ObjectiveTo investigate the possible mechanism of Jishe Qushi Capsule （脊蛇祛湿胶囊， JQC） in treating rheumatoid arthritis （RA） from the perspective of macrophage polarization mediated by neutrophil extracellular traps （NETs）. MethodsTwenty-four female SD rats were randomly divided into four groups， blank control group， model group， JQC group， and peptidylarginine deiminase 4 （PAD4） inhibitor group with 6 rats in each group. All groups but the blank control group were subjected to the induction of collagen-induced arthritis （CIA）. After successful model establishment， rats in the JQC group received intragastric administration of JQC 1.47 g/kg daily； rats in the PAD4 inhibitor group received intraperitoneal injections of the PAD4 inhibitor 4 mg/kg weekly. Rats in the blank， model， and PAD4 inhibitor groups received 2 ml of pure water daily by gavage. All treatments lasted 4 weeks. Joint lesions of each group were assessed on day 7， 14， 21， 28， and 35 after model establishment， and arthritis index （AI） scores were recorded. At 24 h after the final administration， histopathology of knee joints， including HE staining， safranin O-fast green staining， and TRAP staining， was performed. Flow cytometry was used to detect the counts of M1 and M2 macrophages in peripheral blood. ELISA was used to determine serum levels of TRACP， NETs， TNF-α， IL-1β， and iNOS. Western Blotting and qRT-PCR were used to measure MPO， NE， RANKL， OPG， and p65 protein and mRNA expression in knee cartilage tissue. ResultsCompared with the blank control group， the model group showed increased AI scores （P＜0.05）， marked synovial inflammatory infiltration， angiogenesis， and bone-cartilage destruction， increased TRAP-positive osteoclasts， increased M1 macrophages and decreased M2 macrophages， elevated serum TRACP， NETs， TNF-α， IL-1β， and iNOS （P＜0.05）， elevated MPO， NE， RANKL， and p65 protein/mRNA expression and decreased OPG protein/mRNA expression in knee cartilage tissue （P＜0.05）. Compared with the model group， the JQC group exhibited improved synovial inflammation， angiogenesis， and bone-cartilage damage， reduced AI scores on day 21， 28， and 35， decreased osteoclast counts， decreased M1 macrophages and increased M2 macrophages， reduced serum TRACP， NETs， TNF-α， IL-1β， and iNOS （P＜0.05）， decreased MPO， NE， RANKL， and p65 protein/mRNA expression and increased OPG expression （P＜0.05）. Compared with the PAD4 inhibitor group， the JQC group showed significantly lower AI scores， reduced M1 macrophages， increased M2 macrophages （P＜0.05）， reduced serum TRACP， TNF-α， IL-1β， and iNOS， decreased MPO， RANKL， and p65 expression， and increased OPG levels （P＜0.05）. ConclusionThe therapeutic mechanism of JQC for RA may involve inhibition of NETs formation， downregulation of the RANKL/NF-κB signaling pathway， and regulation of macrophage M1/M2 polarization imbalance， thereby suppressing osteoclastogenesis and inflammatory bone destruction.

Objective:To explore the association of serum levels of 41 serum cytokines and growth factors with the risk of sero-negative rheumatoid arthritis(SNRA)by Mendelian randomization.Methods:Genetic instruments for 41 circulating cytokines and growth factors were determined from a genome-wide association study(GWAS)of 8 293 European participants.Summary statistics for the SNRA were obtained from the Finnish database,including 3 877 SNRA cases and 285 035 controls of European ancestry.All of the inverse variance weighted(IVW),weighted median method(WM)and MR-Egger regression were used for MR analysis,while the IVW method was considered as the main analysis.The sensitivity analysis included a heterogeneity test,horizontal pleiotropic test,and leave-one method test to determine the reliability of the MR results.Results:In the IVW method,TNF-α[OR=1.470,95%CI(1.1331～1.910),P=0.004],IP-10[OR=0.794,95%CI(0.660～0.955),P=0.015]and IL-2rα[OR=0.049,95%CI(0.856～0.999),P=0.049].Sensitivity analysis showed no heterogeneity and horizontal pleiotropy.Conclusion:TNF-α,IP-10 and IL-2rα are causally associated to SNRA.TNF-α increases the risk of SNRA,while IP-10 and IL-2rα reduce the risk of SNRA.

Objective"Red butterfly sore","yin-yang toxin"and"Bi disease"are different Chinese medicine diagnoses of systemic lupus erythematosus(SLE).It is not clear whether there are biological differences between these three types of Chinese medicine diagnoses.The aim of this study was to compare the different TCM diagnoses of SLE patients from the perspective of lymphocyte subsets and cytokines.Methods Patients diagnosed with SLE in our hospital from June 1,2021 to December 1,2023 were retrospectively collected,and the differences of T cell subsets,NK cells,B lymphocytes and Th1,Th2 and Th17 cytokines among different groups were compared by one-way ANOVA or nonparametric test.As well as differences in laboratory test indicators such as erythrocyte sedimentation rate(ESR),C-reactive protein(CRP),immunoglobulin,and complement,receiver operating characteristic curve(ROC curve)was used to analyze the value of these indexes in the differential diagnosis of different types of SLE.Results From June 1,2021 to December 1,2023,291 patients diagnosed with SLE in our hospital for the first time were collected,and 104 cases meeting the exclusion criteria of this study were included,including 31 cases of red butterfly sores,30 cases of yin-yang toxin and 43 cases of BI disease.The absolute number and percentage of CD8+T cells,interleukin-10(IL-10)content and tumor necrosis factor α(TNF-α)content were different among the three groups of SLE patients diagnosed by different Chinese medicine,and the absolute number of CD8+T cells in the red butterfly sore group was significantly higher than that in the yin-yang toxicities group(P=0.039)and the disease group(P=0.008).CD8+T cell percentage in red butterfly sore group was significantly higher than that in yin-yang toxin group(P=0.014)and disease group(P=0.004),IL-10 and TNF-α levels in red butterfly sore group were significantly lower than those in disease group(P=0.015,P=0.036),and ROC curve analysis showed that,the absolute number and percentage of CD8+T cells can effectively distinguish red butterfly sores from yin-yang toxins(AUC=0.65,AUC=0.61,P<0.05),and the absolute number and percentage of CD8+T cells,IL-10 and TNF-α can effectively distinguish red butterfly sores from diseases(AUC=0.68,AUC=0.66,P<0.01,AUC=0.67,AUC=0.64,P<0.05).Conclusion Immune lymphocyte subtypes,cytokines,especially the absolute number and percentage of CD8+T cells,IL-10 and TNF-α may play an important role in the identification of different TCM diagnosis of SLE.

OBJECTIVES: To analyze the molecular mechanism of Xixian Pills for treatment of rheumatoid arthritis (RA). METHODS: Forty-eight rats were randomized into 6 groups (n=8), including a normal control group, a collagen-induced arthritis (CIA) model group, 3 Xixian Pills treatment (200, 400 and 800 mg/kg) groups, and a Tripterygium glycosides tablet (TGT) treatment group. In the latter 4 groups, the rats were treated with daily gavage of Xixian Pills or TGT 2 weeks after CIA modeling for 3 consecutive weeks. The differentially expressed proteins in high-dose Xixian Pills group and the model group compared with the normal control group were screened based on the tandem mass spectrometry tag (TMT) technology, and the core targets and signaling pathways were analyzed. The immune cell infiltration and gene expression data were analyzed using ggplot2 and tidyverse packages, and the correlation coefficients between the core targets and the immune cells were calculated. RESULTS: The CIA rats showed significantly increased serum levels of TNF-α and IL-6 and lowered serum IL-10 level. Treatments with high- and medium-dose Xixian Pills and TGT all significantly reduced serum TNF‑α and IL-6 and increased IL-10 levels in CIA rats. Proteomic analysis identified 160 differential proteins between the model group and high-dose Xixian Pills group, and the core targets included CCL5, STAT1, GZMB and IL7R. The areas under the ROC curve of CCL5 and STAT1 were both greater than 0.9. Immunohistochemical and immunofluorescence staining revealed increased levels of CCL5 and STAT1 in the ankle joints of CIA rats, which were significantly decreased after treatment with Xixian Pills. CONCLUSIONS Treatment with Xixian Pills offers protection of the joints in CIA rats possibly by inhibiting joint inflammation via regulating protein expressions of CCL5 and STAT1.
Animals ; Drugs, Chinese Herbal/pharmacology* ; Rats ; Arthritis, Rheumatoid/metabolism* ; Proteomics ; Tripterygium/chemistry* ; Arthritis, Experimental/metabolism* ; Tumor Necrosis Factor-alpha/blood* ; Interleukin-10/blood* ; Interleukin-6/blood* ; Male ; Rats, Sprague-Dawley ; Signal Transduction

Objective"Red butterfly sore","yin-yang toxin"and"Bi disease"are different Chinese medicine diagnoses of systemic lupus erythematosus(SLE).It is not clear whether there are biological differences between these three types of Chinese medicine diagnoses.The aim of this study was to compare the different TCM diagnoses of SLE patients from the perspective of lymphocyte subsets and cytokines.Methods Patients diagnosed with SLE in our hospital from June 1,2021 to December 1,2023 were retrospectively collected,and the differences of T cell subsets,NK cells,B lymphocytes and Th1,Th2 and Th17 cytokines among different groups were compared by one-way ANOVA or nonparametric test.As well as differences in laboratory test indicators such as erythrocyte sedimentation rate(ESR),C-reactive protein(CRP),immunoglobulin,and complement,receiver operating characteristic curve(ROC curve)was used to analyze the value of these indexes in the differential diagnosis of different types of SLE.Results From June 1,2021 to December 1,2023,291 patients diagnosed with SLE in our hospital for the first time were collected,and 104 cases meeting the exclusion criteria of this study were included,including 31 cases of red butterfly sores,30 cases of yin-yang toxin and 43 cases of BI disease.The absolute number and percentage of CD8+T cells,interleukin-10(IL-10)content and tumor necrosis factor α(TNF-α)content were different among the three groups of SLE patients diagnosed by different Chinese medicine,and the absolute number of CD8+T cells in the red butterfly sore group was significantly higher than that in the yin-yang toxicities group(P=0.039)and the disease group(P=0.008).CD8+T cell percentage in red butterfly sore group was significantly higher than that in yin-yang toxin group(P=0.014)and disease group(P=0.004),IL-10 and TNF-α levels in red butterfly sore group were significantly lower than those in disease group(P=0.015,P=0.036),and ROC curve analysis showed that,the absolute number and percentage of CD8+T cells can effectively distinguish red butterfly sores from yin-yang toxins(AUC=0.65,AUC=0.61,P<0.05),and the absolute number and percentage of CD8+T cells,IL-10 and TNF-α can effectively distinguish red butterfly sores from diseases(AUC=0.68,AUC=0.66,P<0.01,AUC=0.67,AUC=0.64,P<0.05).Conclusion Immune lymphocyte subtypes,cytokines,especially the absolute number and percentage of CD8+T cells,IL-10 and TNF-α may play an important role in the identification of different TCM diagnosis of SLE.

Objective:To explore the association of serum levels of 41 serum cytokines and growth factors with the risk of sero-negative rheumatoid arthritis(SNRA)by Mendelian randomization.Methods:Genetic instruments for 41 circulating cytokines and growth factors were determined from a genome-wide association study(GWAS)of 8 293 European participants.Summary statistics for the SNRA were obtained from the Finnish database,including 3 877 SNRA cases and 285 035 controls of European ancestry.All of the inverse variance weighted(IVW),weighted median method(WM)and MR-Egger regression were used for MR analysis,while the IVW method was considered as the main analysis.The sensitivity analysis included a heterogeneity test,horizontal pleiotropic test,and leave-one method test to determine the reliability of the MR results.Results:In the IVW method,TNF-α[OR=1.470,95%CI(1.1331～1.910),P=0.004],IP-10[OR=0.794,95%CI(0.660～0.955),P=0.015]and IL-2rα[OR=0.049,95%CI(0.856～0.999),P=0.049].Sensitivity analysis showed no heterogeneity and horizontal pleiotropy.Conclusion:TNF-α,IP-10 and IL-2rα are causally associated to SNRA.TNF-α increases the risk of SNRA,while IP-10 and IL-2rα reduce the risk of SNRA.

Objective: To investigate the changes of microorganisms and metabolites in joint effusion of patients with Rheumatoid arthritis(RA), Osteoarthritis(OA) and Urarthritis(UA). To provide new ideas for the study of the effect of microbiota on the pathogenesis of arthritis. Methods: Joint effusion samples were collected from 20 patients with RA, 20 patients with OA, and 20 patients with UA. 16S rRNA gene sequencing and untargeted ultra-high performance Liquid chromatography-mass spectrometry(LC-MS) were used to explore the differences in microorganisms and metabolites among the three groups. Pearson correlation analysis was used to detect the correlation between effusion microbiota and metabolites. Results: There were differences in microbial diversity and microbiota composition among the three groups. Combined with VIP>1 from OPLS-DA andP<0.05 from two-tailed Students t-test, 45 differential metabolites(Between RA and OA groups), 38 differential metabolites(Between UA and OA groups) and 16 differential metabolites(Between RA and UA groups), were identified. GO analysis and KEGG pathway analysis showed that the differential metabolic pathways among the three groups were mainly concentrated in citric acid cycle(TCA cycle), nucleotide metabolism, amino acid metabolism and glycolysis pathway. Correlation analysis of joint effusion microbiota and metabolites suggested that bacteria enriched in the three groups of joint effusion, such asPrevotella,Clostridium ruminosus,Prevotellaceae＿UCG-001, were related to many key metabolites such as lysozyme, uric acid, glucose, and L-glutamine. Conclusion This study shows that there are a variety of bacterial flora in joint cavity effusion of RA, OA, and UA patients, and the differential metabolites produced by them are involved in the pathogenesis of the three types of arthritis by affecting a variety of metabolic pathways.

Objective To use the GEO dataset and bioinformatics techniques,such as LASSO logistic regression,ssGSEA,and WGCNA,to screen for RA diagnostic markers and investigate the impact of earthly flavonoids in Smi-lax glabra Roxb.on specific immune cell infiltration,to screen for rheumatoid arthritis(RA)diagnostic markers on specific immune cell infiltration and to analyze the combination of flavonoids in Smilax glabra Roxb.and diagnostic markers.Methods The normal control group and RA gene chip were obtained from the Gene Expression Omnibus database.The R 4.3.0 WGCNA software package was used to integrate and analyze the dataset,identify co-expres-sion modules and associated trait information,and screen key modules closely related to RA.LASSO regression a-nalysis was performed using the glmnet package in R to identify characteristic genes for RA.The area under the re-ceiver operating characteristic(ROC)curve was used to evaluate the diagnostic value of the characteristic genes in RA.The gene expression data of the normal control group and RA group were subjected to quantitative immune cell infiltration analysis using the GSVA,limma,and GSEABase packages in R.The chemical components of earth-worm flavonoids in Smilax glabra Roxb.were analyzed based on UHPLC-Q-Exactive Orbitrap MS.The correlation between flavonoids and characteristic genes was assessed through molecular docking.Results The LASSO regres-sion algorithm selected 5 characteristic genes(apolipoprotein D,zinc finger and BTB domain containing 16,C-C chemokine receptor type 5,matrix metalloproteinase 1,coronin-1A).The area under ROC curve of all 5 character-istic genes was greater than 0.85,which exhibited positive correlations with various immune cells.Twenty earth-worm flavonoids of Smilax glabra Roxb.were identified using UHPLC-Q-Exactive/MS,and Mulberrin and Neobavaisoflavone were well combined with 5 immune characteristic genes.Conclusion Flavonoids compounds of Smilax glabra Roxb.have good combination with RA immune characteristic genes,providing a scientific basis for RA immunomodulation therapy and early diagnosis.

Objective To explore the causal relationship between rheumatoid arthritis(RA)and iron deficiency a-nemia(IDA)in European population by two-sample Mendelian randomization analysis.Methods The single nu-cleotide polymorphisms(SNPs)of RA and IDA were analyzed using public genome-wide association studies(GWAS).The inverse variance weighting method(IVW)was used as the main analysis method to evaluate the causal effect of RA on IDA.MR-Egger method,weighted median method(WM),weighted model method and simple model method were used as regression supplements to evaluate the robustness of sensitivity analysis results.The het-erogeneity function was used to calculate the P-value to test the heterogeneity,and the intercept term intercept was used to test the level pleiotropy.Results In the FINNGEN database at the genome-wide level,strong-related SNPs that removed linkage disequilibrium and met the P＜5.0 × 10-8 by Mendelian randomization analysis were select-ed.After integrating exposure and outcome data,31 SNPs were obtained as the final effective instrumental variables.IVW showed that RA was a risk factor for IDA(the risk of IDA in RA patients was 1.064 times higher than that in non-RA patients,OR=1.064,95％CI:1.028-1.103).The weighted median method and MR-Egger method re-sults supported the positive correlation between RA and IDA.The intercept value was close to 0,indicating that there was no horizontal pleiotropy between exposure and outcome.The heterogeneity function's P＜0.05 indicated that there was heterogeneity between exposure and outcome,but the random effect model test showed P＜0.05,indi-cating that even if there was heterogeneity in causality,the overall trend was stable.Conclusion RA is a risk factor for IDA,and there is a positive correlation between RA and IDA.

Objective To investigate the effect of leflunomide(LEF)on the expression of associated autophagy genes in synoviocytes of rheumatoid arthritis(RA)by regulating HIF-1α signal pathway.Methods Three genera-tions of RA synovial cells were divided into blank control group,LEF group and Tripterygium wilfordii polyglyco-sides group.The blank control group was added with the same volume of DMEM culture medium.The drug group was treated with LEF(concentration 0.2 mg/ml)and Tripterygium wilfordii polyglycosides(concentration 0.03 mg/ml),the proliferation and apoptosis of synovial cells were detected by flow cytometry,the expression of IL-1 β,TNF-α,ANGPTL-4 and VEGF was detected by ELISA,the expression of HIF-1α mRNA was detected by qRT-PCR,and the expression of HIF-1 α,Beclin-1 and BNIP3 protein was detected by Western blot.Results Com-pared with Tripterygium wilfordii polyglycosides group,the expression of IL-1 α,TNF-α,ANGPTL-4 and VEGF in synovial supernatant of LEF group decreased;compared with the blank control group,the expression of HIF-1αmRNA in synovial cells of LEF group and Tripterygium wilfordii polyglycosides group decreased,and the effect of LEF group was the most obvious;compared with the blank control group,the protein expressions of HIF-1α,Bec-lin-1 and BNIP3 in synovial cells of LEF group and Tripterygium wilfordii polyglycosides group decreased,and the effect of LEF group was the most obvious.Conclusion LEF can inhibit the expression of inflammatory factors in RA synovial cells,inhibit HIF-1α signaling pathway,inhibit the expression of autophagy-related genes Beclin-1 and BNIP3,and improve the pathological state of synovitis.