BackgroundAt present, specific immune targeted therapeutics including biologics and kinase inhibitors has made significant progress in Rheumatoid arthritis (RA). However, the irresponse to targeted therapies in some refractory RA patients suggests more attention should be paid on cell death pathways, such as pyroptosis. TNF-α is a proinflammatory cytokine that plays a fundamental role in the pathogenesis of RA, which could induce pyroptosis in monocytes and macrophages and cause the destruction of bone and cartilage. Iguratimod (IGU) has been confirmed as a highly efficacious and safe conventional synthetic disease-modifying anti-rheumatic drug for RA in Asia. Whether the combination of IGU and Tofacitinib (TOF), the Janus kinase inhibitor, would be better partner need to be elucidated.ObjectivesTo evaluate the therapeutic effect of IGU and TOF on active RA and secondary osteoporosis in collagen-induced arthritis (CIA)+TNF model.MethodsIn this study, hematoxylin and eosin (HE) staining were used to evaluate the pathological changes in ankle joints of CIA+TNF model. Immunohistochemistry (IHC) were used to evaluate the level of pyroptosis related proteins in synovial tissue. We performed Micro-computed tomography (Micro-CT), HE staining and IHC to analyze the trabecular bone changes in distal femoral metaphyses to investigate the destruction of knee joint.ResultsAfter 6 weeks treatment of IGU and/or TOF, the diameter of ankle joint and the level of interleukin (IL)-18, IL-1 of CIA+TNF model. Immunohistochemistry (IHC) were used to evaluate the level of pyroptosis related proteins in synovial tissue. We performed M group. HE staining showed that only a small amount of inflammatory cell infiltration and less pannus were seen in synovial tissue of both monotherapy and combination group, when compared with the CIA+TNF group. Of importance, the pyroptosis related proteins, such as gasdermin D (GSDMD), nucleotide-binding domain (NOD)-like receptor protein 3 (NLRP3), caspase-1, and IL-1β were significantly less expression in synovial tissue of combination group compared with the CIA+TNF group. Both the osteoblast bone formation and osteoclast bone absorption were sharply ruined in CIA+TNF model. However the bone destruction was significantly alleviated and bone turnover rate was remarkably increased in combination group, detected by Micro-CT, HE staining and IHC.ConclusionThe TOF-IGU combination synergistically alleviated the disease severity of the CIA model, including relieving joint inflammation and bone erosion, with suppressing the activation of the NLRP3 inflammasome and reducing GSDMD-mediated pyroptosis. The combined application of TOF and IGU may have a stronger therapeutic effect on RA and secondary osteoporosis bone remodeling.Figure 1.Therapeutic effects of TOF and IGU on the disease severity in CIA+TNF model.(A) The diameter of ankle joint; (B) HE staining in synovial tissue; (C) IHC staining of pyroptosis related proteins in synovial tissue; (D) Micro-CT analysis in distal femurs; (E) HE staining in knee joint; (F) IHC staining of ALP and TRAP staining in knee joint.Acknowledgementsthe ECCM Program of Clinical Research Center of Shandong University (No. 2021SDUCRCB010) and the Natural Science Foundation of Shandong Province (ZR2022MH177)Disclosure of InterestsNone Declared.
BackgroundEULAR guideline in rheumatoid arthritis (RA) recommended the primary failure of the first-line conventional synthetic modifying anti-rheumatic drugs (csDMARDs) patients switching to tumor necrosis factor alpha inhibitors (TNFi) [1]. Nevertheless, approximately 30-40% csDMARDs-IR patients also experience inefficacy of TNFi [2]. There is still no index to predict whether TNFi would be responded or not. Moreover, only few studies had focused on the relationship between TNFi nonresponse and other cell programmed deaths except apoptosis.ObjectivesTo predict the possibility of TNFi response prior to prescript in RA patients with the biomarkers of non-apoptotic programmed cell death in synovial cells.MethodsThe datasets of 22 TNFi treated RA synovial samples were enrolled from the Gene Expression Omnibus (GEO) database (GSE140036 and GSE15602). And the differentially expressed genes (DEGs) and modules related to TNFi treatment through weight gene correlation network analysis (WGCNA) were identified with the R packages “limma” and “WGCNA”. Then the enrichment analysis among the shared genes was performed through the R.4.1.2, Metascape website, and WebGestalt website. Following with the confirmation of the non-apoptotic programmed cell death (NAPCD) genes in the shared genes with Student’s T-Test. Furthermore, the TNFi treatment cohort was clustered based on the hub genes, making the receiver operating characteristic (ROC) curve analysis. Moreover, the least absolute shrinkage and selection operator (LASSO) model was constructed to identify the predictive genes.Results2624 DEGs were identified significantly, including 161 upregulated genes and 2463 downregulated genes. One module with TNFi treatment was constructed in WGCNA, significant in both response and nonresponse. Then the gene signatures for TNFi nonresponse were collected from overlaps 2260 genes in above. And we found 38 NAPCD genes might play role in TNFi nonresponse, but reserved 33 genes which statistically significant with T-Test. 22 TNFi treated synovial samples in GEO database could be classified into response or nonresponse subgroups. The ROC curve showed that the AUC for 32 genes in these samples ranged from 0.7 to 0.9, expected for CD46. At last, the LASSO model indicated that CASP5, CAPN10, ITGB4, NLRP2, and SLC2A8 could predict the TNFi nonresponse, as the risk score = CASP5 × 0.028 + CAPN10 × 0.064 + ITGB4× 0.080+ NLRP2 × 0.317+ SLC2A8 × 0.090 (Figure 1).Figure 1.Predictive model of TNFi nonresponse based on NAPCD genes. (A) Volcano map of differential expressed genes; (B) Correlation heat map of gene modules and phenotypes, the red is positively correlated with the phenotype, blue is negatively correlated with the phenotype; (C) The shared 38 genes of TNFi response & nonresponse DEGs, among the WGCNA turquoise module and cell programmed death genes; (D) Consensus clustering matrix for k = 2; (E) The ROC curve of 33 genes; (F) LASSO regression of the 32 genes,except for CD46; (G) Nomogram for predicting TNFi nonresponse in TNFi treatment RA cohort, indicated five possible indicators (CASP5, CAPN10, ITGB4, NLRP2, and SLC2A8) were closely related to TNFi nonresponse.ConclusionOur study firstly screened out the 38 NAPCD candidate genes signatures in RA synovial tissues which took part in TNFi nonresponse through WGCNA and DEGs. Further analysis confirmed that five possible indicators (CASP5, CAPN10, ITGB4, NLRP2, and SLC2A8) were closely related to TNFi nonresponse.References[1]van Delft MAM, Huizinga TWJ: An overview of autoantibodies in rheumatoid arthritis. Journal of autoimmunity 2020, 110:102392.[2]Aletaha D: Precision medicine and management of rheumatoid arthritis. Journal of autoimmunity 2020, 110:102405.AcknowledgementsFunded by ECCM Program of Clinical Research Center of Shandong University.Disclosure of InterestsNone declared.
BackgroundIguratimod (IGU) is widely used in the treatment of rheumatoid arthritis (RA) and has been considered the first-line treatment option in many countries. The efficacy and safety of IGU combination with conventional synthetic disease-modifying antirheumatic drugs (csDMARDs) therapy have been confirmed by many studies[1]. Only a few studies focused on the long-term efficacy and safety of IGU treatment, and the maintenance strategy after short-term remission (REM) still needs to be explored.ObjectivesTo observe the long-term efficacy, safety, and recurrence of IGU combination with csDMARDs in the treatment of active RA and the characteristic of patients who obtain the most benefit from IGU.MethodsThe study was conducted in China (ClinicalTrials.gov ID:NCT03855007). Patients who matched the 2010 ACR/EULAR RA criteria or the 2014 early rheumatoid arthritis (ERA) criteria[2]were recruited. According to patientsicalTrials.gov ID:NCT03855007). Patients who matched the 2010 ACR/EULAR RA criteria or the 2014 early rheumatoid arthritis (ERA) criteria enefit from IGU.on with conventiond follow-up from baseline to week 96 regularly. We divided them into three groups according to whether and when to reduce the drugs: maintained (MA), reducing the dose of IGU, MTX, and LEF after (AF) or before (BF) low disease activity (LDA), or REM was achieved. The proportion of LDA or REM of DAS28-ESR achieved at week 96, the ratio of ACR20, ACR50, ACR70, the recurrence (loss of LDA status and the increase of DAS28-ESR ≥ 0.6[3]), and the occurrence of adverse events (AE) were investigated. The chi-square test or long-rank test analyzed the corresponding statistical differences.ResultsA total of 246 patients were enrolled in this study, and 131 (53.3%) participants completed the 96-week follow-up. Efficacy analysis found that the proportion of achieving ACR20 reached a plateau after 24 weeks, while there was a sustained increase in the LDA and REM ratio of DAS28-ESR (week 24: 57.25% vs. week 96: 68.70%, p = 0.06), ACR50 (66.41% vs. 77.86%, p = 0.01) and ACR70 (22.90% vs. 41.98%, p < 0.01) (Figure 1 A, B).Further subgroup analysis revealed the expected effects of age, gender, and disease duration on whether achieved the LDA or REM at 96 weeks. Moreover, the time of IGU, MTX, and LEF reduction also played an essential role in long-term treatment. There was no difference in the improvement of DAS28-ESR between the MA (n=15) and reduction (AF+BE) groups. We found that patients in the AF group (n=73) were able to achieve and maintain LDA or REM and ACR50 responses better than the BE group (n=43) (Figure 1 C). There was no statistical difference in the incidence of AE between BE and AF groups (p = 0.44).During the follow-up period, 48 patients had a relapse of the disease in the AF and BE groups. The subgroup analysis found that drug reduction after REM was more recommended in patients with a diagnosis of RA (vs. ERA), disease duration ≥ 2 years, age < 45 years, and morning stiffness <1 hour (Figure 1 D) for maintaining long-term LDA or REM (p < 0.05).ConclusionThe RA patients could achieve deeper REM and sustained after long-term treatment of IGU combination with csDMARDs. LDA or REM should be achieved before reducing the dose of IGU and csDMARDs, especially for the long-course, younger, with shorter morning stiffness RA patients.References[1]Mu R, Li C et al. Lancet Reg Health West Pac 2021, 10:100128.[2]Zhao J, Su Y et al. Clin Exp Rheumatol 2014, 32(5):667-673.[3]Smolen JS, Nash P et al. Lancet 2013, 381(9870):918-929.Figure 1.The efficacy and recurrence of IGU combination treatment for patients who completed the 96-week follow-up.(A) The rate of achieving LDA or REM of DAS28-ESR; (B) The rate of achieving ACR20, ACR50, and ACR70, * p < 0.05, compared to week 24; (C) The rate of achieving LDA or REM of DAS28-ESR, ACR50 in AF and BE groups, * p < 0.05, compared to the BE group; (D) The Kaplan-Meier curves of AF and BE subgroup analysis according to the disease stage, duration, age and morning stiffness.AcknowledgementsFunded by the ECCM Program of Clinical Research Center of Shandong University (No. 2021SDUCRCB010), the Horizontal Project of Shandong University (No. 6010120019) and the Natural Science Foundation of Shandong Province (ZR2022MH177).Disclosure of InterestsQincheng Che: None declared, XinLei Liu: None declared, Xiaoyan Qi: None declared, Yanmei Li: None declared, Xiaojie Zhang: None declared, Hui Wang Employee of: Jiangsu Simcere Pharmaceutical Co., Ltd., Qiang Shu: None declared.
The organic dyes used in printing and dyeing wastewater have complex components, diverse structures and strong chemical stability, which make them not suitable for treatment and difficult to degrade in the environment. Porphyrins are macromolecules with 18 π electrons formed by four pyrrole molecules connected with a methylene bridge that has a stable structure. Porphyrin combines with iron to form an active intermediate with a structure similar to the cytochrome P450 enzyme, so they are widely used in the biomimetic field. In the current study, 5,10,15,20-tetra (4-carboxyphenyl) porphine ferric chloride (III) (Fe(III)TCPP) was used as a catalyst and iodosobenzene was used as an oxidant to explore the catalytic degradation of triphenylmethane dyes, such as rhodamine B (RhB) and malachite green (MG). The results of UV-Vis spectral analysis have shown that the conversion rate of the rhodamine B was over 90% when the amount of Fe(III)TCPP was 0.027 mM and the amount of iodosobenzene was eight equivalents. When the catalyst was 0.00681 mM and the amount of the oxidant was five equivalents, the conversion rate of the malachite green reached over 95%. This work provides a feasible method for the degradation of triphenylmethane dyes.
BackgroundPyroptosis is crucial to rheumatoid arthritis (RA) by inducing and aggravating inflammation. TNF-α is abundant in fibroblast-like synoviocytes of RA (RA-FLSs) and plays a key role in pyroptosis by inducing nuclear factor (NF)-κB activation. Additionally, interleukin (IL)-37 is involved in autoimmune diseases as an anti-inflammatory cytokine and innate and acquired immune response inhibitor. However, the effect of IL-37 on pyroptosis in RA-FLSs remains unclear.ObjectivesTo investigate the effects and mechanism of IL-37 on RA-FLS pyroptosis induced by TNF-α and the effect in collagen-induced arthritis (CIA)+TNF model.MethodsIn this study, the serum cytokines in patients with RA and healthy controls were detected using ELISA. The RA-FLSs were then cultured with TNF-α, with or without various IL-37 concentrations, to test the cytokine levels in the cell supernatant. 5-Ethynyl-2′-Deoxyuridine (EdU) assay assessed the effects of IL-37 on RA FLS proliferation. RA-FLS apoptosis was assessed using flow cytometry and mitochondrial membrane potential (MMP) measurement. In addition, transmission electron microscopy (TEM) was used to examine cell pyroptosis. We selected the optimal concentration for the following experiments and detected the signal pathway of IL-37 on pyroptosis of RA FLSs by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and Western blotting. Finally, we validated the therapeutic effects of IL-37 on CIA rat model in vivo.ResultsIL-37 inhibited inflammation in vitro and in vivo and reduced pyroptosis-related protein expression in RA FLSs. Furthermore, we determined that nuclear factor κB (NF-κB) signaling is required for GSDMD-mediated pyroptosis in RA FLSs.ConclusionIL-37 alleviates TNF-α-induced pyroptosis of RA FLSs by inhibiting NF-κB/GSDMD signaling. Additionally, our data revealed a novel mechanism for IL-37 in RA FLSs, suggesting a new potential therapy for IL-37 to treat RA.Figure 1.Serum levels of IL-37,IL-1β,IL-18, the mechanism of IL-37 on RA-FLSs and the therapetic effects in CIA-TNF model. (A)IL-37,(C)IL-1β,(D) IL-18 in RA patients and healthy controls. (B) Levels of IL-37 in RA patients with low,moderate and severe DAS28.(E)The expression of pyroptosis-related proteins analyzed by Western blot.(F) Cell proliferation activity in TNF-α and/or IL-37 stimulated groups..(G) Detection of mitochondrial membrane potential by JC-1 (H) Flow cytometry analysis of each group.(I) Representative Western blots of the NF-κB signaling pathway and the protein of pyroptosis(J)Pyroptosis of RA-FLSs colltected by transmission electron microscopy in each group.(K)The expression of p65 in each group of cells was determined by qRT-PCR. (L) Representative Western blots of p-p65,p65,Gsdmd,Caspase-1.(M) Representative images of arthritis in rat paws. (N) The arthritis score of different groups.REFERENCES:NIL.Acknowledgements:NIL.Disclosure of InterestsNone Declared.
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