| BackgroundRheumatoid Arthritis(RA)is the most common inflammatory arthritis that is characterized by disruption of immune tolerance,and production of various autoantibodies and inflammatory cytokines.The prevalence rate of RA is 0.5%-1%in worldwide,but approximately 0.2%-0.4%in China.Prior study reported that the joint of RA patients might be destructed irreversibly in two years and nearly half patients lose their ability to work in 10 years without early and effective treatments.Current pharmacological therapies of RA include non-steroidal anti-inflammatory drugs(NSAIDs),disease-modifying antirheumatic drugs(DMARDs),glucocorticoids and biological agents.NSAIDs can suppress the inflammation and relieve joint pain,but not effectively control symptoms and disease progression.DMARDs,mainly inhibiting immune system,exert slow effect,large side effects and high risk of infection.Additionally the efficacy was reduced and drug resistance may occur for long-term use.Current biological agents of RA are designed to ameliorate disease by blocking the activity of inflammatory cytokines such as tumor necrosis factor-?(TNF-?),interleukin 1?(IL-1?),IL-6 to exert a quick therapeatic effect,but have high risk of infection and high price.Above all,it is essential to further investigate the mechanism of RA and explore new pharmacological drugs or agents with remarkable curative effect but little side effects for RA.Nucleotide-binding oligomerization domain-like receptor protein3(NLRP3)inflammasome is a kind of multiprotein complex,which contains NLR,ASC(apoptosis-associated speck-like protein containing a CARD)adaptor and caspase-1.The activation of NLRP3 inflammasome mediated by the cleavage of the precursor of pro-IL-1? and pro-IL-18 into active IL-1? and IL-18,which result in the production of inflammation.NLRP3 exert in various inflammatory cells,including monocytes,dendritic cells(DCs)and neutrophils.Previous study reported that the activation of NLRP3 inflammasome play an important role in many clinical inflammatory disorders and autoimmune diseases.Deficiency of NLRP3 gene or caspase-1 gene in encephalomyelitis(EAE)mice showed the significance reduction of neurons demyelinating.Non-specifically inhibiting NLRP3 inflammasome can relief the severity of MRL/lpr lupus-like mice.Nearly half macrophages in the synovium of RA joint secret IL-1?,suggesting that IL-1? has involved in the pathogenesis of RA.It has been found that significance increase of NLRP3 in serum of RA patients induced IL-1? secretion via the activation of caspase-1,which aggravate the disease.Aslo gene expression of ASC,NLRP3-FL in serum from patients with RA are much higher than that in the health control group,which is accompanied by the increase of caspase-1 and IL-18.Accumulation studies showed that inhibiting the secretion of IL-1? prevented the development of RA.And anakinra,a IL-1 receptor inhibitor,can effectively relieve the symptoms and control RA progression.In conclusion,NLRP3 inflammasome play an important role in thein the initiation and development of RA,and NLRP3 inflammasome may be a potential target for the treatment of RA.A report from the journal of nature medicine in 2015,found that a small molecule named MCC950 can specificallyinhibit the activation of NLRP3 inflammasome by interaction with ASC and blocking caspase-1 activation,which lead to the reduction of the release of IL-1?.And MCC950 Treatment in EAE model can alleviate EAE by supressing NLRP3 inflammasome.In order to investigated the role of NLRP3 inflammasome in RA,we studied the effect of MCC950 treatment on the clinical features in a collagen-induced arthritis(CIA)mouse model.Cytokines are kinds of small molecular peptides secreted by immune cells.They play an important role in signal transmission within various kinds of immune cells,chemotaxis immune cells,induce cell growth,and regulate the function of immune cell.Therefore,cytokine network as the executor and embodiment of immune system can reflect the overall function of the body's immune system effectively.Our previous studies suggested that cytokines can be used to evaluate the immune status and reflect the function of immune system.Many cytokines also play an important role in the pathogenesis of rheumatoid arthritis.It can be generally divided into monocytes factors,lymphocytes factors and chemokines and so on.Different cytokines have different functions.Many cytokines play an important role in the pathogenesis of RA,such as IL-1?,IL-6,IL-10,IL-18,IL-33,IFN-?,TNF-?,IL-17,GM-CSF,MCP-1 and MIP-1? and so on.And many cytokine inhibitors such as TNF-? inhibitors and L-1?blockers have been applied in RA therapy.All of these suggest that inhibit cytokines can control the immune system effectively.Many articles have reported that MCC950 can inhibit IL-1? secretion by suppressing NLRP3 inflammasome.However,the effect of MCC950 on other cytokines for example,monokines,Iymphokines and chemokines remain unknown.Helper T lymphocyte(Th)cells are very important in regulating immune response.Th cells can be divided into Th1,Th2,Th17 and Treg cells according to different functions.Various Th cells have different functions in the immune system.However,the most important function is the balance between Th cells.Traditional viewpoint holds that there is a balance between Th1/Th2 cells in the normal immune system.The break of balance cause to disorder of the immune system.It has been reported that the break of balance in Th1/Th2 cells and switching to Thl play an important role in the pathogenesis of RA.Thl cells and their associated cytokines play a critical role in the pathogenesis and progression of RA.During the study of Th17 cells and Treg cells,the balance between Thl7/Treg also plays an important role in the development of RA.Many studies have found that the number and function of Tree cells are defective,which involve in the pathogenesis of RA.The number of Treg was reduced in early RA patients.Thus suggesting that Treg may play a protective role in RA progression,Whereas,Th17 play an promoting role in the pathogenesis of RA through the secretion of IL-17.Th17 cells and Treg cells play a contrast role in immunoregulation.The above studies have shown that Thl7 cells and IL-17 are predominate in RA patients,but deficiency in Treg cells,lead to Treg/Th17 imbalance and aggravation of inflammation.Additionally,Th cell subsets and the balance between Th1/Th2 or Thl7/Treg play an important role in the pathogenesis of RA.MCC950 can inhibit NLRP3 inflammasome and act on the monocyte-macrophage cells,DC cells and most myeloid cells.The effects of MCC950 on Th cells,and the regulation of balance between Th1/Th2 or between Treg/Th17 are still unclear.ObjectiveIn order to investigated the role of NLRP3 inflammasome in RA,we studied the effect of MCC950 treatment on the clinical features in a collagen-induced arthritis(CIA)mouse model.In Vitro Experiment:1.To clarify the effect of MCC950 on the expression of IL-1?and the cytotoxicity of MCC950 in peripheral blood mononuclear cell.2.To clarify the effect of MCC950 on the secretion of cytokines from peripheral blood in healthy volunteers.3.To clarify the effect of MCC950 on Th subsets in healthy volunteers and RA patients.Animal Experiments:1.To clarify the therapeutic effect of MCC950 on CIA mouse model.2.To clarify the effect of MCC950 on cytokines secretion in peripheral blood in CIA mice.3.To clarify the effect of MCC950 on T lymphocyte subsets in CIA mice.Method1.The cytotoxicity of MCC950 in mononuclear cell and its influence on cytokines of whole blood and Th subsets.(1)Peripheral blood mononuclear cell sorting:Centrifuge the whole blood of the healthy volunteers with the lymphocyte separation medium,and the centrifugal rotational speed is 400 g/min,suck out gray layer of mononuclear cell suspension,then add in RPMI-1640,mixing the cells fully.(2)Determine the apoptosis/necrosis ratio of peripheral blood mononuclear cells(PBMCs):for MCC950 cytotoxicity test,adding various concentrations of MCC950 into PBMCs,then check the rate of apoptosis/necrosis with Flow cytometry(FCM)24 hours later.(3)Cell Counting Kit-8(CCK-8)method tests the cytotoxic effect of mcc950.Adding various concentrations of MCC950 into PBMCs,and then add the CCK-8,at last test the optical density(OD)value of each groups.(4)Enzyme-linked immunosorbent assay(ELISA)to detect the expression of IL-1?in supernatant:Peripheral PBMCs or THP-1 stimulated with LPS and ATP,then adding MCC950(10?M)to detect the effect of MCC950 on IL-1? expression.(5)Cytokines in peripheral whole blood were detected by LUMINEX system.LUMINEX system can simultaneously detect various cytokines in a small sample(15?l).There are shorter experiment times and less error compared with ELISA.Take the venous blood from healthy volunteer,then add MCC950 under the stimulus of LPS+ATP,then serum was separated by centrifugation at 6,12 and 24 hours respectively.We detect 17 kinds of cytokines in the serum by LUMINEX system.(6)Get the venous blood from healthy volunteers and separate PBMCs,add MCC950 and cultivate for 72 hours,then detect Th1,Th2,Thl7 under the stimulus of PMA+IONO and Treg cell under the stimulus of ConA.Detect Th1(CD4+IFN-?+),Th2(CD4+IL-4+),Thl7(CD4+IL-17A+)and Treg(CD4+CD25+D127low)subsets by flow cytometry.(7)Get the venous blood from RA patients and separate PBMCs,add MCC950 and cultivate for 72 hours,then detect Th1,Th2 and Th17 under the stimulus of PMA+IONO and Treg cell under the stimulus of ConA.Detect Th1(CD4+IFN-?+),Th2(CD4+IL-4+),Th17(CD4+IL-17A+)and Treg(CD4+ CD25+D127low)subsets by flow cytometry.2.The effect of MCC950 on the pathogenesis of arthritis on collagen-induced arthritis(CIA)mice(1)Male DBA/1J mice(6-8 weeks old)were immunized with lyophilized bovine type? collagen.Male 6 to 8 week-old DBA/1J mice were intradermally inoculated at the base of the tail with 100?g of bovine type ? collagen(Chondrex)mixed with 0.1 ml Freund Complete Adjuvant(Chondrex).Twenty-one days after the primary inoculation,the mice were boosted once with 100?g of bovine type ? collagen in 0.1 ml Freund Incomplete Adjuvant(Chondrex).Mice were divided into three groups,including control group,model groups and MCC950 treatment group.The mice were carefully monitored for the development and severity of joint inflammation after the second booster immunization,Arthritis was scored by two trained examiners via a visual assessment scoring(VAS)system for the degree of inflammation on a scale from 0 to 4.0,no evidence of erythema and swelling;1,erythema on one or two toes or mild swelling confined to the midfoot(tarsals)or ankle joint;2,erythema and mild swelling extending from ankle to the mid foot;3,erythema and mild swelling extending from ankle to metatarsal joints;and 4,erythema and severe swelling encompassing the ankle,foot,and digits.The clinical score for each mouse was determined by summing the scores for all individual limbs and divided by four.At the termination of the experiment,the mice were sacrificed on day 54.The MCC950 treatment group administered intraperitoneally(i.p.20mg/kg)everyday.One hour before the first collagen injection,with treatment continued daily until the end of the experiment.(2)The body weight of mice were recorded at the termination of the experiment and the mice were sacrificed on day 54.(3)Joints of the mice were removed,fixed in 10%formalin,decalcified in 10%EDTA for 14 days and embedded in paraffin.Sections of 5 ?m were stained with hematoxylin and eosin(H&E),and analyzed for histological signs of inflammation,pannus formation,and cartilage and bone destruction.(4)The cytokines levels of serum from CIA mice were assayed by LUMINEX system.First,whole blood of mice was collected and centrifuged.Then collected the serum for cytokines analysis.(5)The ratio of T and B lymphocytes and T cell subsets in whole blood were detected by Flow Cytometry analysis.PBMCs from the whole blood were collected and stained with the following monoclonal antibodies:FITC-anti-CD3 for T lymphocytes and APC-anti-CD19 for B lymphocytes.For analysis of T lymphocytes subsets in PBMCs,cells were stained for 30 min at 4?with the following antibodies:Thl(CD4+IFN-?+),Th2(CD4+IL-4+),Th17(CD4+IL-17A+)and Treg(CD4+Foxp3+).3.Statistic analysisStatistical analysis was performed using SPSS 16.0.All the results were shown as MEANsąSEM.Factorial design for multiple factors and intergroup comparisons were made using one-way analysis of variance(ANOVA)and Least Significant Difference(LSD)test.Welch Test and the Dunnett T3 analysis were used to compare all pairs of means following one-way ANOVA.Statistical significance was accepted when P<0.05.We used Graph Prism 5.0 to make the chart.Results:1.The results for vitro experiment:(1)Different concentrations of MCC950(1Mm,10?M and 50?M)had no effect on the rate of apoptosis/necrosis of PBMCs separated from human peripheral whole blood,suggesting that MCC950 has no cytotoxicity in PBMCs.(2)Co-cultured with LPS+ATP,The secretion of IL-1? in THP-1 cell line and human PBMCs significantly increased.But MCC950 dramatically reduced the IL-1? release in LPS+ATP stimulated THP-1 cell line and human PBMCs.(3)The whole blood from healthy volunteers treated with LPS and ATP for 6 hours,cytokines including G-CSF,GM-CSF,IFN-?,IL-2,IL-4,IL-5,IL-6,IL-12,IL-13,IL-17,MCP-1,MIP-1? and TNF-? in the serum were significantly increased The serum concentration of TNF-? and IL-1? in LPS+ATP+MCC950(10?M)group was reduced distinctly as compared with LPS+ATP group.(4)The whole blood from healthy volunteers treated with LPS and ATP for 12 hours,cytokines including G-CSF,GM-CSF,IFN-?,IL-2,IL-4,IL-5,IL-6,IL-12,IL-13,IL-17,MCP-1,MIP-1? and TNF-? in the serum were significantly increased compared with the control group.The serum concentrations of IFN-?,IL-1?,TNF-?were significantly lower in LPS+ATP+MCC950(10?M)group than that in LPS+ATP group,and the difference was statistically significant.(5)The whole blood from healthy volunteers treat with LPS and ATP for 24 hours,cytokines including G-CSF,GM-CSF,IFN-Y,IL-2,IL-4,IL-5,IL-6,IL-12,IL-13,IL-17,MCP-1,MIP-1? and TNF-? in the serum were increased significantly.The serum concentrations of G-CSF,GM-CSF,IFN-?,IL-1?,IL-2,IL-8 and IL-17were clearly reduced in LPS+ATP+MCC950(10?M)group as compared with LPS+ATP group,and has significant statistically difference.(6)The effect of MCC950 on Th subsets in the PBMCs from healthy volunteers:Compared with control group,there is no significant diffenece in the ratio of Thl and Th2 of PBMCs from healthy volunteers in MCC950 treatment group.However the Th1/Th2 in PBMCs reduced significantly in MCC950 treatment group.Aslo MCC950 dramatically reduced Thl7 cells in peripheral whole blood as compared with control group.In contrary Treg cells in peripheral whole blood significantly increased in MCC950 treatment group.AndTreg/Th17 in peripheral whole blood aslo increased in MCC950 treatment groups.(7)The effect of MCC950 on Th subsets in the PBMCs from patients with RA:Compared with control group of RA patients,there is no significant diffemece in the ratio of Th1 and Th2 ratio in peripheral whole blood in MCC950 treatment group,but ratio of Th1/Th2 in peripheral whole blood was reduced in MCC950 treatment group.And compared with control group of RA patients,the ratio of Th17 and Treg cells in in the PBMCs had no change in MCC950 treatment group.However,the ratio of Treg/Th17 in the PBMCs significantly increased in MCC950 treatment group.2.The results for vivo experiment:(1)There was no different of body weight among the groups of mice before or after treatment.Meaning that MCC950 had no effect on body weight of mice.(2)MCC950 treatment delayed the onset and reduced the severity of joint arthritis in a CIA model.Our results showed that joint arthritis began to day 25 after the first immunization and reach peak on day 41 in CIA model group,and the rate of incidence was 100%,which showed that collagen induced arthritis model was successfully established.Compared with CIA model control group,MCC950 treatment group significantly delayed the onset of arthritis,and arthritis symptoms occur on day 28 after the first immunization and joints of some mice remained normal until they were sacrificed on day 54.And incidence of arthritis in MCC950 treatment group was 83.3%and The arthritis score was significantly reduced.MCC950 treatment group show lower incidence of arthritis and less joint swelling as determined by the clinical scores at 54 days,suggesting that MCC950 had a therapeutic effect on CIA mice.(3)Histological examinations also indicated that MCC950 treatment suppressed accumulation of inflammatory cells in the joint space,synovial hyperplasia,articular cartilage destruction and bone erosion compared with CIA model group.(4)Compared with control group,there is a significant increase of levels of IFN-?,TNF-? and IL-1? in CIA model group,While mice with MCC950 treatment showed a dramatic decrease in the serum cytokines secretion of IL-17,IFN-? and TNF-? and IL-1?.There levels of the rest cytokines have no significant difference between CIA model group and MCC950 treatment group.(5)The rate of Th1 and Th17 in peripheral whole blood increased significantly in the CIA model group.However compared with the control group,there is no significance difference in ratio of Th2 and Treg.And there are significantly increase of Thl/Th2 and decrease of Treg/Th17 in peripheral whole blood in the CIA model group mice,.(6)Compared with the CIA model group,MCC950 treatment increased the percentage of Th2 and Treg cells,but have no effect on Thl and Th17.The rate of Th17/Treg increasd while the rate of Thl/Th2 in peripheral whole blood deceased in MCC950 treatment group mice..Conclusion:MCC950 inhibits the production of IL-1? in human THP-1 cell line and PBMCs.LPS+ATP can stimulate whole blood cell secretion many cytokines and The concentration of cytokines changes over time.MCC950 inhibits many cytokiens secretion such as IL-1? in whole blood.MCC950 increases the rate of Treg/Th17 and decreases the rate of Th1/Th2 in PBMCs from RA patients or healthy volunteers.In CIA model,MCC950 treatment delays the onset and reduces the incidence of arthritis and severity of joint arthritis,pathological damage.Compared with the CIA model group,IL-17,IFN-?,TNF-? and IL-1? can be significantly reduce in MCC950 treatment group.Aslo,Compared with the CIA model group,the percentage of Th2 and Treg in PBMCs increased significantly in MCC950 treatment group,while Thl and Th 17 had no change;meanwhile the ratio of Treg/Th 17 increased and the Th1/Th2 decreased.In summary,MCC950 reduces the severity of joint arthritis and the inflammation of joints in a CIA model via inhibit the activation of NLRP3 inflammasome,which was associated with reducing the inflammatory cytokines such as IL-1?,TNF-?,IL-17 and IFN-?,elevating the proportion of Treg/Th17 and reducing the Th1/Th2. |
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