桂皮醛對miRNA-146a干擾的骨關節炎滑膜炎性反應影響的實驗研究

王 歡 唐學章 丁海濤 張美麗 楊黎黎 張 棟 劉思婷 甘 穩 徐銘康 郭玉茹 許 晶 王慶甫

(1 中日友好醫院,北京,100029; 2 北京中醫藥大學,北京,100029; 3 北京中醫藥大學第三附屬醫院,北京,100000)

桂皮醛對miRNA-146a干擾的骨關節炎滑膜炎性反應影響的實驗研究

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

(1 中日友好醫院,北京,100029; 2 北京中醫藥大學,北京,100029; 3 北京中醫藥大學第三附屬醫院,北京,100000)

目的:研究miRNA-146a基因及桂皮醛對miRNA-146a干擾的滑膜成纖維細胞釋放TLR4、NO、MMP-13的影響,探尋其在OA滑膜炎性反應分子學機制中的作用。方法:采用脂質體轉染法將miR-146a基因模擬及抑制性質粒載體對經LPS誘導后的OA滑膜炎性反應效應細胞滑膜成纖維細胞進行基因導入,并用桂皮醛對干擾后的細胞進行干預,檢測不同組間TLR4、NO及MMP-13表達的差異。結果:miRNA-146a相關質粒轉染后的TLR4、NO、MMP-13濃度的變化趨勢大致相同:與對照組比較,三者的inhibitor組均升高,mimics組則均呈下降趨勢(P<0.05)。桂皮醛單獨作用于miRNA-146a干擾后的細胞時,與對照組比較,TLR4、NO、MMP-13濃度均有明顯下降(P<0.05),而當mimics和CA聯合干預時,三者釋放量顯著下降(P<0.05)。結論:miRNA-146a及桂皮醛均對骨關節滑膜炎性反應產生影響,當mimics和桂皮醛聯合作用時,抑制炎性反應效果最好。為進一步闡明骨關節炎分子機制和藥物靶點的識別與開發提供實驗依據。

骨關節炎;microRNA-146a;toll樣受體;滑膜炎性反應

骨關節炎(Osteoarthritis,OA)普遍被認為是一種以軟骨損傷為主,伴骨贅生成的多發性退行性疾病,而一些較新的參數表明,OA還是一種由固有免疫誘發,以滑膜炎性反應為主要病理變化的受微小RNA(microRNA,miRNA,miR)異常影響的多因素復雜疾病[1-5]。miRNA是一類能通過在轉錄后水平介導基因沉默來調控目的基因的表達,廣泛參與各種生物進程,在維持人體功能的細胞基因網絡中起著至關重要作用的“調節器”[6-7]。近年來,隨著對miRNA認識的逐步深入,一些它們在OA滑膜炎性反應等自身免疫性疾病中作為重要的調節因子參與疾病的直接或間接的證據逐漸被發現,科研人員將研究焦點越來越多的放在miRNA與固有免疫中一類重要的模式識別受體(Pattern Recognition Receptor,PRRs)——TLRs(Toll-like Receptors)相互作用及機制的探索上[8-14]。

本研究采用脂質體轉染法將固有免疫反應中的重要調節因子miR-146a基因模擬及抑制性質粒載體對經LPS誘導后的OA滑膜炎性反應效應細胞滑膜成纖維細胞(Fibroblast-like Synoviocytes,FLS)進行基因導入,并用具有抗炎、鎮痛等作用的新興中藥提取物桂皮醛(Cinnamic Aldehyde,CA)對干擾后的細胞進行干預,通過對不同組間TLR4、NO及MMP-13的表達差異進行檢測,研究桂皮醛對miR-146a干擾的OA滑膜炎性反應的影響,旨在對OA的分子學機制有更明晰的了解,以期為藥物靶點的識別和開發提供新思路。

1 材料與方法

1.1 材料

1.1.1 組織采集 無菌滑膜組織取自于2016年1月—3月北京中醫藥大學第三附屬醫院行膝關節置換術的原發性膝OA患者(男、女各2例,年齡55～75歲),樣例均符合美國風濕病學會制訂的OA診斷標準。

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 滑膜成纖維細胞的體外分離和培養 術后2 h內將所取樣本在盛有Dulbecco′s Phosphate Buffered Saline的無菌培養皿中漂洗3次,手術剪修整留用滑膜層,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的飽和濕度培養箱中消化培養1.5～2 h,100目不銹鋼篩網過濾,以1 500 r/min離心10 min后,收集細胞懸液接種到培養瓶中,當細胞生長至約瓶底面積的80%時,以1∶(2～3)傳代比例進行傳代。取對數生長期的細胞(3～5代)用于實驗。

1.2.2 細胞轉染及藥物干預 將對數生長期的FLS制備成細胞懸液以5×105個/孔的細胞密度接種于6孔細胞培養板,37 ℃、5% CO2的飽和濕度培養箱中過夜。1 μg/mL Lipopolysaccharides from Escherichia coli(LPS)誘導24 h后進行細胞轉染,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)對照組;7)空白組:細胞培養液(未經LPS誘導)。相同條件的細胞培養箱孵育6 h進行轉染反應,換含血清的培養基后,繼續孵育48 h收集各孔內上清液進行檢測。上述miR-146a干擾后的細胞加入受試液: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)對照組:LPS(1 μg/mL);5)空白組:細胞培養液。相同條件再孵育48 h后,收集各孔內上清液進行檢測。

1.2.3 相關檢測 用Griess法對NO的釋放量測定: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,用于標準梯度曲線的繪制。A工作液(1%的對氨基苯磺酸溶液)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,加入標準貯備液或待測樣品室溫靜置30 min,酶標儀540 nm處測得各孔吸光值。用ELISA Kit對TLR4及MMP-13的含量進行測定(按試劑盒說明書操作)。

2 結果

2.1 miR-146a表達水平對TLR4、NO和MMP-13釋放量的影響 經過miR-146a干擾后的細胞表現出TLR及通路下游炎性遞質的差異性釋放:1)首先,對照組炎性遞質的釋放量顯著高于空白組,證明LPS誘導炎性反應成功;2)巧合的是,TLR4、NO、MMP-13在miR-146a干擾各組的變化趨勢大致相同;3)與對照組比較,三者的inhibitor組均升高,mimics組則均呈下降趨勢(P<0.05);4)另外作為對照參數的3組(inhibitor N.C組、N.C組和lipo2 000組)與對照組數值均較接近,差異基本無統計學意義(P>0.05),可認為幾組之間不存在差別,說明用于本實驗的質粒(miR-146a inhibitor、miR-146a mimics)及轉染劑(Lipofetctamine2 000)均起效并且對細胞無明顯細胞毒性等影響。見表1。

表1 miR-146a表達水平對TLR4、NO和MMP-13釋放量的影響

3.2 CA對miR-146a干擾的FLS TLR4濃度的影響 CA單獨作用于FLS后,與對照組比較,TLR4濃度明顯下降(P<0.05),說明CA能抑制TLR4的釋放;但是,與空白組比較可見,TLR4濃度仍較高,無法降至未做誘導的細胞水平。當inhibitor和CA聯合干預時,TLR4濃度與對照組比較略微下降;而當mimics和CA聯合干預時,TLR4濃度下降程度最高。見圖1。

圖1 CA對miR-146a干擾的FLS TLR4濃度的影響

2.3 CA對miR-146a干擾的FLS NO、MMP-13釋放量的影響 CA對miR-146a干擾的FLS NO、MMP-13釋放量的影響趨勢大致相同:首先,與TLR4相似地,和對照組比較,CA單獨作用時的FLS NO、MMP-13釋放量均有明顯下降(P<0.05),說明CA對NO、MMP-13釋放有顯著的抑制作用,但與空白組比較可見,仍無法降至未做誘導的細胞水平。當inhibitor和CA聯合干預時,NO、MMP-13釋放量仍維持在相對較高的水平;而當mimics和CA聯合干預時,NO、MMP-13釋放量出現極其顯著的下降(P<0.05)。結果見圖2、3。

圖2 CA對miR-146a干擾的FLS NO濃度的影響

圖3 CA對miR-146a干擾的FLS MMP-13濃度的影響

3 討論

OA是一種在長期機械作用和炎性反應等壓力下由一系列復雜機制驅動的涉及關節軟骨(Articular Cartilage,AC)、滑膜等多組織損傷導致體內穩態失衡的慢性臨床綜合征[16-18]。相對于其他疾病,OA以其高患病率、進展的致殘率和疼痛為特點成為最常見的關節疾病[19-20],影響著世界各地超過20%的人群,預計到2020年,OA的患病率可能會飆升至57%,是最亟待解決的醫療和社會問題[21-22]。但目前臨床上普遍應用的治療OA的方法對緩解疼痛、修復相關組織損傷和改善關節功能的治療目標尚未能達到滿意的療效。保守治療包括物理治療配合常用的藥物如鎮痛劑、非甾體類抗炎藥(Non-steroidal Anti-inflammatory Drugs,NSAIDs)、選擇性的環加氧酶-2(Cyclo-oxygenase-2,COX-2)抑制劑,類固醇抗炎藥和糖皮質激素注射等。然而,它們只能暫時的對OA的某些臨床癥狀起到有限的緩解,卻不能對損傷的AC等組織行使修復與保護等功能。此外,長期的大劑量的使用(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、肝臟損傷、心血管疾病等不良反應發生的風險[23-26]。關節置換手術是對保守治療療效欠佳的重度OA患者的最終選擇也是公認有效的治療方法。但不易被患者普遍接受,而且手術相關的一些嚴重的并發癥如感染、深靜脈血栓形成和后續使用中的假體松脫也是應該被考慮到的問題[27-29]。因此,開發一種新的藥物既能對終止或逆轉OA進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不良反應是值得關注的要點問題。最近,研究人員發現,一些從天然產物分離出的小分子或生物活性成分可通過各種途徑有效的減輕疼痛、緩解OA炎性反應。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,也是具有發汗解肌、溫經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節性能,它能通過對相關炎性反應通路的調節減少LPS誘導的巨噬細胞促炎性遞質表達,抑制NF-κB的激活和相關下游炎性細胞因子的分泌[30];還能減少IL-1β誘導的COX-2的激活和PGE2的產生[31];在TNF-α處理的內皮細胞中可抑制單核細胞與內皮細胞的黏附[32]。而且細胞毒性小,并有一定的組織器官保護作用,是一種新型的抗炎劑[33]。

我們逐漸認識到,加深對OA發生發展的分子機制的理解,有助于幫助發現導致OA的關鍵途徑和分子,進一步推動新療法的發展。越來越多的研究發現,滑膜細胞的固有免疫應答反應是OA進程中的關鍵環節[34-35]。健壯的固有免疫系統是對感染的即時防御及對病原體的持久抵抗至關重要的屏障。然而,如果它被不適當的激活和終止,就會危害宿主健康,導致一系列急性和慢性炎性反應紊亂的病理狀態的臨床表現[36]。因此,眾多復雜的分子機制從多個層次共同調節以保持炎性反應受到抑制,這些“調節器”種類眾多,從可溶性受體到誘導的細胞內蛋白質[37-40]。Toll樣受體(TLRs)是固有免疫系統中一類分子量在90-15OkDa之間的PRRs,在啟動和調控固有免疫系統中扮演著重要的角色[41-42]。這種跨膜糖蛋白受體大量表達于OA滑膜細胞上[43-44],其中,于1997年由Medzhitov.R等發現的第一個哺乳動物TLR——TLR4在誘導炎性反應及相關基因的表達和修復受損關節組織上有著不可忽視的作用[45-46]。越來越多的研究試圖通過對TLRs的調控進而干預由其參與激活的炎性反應通路的下游炎性遞質的釋放,最終達到緩解OA炎性反應的目的。

miRNA是一種能幾乎在各個水平對TLRs及其介導的相關通路進行調控,廣泛參與到OA的生理病理學,維持AC、滑膜等組織各項功能的負向調機器[47-49]。這類長度約為20～25個核苷酸(Nucleotide,nt)的內源性非編碼單鏈小分子RNA分子能識別靶mRNA上的3′非翻譯區(Untranslated Regions,UTR)并對其部分互補的位點進行結合綁定,通過對靶mRNA降解或阻礙翻,調節靶基因的蛋白質生成[50-51]。近年來,人們逐漸發現細胞在免疫系統的發育和功能上尤其受到miRNA的調控[52-54]。作為OA進程中強勁的轉錄后調節器,從細胞命運決定到信號活動的參與,miRNA構成了一個新的固有免疫應答的調節層[55-57]。尤其是第一個被發現可調節免疫系統的miRNA——miR-146,其中位于其5號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介導的NF-κB炎性反應通路上直接調節2個關鍵的下游適配器分子——腫瘤壞死因子相關受體相關因子6(TNF Receptor-associated Factor,TRAF6)和白介素1受體相關激酶(interleukin 1 receptor associated kinase,IRAK1)的蛋白水平,負反饋循環控制TLRs和細胞因子促炎性信號作用[58-61]。作為一種新興的可獨立影響OA發生發展的基因,其表達的抑制可能是促發OA的重要影響因素,該基因也可作為診斷OA的生物學標志[62-63]。

越來越多的證據表明對miRNA的管制可以有效調控OA,研究者們試圖通過干擾某些miRNA在細胞中的表達和活動以明確它們在疾病進程中的具體作用,一種將外源性的miRNA的模擬劑(Mimics)和抑制劑(Inhibitors)利用轉染試劑人工的參入宿主細胞,使其獲得新的遺傳標志的方法應運而生。Lipofetamin2 000是一種當前應用廣泛且毒性較低的高效陽離子脂質體轉染試劑,可瞬時的將人工合成的外源性基因序列導入細胞內,影響相關基因的表達。

本研究利用Lipofetamin2 000轉染法將促進和抑制性miR-146a質粒轉染入FLS,選擇一種代表性的TLRs TLR4及其介導的信號通路下游釋放的典型炎性遞質NO和OA進程中誘發膠原蛋白降解和破壞的主要物質MMP-13,幾種重要因子聯合檢測,有助于更全面、高效的證實它們在OA滑膜炎性反應中的變化趨勢和交互作用,以推測OA的進展,提供合理的病情評估以指導臨床治療用藥。結果顯示,miR-146a inhibitor可上調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相似。以上結果表明,miR-146a基因在FLS的目標干擾(上調或下降)可以對TLRs及其信號通路下游的炎性遞質和金屬蛋白酶進行調節,進一步影響OA滑膜炎性反應。

現有研究對miR-146a在OA中大致的調節機制及其所發揮的調節作用已有一定的了解,但它們之間進一步的調控機制和聯合藥物使用對OA影響的研究仍處于初期階段。所以在上述研究的基礎上,我們基于已有數據的提示發現,miR-146a作為一個OA滑膜炎性反應炎性反應的分子抑制劑扮演著重要角色。隨后,我們在miR-146a表達的消融導致在一些免疫相關的改變的基礎上,選取一種新型的具有抗炎活性及潛在保護作用的生物活性化合物CA結合miRNA干擾技術來共同治療OA滑膜炎性反應。結果顯示,CA本身就有抑制TLR4、NO、MMP-13表達的效果,當inhibitor和CA聯合干預時,CA可抵消部分miR-146a缺失所帶來的炎性反應問題;而當mimics和CA聯合干預時,就顯示出了對OA滑膜炎性反應優勢的抑制作用,考慮它們很可能能協同通過TLRs調控其信號通路上的其他組件共同抑制滑膜炎性反應。該研究結果在明確miR-146a干擾作用的同時,對其聯合中藥有效成分的抗炎作用進行評估,從基因層面為進一步了解OA滑膜炎性反應機制、選擇合理的藥物進行靶向治療以控制病情進展及組織修復提供了新的方案。

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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

國家自然科學基金面上項目(81373662);北京中醫藥大學自主選題項目(2015-JYB-XS202)

王歡(1986.04—),女,博士研究生,醫師,研究方向:中醫藥防治退行性骨關節病,E-mail：pulongqi@126.com

王慶甫(1956.10—),男,碩士,主任醫師,博士研究生導師,教授,研究方向:中醫藥防治退行性骨關節病,E-mail:qingpu-wang@sohu.com

R274.3

A

10.3969/j.issn.1673-7202.2017.10.034

(2016-08-16收稿 責任編輯：王明)