Inhibition of miR-665 alleviates lipopolysaccharide-induced inflammation via up-regulation of SOCS7 in chondrogenic ATDC5 cells

Purpose: To examine the effect and mechanism of action of miR-665 in osteoarthritis.Methods: An in vitro inflammatory injury model of osteoarthritis was established using chondrogenic ATDC5 cells with lipopolysaccharide (LPS) treatment. The expression levels of inflammatory cytokines were determined by enzyme-linked immunosorbent assays (ELISAs) and by quantitative real-time polymerase chain reaction (qRT-PCR). A binding target for miR-665 was predicted using TargetScan and then evaluated using a dual-luciferase reporter assay.Results: Treatment with LPS significantly up-regulated the inflammatory cytokine expressions of interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-alpha (TNF-α), in ATDC5 cells (p < 0.01), and the expression of miRNA-665 was significantly increased in LPS-treated ATDC5 cells (p < 0.01).Knockdown of miR-665 down-regulated the expression levels of these inflammatory cytokines. Suppressor of cytokine signaling 7 (SOCS7) was identified as a target of miR-665. Data from qRT-PCR and western-blot analyses indicated that SOCS7 expression was promoted by miR-665  inhibition and inhibited by miR-665 over-expression. LPS treatment significantly decreased the expression of SOCS7 protein in ATDC5 cells (p < 0.01), and over-expression of SOCS7 attenuated the LPS-stimulated inflammatory injury. In addition, over-expression of miR-655 enhanced the inflammatory injury and reversed the protective effect of SOCS7 against LPS-stimulated inflammation.Conclusion: Inhibition of miR-665 alleviated LPS-stimulated inflammatory injury in ATDC5 cells via the up-regulation of SOCS7, suggesting a potential therapeutic target for osteoarthritis. Keywords: MiR-665, Lipopolysaccharide, Inflammation, SOCS7, Chondrogenic, ATDC

3-O-Caffeoylquinic acid in Periploca forrestii Schltr extract ameliorates collagen-induced arthritis by inducing IL17/IL23 cells in rats

Purpose: To study the therapeutic effect of 3-O-caffeoylquinic acid (3-O-CQA) from Periploca forrestii extract (PFE) on collagen-mediated arthritis (CIA) in rats, as well as the potential underlying mechanism of action. Methods: PFE and 3-O-CQA were successively and intragastrically administered to CIA rats. Paw swelling, arthritic scores and H & E staining were used to evaluate the therapeutic effect of 3-O-CQA. Moreover, to determine the effects of PFE and 3-O-CQA on fibroblast-resembling synoviocytes obtained from arthritic subjects (RAFLS), the viability of RAFLS cultured in vitro was measured with MMT, while apoptotic lesions were analyzed by flow cytometry. The levels of IL-6 in CIA and RAFLS were determined by enzyme-linked immunosorbent assay (ELISA), while quantitative reverse transcriptionpolymerase chain reaction (qRT-PCR) and immunoblotting were used to assess their mRNA and polypeptide levels, respectively. Results: PFE in 3-O-CQA ameliorated swelling and reduced arthritic scores in CIA rat model, and also decreased cytokine levels (p < 0.05). By decreasing mRNA and protein expressions, 3-O-CQA repressed the phosphorylation of STAT3 and JAK2 as well as the protein levels of IL-23 and RORγt (p < 0.05). Conclusion: The results of this study show that CIA and RAFLS are ameliorated in rats by 3-O-CQA in PFE through regulation of IL17/ IL23 and Th17 cells. Thus, 3-O-CQA affords a therapeutic strategy for the management of collagen-induced arthritis. Keywords: Arthriti; Periploca forrestii Schltr extract; 3-O-Caffeoylquinic acid; Interleukin (IL)-17; IL-23; Th17 cell

Effect of Miao medicine, Jinwujiangu decoction, on IL- 17/IL-23 inflammatory axis of fibroblast-like synoviocytes in rheumatoid arthritis

Purpose: To explore the influence of the Miao medicine, Jinwujiangu decoction, on the interleukin (IL)- 17/IL-23 inflammatory axis of fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA).Methods: Synovial tissue samples were randomly divided into a blank control group, high-dose (0.06mg/mL), medium-dose (0.6mg/mL), and low-dose (6.0mg/mL) groups of Jinwujiangu decoction, a leflunomide group, and a tripterygium glycosides group. Proliferation of RA synovial cells was detected by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. Enzyme-linked immunosorbent assay (ELISA) was used to determine the secretion of IL-6, transforming growth factor beta (TGF-β), and IL-17. Real-time polymerase chain reaction was used to evaluate the expression of IL-23R, IL-17R, RAR-related orphan receptor alpha (RORα), RORγt, and signal transducer and activator of transcription (STAT3) mRNA. The protein activities of IL-17R, STAT3 and pSTAT3 were assessed by Western blot assay.Results: Jinwujiangu decoction inhibited the proliferation of RA synovial cells. Treatment with different drug concentrations resulted in downregulation of IL-6, TGF-β, and IL-17 secretion. The expression levels of IL-23R, IL-17R, RORα, RORγt, and STAT3 mRNA in RA-FLS were significantly reduced after intervention with different drugs. Protein expression levels of STAT3, pSTAT3, and IL-17 in the different drug treatment groups were significantly decreased.Conclusion: Jinwujiangu decoction inhibits the secretion of IL-6 and TGF-β in RA-FLS, and intervenes to regulate gene expression of IL-23/IL-17 inflammation axis and suppress immune inflammation. The results of this study provide new evidence for the study of anti-inflammatory mechanism of TCM compound prescription.Keywords: Jinwujiangu decoction, IL-17/IL-23, Fibroblast-like synoviocytes, Rheumatoid arthritis, Ethnomedicin

Prediction and Verification of the Major Ingredients and Molecular Targets of Tripterygii Radix Against Rheumatoid Arthritis

Tripterygii Radix exhibits good clinical efficacy and safety in rheumatoid arthritis (RA) patients, but its effective components and mechanism of action are still unclear. The purpose of this study was to explore and verify the major ingredients and molecular targets of Tripterygii Radix in RA using drug-compounds-biotargets-diseases network and protein-protein interaction (PPI) network analyses. The processes and pathways were derived from Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. The most important compounds and biotargets were determined based on the degree values. RA fibroblast-like synoviocytes (RA-FLS) were separated from RA patients and identified by hematoxylin and eosin (HE) staining and immunohistochemistry. The purity of RA-FLS was acquired by flow cytometry marked with CD90 or VCAM-1. RA-FLS were subjected to control, dimethyl sulfoxide (control), kaempferol, or lenalidomide treatment. Cell migration was evaluated by the transwell assay. The relative expression of biotarget proteins and cytokines was analyzed by western blotting and flow cytometry. In total, 144 chemical components were identified from Tripterygii Radix; kaempferol was the most active ingredient among 33 other components. Fourteen proteins were found to be affected in RA from 285 common biotargets. The tumor necrosis factor (TNF) signaling pathway was predicted to be one of the most latent treatment pathways. Migration of RA-FLS was inhibited and the expression of protein kinase B (AKT1), JUN, caspase 3 (CASP3), TNF receptor 1 and 2 (TNFR1 and TNFR2), interleukin-6 (IL-6), and TNF-α was significantly affected by kaempferol. Thus, this study confirmed kaempferol as the effective component of Tripterygii Radix against RA-FLS and TNF signaling pathway and its involvement in the regulation of AKT1, JUN, CASP3, TNFR1, TNFR2, IL-6, and TNF-α expression

Case report: Successful treatment of anti‐MDA5‐positive to negative dermatomyositis‐associated interstitial lung disease with the JAK inhibitor tofacitinib

Abstract Objective Anti‐MDA5 antibody‐positive dermatomyositis (DM) is a rare clinical autoimmune disease, and anti‐MDA5‐positive DM with interstitial lung disease (ILD) is the most important cause of death in DM patients. We reported the efficacy of the JAK1/3 inhibitor tofacitinib as an anti‐MDA5‐negative treatment option for patients with anti‐MDA5‐positive DM‐ILD. Method and process Here we report a 51‐year‐old female patient with cough, sputum, shortness of breath for 5 months, rash for 3 months, and muscle pain in the extremities for 1 month. After conventional immunosuppressive therapy plus hormone therapy, the remission was slow. Methylprednisolone was successfully reduced after we administered tofacitinib and tacrolimus. After 132 weeks of follow‐up, anti‐MDA5 antibody turned negative, clinical symptoms were relieved, and lung Imaging tests were successfully reversed. Results and Conclusion There is currently no report of tofacitinib supplement therapy for anti‐MDA5 positive to negative DM. With this case report, tofacitinib is an option for the treatment of anti‐MDA5‐positive DM‐ILD, which deserves attention

Table_1_The causal impact of saturated fatty acids on rheumatoid arthritis: a bidirectional Mendelian randomisation study.XLS

ObjectiveThe causal relationship between saturated fatty acids (SFAs) and rheumatoid arthritis (RA) remains poorly understood. This study aimed to determine whether SFAs are causally related to RA using Mendelian randomisation (MR) analyses.MethodsGenome-wide association study (GWAS) summary data for RA (ukb-d-M13_RHEUMA) and SFAs (met-d-SFA) were obtained from the Integrative Epidemiology Unit OpenGWAS database. A bidirectional MR analysis was performed using a suite of algorithms, namely the MR-Egger, weighted median, simple mode, weighted mode, and inverse-variance weighted (IVW) algorithms, all integrated using the “MR” function. The robustness of the MR findings was further evaluated through sensitivity analyses, including heterogeneity, horizontal pleiotropy, and leave-one-out tests.ResultsThe IVW algorithm in the forward MR analysis indicated a causal link between SFAs and RA (p = 0.025), identifying SFAs as a risk factor for RA (odds ratio = 1.001). Sensitivity analyses indicated no significant heterogeneity, horizontal pleiotropy, or severe bias, reinforcing the credibility of the forward MR results. However, the reverse MR analysis revealed that RA does not causally affect SFA levels (p = 0.195), and this finding was supported by corresponding sensitivity analyses.ConclusionThe findings of this study substantiate the positive causal effect of SFAs on the incidence of RA through bidirectional MR analysis, thereby offering a consequential direction for future research on the diagnosis and treatment of RA.</p

Table_2_The causal impact of saturated fatty acids on rheumatoid arthritis: a bidirectional Mendelian randomisation study.XLS

ObjectiveThe causal relationship between saturated fatty acids (SFAs) and rheumatoid arthritis (RA) remains poorly understood. This study aimed to determine whether SFAs are causally related to RA using Mendelian randomisation (MR) analyses.MethodsGenome-wide association study (GWAS) summary data for RA (ukb-d-M13_RHEUMA) and SFAs (met-d-SFA) were obtained from the Integrative Epidemiology Unit OpenGWAS database. A bidirectional MR analysis was performed using a suite of algorithms, namely the MR-Egger, weighted median, simple mode, weighted mode, and inverse-variance weighted (IVW) algorithms, all integrated using the “MR” function. The robustness of the MR findings was further evaluated through sensitivity analyses, including heterogeneity, horizontal pleiotropy, and leave-one-out tests.ResultsThe IVW algorithm in the forward MR analysis indicated a causal link between SFAs and RA (p = 0.025), identifying SFAs as a risk factor for RA (odds ratio = 1.001). Sensitivity analyses indicated no significant heterogeneity, horizontal pleiotropy, or severe bias, reinforcing the credibility of the forward MR results. However, the reverse MR analysis revealed that RA does not causally affect SFA levels (p = 0.195), and this finding was supported by corresponding sensitivity analyses.ConclusionThe findings of this study substantiate the positive causal effect of SFAs on the incidence of RA through bidirectional MR analysis, thereby offering a consequential direction for future research on the diagnosis and treatment of RA.</p

Data_Sheet_1_The causal impact of saturated fatty acids on rheumatoid arthritis: a bidirectional Mendelian randomisation study.PDF

ObjectiveThe causal relationship between saturated fatty acids (SFAs) and rheumatoid arthritis (RA) remains poorly understood. This study aimed to determine whether SFAs are causally related to RA using Mendelian randomisation (MR) analyses.MethodsGenome-wide association study (GWAS) summary data for RA (ukb-d-M13_RHEUMA) and SFAs (met-d-SFA) were obtained from the Integrative Epidemiology Unit OpenGWAS database. A bidirectional MR analysis was performed using a suite of algorithms, namely the MR-Egger, weighted median, simple mode, weighted mode, and inverse-variance weighted (IVW) algorithms, all integrated using the “MR” function. The robustness of the MR findings was further evaluated through sensitivity analyses, including heterogeneity, horizontal pleiotropy, and leave-one-out tests.ResultsThe IVW algorithm in the forward MR analysis indicated a causal link between SFAs and RA (p = 0.025), identifying SFAs as a risk factor for RA (odds ratio = 1.001). Sensitivity analyses indicated no significant heterogeneity, horizontal pleiotropy, or severe bias, reinforcing the credibility of the forward MR results. However, the reverse MR analysis revealed that RA does not causally affect SFA levels (p = 0.195), and this finding was supported by corresponding sensitivity analyses.ConclusionThe findings of this study substantiate the positive causal effect of SFAs on the incidence of RA through bidirectional MR analysis, thereby offering a consequential direction for future research on the diagnosis and treatment of RA.</p

Associations of the circulating levels of cytokines with risk of systemic sclerosis: a bidirectional Mendelian randomized study

ObjectiveSystemic sclerosis(SSc) remains unclear, studies suggest that inflammation may be linked to its pathogenesis. Hence, we conducted a bidirectional Mendelian randomization (MR) analysis to evaluate the association between cytokine and growth factor cycling levels and the risk of SSc onset.MethodsIn our study, the instrumental variables(IVs) for circulating cytokines were sourced from the genome-wide association study (GWAS) dataset of 8293 Finnish individuals. The SSc data comprised 302 cases and 213145 controls, and was included in the GWAS dataset. We employed four methods for the MR analysis: MR Egger, Inverse variance weighted (IVW), Weighted medium, and Weighted Mode, with IVW being the primary analytical method. Sensitivity analyses were performed using heterogeneity testing, horizontal pleiotropy testing, and the Leave One Out (LOO) method. We also conducted a reverse MR analysis to determine any reverse causal relationship between SSc and circulating cytokines.ResultsAfter Bonferroni correction, MR analysis revealed that the Interleukin-5 (IL-5) cycle level was associated with a reduced risk of SSc [odds ratio (OR)=0.48,95% confidence interval (CI): 0.27-0.84, P=0.01]. It also indicated that the Stem cell growth factor beta (SCGF-β) cycling level might elevate the risk of SSc (OR = 1.36, 95% CI: 1.01-1.83, P = 0.04). However, the reverse MR analysis did not establish a causal relationship between SSc and circulating cytokine levels. Additionally, sensitivity analysis outcomes affirm the reliability of our results.ConclusionOur MR study suggests potential causal relationships between IL-5, SCGF-β, and the risk of SSc. Further research is essential to determine how IL-5 and SCGF-β influence the development of SSc

DataSheet_2_Associations of the circulating levels of cytokines with risk of systemic sclerosis: a bidirectional Mendelian randomized study.pdf

ObjectiveSystemic sclerosis(SSc) remains unclear, studies suggest that inflammation may be linked to its pathogenesis. Hence, we conducted a bidirectional Mendelian randomization (MR) analysis to evaluate the association between cytokine and growth factor cycling levels and the risk of SSc onset.MethodsIn our study, the instrumental variables(IVs) for circulating cytokines were sourced from the genome-wide association study (GWAS) dataset of 8293 Finnish individuals. The SSc data comprised 302 cases and 213145 controls, and was included in the GWAS dataset. We employed four methods for the MR analysis: MR Egger, Inverse variance weighted (IVW), Weighted medium, and Weighted Mode, with IVW being the primary analytical method. Sensitivity analyses were performed using heterogeneity testing, horizontal pleiotropy testing, and the Leave One Out (LOO) method. We also conducted a reverse MR analysis to determine any reverse causal relationship between SSc and circulating cytokines.ResultsAfter Bonferroni correction, MR analysis revealed that the Interleukin-5 (IL-5) cycle level was associated with a reduced risk of SSc [odds ratio (OR)=0.48,95% confidence interval (CI): 0.27-0.84, P=0.01]. It also indicated that the Stem cell growth factor beta (SCGF-β) cycling level might elevate the risk of SSc (OR = 1.36, 95% CI: 1.01-1.83, P = 0.04). However, the reverse MR analysis did not establish a causal relationship between SSc and circulating cytokine levels. Additionally, sensitivity analysis outcomes affirm the reliability of our results.ConclusionOur MR study suggests potential causal relationships between IL-5, SCGF-β, and the risk of SSc. Further research is essential to determine how IL-5 and SCGF-β influence the development of SSc.</p