桂皮醛對(duì)miRNA-146a干擾的骨關(guān)節(jié)炎滑膜炎性反應(yīng)影響的實(shí)驗(yàn)研究

王 歡 唐學(xué)章 丁海濤 張美麗 楊黎黎 張 棟 劉思婷 甘 穩(wěn) 徐銘康 郭玉茹 許 晶 王慶甫

(1 中日友好醫(yī)院,北京,100029; 2 北京中醫(yī)藥大學(xué),北京,100029; 3 北京中醫(yī)藥大學(xué)第三附屬醫(yī)院,北京,100000)

桂皮醛對(duì)miRNA-146a干擾的骨關(guān)節(jié)炎滑膜炎性反應(yīng)影響的實(shí)驗(yàn)研究

王 歡1唐學(xué)章1丁海濤1張美麗1楊黎黎2張 棟2劉思婷2甘 穩(wěn)2徐銘康2郭玉茹2許 晶2王慶甫3

(1 中日友好醫(yī)院,北京,100029; 2 北京中醫(yī)藥大學(xué),北京,100029; 3 北京中醫(yī)藥大學(xué)第三附屬醫(yī)院,北京,100000)

目的:研究miRNA-146a基因及桂皮醛對(duì)miRNA-146a干擾的滑膜成纖維細(xì)胞釋放TLR4、NO、MMP-13的影響,探尋其在OA滑膜炎性反應(yīng)分子學(xué)機(jī)制中的作用。方法:采用脂質(zhì)體轉(zhuǎn)染法將miR-146a基因模擬及抑制性質(zhì)粒載體對(duì)經(jīng)LPS誘導(dǎo)后的OA滑膜炎性反應(yīng)效應(yīng)細(xì)胞滑膜成纖維細(xì)胞進(jìn)行基因?qū)?并用桂皮醛對(duì)干擾后的細(xì)胞進(jìn)行干預(yù),檢測(cè)不同組間TLR4、NO及MMP-13表達(dá)的差異。結(jié)果:miRNA-146a相關(guān)質(zhì)粒轉(zhuǎn)染后的TLR4、NO、MMP-13濃度的變化趨勢(shì)大致相同:與對(duì)照組比較,三者的inhibitor組均升高,mimics組則均呈下降趨勢(shì)(P<0.05)。桂皮醛單獨(dú)作用于miRNA-146a干擾后的細(xì)胞時(shí),與對(duì)照組比較,TLR4、NO、MMP-13濃度均有明顯下降(P<0.05),而當(dāng)mimics和CA聯(lián)合干預(yù)時(shí),三者釋放量顯著下降(P<0.05)。結(jié)論:miRNA-146a及桂皮醛均對(duì)骨關(guān)節(jié)滑膜炎性反應(yīng)產(chǎn)生影響,當(dāng)mimics和桂皮醛聯(lián)合作用時(shí),抑制炎性反應(yīng)效果最好。為進(jìn)一步闡明骨關(guān)節(jié)炎分子機(jī)制和藥物靶點(diǎn)的識(shí)別與開(kāi)發(fā)提供實(shí)驗(yàn)依據(jù)。

骨關(guān)節(jié)炎;microRNA-146a;toll樣受體;滑膜炎性反應(yīng)

骨關(guān)節(jié)炎(Osteoarthritis,OA)普遍被認(rèn)為是一種以軟骨損傷為主,伴骨贅生成的多發(fā)性退行性疾病,而一些較新的參數(shù)表明,OA還是一種由固有免疫誘發(fā),以滑膜炎性反應(yīng)為主要病理變化的受微小RNA(microRNA,miRNA,miR)異常影響的多因素復(fù)雜疾病[1-5]。miRNA是一類能通過(guò)在轉(zhuǎn)錄后水平介導(dǎo)基因沉默來(lái)調(diào)控目的基因的表達(dá),廣泛參與各種生物進(jìn)程,在維持人體功能的細(xì)胞基因網(wǎng)絡(luò)中起著至關(guān)重要作用的“調(diào)節(jié)器”[6-7]。近年來(lái),隨著對(duì)miRNA認(rèn)識(shí)的逐步深入,一些它們?cè)贠A滑膜炎性反應(yīng)等自身免疫性疾病中作為重要的調(diào)節(jié)因子參與疾病的直接或間接的證據(jù)逐漸被發(fā)現(xiàn),科研人員將研究焦點(diǎn)越來(lái)越多的放在miRNA與固有免疫中一類重要的模式識(shí)別受體(Pattern Recognition Receptor,PRRs)——TLRs(Toll-like Receptors)相互作用及機(jī)制的探索上[8-14]。

本研究采用脂質(zhì)體轉(zhuǎn)染法將固有免疫反應(yīng)中的重要調(diào)節(jié)因子miR-146a基因模擬及抑制性質(zhì)粒載體對(duì)經(jīng)LPS誘導(dǎo)后的OA滑膜炎性反應(yīng)效應(yīng)細(xì)胞滑膜成纖維細(xì)胞(Fibroblast-like Synoviocytes,FLS)進(jìn)行基因?qū)?并用具有抗炎、鎮(zhèn)痛等作用的新興中藥提取物桂皮醛(Cinnamic Aldehyde,CA)對(duì)干擾后的細(xì)胞進(jìn)行干預(yù),通過(guò)對(duì)不同組間TLR4、NO及MMP-13的表達(dá)差異進(jìn)行檢測(cè),研究桂皮醛對(duì)miR-146a干擾的OA滑膜炎性反應(yīng)的影響,旨在對(duì)OA的分子學(xué)機(jī)制有更明晰的了解,以期為藥物靶點(diǎn)的識(shí)別和開(kāi)發(fā)提供新思路。

1 材料與方法

1.1 材料

1.1.1 組織采集 無(wú)菌滑膜組織取自于2016年1月—3月北京中醫(yī)藥大學(xué)第三附屬醫(yī)院行膝關(guān)節(jié)置換術(shù)的原發(fā)性膝OA患者(男、女各2例,年齡55～75歲),樣例均符合美國(guó)風(fēng)濕病學(xué)會(huì)制訂的OA診斷標(biāo)準(zhǔn)。

1.1.2 試劑 桂皮醛(HPLC≥98%),源葉生物,LOT:H02M6Q1;Lipopolysaccharides from Escherichia coli,Sigma,LOT:084M4107V;Collagenase Type II,Gibco,LOT:1430519;0.25% Trypsin-EDTA,Gibco,LOT:1737903;DMEM/F12(1∶1)培養(yǎng)基,Gibco,LOT:1737884;FBS,Gibco,LOT:1036489;Dulbecco′s Phosphate Buffered Saline,Gibco,LOT:8115155;Lipofetctamine2000,invitrogen,LOT:1612516;has-miR-146a mimics/inhibitor/N.C,吉瑪基因;TLR4(Human)ELISA Kit,Abnova,LOT:5C106L;MMP-13(Human)ELISA Kit,Abcam,Lot:GR188683-1。

1.2 方法

1.2.1 滑膜成纖維細(xì)胞的體外分離和培養(yǎng) 術(shù)后2 h內(nèi)將所取樣本在盛有Dulbecco′s Phosphate Buffered Saline的無(wú)菌培養(yǎng)皿中漂洗3次,手術(shù)剪修整留用滑膜層,再次漂洗后將組織剪成糊狀,加入組織量20倍體積的Collagenase Type II,37 ℃、5% CO2的飽和濕度培養(yǎng)箱中消化培養(yǎng)1.5～2 h,100目不銹鋼篩網(wǎng)過(guò)濾,以1 500 r/min離心10 min后,收集細(xì)胞懸液接種到培養(yǎng)瓶中,當(dāng)細(xì)胞生長(zhǎng)至約瓶底面積的80%時(shí),以1∶(2～3)傳代比例進(jìn)行傳代。取對(duì)數(shù)生長(zhǎng)期的細(xì)胞(3～5代)用于實(shí)驗(yàn)。

1.2.2 細(xì)胞轉(zhuǎn)染及藥物干預(yù) 將對(duì)數(shù)生長(zhǎng)期的FLS制備成細(xì)胞懸液以5×105個(gè)/孔的細(xì)胞密度接種于6孔細(xì)胞培養(yǎng)板,37 ℃、5% CO2的飽和濕度培養(yǎng)箱中過(guò)夜。1 μg/mL Lipopolysaccharides from Escherichia coli(LPS)誘導(dǎo)24 h后進(jìn)行細(xì)胞轉(zhuǎn)染,分組如下:1)inhibitor組:Lipofetctamine2 000+miR-146a inhibitor;2)mimics組:Lipofetctamine2 000+miR-146a mimics;3)inhibitor N.C組:Lipofetctamine2 000+miR-146a inhibitor N.C;4)N.C組:Lipofetctamine2 000+N.C;5)lipo2 000組:Lipofetctamine2 000;6)對(duì)照組;7)空白組:細(xì)胞培養(yǎng)液(未經(jīng)LPS誘導(dǎo))。相同條件的細(xì)胞培養(yǎng)箱孵育6 h進(jìn)行轉(zhuǎn)染反應(yīng),換含血清的培養(yǎng)基后,繼續(xù)孵育48 h收集各孔內(nèi)上清液進(jìn)行檢測(cè)。上述miR-146a干擾后的細(xì)胞加入受試液:1)inhibitor+CA組:上述inhibitor組+LPS(1 μg/mL)+CA(10 μg/mL);2)mimics+CA組:上述mimics組+LPS(1 μg/mL)+CA(10 μg/mL);3)CA組:LPS(1 μg/mL)+CA(10 μg/mL);4)對(duì)照組:LPS(1 μg/mL);5)空白組:細(xì)胞培養(yǎng)液。相同條件再孵育48 h后,收集各孔內(nèi)上清液進(jìn)行檢測(cè)。

1.2.3 相關(guān)檢測(cè) 用Griess法對(duì)NO的釋放量測(cè)定:用雙蒸餾水溶解并稀釋亞硝酸鈉溶液,以100 μmol/L為起始濃度2倍稀釋,用于標(biāo)準(zhǔn)梯度曲線的繪制。A工作液(1%的對(duì)氨基苯磺酸溶液)和B工作液(0.1%的N-(1-萘基)乙二胺二鹽酸鹽溶液)各50 μL/孔,加入標(biāo)準(zhǔn)貯備液或待測(cè)樣品室溫靜置30 min,酶標(biāo)儀540 nm處測(cè)得各孔吸光值。用ELISA Kit對(duì)TLR4及MMP-13的含量進(jìn)行測(cè)定(按試劑盒說(shuō)明書(shū)操作)。

2 結(jié)果

2.1 miR-146a表達(dá)水平對(duì)TLR4、NO和MMP-13釋放量的影響 經(jīng)過(guò)miR-146a干擾后的細(xì)胞表現(xiàn)出TLR及通路下游炎性遞質(zhì)的差異性釋放:1)首先,對(duì)照組炎性遞質(zhì)的釋放量顯著高于空白組,證明LPS誘導(dǎo)炎性反應(yīng)成功;2)巧合的是,TLR4、NO、MMP-13在miR-146a干擾各組的變化趨勢(shì)大致相同;3)與對(duì)照組比較,三者的inhibitor組均升高,mimics組則均呈下降趨勢(shì)(P<0.05);4)另外作為對(duì)照參數(shù)的3組(inhibitor N.C組、N.C組和lipo2 000組)與對(duì)照組數(shù)值均較接近,差異基本無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05),可認(rèn)為幾組之間不存在差別,說(shuō)明用于本實(shí)驗(yàn)的質(zhì)粒(miR-146a inhibitor、miR-146a mimics)及轉(zhuǎn)染劑(Lipofetctamine2 000)均起效并且對(duì)細(xì)胞無(wú)明顯細(xì)胞毒性等影響。見(jiàn)表1。

表1 miR-146a表達(dá)水平對(duì)TLR4、NO和MMP-13釋放量的影響

3.2 CA對(duì)miR-146a干擾的FLS TLR4濃度的影響 CA單獨(dú)作用于FLS后,與對(duì)照組比較,TLR4濃度明顯下降(P<0.05),說(shuō)明CA能抑制TLR4的釋放;但是,與空白組比較可見(jiàn),TLR4濃度仍較高,無(wú)法降至未做誘導(dǎo)的細(xì)胞水平。當(dāng)inhibitor和CA聯(lián)合干預(yù)時(shí),TLR4濃度與對(duì)照組比較略微下降;而當(dāng)mimics和CA聯(lián)合干預(yù)時(shí),TLR4濃度下降程度最高。見(jiàn)圖1。

圖1 CA對(duì)miR-146a干擾的FLS TLR4濃度的影響

2.3 CA對(duì)miR-146a干擾的FLS NO、MMP-13釋放量的影響 CA對(duì)miR-146a干擾的FLS NO、MMP-13釋放量的影響趨勢(shì)大致相同:首先,與TLR4相似地,和對(duì)照組比較,CA單獨(dú)作用時(shí)的FLS NO、MMP-13釋放量均有明顯下降(P<0.05),說(shuō)明CA對(duì)NO、MMP-13釋放有顯著的抑制作用,但與空白組比較可見(jiàn),仍無(wú)法降至未做誘導(dǎo)的細(xì)胞水平。當(dāng)inhibitor和CA聯(lián)合干預(yù)時(shí),NO、MMP-13釋放量仍維持在相對(duì)較高的水平;而當(dāng)mimics和CA聯(lián)合干預(yù)時(shí),NO、MMP-13釋放量出現(xiàn)極其顯著的下降(P<0.05)。結(jié)果見(jiàn)圖2、3。

圖2 CA對(duì)miR-146a干擾的FLS NO濃度的影響

圖3 CA對(duì)miR-146a干擾的FLS MMP-13濃度的影響

3 討論

OA是一種在長(zhǎng)期機(jī)械作用和炎性反應(yīng)等壓力下由一系列復(fù)雜機(jī)制驅(qū)動(dòng)的涉及關(guān)節(jié)軟骨(Articular Cartilage,AC)、滑膜等多組織損傷導(dǎo)致體內(nèi)穩(wěn)態(tài)失衡的慢性臨床綜合征[16-18]。相對(duì)于其他疾病,OA以其高患病率、進(jìn)展的致殘率和疼痛為特點(diǎn)成為最常見(jiàn)的關(guān)節(jié)疾病[19-20],影響著世界各地超過(guò)20%的人群,預(yù)計(jì)到2020年,OA的患病率可能會(huì)飆升至57%,是最亟待解決的醫(yī)療和社會(huì)問(wèn)題[21-22]。但目前臨床上普遍應(yīng)用的治療OA的方法對(duì)緩解疼痛、修復(fù)相關(guān)組織損傷和改善關(guān)節(jié)功能的治療目標(biāo)尚未能達(dá)到滿意的療效。保守治療包括物理治療配合常用的藥物如鎮(zhèn)痛劑、非甾體類抗炎藥(Non-steroidal Anti-inflammatory Drugs,NSAIDs)、選擇性的環(huán)加氧酶-2(Cyclo-oxygenase-2,COX-2)抑制劑,類固醇抗炎藥和糖皮質(zhì)激素注射等。然而,它們只能暫時(shí)的對(duì)OA的某些臨床癥狀起到有限的緩解,卻不能對(duì)損傷的AC等組織行使修復(fù)與保護(hù)等功能。此外,長(zhǎng)期的大劑量的使用(The Epidemiology,Etiology,Diagnosis,and Treatment of Osteoarthritis of the Knee)這些藥物還存在著一些潛在的安全隱患,如增加胃腸道反應(yīng)、肝臟損傷、心血管疾病等不良反應(yīng)發(fā)生的風(fēng)險(xiǎn)[23-26]。關(guān)節(jié)置換手術(shù)是對(duì)保守治療療效欠佳的重度OA患者的最終選擇也是公認(rèn)有效的治療方法。但不易被患者普遍接受,而且手術(shù)相關(guān)的一些嚴(yán)重的并發(fā)癥如感染、深靜脈血栓形成和后續(xù)使用中的假體松脫也是應(yīng)該被考慮到的問(wèn)題[27-29]。因此,開(kāi)發(fā)一種新的藥物既能對(duì)終止或逆轉(zhuǎn)OA進(jìn)程有足夠的治療效果,又能盡可能地避免幾乎可以忽略不計(jì)不良反應(yīng)是值得關(guān)注的要點(diǎn)問(wèn)題。最近,研究人員發(fā)現(xiàn),一些從天然產(chǎn)物分離出的小分子或生物活性成分可通過(guò)各種途徑有效的減輕疼痛、緩解OA炎性反應(yīng)。桂皮醛是從樟科植物桂皮精油中分離出的主要生物活性化合物,也是具有發(fā)汗解肌、溫經(jīng)通脈功能的中藥桂枝的主要有效成分。研究表明,CA及其衍生物顯示出良好的免疫調(diào)節(jié)性能,它能通過(guò)對(duì)相關(guān)炎性反應(yīng)通路的調(diào)節(jié)減少LPS誘導(dǎo)的巨噬細(xì)胞促炎性遞質(zhì)表達(dá),抑制NF-κB的激活和相關(guān)下游炎性細(xì)胞因子的分泌[30];還能減少IL-1β誘導(dǎo)的COX-2的激活和PGE2的產(chǎn)生[31];在TNF-α處理的內(nèi)皮細(xì)胞中可抑制單核細(xì)胞與內(nèi)皮細(xì)胞的黏附[32]。而且細(xì)胞毒性小,并有一定的組織器官保護(hù)作用,是一種新型的抗炎劑[33]。

我們逐漸認(rèn)識(shí)到,加深對(duì)OA發(fā)生發(fā)展的分子機(jī)制的理解,有助于幫助發(fā)現(xiàn)導(dǎo)致OA的關(guān)鍵途徑和分子,進(jìn)一步推動(dòng)新療法的發(fā)展。越來(lái)越多的研究發(fā)現(xiàn),滑膜細(xì)胞的固有免疫應(yīng)答反應(yīng)是OA進(jìn)程中的關(guān)鍵環(huán)節(jié)[34-35]。健壯的固有免疫系統(tǒng)是對(duì)感染的即時(shí)防御及對(duì)病原體的持久抵抗至關(guān)重要的屏障。然而,如果它被不適當(dāng)?shù)募せ詈徒K止,就會(huì)危害宿主健康,導(dǎo)致一系列急性和慢性炎性反應(yīng)紊亂的病理狀態(tài)的臨床表現(xiàn)[36]。因此,眾多復(fù)雜的分子機(jī)制從多個(gè)層次共同調(diào)節(jié)以保持炎性反應(yīng)受到抑制,這些“調(diào)節(jié)器”種類眾多,從可溶性受體到誘導(dǎo)的細(xì)胞內(nèi)蛋白質(zhì)[37-40]。Toll樣受體(TLRs)是固有免疫系統(tǒng)中一類分子量在90-15OkDa之間的PRRs,在啟動(dòng)和調(diào)控固有免疫系統(tǒng)中扮演著重要的角色[41-42]。這種跨膜糖蛋白受體大量表達(dá)于OA滑膜細(xì)胞上[43-44],其中,于1997年由Medzhitov.R等發(fā)現(xiàn)的第一個(gè)哺乳動(dòng)物TLR——TLR4在誘導(dǎo)炎性反應(yīng)及相關(guān)基因的表達(dá)和修復(fù)受損關(guān)節(jié)組織上有著不可忽視的作用[45-46]。越來(lái)越多的研究試圖通過(guò)對(duì)TLRs的調(diào)控進(jìn)而干預(yù)由其參與激活的炎性反應(yīng)通路的下游炎性遞質(zhì)的釋放,最終達(dá)到緩解OA炎性反應(yīng)的目的。

miRNA是一種能幾乎在各個(gè)水平對(duì)TLRs及其介導(dǎo)的相關(guān)通路進(jìn)行調(diào)控,廣泛參與到OA的生理病理學(xué),維持AC、滑膜等組織各項(xiàng)功能的負(fù)向調(diào)機(jī)器[47-49]。這類長(zhǎng)度約為20～25個(gè)核苷酸(Nucleotide,nt)的內(nèi)源性非編碼單鏈小分子RNA分子能識(shí)別靶mRNA上的3′非翻譯區(qū)(Untranslated Regions,UTR)并對(duì)其部分互補(bǔ)的位點(diǎn)進(jìn)行結(jié)合綁定,通過(guò)對(duì)靶mRNA降解或阻礙翻,調(diào)節(jié)靶基因的蛋白質(zhì)生成[50-51]。近年來(lái),人們逐漸發(fā)現(xiàn)細(xì)胞在免疫系統(tǒng)的發(fā)育和功能上尤其受到miRNA的調(diào)控[52-54]。作為OA進(jìn)程中強(qiáng)勁的轉(zhuǎn)錄后調(diào)節(jié)器,從細(xì)胞命運(yùn)決定到信號(hào)活動(dòng)的參與,miRNA構(gòu)成了一個(gè)新的固有免疫應(yīng)答的調(diào)節(jié)層[55-57]。尤其是第一個(gè)被發(fā)現(xiàn)可調(diào)節(jié)免疫系統(tǒng)的miRNA——miR-146,其中位于其5號(hào)染色體上的家族成員之一miR-146a是一種NF-kB依賴型基因,在TLRs介導(dǎo)的NF-κB炎性反應(yīng)通路上直接調(diào)節(jié)2個(gè)關(guān)鍵的下游適配器分子——腫瘤壞死因子相關(guān)受體相關(guān)因子6(TNF Receptor-associated Factor,TRAF6)和白介素1受體相關(guān)激酶(interleukin 1 receptor associated kinase,IRAK1)的蛋白水平,負(fù)反饋循環(huán)控制TLRs和細(xì)胞因子促炎性信號(hào)作用[58-61]。作為一種新興的可獨(dú)立影響OA發(fā)生發(fā)展的基因,其表達(dá)的抑制可能是促發(fā)OA的重要影響因素,該基因也可作為診斷OA的生物學(xué)標(biāo)志[62-63]。

越來(lái)越多的證據(jù)表明對(duì)miRNA的管制可以有效調(diào)控OA,研究者們?cè)噲D通過(guò)干擾某些miRNA在細(xì)胞中的表達(dá)和活動(dòng)以明確它們?cè)诩膊∵M(jìn)程中的具體作用,一種將外源性的miRNA的模擬劑(Mimics)和抑制劑(Inhibitors)利用轉(zhuǎn)染試劑人工的參入宿主細(xì)胞,使其獲得新的遺傳標(biāo)志的方法應(yīng)運(yùn)而生。Lipofetamin2 000是一種當(dāng)前應(yīng)用廣泛且毒性較低的高效陽(yáng)離子脂質(zhì)體轉(zhuǎn)染試劑,可瞬時(shí)的將人工合成的外源性基因序列導(dǎo)入細(xì)胞內(nèi),影響相關(guān)基因的表達(dá)。

本研究利用Lipofetamin2 000轉(zhuǎn)染法將促進(jìn)和抑制性miR-146a質(zhì)粒轉(zhuǎn)染入FLS,選擇一種代表性的TLRs TLR4及其介導(dǎo)的信號(hào)通路下游釋放的典型炎性遞質(zhì)NO和OA進(jìn)程中誘發(fā)膠原蛋白降解和破壞的主要物質(zhì)MMP-13,幾種重要因子聯(lián)合檢測(cè),有助于更全面、高效的證實(shí)它們?cè)贠A滑膜炎性反應(yīng)中的變化趨勢(shì)和交互作用,以推測(cè)OA的進(jìn)展,提供合理的病情評(píng)估以指導(dǎo)臨床治療用藥。結(jié)果顯示,miR-146a inhibitor可上調(diào)FLS釋放TLR4、NO、MMP-13水平;miR-146a mimics則可使三者濃度均減低,且miR-146a干擾后三者的變化趨勢(shì)相似。以上結(jié)果表明,miR-146a基因在FLS的目標(biāo)干擾(上調(diào)或下降)可以對(duì)TLRs及其信號(hào)通路下游的炎性遞質(zhì)和金屬蛋白酶進(jìn)行調(diào)節(jié),進(jìn)一步影響OA滑膜炎性反應(yīng)。

現(xiàn)有研究對(duì)miR-146a在OA中大致的調(diào)節(jié)機(jī)制及其所發(fā)揮的調(diào)節(jié)作用已有一定的了解,但它們之間進(jìn)一步的調(diào)控機(jī)制和聯(lián)合藥物使用對(duì)OA影響的研究仍處于初期階段。所以在上述研究的基礎(chǔ)上,我們基于已有數(shù)據(jù)的提示發(fā)現(xiàn),miR-146a作為一個(gè)OA滑膜炎性反應(yīng)炎性反應(yīng)的分子抑制劑扮演著重要角色。隨后,我們?cè)趍iR-146a表達(dá)的消融導(dǎo)致在一些免疫相關(guān)的改變的基礎(chǔ)上,選取一種新型的具有抗炎活性及潛在保護(hù)作用的生物活性化合物CA結(jié)合miRNA干擾技術(shù)來(lái)共同治療OA滑膜炎性反應(yīng)。結(jié)果顯示,CA本身就有抑制TLR4、NO、MMP-13表達(dá)的效果,當(dāng)inhibitor和CA聯(lián)合干預(yù)時(shí),CA可抵消部分miR-146a缺失所帶來(lái)的炎性反應(yīng)問(wèn)題;而當(dāng)mimics和CA聯(lián)合干預(yù)時(shí),就顯示出了對(duì)OA滑膜炎性反應(yīng)優(yōu)勢(shì)的抑制作用,考慮它們很可能能協(xié)同通過(guò)TLRs調(diào)控其信號(hào)通路上的其他組件共同抑制滑膜炎性反應(yīng)。該研究結(jié)果在明確miR-146a干擾作用的同時(shí),對(duì)其聯(lián)合中藥有效成分的抗炎作用進(jìn)行評(píng)估,從基因?qū)用鏋檫M(jìn)一步了解OA滑膜炎性反應(yīng)機(jī)制、選擇合理的藥物進(jìn)行靶向治療以控制病情進(jìn)展及組織修復(fù)提供了新的方案。

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ExperimentalStudyonEffectsofCinnamicAldehydeonSynovialInflammationinOsteoarthriticBasedontheMicroRNA-146aInterference

Wang Huan1，Tang Xuezhang1，Ding Haitao1，Zhang Meili1，Yang Lili2，Zhang Dong2，Liu Siting2，Gan Wen2，Xu Mingkang2，Guo Yuru2，Xu Jing2，Wang Qingfu3

(1China-JapanFriendshipHospital，Beijing100029，China; 2BeijingUniversityofChineseMedicine，Beijing100029，China; 3TheThirdAffiliatedHospitalofBeijingUniversityofChineseMedicine，Beijing100029，China)

Objective:To investigate the influence of miRNA-146a gene disturbances and cinnamic aldehyde (CA) on TLR4，NO，and MMP-13，which was released by synovioblast，and to explore their roles in the molecular mechanism of synovial inflammation in osteoarthritis (OA).MethodsPlasmids of miR-146a gene mimics and inhibitors were loaded into LPS-induced FLS using lipofection transfection.Then the interferential FLS were treated with CA，and the release amount of TRL4，NO and MMP-13 were detected.ResultsAfter the miRNA-146a related plasmid transfection，the variation tendency of the concentrations of TLR4，NO and MMP-13 were almost the same.Compared with control group，the inhibitor group was elevated，and mimics group was declined (P<0.05).Compared with the control group，when the interferential FLS was treated with CA alone，TLR4，its release of TLR4，NO and MMP-13 were significantly decreased (P<0.05)，whereas the mimics and CA coordinated intervention，the release was significantly decreased (P<0.05).ConclusionBoth of the miR-146a and CA have effects on synovial inflammation in OA，especially when mimics and CA were combined，the inhibition of inflammation was best.It could provide an experimental basis to clarify molecular mechanism，identify and develop novel drug targets of OA.

Osteoarthritis; MicroRNA-146a; Cinnamic aldehyde; Toll-like receptors; Synovial inflammation

國(guó)家自然科學(xué)基金面上項(xiàng)目(81373662);北京中醫(yī)藥大學(xué)自主選題項(xiàng)目(2015-JYB-XS202)

王歡(1986.04—),女,博士研究生,醫(yī)師,研究方向:中醫(yī)藥防治退行性骨關(guān)節(jié)病,E-mail：pulongqi@126.com

王慶甫(1956.10—),男,碩士,主任醫(yī)師,博士研究生導(dǎo)師,教授,研究方向:中醫(yī)藥防治退行性骨關(guān)節(jié)病,E-mail:qingpu-wang@sohu.com

R274.3

A

10.3969/j.issn.1673-7202.2017.10.034

(2016-08-16收稿 責(zé)任編輯：王明)