| Rheumatoid arthritis(Rheumatoid arthritis, RA) is a heterogenic and systemic autoimmune disease characterized by synovitis and joint structural damage, the pathogenesis is still unknown. Studies indicated that fibroblast-like synovial cells(FLS) play a vital role in the genesis and development of RA. Abnormal proliferation and invasion of FLS result in pannus formation, and further inducing the destruction of articular cartilage.IL-1β is considered to be the mainly inflammatory cytokine related to FLS activation.NLRP3 is a protein from pattern recognition receptors, NOD-like receptor(NLR) family. NLRP3 inflammasome can activate caspase-1, further promoting the maturation and release of IL-1β.As a redox regulator, thioredoxin-interacting protein(TXNIP) plays an important role in ROS activation pathways of NLRP3 inflammasome, however, the underlying mechanisms still under investigation.Recent studies have shown that microRNAs participated in the regulation of post-transcription and involved in various important biological processes such as cell proliferation, differentiation and apoptosis etc, which plays a pivotal regulatory role in the development of inflammation.This study is aimed at investigating the NLRP3, TXNIP and miR-20 a expression in FLS cells derived from adjuvant arthritis(AA) rat models and making an in-depth discussion on the mechanisms of inflammatory factors released by NLRP3 inflammasome activation as well as the effect of NLRP3 inflammasome activation induced by mi R-20 a in order to seeking the new targets of RA. The main contents are summarized as follows 1. Establishment of rat models of adjuvant arthritisAthological features between AA and RA. AA rat models were established for animal model studies, and FLS were chose as celluar objects. Freund’s complete adjuvant was used to construct rat models of AA. A successful AA rat model was confirmed by measuring rat secondary paw edema, polyarthritis index, spleen and thymus index and joint histopathology staining. The result indicated that the former three indexs in model groups were significant higher compared to controls. Joint histopathology staining showed that significant synovial hyperplasia and increasing cells of synovial lining layer to 3-5 layers or even more in model groups, accompanied by vast inflammatory cell infiltration and pannus generation. These results suggested: AA model was successfully established. 2. The effect of NLRP3 inflammasome on inflammatory cytokine release and its mechanism in FLS of AAThe level of rat serum IL-1β and IL-1β, MMP-1, TIMP-1 release in culture medium supernatant were detected by enzyme-linked immunosorbent assay(ELISA). In order to investigate the role of IL-1β in the development of AA, fibroblast-like synovial cells(FLS) proliferation was observed after IL-1β(10ng / ml) stimulation. The result provided increasing trends of the level of rat serum IL-1β in AA models and IL-1β, MMP-1, TIMP-1 release in culture medium supernatant of FLS. The inflammatory cytokine stimulation test also proved that IL-1β can promote the proliferation of FLS which is closely associated with the progression of AA.The activation of NLRP3 inflammasome can promote IL-1β maturation and release. For the sake of investigating the role of NLRP3 inlammasome in rheumatoid arthritis(RA), NLRP3 expression in rat synovial tissues and FLS were measured by western blotting; The transcription levels of NLRP3 / ASC / casp-1 genes were analysed by qRT-PCR; Adopting lipofectamineTM 2000 transiently transfected NLRP3-siRNA into the FLS then silenced NLRP3, qRT-PCR and western blotting were used to measure the gene and protein expressions of NLRP3 / ASC / casp-1 respectively. The expression of IL-1β, MMP-1, and TIMP-1 were detected by ELISA in order to observe the effect on NLRP3 inflammasome expression and IL-1β, MMP-1, TIMP-1 release by NLRP3 / ASC / casp-1proteins regulation. The result demonstrated that NLRP3 expression in synovial tissue of rat model group was significantly increased, protein and mRNA levels of NLRP3 / ASC / casp-1 also showed an abnormal expression in the extraction of FLS; NLRP3 Silencing by small interfering RNA can decline the NLRP3/ ASC/ casp-1 expression and inhibit inflammatory cytokines IL-1β, MMP-1, TIMP-1 secretion. These all indicated that NLRP3 presents high expression in AA models and is closely associated with the release of inflammatory cytokines such as IL-1β, MMP-1 and TIMP-1. 3. Regulation of NLRP3 expression and inflammatory cytokines release by TXNIPIn order to investigate whether TXNIP plays a regulating role on NLRP3 expression and relation with abnormal release of inflammatory cytokines, we detected the TXNIP expression in rat synovial tissues and FLS by western blotting. The transcription levels of TXNIP gene in FLS was analysed by qRT-PCR. Using siRNA technology, transiently transfected TXNIP-siRNA via lipofectamineTM 2000, qRT-PCR and western blotting were used to measure the gene and protein expressions of NLRP3 / ASC / casp-1 respectively. The expression of IL-1β, MMP-1, and TIMP-1 were detected by ELISA in order to observe the effect on NLRP3 inlammasome expression and IL-1β, MMP-1, TIMP-1 release by TXNIP regulation. The result provided that TXNIP expression in synovial tissue of rat model group was significantly increased, and mRNA level of TXNIP also showed an increased trend in the extraction of FLS; TXNIP Silencing can down-regulate NLRP3/ ASC/ casp-1 genes and proteins expression and inhibit inflammatory cytokines IL-1β, MMP-1, TIMP-1 release. These all suggested that TXNIP may promote NLRP3 inflammasome activation and further increasing the inflammatory cytokines IL-1β, MMP-1, TIMP-1 expression. 4. Regulation of NLRP3 and TXNIP expressions and inflammatory cytokines release by miR-20 a in FLS of rat AA modelsMiR-20 a expression was detected by qRT-PCR; the potential targeted relationship between miR-20 a and TXNIP was explored by bioinformatics and verified by luciferase reporter gene system. Co-transfection of mi R-20 a mimics and TXNIP-siRNA was used to overexpress miR-20 a and silence TXNIP. NLRP3 expression was analysed by western blotting, in order to verifying the targeting relationship between miR-20 a and TXNIP. Using siRNA technology, transiently transfected miR-20 a mimics via lipofectamineTM 2000, qRT-PCR and western blotting were used to measure the gene and protein expressions of TXNIP and NLRP3 / ASC / casp-1 respectively. The expressions of IL-1β, MMP-1, and TIMP-1 were detected by ELISA in order to observe the effect on TXNIP and NLRP3 inlammasome expression and IL-1β, MMP-1, TIMP-1 release by miR-20 a regulation. The result showed that miR-20 a decreased significantly in FLS. Bioinformatics software and luciferase reporter gene system also proved that the presence of targeted relation between miR-20 a and TXNIP. There is no significantly statistical difference of NLRP3 expression between co-transfection group and separated transfection group. This figures suggested that TXNIP may plays a mediate role while mi R-20 regulating NLRP3. Over-expressed miR-20 a could down-regulate TXNIP expression then further reduce NLRP3 / ASC / casp-1 expression and IL-1β, MMP-1, TIMP-1 release. It indicated that mi R-20 a possibly reduces TXNIP levels after transcription then further bring down NLRP3 inflammatory expression and inflammatory cytokines release.In conclusion, miR-20 a acts as a regulator on NLRP3 inflammasome-mediated release of inflammatory factors, the potential mechanism is seemed to be negatively regulated TXNIP, thereby inhibiting NLRP3 inflammasome activation and inflammatory cytokines IL-1β, MMP-1,TIMP-1 release, which could become a potential target for the improvement on development of RA. |
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