guān)節(jié)炎患者滑膜液檢測(cè)到IL-18在患者體內(nèi)大量表達(dá)并促進(jìn)干擾素-γ(IFN-γ)的產(chǎn)生。通過(guò)以上研究可推測(cè)出IL-18與關(guān)節(jié)炎有密切聯(lián)系，但關(guān)于IL-18在顳下頜關(guān)節(jié)炎中的作用目前鮮有研究。白介素18受體(IL-18R)屬于IL-1受體/Toll樣受體(IL-1R/TLR)家族，是由IL-18Rα亞基和IL-18Rβ亞基組成的異質(zhì)二聚體，其中IL-18Rα亞基直接與IL-18特異性結(jié)合。IL-18結(jié)合蛋白(IL-18BP)可由單核細(xì)胞與巨噬細(xì)胞等細(xì)胞產(chǎn)生[15]，其通過(guò)與IL-18功能性結(jié)合，阻斷IL-18與IL-18R的相互作用進(jìn)而發(fā)揮抑制IL-18生理功能的作用[16]。因此，本實(shí)驗(yàn)通過(guò)檢測(cè)IL-18對(duì)單核細(xì)胞/巨噬細(xì)胞中IL-18R陽(yáng)性表達(dá)率及促炎性細(xì)胞因子水平的影響來(lái)探究其在顳下頜關(guān)節(jié)炎中的作用，以期為顳下頜關(guān)節(jié)炎的臨床治療提供借鑒及新靶點(diǎn)。

Selvester QRS積分系統(tǒng)在評(píng)估心肌梗死后心肌瘢痕負(fù)荷及預(yù)測(cè)遠(yuǎn)期預(yù)后方面具有較高的臨床價(jià)值。本研究結(jié)果表明，Selvester QRS積分在評(píng)估急性STEMI患者再灌注治療后3個(gè)月內(nèi)時(shí)，前壁心梗組的積分是逐步下降的，而下壁心梗組的積分則較為穩(wěn)定，因此，為了得到更準(zhǔn)確的評(píng)估結(jié)果，對(duì)于急性前壁心肌梗死患者，可在再灌注治療后3個(gè)月后實(shí)施，而對(duì)下壁心肌梗死的積分評(píng)估時(shí)間則無(wú)明確的限制。由于本研究為單中心研究，入選樣本量相對(duì)較小，且多為男性患者，因此研究結(jié)果可能存在偏倚，尚有待多中心或大樣本的臨床研究進(jìn)一步證實(shí)。

1 材料與方法

1.1 實(shí)驗(yàn)動(dòng)物 SPF級(jí)雄性BALB/c小鼠30只，8周齡，體質(zhì)量18～22 g，購(gòu)自北京維通利華實(shí)驗(yàn)動(dòng)物技術(shù)有限公司，批號(hào)：11400700056942。

1.2 實(shí)驗(yàn)藥物 Ⅱ型膠原酶(北京索萊寶科技有限公司，批號(hào)：17101-015)；IL-18(R&D Systems，批號(hào)：B004-5)；IL-18BP(R&D Systems，批號(hào)：122-BP-100)。實(shí)驗(yàn)前稱?、蛐湍z原酶1 g，完全溶于500 μl磷酸鹽緩沖液(PBS)中，配制出濃度為2 g/ml的Ⅱ型膠原酶；稱取IL-18、IL-18BP各1 μg，分別溶于1 ml PBS中，得到1 ml(1 μg/ml)原液，分別抽取1 μl原液加入99 μl PBS中，得到100 μl濃度為10 ng/ml的IL-18、IL-18BP，隨用隨配。

1.3 實(shí)驗(yàn)試劑 BV421-CD11b、APC/Cy7-Ly-6c、PE-F4-80、APC/Cy7-CD11c表面抗體(BioLegend)，APC-IL-18R細(xì)胞內(nèi)抗體(R&D Systems)，腫瘤壞死因子(TNF)-α、IL-1β ELISA試劑盒(北京達(dá)科為生物技術(shù)有限公司)，TNF-α抗體(Rockland公司)，IL-1β抗體(美國(guó)PeproTech公司)。

1.4 實(shí)驗(yàn)儀器 酶標(biāo)儀、普通臺(tái)式離心機(jī)、全自動(dòng)洗板機(jī)、-80 ℃冰箱(美國(guó)Thermo公司)，流式細(xì)胞儀(美國(guó)BD公司)，水浴鍋(上海精宏實(shí)驗(yàn)設(shè)備有限公司)。

1.5 研究方法

1.5.1 小鼠分組及處理 2015年11月—2016年4月，將30只小鼠置于室內(nèi)溫度為23～28 ℃、相對(duì)濕度為60%～75%的環(huán)境中，自由攝取標(biāo)準(zhǔn)嚙齒動(dòng)物的食物和水，適應(yīng)性喂養(yǎng)1周后，采用隨機(jī)數(shù)字表法將其分為A、B、C、D、E組，各6只。B組～E組在第1、21天時(shí)分別注射Ⅱ型膠原酶100 μl；從第1天開(kāi)始，A組僅注射0.9%氯化鈉溶液100 μl，B組注射0.9%氯化鈉溶液100 μl，C組注射IL-18 100 μl，D組注射IL-18BP 100 μl，E組注射IL-18、IL-18BP各100 μl；每3 d注射1次。具體操作如下：用2%異氟烷短暫麻醉后，修剪小鼠雙側(cè)顳下頜關(guān)節(jié)區(qū)域周圍毛發(fā)，注射器針尖透過(guò)皮膚向前直達(dá)顴弓，然后逐漸向前插入顴弓后下邊緣的下方，最終進(jìn)入關(guān)節(jié)腔，注入不同刺激劑，注射速度約為2 μl/s[17]。第25天，B組～E組小鼠表現(xiàn)為明顯的刮擦頭面部及縮頭反應(yīng)，提示炎癥出現(xiàn)，造模成功[18]。

1.5.2 標(biāo)本采集

1.5.2.2 組織灌洗液采集 第25天采集小鼠顳下頜關(guān)節(jié)組織灌洗液200 μl，提取顳下頜關(guān)節(jié)組織灌洗液上清液，-80 ℃保存?zhèn)溆谩?/p>

1.5.2.3 流式細(xì)胞術(shù)(flow cytometry，F(xiàn)CM)檢測(cè)外周血單核細(xì)胞/顳下頜關(guān)節(jié)組織灌洗液巨噬細(xì)胞中IL-18R陽(yáng)性表達(dá)率 取外周血/顳下頜關(guān)節(jié)組織灌洗液200 μl，加入BV421-CD11b、APC/Cy7-Ly-6c、PE-F4-80、APC/Cy7-CD11c表面抗體，室溫避光孵育15 min，加入1.5 ml紅細(xì)胞裂解液裂解紅細(xì)胞，室溫避光孵育12 min，1 200 r/min離心6 min(離心半徑5 cm)，棄上清液；加入1 ml 1×PBS洗掉被裂解過(guò)的破碎紅細(xì)胞，1 200 r/min離心6 min(離心半徑5 cm)，棄上清液；加入250 μl透膜固定液重懸細(xì)胞，4 ℃避光孵育20 min，加入洗液500 μl，洗掉透膜固定液，1 200 r/min離心6 min(離心半徑5 cm)，棄上清液；加入100 μl洗液重懸細(xì)胞，加入APC-IL-18R細(xì)胞內(nèi)抗體，4 ℃避光孵育30 min，加入洗液500 μl，1 200 r/min離心6 min(離心半徑5 cm)，棄上清液；加入300 μl 1×PBS重懸細(xì)胞沉淀，使細(xì)胞混勻，采用流式細(xì)胞儀檢測(cè)單核細(xì)胞/巨噬細(xì)胞中IL-18R陽(yáng)性表達(dá)率。實(shí)驗(yàn)獨(dú)立重復(fù)6次。

1.5.2.4 ELISA法檢測(cè)外周血血漿/顳下頜關(guān)節(jié)組織灌洗液上清液中TNF-α、IL-1β水平 設(shè)置標(biāo)準(zhǔn)孔、樣本孔和空白孔(標(biāo)準(zhǔn)孔和空白孔均設(shè)1個(gè)復(fù)孔)，加入100 μl提前稀釋好的標(biāo)準(zhǔn)液與樣本到各對(duì)應(yīng)孔中，輕輕混勻，封板，室溫孵育90 min；洗板3次，加入TNF-α、IL-1β一抗，輕輕混勻，封板，室溫孵育60 min；洗板3次，每孔加入鏈霉親和素標(biāo)記的辣根過(guò)氧化物酶結(jié)合一抗抗體，輕輕混勻，封板，室溫孵育30 min；洗板3次，各孔加入顯色液，室溫避光15 min，加入終止液，采用酶標(biāo)儀檢測(cè)450 nm處吸光度。實(shí)驗(yàn)獨(dú)立重復(fù)6次。

2 結(jié)果

2.1 各組小鼠外周血單核細(xì)胞中IL-18R陽(yáng)性表達(dá)率比較 5組小鼠外周血單核細(xì)胞中IL-18R陽(yáng)性表達(dá)率比較，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。其中B組、C組小鼠外周血單核細(xì)胞中IL-18R陽(yáng)性表達(dá)率均大于A組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)；C組小鼠外周血單核細(xì)胞中IL-18R陽(yáng)性表達(dá)率大于B組、D組、E組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05，見(jiàn)表1)。

Table 1 Comparison of positive expression rate of IL-18R in peripheral monocytes of mice among the five groups

組別IL-18R陽(yáng)性表達(dá)率A組0.30±0.06B組4.76±1.39aC組19.14±6.13abD組2.44±0.97cE組2.65±0.80cF值42.01P值<0.001

注：IL-18R=白介素18受體；與A組比較，aP<0.05；與B組比較，bP<0.05；與C組比較，cP<0.05

2.2 各組小鼠顳下頜關(guān)節(jié)組織灌洗液巨噬細(xì)胞中IL-18R陽(yáng)性表達(dá)率比較 5組小鼠顳下頜關(guān)節(jié)組織灌洗液巨噬細(xì)胞中IL-18R陽(yáng)性表達(dá)率比較，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。其中B組～E組小鼠顳下頜關(guān)節(jié)組織灌洗液巨噬細(xì)胞中IL-18R陽(yáng)性表達(dá)率大于A組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)；C組小鼠顳下頜關(guān)節(jié)組織灌洗液巨噬細(xì)胞中IL-18R陽(yáng)性表達(dá)率大于B組、D組、E組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05，見(jiàn)表2)。

Table 2 Comparison of positive expression rate of IL-18R in macrophages in temporomandibular joint lavage fluid of mice among the five groups

組別IL-18R陽(yáng)性表達(dá)率A組0.19±0.04B組5.00±0.95aC組15.69±1.76abD組4.44±0.68acE組4.78±0.64acF值204.27P值<0.001

注：與A組比較，aP<0.05；與B組比較，bP<0.05；與C組比較，cP<0.05

2.3 各組小鼠外周血血漿中TNF-α、IL-1β水平比較 5組小鼠外周血血漿中TNF-α、IL-1β水平比較，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。其中B組～E組小鼠外周血血漿中TNF-α、IL-1β水平高于A組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)；C組小鼠外周血血漿中TNF-α、IL-1β水平高于B組、D組、E組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05，見(jiàn)表3)。

Table 3 Comparison of levels of TNF-α and IL-1β in peripheral plasma of mice among the five groups

組別TNF-αIL-1βA組26.0±2.84.3±2.5B組71.7±8.0a11.2±2.2aC組141.4±7.6ab23.6±4.9abD組69.3±8.5ac11.8±2.2acE組69.7±3.5ac12.4±1.8acF值239.7733.28P值<0.001<0.001

注：TNF=腫瘤壞死因子，IL=白介素；與A組比較，aP<0.05；與B組比較，bP<0.05；與C組比較，cP<0.05

2.4 各組小鼠顳下頜關(guān)節(jié)組織灌洗液上清液中TNF-α、IL-1β水平比較 5組小鼠顳下頜關(guān)節(jié)組織灌洗液上清液中TNF-α、IL-1β水平比較，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。其中B組～E組小鼠顳下頜關(guān)節(jié)組織灌洗液上清液中TNF-α、IL-1β水平高于A組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)；C組小鼠顳下頜關(guān)節(jié)組織灌洗液上清液中TNF-α、IL-1β水平高于B組、D組、E組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)；D組小鼠顳下頜關(guān)節(jié)組織灌洗液上清液中TNF-α水平低于B組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)；E組小鼠顳下頜關(guān)節(jié)組織灌洗液上清液中TNF-α水平高于D組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05，見(jiàn)表4)。

Table 4 Comparison of levels of TNF-α and IL-1β in liquid supernatant of temporomandibular joint lavage fluid of mice among the five groups

組別TNF-αIL-1βA組25.9±2.54.3±2.4B組138.2±2.5a24.2±3.6aC組250.1±7.0ab68.3±7.8abD組132.1±4.6abc24.2±3.4acE組139.1±2.4acd24.4±3.6acF值2101.76161.03P值<0.001<0.001

注：與A組比較，aP<0.05；與B組比較，bP<0.05；與C組比較，cP<0.05；與D組比較，dP<0.05

3 討論

近年來(lái)越來(lái)越多研究顯示，IL-18在炎癥中有重要作用，在骨關(guān)節(jié)炎如肩關(guān)節(jié)炎[12]、膝關(guān)節(jié)炎[13]、類風(fēng)濕關(guān)節(jié)炎[14]等中有重要作用。KANEYAMA等[19-20]、SUZUKI等[21]發(fā)現(xiàn)，顳下頜關(guān)節(jié)炎患者的顳下頜關(guān)節(jié)組織灌洗液中TNF水平升高。BRENNAN等[22]、WATKINS等[23]、DUFF[24]發(fā)現(xiàn)，在關(guān)節(jié)滑膜組織中，TNF可通過(guò)破壞軟骨和骨組織、感受器致敏等途徑來(lái)促進(jìn)關(guān)節(jié)炎的發(fā)生發(fā)展。NORDAHL等[25-26]、NORTH等[27]發(fā)現(xiàn)，與檢測(cè)不到IL-1β的慢性關(guān)節(jié)炎患者相比，可檢測(cè)到IL-1β的慢性關(guān)節(jié)炎患者在X線片上的關(guān)節(jié)組織改變程度更大。KONTZIAS等[28]發(fā)現(xiàn)，IL-18對(duì)促炎性細(xì)胞因子的產(chǎn)生起到了至關(guān)重要的作用。

本研究結(jié)果顯示，B組、C組小鼠外周血單核細(xì)胞和B組～E組小鼠顳下頜關(guān)節(jié)組織灌洗液巨噬細(xì)胞中IL-18R陽(yáng)性表達(dá)率大于A組，表示Ⅱ型膠原酶可以引起小鼠顳下頜關(guān)節(jié)炎，造模成功。C組小鼠外周血單核細(xì)胞、顳下頜關(guān)節(jié)組織灌洗液巨噬細(xì)胞中IL-18R陽(yáng)性表達(dá)率大于B組、D組、E組，表示IL-18在顳下頜關(guān)節(jié)炎中起到促進(jìn)炎癥的作用；E組小鼠外周血單核細(xì)胞、顳下頜關(guān)節(jié)組織灌洗液巨噬細(xì)胞中IL-18R陽(yáng)性表達(dá)率與D組無(wú)明顯差異，表示IL-18BP預(yù)處理小鼠后，即使再有IL-18的刺激也不會(huì)使IL-18R表達(dá)水平增高。B組、C組小鼠外周血血漿、顳下頜關(guān)節(jié)組織灌洗液上清液中TNF-α、IL-1β水平高于A組，C組小鼠外周血血漿、顳下頜關(guān)節(jié)組織灌洗液上清液中TNF-α、IL-1β水平高于B組、D組、E組，提示IL-18在炎癥中起到激活單核細(xì)胞、巨噬細(xì)胞產(chǎn)生炎癥因子的作用；B組、E組小鼠顳下頜關(guān)節(jié)組織灌洗液上清液中TNF-α水平高于D組，推測(cè)炎癥產(chǎn)生后，即使加入IL-18BP，IL-18BP也未能競(jìng)爭(zhēng)性結(jié)合IL-18，從而使IL-18在炎癥中仍舊起到激活單核細(xì)胞/巨噬細(xì)胞產(chǎn)生炎性因子的作用。以上結(jié)果表示，IL-18不僅是促炎性細(xì)胞因子，更通過(guò)單核細(xì)胞/巨噬細(xì)胞上其相應(yīng)的受體IL-18R激活單核細(xì)胞/巨噬細(xì)胞，從而產(chǎn)生TNF-α與IL-1β等炎性因子，促進(jìn)并加快顳下頜關(guān)節(jié)炎的形成。

顳下頜關(guān)節(jié)炎是臨床較常見(jiàn)的口腔疾病之一，其總體預(yù)后較差。有報(bào)道以不同的診斷方法對(duì)顳下頜關(guān)節(jié)炎進(jìn)行評(píng)估發(fā)現(xiàn)，顳下頜關(guān)節(jié)炎在青少年中尤為普遍，而且17%～87%的人群有不同的亞型與分類[29-30]。顳下頜關(guān)節(jié)炎的治療方法多種多樣，如物理療法、社會(huì)心理支持療法、藥物治療、外科手術(shù)等。藥物治療包括非甾體抗炎藥(NSAIDs)、皮質(zhì)類固醇、抗風(fēng)濕藥物(DMARDs)等，均有療效，但各有不足，如NSAIDs主要對(duì)少數(shù)嚴(yán)重關(guān)節(jié)炎有效[31]；皮質(zhì)類固醇的使用目前僅在兔身上做過(guò)實(shí)驗(yàn)，還未曾在人及其他動(dòng)物身上獲取可靠結(jié)果[32]；DMARDs也不常應(yīng)用于顳下頜關(guān)節(jié)炎的治療。此外，還有地塞米松電離子透入療法(IP)，但會(huì)出現(xiàn)短暫性紅斑等不良反應(yīng)；TNF-α抑制劑可緩解顳下頜關(guān)節(jié)炎的疼痛，改善口腔功能[33]，但如何根治還鮮有報(bào)道。也有醫(yī)生采用手術(shù)或利用關(guān)節(jié)刺穿術(shù)治療顳下頜關(guān)節(jié)炎，但手術(shù)治療造成的失誤[34]和關(guān)節(jié)炎晚期[35]使其均不能成為最佳治療方法。本研究結(jié)果提示，可以通過(guò)特異性抑制或阻斷IL-18，防止其與IL-18R結(jié)合，抑制單核細(xì)胞/巨噬細(xì)胞的激活，下調(diào)IL-18等促炎性細(xì)胞因子的水平，從而降低炎癥發(fā)生率，推測(cè)IL-18可作為顳下頜關(guān)節(jié)炎的治療新靶點(diǎn)。

綜上所述，IL-18能通過(guò)其受體IL-18R刺激單核細(xì)胞/巨噬細(xì)胞產(chǎn)生TNF-α與IL-1β等炎性因子，從而參與顳下頜關(guān)節(jié)炎的發(fā)生發(fā)展，推測(cè)IL-18R可能成為顳下頜關(guān)節(jié)炎的治療靶點(diǎn)。本研究為動(dòng)物實(shí)驗(yàn)，今后應(yīng)進(jìn)一步行人體試驗(yàn)對(duì)本研究結(jié)果進(jìn)行驗(yàn)證與探究；且IL-18是否能通過(guò)其他形式刺激炎癥的產(chǎn)生，有待進(jìn)一步研究。

作者貢獻(xiàn)：黃芳、何韶衡、李志剛進(jìn)行文章的構(gòu)思與設(shè)計(jì)；黃芳進(jìn)行研究的施行、數(shù)據(jù)整理、統(tǒng)計(jì)學(xué)處理、撰寫(xiě)論文；何韶衡、李志剛進(jìn)行可行性分析；胡雅琳、姜程今、劉婧芳進(jìn)行數(shù)據(jù)收集；何韶衡、李志剛對(duì)文章整體負(fù)責(zé)，監(jiān)督管理。

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[1]SAURENMANN R.Clinical diagnosis of temporomandibular joint arthritis:a difficult task[J].J Rheumatol,2014,41(9):1734-1736.

[2]WEISS P F,ARABSHAHI B,JOHNSON A,et al.High prevalence of temporomandibular joint arthritis at disease onset in children with juvenile idiopathic arthritis,as detected by magnetic resonance imaging but not by ultrasound[J].Arthritis Rheum,2008,58(4):1189-1196.

[3]OLSON L,ECKERDAL O,HALLONSTEN A L,et al.Craniomandibular function in juvenile chronic arthritis.A clinical and radiographic study[J].Swed Dent J,1991,15(2):71-83.

[4]TWILT M,MOBERS S M,ARENDS L R,et al.Temporomandibular involvement in juvenile idiopathic arthritis[J].J Rheumatol,2004,31(7):1418-1422.

[5]PEDERSEN T K,KüSELER A,GELINECK J,et al.A prospective study of magnetic resonance and radiographic imaging in relation to symptoms and clinical findings of the temporomandibular joint in children with juvenile idiopathic arthritis[J].J Rheumatol,2008,35(8):1668-1675.

[6]DAO T,MEHAL W Z,CRISPE I N.IL-18 augments perforin-dependent cytotoxicity of liver NK-T cells[J].J Immunol,1998,161(5):2217-2222.

[7]YOSHIMOTO T,OKAMURA H,TAGAWA Y I,et al.Interleukin 18 together with interleukin 12 inhibits IgE production by induction of interferon-gamma production from activated B cells[J].Proc Natl Acad Sci U S A,1997,94(8):3948-3953.

[8]KRISHNAN S M,SOBEY C G,LATZ E,et al.IL-1β and IL-18:inflammatory markers or mediators of hypertension?[J].Br J Pharmacol,2014,171(24):5589-5602.DOI:10.1111/bph.12876.

[9]VENKATACHALAM K,PRABHU S D,REDDY V S,et al.Neutralization of interleukin-18 ameliorates ischemia/reperfusion-induced myocardial injury[J].J Biol Chem,2009,284(12):7853-7865.DOI:10.1074/jbc.M808824200.

[10]DUDLER J,SIMEONI E,FLEURY S,et al.Gene transfer of interleukin-18-binding protein attenuates cardiac allograft rejection[J].Transpl Int,2007,20(5):460-466.

[11]SHIMIZU C,MATSUMOTO K,FUJITA T,et al.Imbalance of interleukin-18 and interleukin-18 binding protein in patients with IgA nephropathy implicating renal vasculopathy[J].Clin Lab,2015,61(1/2):23-30.

[12]WANNER J,SUBBAIAH R,SKOMOROVSKA-PROKVOLIT Y,et al.Proteomic profiling and functional characterization of early and late shoulder osteoarthritis[J].Arthritis Res Ther,2013,15(6):R180.DOI:10.1186/ar4369.

[13]李勇,江建明,楊德鴻,等.骨關(guān)節(jié)炎關(guān)節(jié)滑液中白細(xì)胞介素18及其他相關(guān)因子含量測(cè)定[J].南方醫(yī)科大學(xué)學(xué)報(bào),2009,29(4):729-731. LI Y,JIANG J M,YANG D H,et al.Determination of the concentrations of interleukin-18 and other cytokines in the synovial fluid in patients with osteoarthritis[J].Journal of Southern Medical University,2009,29(4):729-731.

[14]GOUDA E A,ABOULATA A A,ELHAROUN A S,et al.Interleukin-18 expression in rheumatoid artheritis synovial tissue and its relation to disease activity[J].Egypt J Immunol,2007,14(2):1-10.

[15]VEENSTRA K G,JONAK Z L,TRULLI S,et al.IL-12 induces monocyte IL-18 binding protein expression via IFN-gamma[J].J Immunol,2002,168(5):2282-2287.

[16]NOVICK D,KIM S H,FANTUZZI G,et al.Interleukin-18 binding protein:a novel modulator of the Th1 cytokine response[J].Immunity,1999,10(1):127-136.

[17]KIM S H,SON C N,LEE H J,et al.Infliximab partially alleviates the bite force reduction in a mouse model of temporomandibular joint pain[J].J Korean Med Sci,2015,30(5):552-558.DOI:10.3346/jkms.2015.30.5.552.

[18]李波,盧利,譚學(xué)新,等.代謝型谷氨酸受體5在大鼠顳下頜關(guān)節(jié)疼痛中的作用[J].中國(guó)醫(yī)科大學(xué)學(xué)報(bào),2012,41(10):912-914. LI B,LU L,TAN X X,et al.Function of metabtrophic glutamate receptor subtype 5 for temporomandibular joint pain in rats[J].Journal of China Medical University,2012,41(10):912-914.

[19]KANEYAMA K,SEGAMI N,SUN W,et al.Levels of soluble cytokine factors in temporomandibular joint effusions seen on magnetic resonance images[J].Oral Surg Oral Med Oral Pathol Oral Radiol Endod,2005,99(4):411-418.DOI:10.1016/j.tripleo.2004.08.012.

[20]KANEYAMA K,SEGAMI N,NISHIMURA M,et al.Importance of proinflammatory cytokines in synovial fluid from 121 joints with temporomandibular disorders[J].Br J Oral Maxillofac Surg,2002,40(5):418-423.

[21]SUZUKI T,SEGAMI N,NISHIMURA M,et al.Co-expression of interleukin-1beta and tumor necrosis factor alpha in synovial tissues and synovial fluids of temporomandibular joint with internal derangement:comparison with histological grading of synovial inflammation[J].J Oral Pathol Med,2002,31(9):549-557.

[22]BRENNAN F M,MAINI R N,FELDMANN M.Role of pro-inflammatory cytokines in rheumatoid arthritis[J].Springer Semin Immunopathol,1998,20(1/2):133-147.

[23]WATKINS L R,WIERTELAK E P,GOEHLER L E,et al.Characterization of cytokine-induced hyperalgesia[J].Brain Res,1994,654(1):15-26.

[24]DUFF G W.Cytokines and acute phase proteins in rheumatoid arthritis[J].Scand J Rheumatol Suppl,1994,100:9-19.

[25]NORDAHL S,ALSTERGREN P,ELIASSON S,et al.Radiographic signs of bone destruction in the arthritic temporomandibular joint with special reference to markers of disease activity.A longitudinal study[J].Rheumatology (Oxford),2001,40(6):691-694.

[26]NORDAHL S,ALSTERGREN P,ELIASSON S,et al.Interleukin-1beta in plasma and synovial fluid in relation to radiographic changes in arthritic temporomandibular joints[J].Eur J Oral Sci,1998,106(1):559-563.

[27]NORTH J,SITUNAYAKE R D,TIKLY M,et al.Interleukin 1 beta,hand and foot bone mineral content and the development of joint erosions in rheumatoid arthritis[J].Ann Rheum Dis,1994,53(8):543-546.

[28]KONTZIAS A,EFTHIMIOU P.Adult-onset Still′s disease:pathogenesis,clinical manifestations and therapeutic advances[J].Drugs,2008,68(3):319-337.[29]TWILT M,MOBERS S M,ARENDS L R,et al.Temporomandibular involvement in juvenile idiopathic arthritis[J].J Rheumatol,2004,31(7):1418-1422.

[30]PEDERSEN T K,KUSELER A,GELINECK J,et al.A prospective study of magnetic resonance and radiographic imaging in relation to symptoms and clinical findings of the temporomandibular joint in children with juvenile idiopathic arthritis[J].J Rheumatol,2008,35(8):1668-1675.

[31]HASHKES P J,LAXER R M.Medical treatment of juvenile idiopathic arthritis[J].JAMA,2005,294(13):1671-1684.

[32]STOUSTRUP P,KRISTENSEN K D,KUSELER A,et al.Reduced mandibular growth in experimental arthritis in the temporomandibular joint treated with intra-articular corticosteroid[J].Eur J Orthod,2008,30(2):111-119.

[33]LAMAZZA L,GUERRA F,PEZZA M,et al.The use of etanercept as a non-surgical treatment for temporomandibular joint psoriatric arthritis:a case report[J].Aust Dent J,2009,54(2):161-165.DOI:10.1111/j.1834-7819.2009.01110.x.

[34]DOLWICK M F,DIMITROULIS G.Is there a role for temporomandibular joint surgery?[J].Br J Oral Maxillofac Surg,1994,32(5):307-313.

[35]AL-BELASY F A,DOLWICK M F.Arthrocentesis for the treatment of temporomandibular joint closed lock:a review article[J].Int J Oral Maxillofac Surg,2007,36(9):773-782.

(修回日期：崔麗紅)

Role for IL-18 in Temporomandibular Joint Arthritis

HUANGFang1,HUYa-lin1,JIANGCheng-jin1,LIUJing-fang1,HEShao-heng2*,LIZhi-gang3*

Objective To explore the role of interleukin-18 (IL-18) in temporomandibular joint arthritis.Methods This study was conducted from November 2015 to April 2016.After being adaptively fed for 1 week,30 male BALB/c mice (SPF grade) were randomized into 5 groups (group A,group B,group C,group D and group E) with 6 mice in each.Group B,group C,group D and group E were injected collagenase Ⅱ on the 1st and 21st days of intervention,respectively.From the 1st to the 24th days of intervention,group A and group B were injected 0.9% sodium chloride solution,group C,group D and group E were injected IL-18,IL-18 binding protein (IL-18BP),IL-18 and IL-18BP,respectively.On the 25th day of intervention,peripheral blood and temporomandibular joint lavage fluid of mice were collected.The positive expression rates of the interleukin-18 receptor (IL-18R) in monocytes in peripheral blood and macrophages in temporomandibular joint lavage fluid were detected by flow cytometry (FCM).The levels of TNF-α and IL-1β in peripheral plasma/liquid supernatant of temporomandibular joint lavage fluid were detected by enzyme-linked immunosorbent assay (ELISA).Results The positive expression rate of IL-18R in peripheral blood monocytes in group B and group C was significantly higher than that in group A (P<0.05);the positive expression rate of IL-18R in peripheral blood monocytes in group C was significantly higher than that in group B,group D and group E (P<0.05).The positive expression rate of IL-18R in macrophages in temporomandibular joint lavage fluid in group B,group C,group D and group E was higher than that in group A (P<0.05);the positive expression rate of IL-18R in macrophages in temporomandibular joint lavage fluid in group C was higher than that in group B,group D and group E (P<0.05).The levels of TNF-α and IL-1β in peripheral plasma in group B,group C,group D and group E were higher than those in group A (P<0.05);the levels of TNF-α and IL-1β in peripheral plasma in group C were higher than those in group B,group D and group E (P<0.05).The levels of TNF-α and IL-1β in liquid supernatant of temporomandibular joint lavage fluid in group B,group C,group D and group E were higher than those in group A (P<0.05);the levels of TNF-α and IL-1β in liquid supernatant of temporomandibular joint lavage fluid in group C were higher than those in group B,group D and group E (P<0.05);the level of TNF-α in liquid supernatant of temporomandibular joint lavage fluid in group D was lower than that in group B (P<0.05);the level of TNF-α in liquid supernatant of temporomandibular joint lavage fluid in group E was higher than that in group D (P<0.05).Conclusion IL-18 can produce pro-inflammatory cytokines such as TNF-α and IL-1β which may play an important role in the occurrence and development of temporomandibular joint arthritis through IL-18R stimulating monocytes/macrophages.IL-18R may be a novel therapy target for temporomandibular joint arthritis.

Temporomandibular joint disorders;Arthritis;Interleukin-18

國(guó)家自然科學(xué)基金資助項(xiàng)目(81471592)

R 782.6

A

10.3969/j.issn.1007-9572.2017.12.010

2016-09-12；

2016-12-30)

1.121000 遼寧省錦州市，錦州醫(yī)科大學(xué)

2.121000 遼寧省錦州市，錦州醫(yī)科大學(xué)附屬第一醫(yī)院變態(tài)反應(yīng)與臨床免疫研究中心

3.121000 遼寧省錦州市，錦州醫(yī)科大學(xué)附屬第二醫(yī)院修復(fù)科

*通信作者：何韶衡，教授；E-mail： shoahenghe@126.com

李志剛，教授；E-mail：ZGLi700103@163.com