| BackgroudRheumatoid 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 happensOxidative 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 methods1. AOPPs-RSA Preparation and DeterminationAOPPs-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) cultureFLSs 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 ROSThe 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 systemFLSs 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 systemFLSs 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 analysisAll 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 FLSsIn 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 systemTo 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 systemIn 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 FLSs4. AOPPs activated the NF-kB system;。5. Blocking the different levels of signal transduction pathway will block AOPPs induce the inflammatory response. |
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