桂皮醛对miRNA-146a干扰的骨关节炎滑膜炎性反应影响的实验研究

王 欢 唐学章 丁海涛 张美丽 杨黎黎 张 栋 刘思婷 甘 稳 徐铭康 郭玉茹 许 晶 王庆甫

(1 中日友好医院,北京,100029; 2 北京中医药大学,北京,100029; 3 北京中医药大学第三附属医院,北京,100000)

桂皮醛对miRNA-146a干扰的骨关节炎滑膜炎性反应影响的实验研究

王 欢1唐学章1丁海涛1张美丽1杨黎黎2张 栋2刘思婷2甘 稳2徐铭康2郭玉茹2许 晶2王庆甫3

(1 中日友好医院,北京,100029; 2 北京中医药大学,北京,100029; 3 北京中医药大学第三附属医院,北京,100000)

目的:研究miRNA-146a基因及桂皮醛对miRNA-146a干扰的滑膜成纤维细胞释放TLR4、NO、MMP-13的影响,探寻其在OA滑膜炎性反应分子学机制中的作用。方法:采用脂质体转染法将miR-146a基因模拟及抑制性质粒载体对经LPS诱导后的OA滑膜炎性反应效应细胞滑膜成纤维细胞进行基因导入,并用桂皮醛对干扰后的细胞进行干预,检测不同组间TLR4、NO及MMP-13表达的差异。结果:miRNA-146a相关质粒转染后的TLR4、NO、MMP-13浓度的变化趋势大致相同:与对照组比较,三者的inhibitor组均升高,mimics组则均呈下降趋势(P<0.05)。桂皮醛单独作用于miRNA-146a干扰后的细胞时,与对照组比较,TLR4、NO、MMP-13浓度均有明显下降(P<0.05),而当mimics和CA联合干预时,三者释放量显著下降(P<0.05)。结论:miRNA-146a及桂皮醛均对骨关节滑膜炎性反应产生影响,当mimics和桂皮醛联合作用时,抑制炎性反应效果最好。为进一步阐明骨关节炎分子机制和药物靶点的识别与开发提供实验依据。

骨关节炎;microRNA-146a;toll样受体;滑膜炎性反应

骨关节炎(Osteoarthritis,OA)普遍被认为是一种以软骨损伤为主,伴骨赘生成的多发性退行性疾病,而一些较新的参数表明,OA还是一种由固有免疫诱发,以滑膜炎性反应为主要病理变化的受微小RNA(microRNA,miRNA,miR)异常影响的多因素复杂疾病[1-5]。miRNA是一类能通过在转录后水平介导基因沉默来调控目的基因的表达,广泛参与各种生物进程,在维持人体功能的细胞基因网络中起着至关重要作用的“调节器”[6-7]。近年来,随着对miRNA认识的逐步深入,一些它们在OA滑膜炎性反应等自身免疫性疾病中作为重要的调节因子参与疾病的直接或间接的证据逐渐被发现,科研人员将研究焦点越来越多的放在miRNA与固有免疫中一类重要的模式识别受体(Pattern Recognition Receptor,PRRs)——TLRs(Toll-like Receptors)相互作用及机制的探索上[8-14]。

本研究采用脂质体转染法将固有免疫反应中的重要调节因子miR-146a基因模拟及抑制性质粒载体对经LPS诱导后的OA滑膜炎性反应效应细胞滑膜成纤维细胞(Fibroblast-like Synoviocytes,FLS)进行基因导入,并用具有抗炎、镇痛等作用的新兴中药提取物桂皮醛(Cinnamic Aldehyde,CA)对干扰后的细胞进行干预,通过对不同组间TLR4、NO及MMP-13的表达差异进行检测,研究桂皮醛对miR-146a干扰的OA滑膜炎性反应的影响,旨在对OA的分子学机制有更明晰的了解,以期为药物靶点的识别和开发提供新思路。

1 材料与方法

1.1 材料

1.1.1 组织采集 无菌滑膜组织取自于2016年1月—3月北京中医药大学第三附属医院行膝关节置换术的原发性膝OA患者(男、女各2例,年龄55～75岁),样例均符合美国风湿病学会制订的OA诊断标准。

1.1.2 试剂 桂皮醛(HPLC≥98%),源叶生物,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)培养基,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,吉玛基因;TLR4(Human)ELISA Kit,Abnova,LOT:5C106L;MMP-13(Human)ELISA Kit,Abcam,Lot:GR188683-1。

1.2 方法

1.2.1 滑膜成纤维细胞的体外分离和培养 术后2 h内将所取样本在盛有Dulbecco′s Phosphate Buffered Saline的无菌培养皿中漂洗3次,手术剪修整留用滑膜层,再次漂洗后将组织剪成糊状,加入组织量20倍体积的Collagenase Type II,37 ℃、5% CO2的饱和湿度培养箱中消化培养1.5～2 h,100目不锈钢筛网过滤,以1 500 r/min离心10 min后,收集细胞悬液接种到培养瓶中,当细胞生长至约瓶底面积的80%时,以1∶(2～3)传代比例进行传代。取对数生长期的细胞(3～5代)用于实验。

1.2.2 细胞转染及药物干预 将对数生长期的FLS制备成细胞悬液以5×105个/孔的细胞密度接种于6孔细胞培养板,37 ℃、5% CO2的饱和湿度培养箱中过夜。1 μg/mL Lipopolysaccharides from Escherichia coli(LPS)诱导24 h后进行细胞转染,分组如下:1)inhibitor组:Lipofetctamine2 000+miR-146a inhibitor;2)mimics组:Lipofetctamine2 000+miR-146a mimics;3)inhibitor N.C组:Lipofetctamine2 000+miR-146a inhibitor N.C;4)N.C组:Lipofetctamine2 000+N.C;5)lipo2 000组:Lipofetctamine2 000;6)对照组;7)空白组:细胞培养液(未经LPS诱导)。相同条件的细胞培养箱孵育6 h进行转染反应,换含血清的培养基后,继续孵育48 h收集各孔内上清液进行检测。上述miR-146a干扰后的细胞加入受试液:1)inhibitor+CA组:上述inhibitor组+LPS(1 μg/mL)+CA(10 μg/mL);2)mimics+CA组:上述mimics组+LPS(1 μg/mL)+CA(10 μg/mL);3)CA组:LPS(1 μg/mL)+CA(10 μg/mL);4)对照组:LPS(1 μg/mL);5)空白组:细胞培养液。相同条件再孵育48 h后,收集各孔内上清液进行检测。

1.2.3 相关检测 用Griess法对NO的释放量测定:用双蒸馏水溶解并稀释亚硝酸钠溶液,以100 μmol/L为起始浓度2倍稀释,用于标准梯度曲线的绘制。A工作液(1%的对氨基苯磺酸溶液)和B工作液(0.1%的N-(1-萘基)乙二胺二盐酸盐溶液)各50 μL/孔,加入标准贮备液或待测样品室温静置30 min,酶标仪540 nm处测得各孔吸光值。用ELISA Kit对TLR4及MMP-13的含量进行测定(按试剂盒说明书操作)。

2 结果

2.1 miR-146a表达水平对TLR4、NO和MMP-13释放量的影响 经过miR-146a干扰后的细胞表现出TLR及通路下游炎性递质的差异性释放:1)首先,对照组炎性递质的释放量显著高于空白组,证明LPS诱导炎性反应成功;2)巧合的是,TLR4、NO、MMP-13在miR-146a干扰各组的变化趋势大致相同;3)与对照组比较,三者的inhibitor组均升高,mimics组则均呈下降趋势(P<0.05);4)另外作为对照参数的3组(inhibitor N.C组、N.C组和lipo2 000组)与对照组数值均较接近,差异基本无统计学意义(P>0.05),可认为几组之间不存在差别,说明用于本实验的质粒(miR-146a inhibitor、miR-146a mimics)及转染剂(Lipofetctamine2 000)均起效并且对细胞无明显细胞毒性等影响。见表1。

表1 miR-146a表达水平对TLR4、NO和MMP-13释放量的影响

3.2 CA对miR-146a干扰的FLS TLR4浓度的影响 CA单独作用于FLS后,与对照组比较,TLR4浓度明显下降(P<0.05),说明CA能抑制TLR4的释放;但是,与空白组比较可见,TLR4浓度仍较高,无法降至未做诱导的细胞水平。当inhibitor和CA联合干预时,TLR4浓度与对照组比较略微下降;而当mimics和CA联合干预时,TLR4浓度下降程度最高。见图1。

图1 CA对miR-146a干扰的FLS TLR4浓度的影响

2.3 CA对miR-146a干扰的FLS NO、MMP-13释放量的影响 CA对miR-146a干扰的FLS NO、MMP-13释放量的影响趋势大致相同:首先,与TLR4相似地,和对照组比较,CA单独作用时的FLS NO、MMP-13释放量均有明显下降(P<0.05),说明CA对NO、MMP-13释放有显著的抑制作用,但与空白组比较可见,仍无法降至未做诱导的细胞水平。当inhibitor和CA联合干预时,NO、MMP-13释放量仍维持在相对较高的水平;而当mimics和CA联合干预时,NO、MMP-13释放量出现极其显著的下降(P<0.05)。结果见图2、3。

图2 CA对miR-146a干扰的FLS NO浓度的影响

图3 CA对miR-146a干扰的FLS MMP-13浓度的影响

3 讨论

OA是一种在长期机械作用和炎性反应等压力下由一系列复杂机制驱动的涉及关节软骨(Articular Cartilage,AC)、滑膜等多组织损伤导致体内稳态失衡的慢性临床综合征[16-18]。相对于其他疾病,OA以其高患病率、进展的致残率和疼痛为特点成为最常见的关节疾病[19-20],影响着世界各地超过20%的人群,预计到2020年,OA的患病率可能会飙升至57%,是最亟待解决的医疗和社会问题[21-22]。但目前临床上普遍应用的治疗OA的方法对缓解疼痛、修复相关组织损伤和改善关节功能的治疗目标尚未能达到满意的疗效。保守治疗包括物理治疗配合常用的药物如镇痛剂、非甾体类抗炎药(Non-steroidal Anti-inflammatory Drugs,NSAIDs)、选择性的环加氧酶-2(Cyclo-oxygenase-2,COX-2)抑制剂,类固醇抗炎药和糖皮质激素注射等。然而,它们只能暂时的对OA的某些临床症状起到有限的缓解,却不能对损伤的AC等组织行使修复与保护等功能。此外,长期的大剂量的使用(The Epidemiology,Etiology,Diagnosis,and Treatment of Osteoarthritis of the Knee)这些药物还存在着一些潜在的安全隐患,如增加胃肠道反应、肝脏损伤、心血管疾病等不良反应发生的风险[23-26]。关节置换手术是对保守治疗疗效欠佳的重度OA患者的最终选择也是公认有效的治疗方法。但不易被患者普遍接受,而且手术相关的一些严重的并发症如感染、深静脉血栓形成和后续使用中的假体松脱也是应该被考虑到的问题[27-29]。因此,开发一种新的药物既能对终止或逆转OA进程有足够的治疗效果,又能尽可能地避免几乎可以忽略不计不良反应是值得关注的要点问题。最近,研究人员发现,一些从天然产物分离出的小分子或生物活性成分可通过各种途径有效的减轻疼痛、缓解OA炎性反应。桂皮醛是从樟科植物桂皮精油中分离出的主要生物活性化合物,也是具有发汗解肌、温经通脉功能的中药桂枝的主要有效成分。研究表明,CA及其衍生物显示出良好的免疫调节性能,它能通过对相关炎性反应通路的调节减少LPS诱导的巨噬细胞促炎性递质表达,抑制NF-κB的激活和相关下游炎性细胞因子的分泌[30];还能减少IL-1β诱导的COX-2的激活和PGE2的产生[31];在TNF-α处理的内皮细胞中可抑制单核细胞与内皮细胞的黏附[32]。而且细胞毒性小,并有一定的组织器官保护作用,是一种新型的抗炎剂[33]。

我们逐渐认识到,加深对OA发生发展的分子机制的理解,有助于帮助发现导致OA的关键途径和分子,进一步推动新疗法的发展。越来越多的研究发现,滑膜细胞的固有免疫应答反应是OA进程中的关键环节[34-35]。健壮的固有免疫系统是对感染的即时防御及对病原体的持久抵抗至关重要的屏障。然而,如果它被不适当的激活和终止,就会危害宿主健康,导致一系列急性和慢性炎性反应紊乱的病理状态的临床表现[36]。因此,众多复杂的分子机制从多个层次共同调节以保持炎性反应受到抑制,这些“调节器”种类众多,从可溶性受体到诱导的细胞内蛋白质[37-40]。Toll样受体(TLRs)是固有免疫系统中一类分子量在90-15OkDa之间的PRRs,在启动和调控固有免疫系统中扮演着重要的角色[41-42]。这种跨膜糖蛋白受体大量表达于OA滑膜细胞上[43-44],其中,于1997年由Medzhitov.R等发现的第一个哺乳动物TLR——TLR4在诱导炎性反应及相关基因的表达和修复受损关节组织上有着不可忽视的作用[45-46]。越来越多的研究试图通过对TLRs的调控进而干预由其参与激活的炎性反应通路的下游炎性递质的释放,最终达到缓解OA炎性反应的目的。

miRNA是一种能几乎在各个水平对TLRs及其介导的相关通路进行调控,广泛参与到OA的生理病理学,维持AC、滑膜等组织各项功能的负向调机器[47-49]。这类长度约为20～25个核苷酸(Nucleotide,nt)的内源性非编码单链小分子RNA分子能识别靶mRNA上的3′非翻译区(Untranslated Regions,UTR)并对其部分互补的位点进行结合绑定,通过对靶mRNA降解或阻碍翻,调节靶基因的蛋白质生成[50-51]。近年来,人们逐渐发现细胞在免疫系统的发育和功能上尤其受到miRNA的调控[52-54]。作为OA进程中强劲的转录后调节器,从细胞命运决定到信号活动的参与,miRNA构成了一个新的固有免疫应答的调节层[55-57]。尤其是第一个被发现可调节免疫系统的miRNA——miR-146,其中位于其5号染色体上的家族成员之一miR-146a是一种NF-kB依赖型基因,在TLRs介导的NF-κB炎性反应通路上直接调节2个关键的下游适配器分子——肿瘤坏死因子相关受体相关因子6(TNF Receptor-associated Factor,TRAF6)和白介素1受体相关激酶(interleukin 1 receptor associated kinase,IRAK1)的蛋白水平,负反馈循环控制TLRs和细胞因子促炎性信号作用[58-61]。作为一种新兴的可独立影响OA发生发展的基因,其表达的抑制可能是促发OA的重要影响因素,该基因也可作为诊断OA的生物学标志[62-63]。

越来越多的证据表明对miRNA的管制可以有效调控OA,研究者们试图通过干扰某些miRNA在细胞中的表达和活动以明确它们在疾病进程中的具体作用,一种将外源性的miRNA的模拟剂(Mimics)和抑制剂(Inhibitors)利用转染试剂人工的参入宿主细胞,使其获得新的遗传标志的方法应运而生。Lipofetamin2 000是一种当前应用广泛且毒性较低的高效阳离子脂质体转染试剂,可瞬时的将人工合成的外源性基因序列导入细胞内,影响相关基因的表达。

本研究利用Lipofetamin2 000转染法将促进和抑制性miR-146a质粒转染入FLS,选择一种代表性的TLRs TLR4及其介导的信号通路下游释放的典型炎性递质NO和OA进程中诱发胶原蛋白降解和破坏的主要物质MMP-13,几种重要因子联合检测,有助于更全面、高效的证实它们在OA滑膜炎性反应中的变化趋势和交互作用,以推测OA的进展,提供合理的病情评估以指导临床治疗用药。结果显示,miR-146a inhibitor可上调FLS释放TLR4、NO、MMP-13水平;miR-146a mimics则可使三者浓度均减低,且miR-146a干扰后三者的变化趋势相似。以上结果表明,miR-146a基因在FLS的目标干扰(上调或下降)可以对TLRs及其信号通路下游的炎性递质和金属蛋白酶进行调节,进一步影响OA滑膜炎性反应。

现有研究对miR-146a在OA中大致的调节机制及其所发挥的调节作用已有一定的了解,但它们之间进一步的调控机制和联合药物使用对OA影响的研究仍处于初期阶段。所以在上述研究的基础上,我们基于已有数据的提示发现,miR-146a作为一个OA滑膜炎性反应炎性反应的分子抑制剂扮演着重要角色。随后,我们在miR-146a表达的消融导致在一些免疫相关的改变的基础上,选取一种新型的具有抗炎活性及潜在保护作用的生物活性化合物CA结合miRNA干扰技术来共同治疗OA滑膜炎性反应。结果显示,CA本身就有抑制TLR4、NO、MMP-13表达的效果,当inhibitor和CA联合干预时,CA可抵消部分miR-146a缺失所带来的炎性反应问题;而当mimics和CA联合干预时,就显示出了对OA滑膜炎性反应优势的抑制作用,考虑它们很可能能协同通过TLRs调控其信号通路上的其他组件共同抑制滑膜炎性反应。该研究结果在明确miR-146a干扰作用的同时,对其联合中药有效成分的抗炎作用进行评估,从基因层面为进一步了解OA滑膜炎性反应机制、选择合理的药物进行靶向治疗以控制病情进展及组织修复提供了新的方案。

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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

国家自然科学基金面上项目(81373662);北京中医药大学自主选题项目(2015-JYB-XS202)

王欢(1986.04—),女,博士研究生,医师,研究方向:中医药防治退行性骨关节病,E-mail：pulongqi@126.com

王庆甫(1956.10—),男,硕士,主任医师,博士研究生导师,教授,研究方向:中医药防治退行性骨关节病,E-mail:qingpu-wang@sohu.com

R274.3

A

10.3969/j.issn.1673-7202.2017.10.034

(2016-08-16收稿 责任编辑：王明)