| Rheumatoid arthritis (RA) is a chronic, inflammatory autoimmune disease that targets the synovial membrane of joints, resulting in bone erosion, joint destruction, and joint deformtied. The clear mechanisms of RA pathogenesis have not been fully elucidated. Studies have suggested that inflammation is related to the pathogenesis of RA, by which abnormal cell mediated immune responses and humoral immune responses are considered to play an important role in. In addition, macrophages have been recognize to perform in the pathogenesis of the disease.Innate immune response is the first line of defense against pathogens. Inflammasomes are the important part of innate immunity. They express several specialized suriace receptors known as pattern-recognition receptors (PRRs) such as TLRs, NOD-like receptors, RIG-I-like receptors and C-type lectin receptors (CLRs). These PRRs are able to capture antigens via binding to the pathogen-associated molecular pattern molecules (PAMPs). NLRP3 is one the mostly investigated inflammasomes to date, plays an important role in inflammation, the pathogenesis of cancer and maintenance of homeostasis.Adjuvant arthritis (AA) rat model is the most classic arthritis animal model. In our previous studies, inflammatory cytokines such as IL-1?,IL-18 in serum, peritoneal macrophage were significantly increased in AA model compared with normal controls. Expression of Nlrp3, ASC, caspase-1 were strongly higher in AA model as well, but miR-223 was reduced, suggesting that miR-223 may play a role in the pathogenesis of AA. MiRNAs (miRNAs) are a class of small non-coding RNAs whose mature products are -22 nucleotides long. They negatively regulate gene expression by inducing translational inhibition or transcript degradation. The present study used luciferase reporter gene assay confirmed that mir-223 targeted Nlrp3. Because of abnormal expression of miR-223, Nlrp3 inflammasome and the downstream inflammatory cytokines in AA model, peritoneal macrophage, we proposed that (1) miR-223 may regulate inflammatory cytokines production via regulating Nlrp3? (2) Targeting miR-223 may give a therapeutic target in AA? In view of Nlrp3 and miR-223 expression in rat peritoneal macrophage RAW264.7, THP-1 cells exist in common, therefore, the present study used RAW264.7 cells to discuss the hypothesis. There are four parts in the present study, including:1. Establishment of AA model and examination of inflammatory cytokinesWhen successfully established the arthritis model, weight of AA rat was reduced but the secondary swelling degree of AA rat was elevated compared with the control group since the 12th day. From the day 28 to 32, weight of AA rat was gradually increased and the secondary swelling degree was gradually reduced. Histopathological analysis showed that synovium was significantly thickened compared with the controls. There were 3-4 layers of synovial cells, which extended into the articular cavity. In addition, there were many inflammatory cells infiltrated into the joints, and generated some vessels in the joints.ELISA analysis found that serum levels of IL-1?, IL-18, IL-22, IL-33 were higher in the AA group compared with controls. When collected the cells from the abdomen, cells can bind with CD68, suggesting that these cells were macrophages. Similarly, expression of IL-1?, IL-18 was significantly higher in the cells form AA group in comparison with controls.2. Expression of Nlrp3, miR-223 in AA miceThe number of positive cells expressing Nlrp3, ASC, caspase-1 was significantly higher in the synovial tissue of AA group compared with the control group. Similarly, mRNA and protein expression of Nhp3, ASC, caspase-1 was significantly higher in the peritoneal macrophage of AA group in comparison with the controls, but the level of mir-223 was reduced in the AA group, suggesting that Nlrp3 may interact with miR-223.3. Screening test of macrophage, NLRP3 inflammsome regulate the secretion of cytokines in RAW264.7 cellsWhen LPS stimulated peritoneal macrophage, RAW264.7, THP-1 cells, we found higher expression of IL-1?.IL-18 in the model set compared with the controls. In addition, mRNA and protein expression of Nlrp3 was elevated when primary peritoneal macrophages, RAW264.7, THP-1 cells stimulated with LPS, but mRNA expression of mir-223 was reduced. Owing to the findings that both Nlrp3, miR-223 expressed in RAW264.7 cells similar to the expression in peritoneal macrophage, THP-1 cells when treated with LPS, the present study used RAW264.7 cells to discuss the role of Nhp3 in regulating production of inflammatory cytokines.After RAW264.7 cells stimulated with LPS, ATP, K~+, we found that LPS or ATP stimulation increased expression of IL-1?, IL-18, IL-33, respectively. Combined stimulation with LPS and ATP significantly increased expression of IL-1?,IL-18, IL-33. However, high level of K~+stimulation can markedly reduce the expression of IL-1?, IL-18, IL-33. In addition, western blot analysis showed that LPS, ATP stimulation, respectively, or combined stimulation with LPS and ATP increased expression of Nlrp3, ASC, caspase-1. On the contrary, high level of K~+ stimulation strongly down-regulated protein expression of Nhp3, ASC, caspase-1. To confirm the critical role of Nlrp3 in regulating production of inflammatory cytokines, we used siRNA to interfere nlrp3. Inhibition of nlrp3 can significantly reduce the expression of IL-1?, IL-18, but the change of IL-33 was not significantly. LPS, ATP stimulation increased mRNA and protein expression of Nlrp3, ASC, caspase-1, especially the level of caspase-1. However, inhibition of Nlrp3 reduced the mRNA and protein expression of Nlrp3, ASC, caspase-1. These data indicated thatNlrp3 performs significantly in regulating expression of IL-1? and IL-18.4. miR-223 regulation of Nlrp3To discuss the interaction of miR-223 and Nhp3, the luciferase reporter gene assay confirmed that miR-223 targeted Nlrp3. LPS induction reduced expression of miR-223 but increase expression of Nlrp3 in the cells, but transfecting of mimics significantly up-regulated expression of miR-223 and reduced expression of Nhp3 compared with the controls, respectively. In addition, mimics transfection can reduce mRNA and protein expression of Nlrp3. Moreover, LPS stimulation increased expression of IL-1?, IL-18 in the RAW264.7 cells, but miR-223 mimics transfection significantly reduced levels of IL-1?, IL-18 compared with the controls.LPS induction can reduce expression of miR-223 but increase expression of Nlrp3 in the cells, but transfecting of inhibitors significantly reduced expression of miR-223 and increased expression of Nlrp3 compared with the controls, respectively. In addition, inhibitors transfection can increase the mRNA and protein expression of Nhp3. Moreover, LPS stimulation increased expression of IL-1?, IL-18 in the RAW264.7 cells, but mir-223 inhibitors transfection significantly increased levels of IL-1?, IL-18 compared with the controls.To confirm the role of miR-223 in targeting inflammatory cytokines, combined transfecting of mimics and inhibition of nlrp3 significantly up-regulated expression of miR-223 and reduced expression of Nlrp3 compared with the controls. In addition, LPS stimulation increased expression of IL-1?, IL-18 in RAW264.7 cells, but combined transfecting of mimics and inhibition of Nlrp3 significantly reduced levels of IL-1?p, IL-18 compared with the controls. These findings suggested that miR-223 can target the NIrp3 inflammasome, regulate the generation of inflammatory cytokines, and therefore, may be a potential therapeutic target for AA.Conclusion1. Expression of miR-223 and NLRP3 was abnormal in the AArat model.2. NLRP3 inflammasome may play an important role in the pathogenesis of AA, which regulated the generation of inflammatory cytokines IL-1? IL-18.3. miR-223 can target the NLRP3 inflammasome, therefore, regulate the generation of inflammatory cytokines IL-1?, IL-18, and it may participate in the pathogenesis of AA. |
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