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.

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 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.

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 observe the effects of the tofacitinib on interstitial lung disease( ILD) by regulating the JAK⁃STAT signaling pathway. Methods: Wistar rats were randomly divided into 4 groups:normal group , ILD group ,prednisone acetate group , and tofacitinib group. Except for the normal group , the other three groups were given 3mg/ml bleomycin solution for modeling. After 28 days of intragastric administration , the lung tissues of all rats were collected for hematoxylin⁃eosin staining ( HE) and Western blot ( WB) to detect the protein levels of JAK1 and STAT1;Enzyme⁃linked immunosorbent assay(ELISA) was used to detect tumor necrosis factor in rat serum (TNF) Ⅳ α , interleukin (IL) Ⅳ6 , IL⁃10 , IL⁃1β . Results : HE staining of lung tissue in ILD ,prednisone acetate group and tofacitinib group showed alveolar tissue thickening , alveolar wall capillary congestion , bronchial luminal epithelial cells shedding, and inflammatory cell exudation. The results of WB showed that JAK1 and STAT1 significantly increased in ILD group , and decreased in different degrees compared with ILD group , tofacitinib group and prednisone acetate group (P < 0. 05) . The ELISA results showed that the expressions of serum TNF⁃α , IL⁃6 and IL⁃1β in the ILD group were significantly higher than those in the normal group (P < 0. 01) . The expression of pine group decreased (P < 0. 05) , and the expression of IL⁃10 was the opposite. Conclusion Tofacitinib reduces lung tissue damage and the inflammatory response in the treatment of ILD by inhibiting the JAK⁃STAT pathway and down⁃regulating the expression of inflammatory factors TNF⁃α , IL⁃6 , IL⁃1β and up⁃regulating the anti⁃inflammatory factor IL⁃10.

Stem/progenitor cells are important for salivary gland development, homeostasis maintenance, and regeneration following injury. Keratin-14⁺ (K14⁺) cells have been recognized as bona fide salivary gland stem/progenitor cells. However, K14 is also expressed in terminally differentiated myoepithelial cells; therefore, more accurate molecular markers for identifying salivary stem/progenitor cells are required. The intraflagellar transport (IFT) protein IFT140 is a core component of the IFT system that functions in signaling transduction through the primary cilia. It is reportedly expressed in mesenchymal stem cells and plays a role in bone formation. In this study, we demonstrated that IFT140 was intensively expressed in K14⁺ stem/progenitor cells during the developmental period and early regeneration stage following ligation-induced injuries in murine submandibular glands. In addition, we demonstrated that IFT140⁺/ K14⁺ could self-renew and differentiate into granular duct cells at the developmental stage in vivo. The conditional deletion of Ift140 from K14⁺ cells caused abnormal epithelial structure and function during salivary gland development and inhibited regeneration. IFT140 partly coordinated the function of K14⁺ stem/progenitor cells by modulating ciliary membrane trafficking. Our investigation identified a combined marker, IFT140⁺/K14⁺, for salivary gland stem/progenitor cells and elucidated the essential role of IFT140 and cilia in regulating salivary stem/progenitor cell differentiation and gland regeneration.
Animals ; Carrier Proteins/metabolism* ; Cell Differentiation ; Keratin-14/metabolism* ; Mice ; Osteogenesis ; Salivary Glands/metabolism* ; Stem Cells

Objective : To investigate the changes of intestinal flora in patients with rheumatoid arthritis (RA) before and after treatment with technetium[ 99 Tc] methylenediphosphonate injection ( 99 Tc⁃MDP) . Methods : Thirty⁃two patients with RA were collected the fresh fecal specimens before and after they treated with 99 Tc⁃MDP. The genome DNA of fecal specimens was extracted and the V3 ⁃V4 regions of 16S rRNA were sequenced by Illumina Miseq platform. The sequencing results were analyzed by bioinformatics methodology. Results : The Chao , observed species , Shannon and Simpson index between the two groups in Alpha diversity analysis had no statistical difference (P > 0. 05 ) , but the value in post⁃treatment group were increased compared with the pre⁃treatment group. Compared with the pre⁃treatment of RA , there was no statistical difference in the higher proportion relative abundance on Phylum level of the intestinal microbes of RA patients , and the relative abundance of Tenericutes decreased ( P < 0. 05 ) . At the genus level , the relative abundance of Ruminococcaceae , Butyricicoccus , Christensenellaceae_R ⁃7_group , Holdemania decreased after treatment ( P < 0. 05 ) . And the relative abundances of Pasteurella , Lachnoanaerobaculum and Stomatobaculum increased (P < 0. 05) . Conclusion The abundance and species of gut microbiota in RA patients have some changes after treatment with 99Tc⁃MDP , and the diversity increases after treatment.

Malignant peripheral nerve sheath tumors(MPNST) of the bladder is a very rare malignant tumor, usually secondary to neurofibromatosis type 1 (NF-1), with a few sporadic cases. This paper reports a case of 70-year-old man with bladder MPNST who underwent transurethral resection. Adjuvant intravesical instillation with gemcitabine was given after surgery, and there was no local recurrence and distant metastasis after 12-month follow-up. This paper also made a corresponding literature review.