氧化修饰蛋白质诱导滑膜细胞炎症应答分子机制的研究
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摘要
研究背景
类风湿性关节炎(Rheumatoid arthritis, RA)是以一种以对称性多关节病变为主的慢性自身免疫性疾病。它的基本病理学改变是滑膜组织衬里层的滑膜细胞呈轻中度增生状态,炎性细胞浸润蔓延到血管周围,形成血管翳。关节滑膜炎症及血管翳的形成是其主要的病理特点,表现为侵蚀性血管翳的形成,继而破坏关节软骨、软骨下骨、骨及其周围组织。
RA全世界的发病率约为0.5%-1.0%,小于45岁的患者约占80%,其发病起始5年致残率高达30%。临床上RA主要表现为受累关节的肿胀及疼痛,继而发生关节强直及畸形改变,最终导致关节功能障碍从而使患者丧失劳动能力。RA的发生发展会对患者的生理及心理造成严重影响,影响其日常生活及社会活动,降低患者生活质量。因此,RA是一种严重威胁人类健康的疾病,探讨其真正的发病机理进而进行预防和治疗成为医务工作人员的一项重要使命。
“氧化应激”是体内氧化系统与抗氧化系统的平衡被打破所导致的一种活性氧族物质(Reactive oxygen species, ROS)增多的一种状态。ROS是生物体内有氧代谢产生的含氧自由基,主要包括超氧阴离子(O2-)、羟基(-OH)和过氧化氢(H2O2)。ROS在细胞信号级联反应中起着第二信使分子的作用。烟酰胺腺嘌呤二核苷酸磷酸(Nicotinamide-adenine dinucleotide phosphate, NADPH)氧化酶是细胞内ROS产生的主要来源。氧化应激会造成蛋白质、脂质、DNA等多种生物大分子氧化损伤,而且蛋白质受到的氧化损伤要早于脂质过氧化及其对DNA的损伤。因此,目前普遍认为蛋白质是体内氧化应激损伤的主要原初靶。
氧化修饰蛋白质(Advanced oxidation protein products, AOPPs)是一类含双酪氨酸的蛋白质交联产物,在体内氧化应激过程中,它是由激活的中性粒细胞髓过氧化物酶产生的次氯酸(HCLO)作用于蛋白质(主要是白蛋白)而形成的,是一种蛋白质氧化应激标志物,是体内蛋白质氧化所形成的终末产物的总称。1996年由Witko-Sarsat等人首先从慢性肾衰竭(Chronic renal failure, CRF)患者血浆中分离出来。在体外试验中,用正常人血浆或纯化的人血清白蛋白(Human serum albumin, HSA)与HCIO相作用,亦可得到与慢性肾衰竭病人血浆中类似的AOPPs。
体内氧化应激的状态会造成蛋白质肽链断裂、交联、碳基生成和构象变化等结构和功能改变,形成氧化修饰蛋白质AOPPs。在生理状态下,氧化修饰的蛋白质很易被蛋白水解酶水解,蛋白质氧化修饰有利于体内蛋白质的更新与代谢。但是,在氧化应激状态下,蛋白质修饰的速率会增加,体内AOPPs水平明显增加。AOPPs水平的增高与许多氧化应激相关性疾病如慢性化脓性中耳炎、慢性肾脏疾病、糖尿病、肥胖症、恶性肿瘤的发生发展有很大的关系,并与疾病的严重程度密切相关。因此,AOPPs是氧化应激的重要标志物。
AOPPs不仅是氧化应激的后果,其本身作为致病介质具有广泛的病理作用,参与人类多种重要疾病的发生发展。有研究已经证实AOPPs能够激活单核细胞,诱导呼吸链爆发,增加ROS的生成,刺激TNF-α、IL-6、IL-1等促炎症细胞因子的合成释放,诱发细胞炎症反应,参与慢性肾衰患者全身微炎症状态的发生发展。此外,AOPPs本身就是活性氧族物质ROS的来源,可作为大分子生物介质参与许多病理过程。
RA患者普遍存在以氧化还原失衡为特征的氧化应激。RA患者氧化应激水平会升高会导致体内的血液、关节液中的蛋白质尤其是白蛋白的氧化损伤明显增加,进而增加RA患者体内AOPPs的含量,提升ROS水平。近年来,许多研究证实ROS可激活下游的多条细胞信号转导通路,如促分裂素原活化蛋白激酶(Mitogen-activated protein kinases, MAPK)、核因子-κB (Nuclear factor-kappa B,NF-κB)等信号通路。有研究证实ROS敏感的NF-κB信号途径在滑膜细胞炎症反应过程中起着重要的调节作用。
人类关节滑膜为疏松结缔组织,可分为两层：滑膜衬里层和滑膜衬里下层。滑膜衬里层是由成纤维细胞样滑膜细胞(Fibroblast-like synoviocytes,FLS)和巨噬样细胞组成,而衬里下层是由疏松结缔组织基质构成。FLS与其深层的纤维细胞并无基底膜相隔,直接与纤维结缔组织或脂肪组织相连接,故在关节发生炎症时易蔓延到周围组织,是介导RA患者发生滑膜炎症的重要细胞。
由于AOPPs与氧化应激和炎症反应密切相关,所以本研究认为AOPPs(?)可能通过激活FLS内NADPH氧化酶进而提升ROS水平,激活NF-κB信号转导通路,诱导FLS炎症反应的发生,参与RA疾病的发生发展。
研究方法
1、氧化修饰蛋白质AOPPs的制备：
将鼠血清白蛋白(Rat serum albumin, RSA)溶于无内毒素的PBS中配制成20mg/ml,将RSA溶液与40mmol/L的次氯酸等体积混合,室温下放置30min进行反应,这样制备出AOPPs内的RSA与次氯酸的摩尔比为1：140。将制备好的AOPPs放入透析袋中,将装有AOPPs的透析袋放入无内毒素的4℃C PBS溶液中透析24h,除去游离的次氯酸。接着用0.22μmm的微孔滤膜过滤除菌后,再用Detoxi-Gel内毒素去除胶过滤除去内毒素。用鲎试剂法检测制备好的AOPPs内的内毒素水平,内毒素水平控制在0.025EU/ml以内的AOPPs可以用于后续试验。将成功制备好的AOPPs放入4℃冰箱保存。以氯氨T为标准,酸性条件下,测340nm光吸收,来测量AOPPs的含量。
2、大鼠滑膜细胞的培养
无菌条件下切除SD大鼠双侧后肢膝关节的滑膜组织,用眼科剪及镊子剃去滑膜组织上面的血液污渍及其他组织。用37℃C的PBS缓冲液冲洗剪下来的滑膜组织2-3次。将滑膜组织移至小青霉素瓶,加入lml D-Hanks液,用眼科剪将滑膜组织剪成1mm3的块状组织。将青霉素瓶静止5分钟,待滑膜组织块沉底后,去除上清。根据滑膜组织块的大小及含量的多少加入适量的DMEM完全培养基(含10%胎牛血清和青、链霉素各10万/L双抗液体),晃动,搅匀。将含有完全培养基的组织块均匀地放入培养瓶内,倒置培养瓶,再向培养瓶加入3～4m1完全培养基。拧紧瓶盖(透气培养瓶需拧紧,非透气培养瓶可稍留缝隙)放置于37℃、含有5%CO2培养箱内,4-6小时后将培养瓶缓慢翻转过来,使完全培养基完全浸没滑膜组织块,继续放在培养箱中静置培养。每天在显微镜下每天观察滑膜组织块生长情况。每2-3天换液一次,待组织块中成纤维样细胞长出完全后,传代培养,用第二代或者第三代滑膜细胞进行后续试验。
3、细胞活性的测定：
将FLS接种于96孔板内,用50、100、200μg/ml的AOPPs、200μg/ml未经修饰的RSA以及完全培养基DMEM刺激12小时。用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐[3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide, MTT]法测定细胞活力。
4、AOPPs诱导FLS炎症应答的病理生物学作用
将FLS接种于6孔板中,用50、100、200μg/ml的AOPPs、200μg/ml未经修饰的RSA以及完全培养基DMEM刺激12小时。用肿瘤坏死因子-α(TNF-a)和白细胞介素-1β(IL-1β)、基质金属蛋白酶-3(Matrix Metallo Preteinases-3, MMP-3)、基质金属蛋白酶-13(MMP-13)以及血管内皮生长因子(Vascular Endothelial Growth Factor, VEGF)的ELISA试剂盒检测FLS在不同浓度AOPPs的刺激下分泌TNF-α、IL-1β、MMP-3、MMP-13以及VEGF的情况,用Real-Time PCR法检测AOPPs对滑膜细胞内的TNF-α、IL-1β、MMP-3、MMP-13以及VEGF的mRNA表达水平的研究,进而探讨AOPPs对FLS炎症反应的病理作用。
5、AOPPs提升FLS内活性氧族(ROS)水平的研究
将FLS接种于96孔板中,用50.100.2001μg/ml的AOPPs.200μg/ml未经修饰的RSA以及完全培养基DMEM刺激12小时,用荧光探针二氯荧光黄双乙酸盐(DCFH-DA)检测FLS在不同浓度AOPPs的刺激下产生ROS的水平。接着用200μg/ml的AOPPs分别刺激滑膜细胞5分钟、10分钟、20分钟、40分钟、60分钟、90分钟以及120分钟,再用DCFH-DA检测在相同浓度的AOPPs刺激下,不同时间对FLS产生ROS的影响。
6、AOPPs激活FLS内NADPH氧化酶系统
将FLS接种于6孔板中,用50、100、200μg/ml的AOPPs、200μg/ml未经修饰的RSA以及完全培养基DMEM刺激0-60分钟。用免疫共沉淀法检测p47phox分别与gP91phox和p22hox结合情况,用不同浓度的AOPPs分别刺激FLS3小时、6小时、12小时以及24小时,用Western blot检测AOPPs诱导p47phox磷酸化以及NADPH亚单位p47phox、gP91phow和p22phox含量的表达；
6.AOPPs激活FLS内NF-κB的实验研究
将FLS接种于6孔板中,用50.100.200μg/ml的AOPPs.200μg/ml未经修饰的RSA以及完全培养基DMEM刺激0-60分钟。用Western blot检测AOPPs诱导下滑膜细胞胞浆IκBα及胞核NF-κB亚基p65的表达情况。
7、阻断不同层面受体介导的信号转导通路对AOPPs激活FLS炎症反应的实验研究：
用NADPH氧化酶抑制剂(DPI和apocynin)、ROS清除剂(NAC、catalase和SOD)和NF-κKB特异抑制剂(SN50)预处理FLS一定时间后,再用50、100、200μg/ml的AOPPs.200μg/ml未经修饰的RSA以及完全培养基DMEM刺激一定时间后,分别用ELISA及Real-Time PCR法检测FLS分泌TNF-α、IL-1β、 MMP-3、MMP-13以及VEGF蛋白及mRNA的水平,用用Western blot检钡AOPPs诱导下滑膜细胞胞浆IκBα及胞核NF-κB亚基p65的表达情况。
8、统计学分析：
所有实验均重复3次,每个实验指标取这些数据的平均值,以均数士标准差表示,所有统计结果应用统计软件SPSS13.0完成。多个样本均数的比较采用One-Way ANOVA方法,在做两两比较时先计算方差齐性,当方差齐时两两比较采用LSD法,方差不齐时两两比较采用Dunnetts T3法；多个样本非参数比较采取多个独立样本非参数检验(K Independent Samples Test),当p<0.05时为具有统计学意义。
实验结果：
1.AOPPs诱导滑膜细胞分泌促炎症因子、基质金属蛋白酶及血管内皮生长因子：
ELISA结果显示,伴随着AOPPs浓度的增长,滑膜细胞分泌的TNF-α、 MMP-3、MMP-13和VEGF的水平也逐渐增长,200μg/ml的AOPPs促进滑膜细胞分泌的上述细胞因子含量最多,但是100μg/ml的AOPPs促进滑膜细胞分泌的IL-1β最多,而200μg/ml的AOPPs分泌的比100μg/ml的AOPPs少,但是比50μg/ml的AOPPs要多。AOPPs促进滑膜细胞分泌的细胞因子能够被不同层面的阻断剂如ROS清除剂NAC.NADPH氧化酶抑制剂apocylnin及NF-κB抑制剂SN50所阻断。
Real-Time PCR结果与ELISA的结果相似,伴随着AOPPs浓度的增长,滑膜细胞内的TNF-α、MMP-3、MMP-13和VEGF的mRNA水平也逐渐增长,200μg/ml的AOPPs提升滑膜细胞内的mRNA水平最高,同样地,100μg/ml的AOPPs刺激滑膜细胞内IL-1β的mRNA分泌最多,而200μg/ml的AOPPs分泌的比100μg/ml的AOPPs少,但是比50μg/ml的AOPPs要多。
2.AOPPs提升滑膜细胞内ROS水平：
荧光显微镜下显示AOPPs能够显著提升滑膜细胞内的荧光强度,与RSA及DMEM组有显著的差异。DCFH-DA结果显示,伴随着AOPPs浓度的增长,滑膜细胞内的ROS水平逐渐增高,200μg/ml的AOPPs提升滑膜细胞内ROS水平最高,约为50μg/ml的AOPPs的8倍左右。而用100μg/ml的AOPPs刺激滑膜细胞0-120分钟时,在AOPPs刺激90分钟的时候滑膜细胞内的ROS水平最高,约为正常对照组的6倍左右,但是用100μg/ml的AOPPs刺激滑膜细胞120分钟时滑膜细胞内ROS的水平低于刺激90分钟的水平,约为正常对照组的3.5倍左右。不同层面受体介导的信号转导阻断剂NAC、SOD、DPI及Apocynin都能够显著抑制AOPPs提升滑膜细胞ROS的水平,ROS的水平大约为AOPPs组的二分之一到三分之一左右。
3、AOPPs激活滑膜细胞内NADPH氧化酶系统：
在AOPPs刺激作用下,滑膜细胞内的NADPH氧化酶的胞浆亚单位p47phox快速磷酸化,正常组及未经修饰的RSA组未见到此效应。免疫共沉淀检测到AOPPs促进了p47phox与细胞膜上的亚单位p22phox、gp91phox的结合,形成复合体。与正常组及RAS组相比, AOPPs刺激3、6、12及24小时后明显增加了p47phox、 p22phox及gp91phox亚单元蛋白表达。
4、AOPPs激活滑膜细胞内NF-κB系统：
在AOPPs的刺激作用下,滑膜细胞胞浆内的NF-κB系统内的IκBα逐渐降解,而在细胞核内,NF-κB的亚基p65的表达逐渐升高,正常组及RSA组无此效应。AOPPs诱导滑膜细胞NF-κB系统激活的效应能够被阻断剂DPIapocynin、catalase和SOD所抑制。
结论：
1.AOPPs促进滑膜细胞分泌炎症因子IL-1β、TNF-α、基质金属蛋白酶MMP-3、MMP-13以及血管内皮生长因子VEGF产生。
2.AOPPS激活了滑膜细胞内的NADPH氧化酶系统。
3. AOPPs提升了滑膜细胞内ROS的水平。
4.AOPPs激活了滑膜细胞内的NF-κB系统。
5.NADPH氧化酶系统抑制剂、ROS清除剂及NF-κB系统抑制剂都能够阻断滑膜细胞分泌炎症因子IL-1β、TNF-α、MMP-3、MMP-13以及VEGF产生。
Backgroud
Rheumatoid arthritis (RA) is a kind of chronic autoimmune diseases characterized by symmetrical joints disease. The basic pathological alteration of this disease is inflammatory cell infiltration, the proliferation of fibroblast-like synoviocytes and the formation of pannus. The inflammatory of synovium of the joint and pannus are the main pathological character of the disease. The formation of aggressive pannus will destroy the articular cartilage, subchondral bone and the around tissue.
The morbidity of RA in the world is0.5%-1.0%and the proportion of average age of onset younger than45is80%. The disability rate in the first5years is reach up to30%. The clinical manifestation of RA is sell and pain of the affected joint and followed by ankylosis and deformity of the joint and at last the patient will be incapacitated because of joint dysfunction. The development and progression of RA will do harm to the physiology and mentality of patients followed by affecting the daily life and public activity and at last lower the quality of the patients' life. Therefore, RA is a kind of disease which poses a serious threat the health of humans and protection and treatment of the disease have been an important mission of the medical staff.
Oxidative stress is a kind of state which the balance of oxidative system and antioxidant system is broken result in the increase of reactive oxygen species (ROS). ROS, which consist of O2-,-OH and H2O2, are the production of aerobic metabolism. ROS play a role of second messenger in cell signaling cascade. Nicotinamide-adenine dinucleotide phosphate (NADPH) is the main source of ROS. Oxidative stress will damage the protein, lipid and DNA and the occurrence of protein damage is earlier than that of lipid and DNA. Therefore, it is widely believed that protein damage is the main original target of oxidative stress.
Advanced oxidation protein products (AOPPs) are a kind of protein crosslinked product containing Dtyr. During the procession of oxidative stress they are formed by the reaction of HCLO and albumin. They are a kind of landmark of oxidative stress and the generic terms of protein oxidation. They are separated from patients of chronic renal failure by Witko-Sarsat in1996. And in vitro, AOPPs can be obtained by the reaction of Human serum albumin and HCIO.
The status of oxidative stress will make the protein's construction changed and at last the advanced oxidation protein products were formed. In the physiological status the advanced oxidation protein products were easily hydrolysed by proteolytic enzyme and this is helpful to the update and metabolism of proteins in the body. However, in the status of oxidative stress the rate of protein modification will be increased and the level of AOPPs will be raised. High level of AOPPs is connected with many diseases such as chronic suppurative otitis media, chronic renal disease, diabetes mellitus, obesity and cancer. Therefore, AOPPs are important landmark of oxidative stress.
AOPPs are not the result of oxidative stress but also the pathogenic factor participated in the occurrence and development of many diseases. Many studies have demonstrated that AOPPs participated in the activation of monocyte, induce the break out of respiratory chain, increase the production of ROS and induce the cytokines, such as TNF-α、IL-6、IL-1release, participated in the inflammatory response. Furthermore, AOPPs are the resource of ROS and act as macromolecule take part in many pathological processes.
Oxidative stress is ubiquitous in RA patients. The high level of oxidative stress will cause the damage of protein in blood and synovial fluid and at last elevate the level of AOPPs in RA patients. In recent years, many studies have demonstrated that ROS can activated many cell signal transduction pathways such as Mitogen-activated protein kinases(MAPH) and Nuclear factor-kappa B (NF-κB). A study has demonstrated that NF-KB is important in regulating the inflammatory response of fibroblast like synoviocytes (FLS).
Human's synovium is loose connective tissue consist of synovial lining layer and synovial lining down the lower. The synovial lining layer consist of fibroblast like synoviocytes (FLS) and macrophage. The FLS are important cells which directly connect to the fibrous connective tissue or adipose tissue and therefore the cells are easily spread to the surrounding tissue when the inflammatory reaction happens
Oxidative stress has close relationship with the inflammatory reaction so we believe that AOPPs can activate the NADPH oxidase, raise the level of ROS, activate the NF-κB signal transduction pathway, induce the inflammatory reaction happen and at last particated the occurence and development of RA.
Matedals and methods
1. AOPPs-RSA Preparation and Determination
AOPPs-Rat Serum Albumin (RSA) was prepared according to a described procedure with minor modifications. Briefly, RSA solution (20mg/ml, St Louis, MO, USA) was exposed to200mmol/L of HOCL for30mins at room temperature and then dialyzed against PBS at4℃for24hrs to remove free HOCL. Control incubation was performed in native RSA dissolved in PBS alone. All the preparations were passed through a Detoxi-Gel column (Thermo, Massachusetts,USA) to remove any endotoxin. An amebocyte lysate assay kit (Sigma,USA) was used to determine the level of endotoxin in both AOPPs-RSA and unmodified RSA group and the concentration of endotoxin in them were below0.025EU/ml. AOPPs content in the sample was determined as described previously. Briefly,200μl of sample or chloramine-T was placed in a96-well plate, and then20μl of acetic acid was added. A microplate reader was used to measure the absorbance at340nm immediately.
2. Fibroblast-like synoviocytes (FLSs) culture
FLSs were obtained according to a described procedure with some modifications. Fresh synovial tissues were isolated aseptically from both knee joints of Female Lewis rats (4weeks old,150-200g) and washed with phosphate-buffered saline (PBS). After that they were minced and digested in a solution of0.2%collagenase in Dulbecco's modified Eagle's medium (DMEM, Gibco, Life Technologies, California, USA) at37℃for2.5hrs, the tissue were further digested by0.25%trypsin for2hrs and then the cells were centrifuged at1000g for5mins.
FLSs were seeded in,25cm2flat-bottom culture flasks and supplemented with DMEM containing10%fetal bovine serum (FBS)(Gibco, Life Technologies, California, USA) and antibiotics (100IU/ml penicillin,100IU/ml streptomycin, Irvine Scientific, Santa Ana USA). The cells were cultured by incubating at37℃in a humidified atmosphere with5%CO2. After reaching a subconfluent state, the cells were subcultured after trypsinization with0.25%trypsin/0.02%EDTA. Third to sixth passage cells were used for all the experiments. All animal procedures performed with permission and followed the guidelines laid down by the Animal Use and Care Committee of Southern Medical University.
3. The determination of cell activity:
The FLS were seeded in96-well plates. FLSs were extensively washed with PBS, cultured in DMEM with0.5%serum for12hrs, and then stimulated by adding control medium, various concentrations of AOPPs-RSA (50,100,200μg/ml) or unmodified RSA (200μg/ml).3-(4,5)-dimethylthiahiazo(-z-yl)-3,5-diphenytetrazoliumromide (MTT) is used to determine the cell activity.
4. AOPPs induce the inflammatory response of FLS.
To investigate whether AOPPs induce cytokines production in FLSs, we analyzed the supernatant of the synovial cells cultured with AOPPs. To be specific, cells were cultured in DMEM with FBS (10%) and then synchronized for12hrs with DMEM/0.5%FBS. FLSs were stimulated with increasing concentrations of AOPPs (50,100,200μg/ml) or unmodified RSA (200μg/ml) or control medium respectively. After that, cell supernatants were collected and centrifuged at12000g for15mins. Supernatants were stored at-80℃until experimentation. Levels of MMP-3, MMP-13, TNF-a, IL-1β and VEGF in the supernatant were quantified respectively using the MMP-3ELISA kits, MMP-13ELISA kit, TNF-aELISA kit, IL-1βELISA kit and VEGF ELISA kit according to the manufacturer's protocol. The OD was measured at450nm by a spectrophotometric plate reader. All the experiments were performed and tested in triplicate.
5. AOPPs upraise the level of ROS
The level of intracellular ROS was assessed by fluorescence microplate reader with the probe2',7'-dichlorofluorescein diacetate (DCFH-DA), which oxidizes to fluorescent dichlorofluorescein (DCF) in the presence of ROS, as described previously. Briefly, FLSs were suspended in DMEM at a given concentration of108/L and150μL of cells suspension was added to the96-well plates. Each sample was incubated in10μM DCFH-DA for30mins in darkness followed by AOPPs treatment as described above. Fluorescence intensity was measured on a SpectraMax M5system. The excitation and emission wavelength were488nm and525nm respectively. All the obtained data were normalized with the control values.
6. AOPPs activated the NADPH system
FLSs were extensively washed with PBS, cultured in DMEM with0.5%serum for12hrs, and then stimulated by adding control medium, various concentrations of AOPPs-RSA (50,100,200μg/ml) or unmodified RSA (200μg/ml) for60mins. Immunoprecipitation and immunoblotting were peformed respectively to analyze the phosphorylation of p47phox and interaction of p47phox with p22phox or gp91phox. To determine total p47phox, p22phox or gp91phox, the membranes were washed with an elute buffer, reacted with anti-p47phox monoclonal antibody, anti-p22phox or anti-gp91pbox polyclonal antibodies respectively and then detected by the HRP-conjugated anti-IgG antibody.
7. AOPPs activated the NF-κB system
FLSs were extensively washed with PBS, cultured in DMEM with0.5%serum for12hrs, and then stimulated by adding control medium, various concentrations of AOPPs-RSA (50,100,200μg/ml) or unmodified RSA (200μg/ml) for60mins. Western blotting was performed to detect the specificity of antigen-antibody interaction using total cellular proteins and the nuclear proteins, extracted from FLSs according to manufacturer's instruction of nuclear and cytoplasmic extraction reagents kit. Proteins (35μg) were loaded per lane and separated by10%SDS-PAGE and electrotransferred to PVDF membranes by a semi-dry transfer. Then the PVDF membranes was blocked in5%nonfat milk in TBS-Tween for1hr at room temperature and incubated overnight at4℃with the primary antibodies anti-NF-κB p65, anti-IκBα, respectively. Later the membranes were washed three times for10mins each in TBST and incubated for1hr at room temperature with appropriate HRP-linked secondary antibodies. The relative levels of protein were determined by densitometry using Total Lab2.0software.
8. To explore the effect of blocking the different levels of signal transduction pathway in AOPPs induced the inflammatory response.
FLSs were extensively washed with PBS, cultured in DMEM with0.5%serum for12hrs, and then stimulated by adding control medium, various concentrations of AOPPs-RSA (50,100,200μg/ml) or unmodified RSA (200μg/ml) in the presence of antioxidant N-acetyl-L-cysteine (NAC), superoxide dismutase (SOD), diphenyleneiodonium (DPI), apocynin, catalase and SN50. And then we performed the ELSA, Real-time PCR and western blot again to test the expression of TNF-a, IL-1β, MMP-3, MMP-13, VEGF, IκBα and NF-κB in FLS.
9. Statistical analysis
All the experiments were performed in triplicate. Results were expressed as mean±standard deviation. Statistical differences between means for different groups were compared using one-way ANOVA (analysis of variance). Multiple comparisons were performed using the LSD method or Dunnett's T3method. Statistical differences between nonparametric for different groups were compared using K Independent Samples Test procedure. Comparing several treatments with control group were evaluated by the Statistical analyses were conducted with SPSS13.0software.
Results:
1. Effect of AOPPs stimulation on the expression of IL-1β, TNF-α, MMP-3, MMP-13and VEGF.
Initially, we tested whether AOPPs can induce FLSs to release cytokines. As shown in Fig.1a-c, secretion of cytokine IL-1β, TNF-α, MMP-3, MMP-13and VEGF by FLSs in AOPPs group were in a concentration-dependent manners compared with low levels detected in the conntrol cells and unmodified RSA group (Fig.l b and c). However, it is seen that the expression of IL-1β in200μg/ml AOPPs group was lower than that in100μg/ml AOPPs group but still at a signaficantly higher levels than control cells and RSA group.
Real-time RT-PCR is used to quantify the effect of AOPPs at various concentrations on the expression of IL-1β, TNF-α, MMP-3, MMP-13and VEGF in mRNA. Compared with the control cells and unmodified RSA group, the mRNA levels were significantly increased when FLSs were cultured with AOPPs. And no significant difference was found in the messenger RNA expression between unmodified RSA group and control cells. When the above data is evaluated, it indicates that FLSs can be stimulated by AOPPs to secrete cytokines at both protein and gene level, which may be involved in the pathological progression of RA.
2. AOPPs induce ROS generation in FLSs
In order to study the ROS generation in FLSs, we challenged AOPPs group in FLSs and noticed a siginificant increase of ROS level (3-to8-fold) in AOPPs group compared to both unmodified RSA cells and control cells. When FLSs were incubated with AOPPs at different concentration within90mins it showed a time-dependent increase in ROS generation. Furthermore, the ROS generation in FLSs cultured with AOPPs was observed under a fluorescence microscopy with DCFH-DA.
3. AOPPs activated the NADPH system
To investigate whether AOPPs treatment activate NADPH oxidase, initially we measured the phosphorylation of p47phox, a subunit of NADPH oxidase located in the cytoplasm of the FLSs. AOPPs group showed rapid phosphorylation of p47phox at5mins, and peaked at60mins, whereas control group and RSA group had no siginificant effect. Translocation of p47phox to the cell membrane plays a key role in NADPH oxidase activation. In order to examine the interaction of p47phox with the membrane subunits, we immunoprecipitated p22phox and gp91phox with the specific antibodies and then probed for the coexistence of p47phox in the cells. The amount of p47phox-gp91phox complex rapidly increased in AOPPs group at5mins and p47phox-p22phox complex appeared later than p47phox-gp91phox complex at15mins. Likewise, to determine sustained activity of NADPH oxidase we examined the protein levels of its subunits in FLSs treated with or without AOPPs. AOPPs group showed significant upregulated expression of p47phox, p22phox and gp91phox compared with control cells after6hrs. Only the expression of gp91phox was increased after3hrs in AOPPs group.
4. AOPPs activated the NF-kB system
In order to explore the potential molecular mechanism underlying the AOPPs actions on FLSs, western blot was performed to examine if AOPPs triggers NF-κB activation in the cells. As we know two important steps before NF-κB activation are IκBα degradation in cytoplasm and NF-κB p65translocation to nucleus. In AOPPs group (200μg/ml) IκBα degradation in cytoplasm was observed at15mins, which continued till60mins and NF-κB p65proteins showed an increased level in nucleus from5mins till60mins. These data suggested that AOPPs can induce the degradation of IκBα, which leads to the nuclear translocation of p65and at last activate NF-κB in FLSs.
Conclusions:
1.AOPPs induced the expression of IL-1β、TNF-α、MMP-3、MMP-13and VEGF in FLS.
2. AOPPs activated the NADPH system in FLS;
3. AOPPs induced ROS generation in FLSs
4. AOPPs activated the NF-kB system;。
5. Blocking the different levels of signal transduction pathway will block AOPPs induce the inflammatory response.
类风湿性关节炎(Rheumatoid arthritis, RA)是以一种以对称性多关节病变为主的慢性自身免疫性疾病。它的基本病理学改变是滑膜组织衬里层的滑膜细胞呈轻中度增生状态,炎性细胞浸润蔓延到血管周围,形成血管翳。关节滑膜炎症及血管翳的形成是其主要的病理特点,表现为侵蚀性血管翳的形成,继而破坏关节软骨、软骨下骨、骨及其周围组织。
RA全世界的发病率约为0.5%-1.0%,小于45岁的患者约占80%,其发病起始5年致残率高达30%。临床上RA主要表现为受累关节的肿胀及疼痛,继而发生关节强直及畸形改变,最终导致关节功能障碍从而使患者丧失劳动能力。RA的发生发展会对患者的生理及心理造成严重影响,影响其日常生活及社会活动,降低患者生活质量。因此,RA是一种严重威胁人类健康的疾病,探讨其真正的发病机理进而进行预防和治疗成为医务工作人员的一项重要使命。
“氧化应激”是体内氧化系统与抗氧化系统的平衡被打破所导致的一种活性氧族物质(Reactive oxygen species, ROS)增多的一种状态。ROS是生物体内有氧代谢产生的含氧自由基,主要包括超氧阴离子(O2-)、羟基(-OH)和过氧化氢(H2O2)。ROS在细胞信号级联反应中起着第二信使分子的作用。烟酰胺腺嘌呤二核苷酸磷酸(Nicotinamide-adenine dinucleotide phosphate, NADPH)氧化酶是细胞内ROS产生的主要来源。氧化应激会造成蛋白质、脂质、DNA等多种生物大分子氧化损伤,而且蛋白质受到的氧化损伤要早于脂质过氧化及其对DNA的损伤。因此,目前普遍认为蛋白质是体内氧化应激损伤的主要原初靶。
氧化修饰蛋白质(Advanced oxidation protein products, AOPPs)是一类含双酪氨酸的蛋白质交联产物,在体内氧化应激过程中,它是由激活的中性粒细胞髓过氧化物酶产生的次氯酸(HCLO)作用于蛋白质(主要是白蛋白)而形成的,是一种蛋白质氧化应激标志物,是体内蛋白质氧化所形成的终末产物的总称。1996年由Witko-Sarsat等人首先从慢性肾衰竭(Chronic renal failure, CRF)患者血浆中分离出来。在体外试验中,用正常人血浆或纯化的人血清白蛋白(Human serum albumin, HSA)与HCIO相作用,亦可得到与慢性肾衰竭病人血浆中类似的AOPPs。
体内氧化应激的状态会造成蛋白质肽链断裂、交联、碳基生成和构象变化等结构和功能改变,形成氧化修饰蛋白质AOPPs。在生理状态下,氧化修饰的蛋白质很易被蛋白水解酶水解,蛋白质氧化修饰有利于体内蛋白质的更新与代谢。但是,在氧化应激状态下,蛋白质修饰的速率会增加,体内AOPPs水平明显增加。AOPPs水平的增高与许多氧化应激相关性疾病如慢性化脓性中耳炎、慢性肾脏疾病、糖尿病、肥胖症、恶性肿瘤的发生发展有很大的关系,并与疾病的严重程度密切相关。因此,AOPPs是氧化应激的重要标志物。
AOPPs不仅是氧化应激的后果,其本身作为致病介质具有广泛的病理作用,参与人类多种重要疾病的发生发展。有研究已经证实AOPPs能够激活单核细胞,诱导呼吸链爆发,增加ROS的生成,刺激TNF-α、IL-6、IL-1等促炎症细胞因子的合成释放,诱发细胞炎症反应,参与慢性肾衰患者全身微炎症状态的发生发展。此外,AOPPs本身就是活性氧族物质ROS的来源,可作为大分子生物介质参与许多病理过程。
RA患者普遍存在以氧化还原失衡为特征的氧化应激。RA患者氧化应激水平会升高会导致体内的血液、关节液中的蛋白质尤其是白蛋白的氧化损伤明显增加,进而增加RA患者体内AOPPs的含量,提升ROS水平。近年来,许多研究证实ROS可激活下游的多条细胞信号转导通路,如促分裂素原活化蛋白激酶(Mitogen-activated protein kinases, MAPK)、核因子-κB (Nuclear factor-kappa B,NF-κB)等信号通路。有研究证实ROS敏感的NF-κB信号途径在滑膜细胞炎症反应过程中起着重要的调节作用。
人类关节滑膜为疏松结缔组织,可分为两层：滑膜衬里层和滑膜衬里下层。滑膜衬里层是由成纤维细胞样滑膜细胞(Fibroblast-like synoviocytes,FLS)和巨噬样细胞组成,而衬里下层是由疏松结缔组织基质构成。FLS与其深层的纤维细胞并无基底膜相隔,直接与纤维结缔组织或脂肪组织相连接,故在关节发生炎症时易蔓延到周围组织,是介导RA患者发生滑膜炎症的重要细胞。
由于AOPPs与氧化应激和炎症反应密切相关,所以本研究认为AOPPs(?)可能通过激活FLS内NADPH氧化酶进而提升ROS水平,激活NF-κB信号转导通路,诱导FLS炎症反应的发生,参与RA疾病的发生发展。
研究方法
1、氧化修饰蛋白质AOPPs的制备：
将鼠血清白蛋白(Rat serum albumin, RSA)溶于无内毒素的PBS中配制成20mg/ml,将RSA溶液与40mmol/L的次氯酸等体积混合,室温下放置30min进行反应,这样制备出AOPPs内的RSA与次氯酸的摩尔比为1：140。将制备好的AOPPs放入透析袋中,将装有AOPPs的透析袋放入无内毒素的4℃C PBS溶液中透析24h,除去游离的次氯酸。接着用0.22μmm的微孔滤膜过滤除菌后,再用Detoxi-Gel内毒素去除胶过滤除去内毒素。用鲎试剂法检测制备好的AOPPs内的内毒素水平,内毒素水平控制在0.025EU/ml以内的AOPPs可以用于后续试验。将成功制备好的AOPPs放入4℃冰箱保存。以氯氨T为标准,酸性条件下,测340nm光吸收,来测量AOPPs的含量。
2、大鼠滑膜细胞的培养
无菌条件下切除SD大鼠双侧后肢膝关节的滑膜组织,用眼科剪及镊子剃去滑膜组织上面的血液污渍及其他组织。用37℃C的PBS缓冲液冲洗剪下来的滑膜组织2-3次。将滑膜组织移至小青霉素瓶,加入lml D-Hanks液,用眼科剪将滑膜组织剪成1mm3的块状组织。将青霉素瓶静止5分钟,待滑膜组织块沉底后,去除上清。根据滑膜组织块的大小及含量的多少加入适量的DMEM完全培养基(含10%胎牛血清和青、链霉素各10万/L双抗液体),晃动,搅匀。将含有完全培养基的组织块均匀地放入培养瓶内,倒置培养瓶,再向培养瓶加入3～4m1完全培养基。拧紧瓶盖(透气培养瓶需拧紧,非透气培养瓶可稍留缝隙)放置于37℃、含有5%CO2培养箱内,4-6小时后将培养瓶缓慢翻转过来,使完全培养基完全浸没滑膜组织块,继续放在培养箱中静置培养。每天在显微镜下每天观察滑膜组织块生长情况。每2-3天换液一次,待组织块中成纤维样细胞长出完全后,传代培养,用第二代或者第三代滑膜细胞进行后续试验。
3、细胞活性的测定：
将FLS接种于96孔板内,用50、100、200μg/ml的AOPPs、200μg/ml未经修饰的RSA以及完全培养基DMEM刺激12小时。用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐[3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide, MTT]法测定细胞活力。
4、AOPPs诱导FLS炎症应答的病理生物学作用
将FLS接种于6孔板中,用50、100、200μg/ml的AOPPs、200μg/ml未经修饰的RSA以及完全培养基DMEM刺激12小时。用肿瘤坏死因子-α(TNF-a)和白细胞介素-1β(IL-1β)、基质金属蛋白酶-3(Matrix Metallo Preteinases-3, MMP-3)、基质金属蛋白酶-13(MMP-13)以及血管内皮生长因子(Vascular Endothelial Growth Factor, VEGF)的ELISA试剂盒检测FLS在不同浓度AOPPs的刺激下分泌TNF-α、IL-1β、MMP-3、MMP-13以及VEGF的情况,用Real-Time PCR法检测AOPPs对滑膜细胞内的TNF-α、IL-1β、MMP-3、MMP-13以及VEGF的mRNA表达水平的研究,进而探讨AOPPs对FLS炎症反应的病理作用。
5、AOPPs提升FLS内活性氧族(ROS)水平的研究
将FLS接种于96孔板中,用50.100.2001μg/ml的AOPPs.200μg/ml未经修饰的RSA以及完全培养基DMEM刺激12小时,用荧光探针二氯荧光黄双乙酸盐(DCFH-DA)检测FLS在不同浓度AOPPs的刺激下产生ROS的水平。接着用200μg/ml的AOPPs分别刺激滑膜细胞5分钟、10分钟、20分钟、40分钟、60分钟、90分钟以及120分钟,再用DCFH-DA检测在相同浓度的AOPPs刺激下,不同时间对FLS产生ROS的影响。
6、AOPPs激活FLS内NADPH氧化酶系统
将FLS接种于6孔板中,用50、100、200μg/ml的AOPPs、200μg/ml未经修饰的RSA以及完全培养基DMEM刺激0-60分钟。用免疫共沉淀法检测p47phox分别与gP91phox和p22hox结合情况,用不同浓度的AOPPs分别刺激FLS3小时、6小时、12小时以及24小时,用Western blot检测AOPPs诱导p47phox磷酸化以及NADPH亚单位p47phox、gP91phow和p22phox含量的表达；
6.AOPPs激活FLS内NF-κB的实验研究
将FLS接种于6孔板中,用50.100.200μg/ml的AOPPs.200μg/ml未经修饰的RSA以及完全培养基DMEM刺激0-60分钟。用Western blot检测AOPPs诱导下滑膜细胞胞浆IκBα及胞核NF-κB亚基p65的表达情况。
7、阻断不同层面受体介导的信号转导通路对AOPPs激活FLS炎症反应的实验研究：
用NADPH氧化酶抑制剂(DPI和apocynin)、ROS清除剂(NAC、catalase和SOD)和NF-κKB特异抑制剂(SN50)预处理FLS一定时间后,再用50、100、200μg/ml的AOPPs.200μg/ml未经修饰的RSA以及完全培养基DMEM刺激一定时间后,分别用ELISA及Real-Time PCR法检测FLS分泌TNF-α、IL-1β、 MMP-3、MMP-13以及VEGF蛋白及mRNA的水平,用用Western blot检钡AOPPs诱导下滑膜细胞胞浆IκBα及胞核NF-κB亚基p65的表达情况。
8、统计学分析：
所有实验均重复3次,每个实验指标取这些数据的平均值,以均数士标准差表示,所有统计结果应用统计软件SPSS13.0完成。多个样本均数的比较采用One-Way ANOVA方法,在做两两比较时先计算方差齐性,当方差齐时两两比较采用LSD法,方差不齐时两两比较采用Dunnetts T3法；多个样本非参数比较采取多个独立样本非参数检验(K Independent Samples Test),当p<0.05时为具有统计学意义。
实验结果：
1.AOPPs诱导滑膜细胞分泌促炎症因子、基质金属蛋白酶及血管内皮生长因子：
ELISA结果显示,伴随着AOPPs浓度的增长,滑膜细胞分泌的TNF-α、 MMP-3、MMP-13和VEGF的水平也逐渐增长,200μg/ml的AOPPs促进滑膜细胞分泌的上述细胞因子含量最多,但是100μg/ml的AOPPs促进滑膜细胞分泌的IL-1β最多,而200μg/ml的AOPPs分泌的比100μg/ml的AOPPs少,但是比50μg/ml的AOPPs要多。AOPPs促进滑膜细胞分泌的细胞因子能够被不同层面的阻断剂如ROS清除剂NAC.NADPH氧化酶抑制剂apocylnin及NF-κB抑制剂SN50所阻断。
Real-Time PCR结果与ELISA的结果相似,伴随着AOPPs浓度的增长,滑膜细胞内的TNF-α、MMP-3、MMP-13和VEGF的mRNA水平也逐渐增长,200μg/ml的AOPPs提升滑膜细胞内的mRNA水平最高,同样地,100μg/ml的AOPPs刺激滑膜细胞内IL-1β的mRNA分泌最多,而200μg/ml的AOPPs分泌的比100μg/ml的AOPPs少,但是比50μg/ml的AOPPs要多。
2.AOPPs提升滑膜细胞内ROS水平：
荧光显微镜下显示AOPPs能够显著提升滑膜细胞内的荧光强度,与RSA及DMEM组有显著的差异。DCFH-DA结果显示,伴随着AOPPs浓度的增长,滑膜细胞内的ROS水平逐渐增高,200μg/ml的AOPPs提升滑膜细胞内ROS水平最高,约为50μg/ml的AOPPs的8倍左右。而用100μg/ml的AOPPs刺激滑膜细胞0-120分钟时,在AOPPs刺激90分钟的时候滑膜细胞内的ROS水平最高,约为正常对照组的6倍左右,但是用100μg/ml的AOPPs刺激滑膜细胞120分钟时滑膜细胞内ROS的水平低于刺激90分钟的水平,约为正常对照组的3.5倍左右。不同层面受体介导的信号转导阻断剂NAC、SOD、DPI及Apocynin都能够显著抑制AOPPs提升滑膜细胞ROS的水平,ROS的水平大约为AOPPs组的二分之一到三分之一左右。
3、AOPPs激活滑膜细胞内NADPH氧化酶系统：
在AOPPs刺激作用下,滑膜细胞内的NADPH氧化酶的胞浆亚单位p47phox快速磷酸化,正常组及未经修饰的RSA组未见到此效应。免疫共沉淀检测到AOPPs促进了p47phox与细胞膜上的亚单位p22phox、gp91phox的结合,形成复合体。与正常组及RAS组相比, AOPPs刺激3、6、12及24小时后明显增加了p47phox、 p22phox及gp91phox亚单元蛋白表达。
4、AOPPs激活滑膜细胞内NF-κB系统：
在AOPPs的刺激作用下,滑膜细胞胞浆内的NF-κB系统内的IκBα逐渐降解,而在细胞核内,NF-κB的亚基p65的表达逐渐升高,正常组及RSA组无此效应。AOPPs诱导滑膜细胞NF-κB系统激活的效应能够被阻断剂DPIapocynin、catalase和SOD所抑制。
结论：
1.AOPPs促进滑膜细胞分泌炎症因子IL-1β、TNF-α、基质金属蛋白酶MMP-3、MMP-13以及血管内皮生长因子VEGF产生。
2.AOPPS激活了滑膜细胞内的NADPH氧化酶系统。
3. AOPPs提升了滑膜细胞内ROS的水平。
4.AOPPs激活了滑膜细胞内的NF-κB系统。
5.NADPH氧化酶系统抑制剂、ROS清除剂及NF-κB系统抑制剂都能够阻断滑膜细胞分泌炎症因子IL-1β、TNF-α、MMP-3、MMP-13以及VEGF产生。
Backgroud
Rheumatoid arthritis (RA) is a kind of chronic autoimmune diseases characterized by symmetrical joints disease. The basic pathological alteration of this disease is inflammatory cell infiltration, the proliferation of fibroblast-like synoviocytes and the formation of pannus. The inflammatory of synovium of the joint and pannus are the main pathological character of the disease. The formation of aggressive pannus will destroy the articular cartilage, subchondral bone and the around tissue.
The morbidity of RA in the world is0.5%-1.0%and the proportion of average age of onset younger than45is80%. The disability rate in the first5years is reach up to30%. The clinical manifestation of RA is sell and pain of the affected joint and followed by ankylosis and deformity of the joint and at last the patient will be incapacitated because of joint dysfunction. The development and progression of RA will do harm to the physiology and mentality of patients followed by affecting the daily life and public activity and at last lower the quality of the patients' life. Therefore, RA is a kind of disease which poses a serious threat the health of humans and protection and treatment of the disease have been an important mission of the medical staff.
Oxidative stress is a kind of state which the balance of oxidative system and antioxidant system is broken result in the increase of reactive oxygen species (ROS). ROS, which consist of O2-,-OH and H2O2, are the production of aerobic metabolism. ROS play a role of second messenger in cell signaling cascade. Nicotinamide-adenine dinucleotide phosphate (NADPH) is the main source of ROS. Oxidative stress will damage the protein, lipid and DNA and the occurrence of protein damage is earlier than that of lipid and DNA. Therefore, it is widely believed that protein damage is the main original target of oxidative stress.
Advanced oxidation protein products (AOPPs) are a kind of protein crosslinked product containing Dtyr. During the procession of oxidative stress they are formed by the reaction of HCLO and albumin. They are a kind of landmark of oxidative stress and the generic terms of protein oxidation. They are separated from patients of chronic renal failure by Witko-Sarsat in1996. And in vitro, AOPPs can be obtained by the reaction of Human serum albumin and HCIO.
The status of oxidative stress will make the protein's construction changed and at last the advanced oxidation protein products were formed. In the physiological status the advanced oxidation protein products were easily hydrolysed by proteolytic enzyme and this is helpful to the update and metabolism of proteins in the body. However, in the status of oxidative stress the rate of protein modification will be increased and the level of AOPPs will be raised. High level of AOPPs is connected with many diseases such as chronic suppurative otitis media, chronic renal disease, diabetes mellitus, obesity and cancer. Therefore, AOPPs are important landmark of oxidative stress.
AOPPs are not the result of oxidative stress but also the pathogenic factor participated in the occurrence and development of many diseases. Many studies have demonstrated that AOPPs participated in the activation of monocyte, induce the break out of respiratory chain, increase the production of ROS and induce the cytokines, such as TNF-α、IL-6、IL-1release, participated in the inflammatory response. Furthermore, AOPPs are the resource of ROS and act as macromolecule take part in many pathological processes.
Oxidative stress is ubiquitous in RA patients. The high level of oxidative stress will cause the damage of protein in blood and synovial fluid and at last elevate the level of AOPPs in RA patients. In recent years, many studies have demonstrated that ROS can activated many cell signal transduction pathways such as Mitogen-activated protein kinases(MAPH) and Nuclear factor-kappa B (NF-κB). A study has demonstrated that NF-KB is important in regulating the inflammatory response of fibroblast like synoviocytes (FLS).
Human's synovium is loose connective tissue consist of synovial lining layer and synovial lining down the lower. The synovial lining layer consist of fibroblast like synoviocytes (FLS) and macrophage. The FLS are important cells which directly connect to the fibrous connective tissue or adipose tissue and therefore the cells are easily spread to the surrounding tissue when the inflammatory reaction happens
Oxidative stress has close relationship with the inflammatory reaction so we believe that AOPPs can activate the NADPH oxidase, raise the level of ROS, activate the NF-κB signal transduction pathway, induce the inflammatory reaction happen and at last particated the occurence and development of RA.
Matedals and methods
1. AOPPs-RSA Preparation and Determination
AOPPs-Rat Serum Albumin (RSA) was prepared according to a described procedure with minor modifications. Briefly, RSA solution (20mg/ml, St Louis, MO, USA) was exposed to200mmol/L of HOCL for30mins at room temperature and then dialyzed against PBS at4℃for24hrs to remove free HOCL. Control incubation was performed in native RSA dissolved in PBS alone. All the preparations were passed through a Detoxi-Gel column (Thermo, Massachusetts,USA) to remove any endotoxin. An amebocyte lysate assay kit (Sigma,USA) was used to determine the level of endotoxin in both AOPPs-RSA and unmodified RSA group and the concentration of endotoxin in them were below0.025EU/ml. AOPPs content in the sample was determined as described previously. Briefly,200μl of sample or chloramine-T was placed in a96-well plate, and then20μl of acetic acid was added. A microplate reader was used to measure the absorbance at340nm immediately.
2. Fibroblast-like synoviocytes (FLSs) culture
FLSs were obtained according to a described procedure with some modifications. Fresh synovial tissues were isolated aseptically from both knee joints of Female Lewis rats (4weeks old,150-200g) and washed with phosphate-buffered saline (PBS). After that they were minced and digested in a solution of0.2%collagenase in Dulbecco's modified Eagle's medium (DMEM, Gibco, Life Technologies, California, USA) at37℃for2.5hrs, the tissue were further digested by0.25%trypsin for2hrs and then the cells were centrifuged at1000g for5mins.
FLSs were seeded in,25cm2flat-bottom culture flasks and supplemented with DMEM containing10%fetal bovine serum (FBS)(Gibco, Life Technologies, California, USA) and antibiotics (100IU/ml penicillin,100IU/ml streptomycin, Irvine Scientific, Santa Ana USA). The cells were cultured by incubating at37℃in a humidified atmosphere with5%CO2. After reaching a subconfluent state, the cells were subcultured after trypsinization with0.25%trypsin/0.02%EDTA. Third to sixth passage cells were used for all the experiments. All animal procedures performed with permission and followed the guidelines laid down by the Animal Use and Care Committee of Southern Medical University.
3. The determination of cell activity:
The FLS were seeded in96-well plates. FLSs were extensively washed with PBS, cultured in DMEM with0.5%serum for12hrs, and then stimulated by adding control medium, various concentrations of AOPPs-RSA (50,100,200μg/ml) or unmodified RSA (200μg/ml).3-(4,5)-dimethylthiahiazo(-z-yl)-3,5-diphenytetrazoliumromide (MTT) is used to determine the cell activity.
4. AOPPs induce the inflammatory response of FLS.
To investigate whether AOPPs induce cytokines production in FLSs, we analyzed the supernatant of the synovial cells cultured with AOPPs. To be specific, cells were cultured in DMEM with FBS (10%) and then synchronized for12hrs with DMEM/0.5%FBS. FLSs were stimulated with increasing concentrations of AOPPs (50,100,200μg/ml) or unmodified RSA (200μg/ml) or control medium respectively. After that, cell supernatants were collected and centrifuged at12000g for15mins. Supernatants were stored at-80℃until experimentation. Levels of MMP-3, MMP-13, TNF-a, IL-1β and VEGF in the supernatant were quantified respectively using the MMP-3ELISA kits, MMP-13ELISA kit, TNF-aELISA kit, IL-1βELISA kit and VEGF ELISA kit according to the manufacturer's protocol. The OD was measured at450nm by a spectrophotometric plate reader. All the experiments were performed and tested in triplicate.
5. AOPPs upraise the level of ROS
The level of intracellular ROS was assessed by fluorescence microplate reader with the probe2',7'-dichlorofluorescein diacetate (DCFH-DA), which oxidizes to fluorescent dichlorofluorescein (DCF) in the presence of ROS, as described previously. Briefly, FLSs were suspended in DMEM at a given concentration of108/L and150μL of cells suspension was added to the96-well plates. Each sample was incubated in10μM DCFH-DA for30mins in darkness followed by AOPPs treatment as described above. Fluorescence intensity was measured on a SpectraMax M5system. The excitation and emission wavelength were488nm and525nm respectively. All the obtained data were normalized with the control values.
6. AOPPs activated the NADPH system
FLSs were extensively washed with PBS, cultured in DMEM with0.5%serum for12hrs, and then stimulated by adding control medium, various concentrations of AOPPs-RSA (50,100,200μg/ml) or unmodified RSA (200μg/ml) for60mins. Immunoprecipitation and immunoblotting were peformed respectively to analyze the phosphorylation of p47phox and interaction of p47phox with p22phox or gp91phox. To determine total p47phox, p22phox or gp91phox, the membranes were washed with an elute buffer, reacted with anti-p47phox monoclonal antibody, anti-p22phox or anti-gp91pbox polyclonal antibodies respectively and then detected by the HRP-conjugated anti-IgG antibody.
7. AOPPs activated the NF-κB system
FLSs were extensively washed with PBS, cultured in DMEM with0.5%serum for12hrs, and then stimulated by adding control medium, various concentrations of AOPPs-RSA (50,100,200μg/ml) or unmodified RSA (200μg/ml) for60mins. Western blotting was performed to detect the specificity of antigen-antibody interaction using total cellular proteins and the nuclear proteins, extracted from FLSs according to manufacturer's instruction of nuclear and cytoplasmic extraction reagents kit. Proteins (35μg) were loaded per lane and separated by10%SDS-PAGE and electrotransferred to PVDF membranes by a semi-dry transfer. Then the PVDF membranes was blocked in5%nonfat milk in TBS-Tween for1hr at room temperature and incubated overnight at4℃with the primary antibodies anti-NF-κB p65, anti-IκBα, respectively. Later the membranes were washed three times for10mins each in TBST and incubated for1hr at room temperature with appropriate HRP-linked secondary antibodies. The relative levels of protein were determined by densitometry using Total Lab2.0software.
8. To explore the effect of blocking the different levels of signal transduction pathway in AOPPs induced the inflammatory response.
FLSs were extensively washed with PBS, cultured in DMEM with0.5%serum for12hrs, and then stimulated by adding control medium, various concentrations of AOPPs-RSA (50,100,200μg/ml) or unmodified RSA (200μg/ml) in the presence of antioxidant N-acetyl-L-cysteine (NAC), superoxide dismutase (SOD), diphenyleneiodonium (DPI), apocynin, catalase and SN50. And then we performed the ELSA, Real-time PCR and western blot again to test the expression of TNF-a, IL-1β, MMP-3, MMP-13, VEGF, IκBα and NF-κB in FLS.
9. Statistical analysis
All the experiments were performed in triplicate. Results were expressed as mean±standard deviation. Statistical differences between means for different groups were compared using one-way ANOVA (analysis of variance). Multiple comparisons were performed using the LSD method or Dunnett's T3method. Statistical differences between nonparametric for different groups were compared using K Independent Samples Test procedure. Comparing several treatments with control group were evaluated by the Statistical analyses were conducted with SPSS13.0software.
Results:
1. Effect of AOPPs stimulation on the expression of IL-1β, TNF-α, MMP-3, MMP-13and VEGF.
Initially, we tested whether AOPPs can induce FLSs to release cytokines. As shown in Fig.1a-c, secretion of cytokine IL-1β, TNF-α, MMP-3, MMP-13and VEGF by FLSs in AOPPs group were in a concentration-dependent manners compared with low levels detected in the conntrol cells and unmodified RSA group (Fig.l b and c). However, it is seen that the expression of IL-1β in200μg/ml AOPPs group was lower than that in100μg/ml AOPPs group but still at a signaficantly higher levels than control cells and RSA group.
Real-time RT-PCR is used to quantify the effect of AOPPs at various concentrations on the expression of IL-1β, TNF-α, MMP-3, MMP-13and VEGF in mRNA. Compared with the control cells and unmodified RSA group, the mRNA levels were significantly increased when FLSs were cultured with AOPPs. And no significant difference was found in the messenger RNA expression between unmodified RSA group and control cells. When the above data is evaluated, it indicates that FLSs can be stimulated by AOPPs to secrete cytokines at both protein and gene level, which may be involved in the pathological progression of RA.
2. AOPPs induce ROS generation in FLSs
In order to study the ROS generation in FLSs, we challenged AOPPs group in FLSs and noticed a siginificant increase of ROS level (3-to8-fold) in AOPPs group compared to both unmodified RSA cells and control cells. When FLSs were incubated with AOPPs at different concentration within90mins it showed a time-dependent increase in ROS generation. Furthermore, the ROS generation in FLSs cultured with AOPPs was observed under a fluorescence microscopy with DCFH-DA.
3. AOPPs activated the NADPH system
To investigate whether AOPPs treatment activate NADPH oxidase, initially we measured the phosphorylation of p47phox, a subunit of NADPH oxidase located in the cytoplasm of the FLSs. AOPPs group showed rapid phosphorylation of p47phox at5mins, and peaked at60mins, whereas control group and RSA group had no siginificant effect. Translocation of p47phox to the cell membrane plays a key role in NADPH oxidase activation. In order to examine the interaction of p47phox with the membrane subunits, we immunoprecipitated p22phox and gp91phox with the specific antibodies and then probed for the coexistence of p47phox in the cells. The amount of p47phox-gp91phox complex rapidly increased in AOPPs group at5mins and p47phox-p22phox complex appeared later than p47phox-gp91phox complex at15mins. Likewise, to determine sustained activity of NADPH oxidase we examined the protein levels of its subunits in FLSs treated with or without AOPPs. AOPPs group showed significant upregulated expression of p47phox, p22phox and gp91phox compared with control cells after6hrs. Only the expression of gp91phox was increased after3hrs in AOPPs group.
4. AOPPs activated the NF-kB system
In order to explore the potential molecular mechanism underlying the AOPPs actions on FLSs, western blot was performed to examine if AOPPs triggers NF-κB activation in the cells. As we know two important steps before NF-κB activation are IκBα degradation in cytoplasm and NF-κB p65translocation to nucleus. In AOPPs group (200μg/ml) IκBα degradation in cytoplasm was observed at15mins, which continued till60mins and NF-κB p65proteins showed an increased level in nucleus from5mins till60mins. These data suggested that AOPPs can induce the degradation of IκBα, which leads to the nuclear translocation of p65and at last activate NF-κB in FLSs.
Conclusions:
1.AOPPs induced the expression of IL-1β、TNF-α、MMP-3、MMP-13and VEGF in FLS.
2. AOPPs activated the NADPH system in FLS;
3. AOPPs induced ROS generation in FLSs
4. AOPPs activated the NF-kB system;。
5. Blocking the different levels of signal transduction pathway will block AOPPs induce the inflammatory response.
引文
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