Direct Observation of All Open-Shell Intermediates in a Photocatalytic Cycle.

Molecular photocatalysis has shown tremendous success in sustainable energy and chemical synthesis. However, visualizing the transient open-shell intermediates in photocatalysis is a significant and long-standing challenge. By employing our recently developed innovative time-resolved electron paramagnetic resonance technique, we directly observed all radicals and radical ions involved in the photocatalytic addition of pempidine to tert-butyl acrylate. The full picture of the photocatalytic cycle is vividly illustrated by the fine structures, chemical kinetics, and dynamic spin polarization of all open-shell intermediates directly observed in this prototypical system. Given the universality of this methodology, we believe it greatly empowers the research paradigm of direct observation in both photocatalysis and radical chemistry.

Role and Therapeutic Potential for Targeting Fibroblast Growth Factor 10/FGFR1 in Relapsed Rheumatoid Arthritis.

OBJECTIVE: Fibroblast-like synoviocytes (FLSs) contribute to inflammation and joint damage in rheumatoid arthritis (RA). However, the regulatory mechanisms of FLSs in relapse and remission of RA remain unknown. Identifying FLS heterogeneity and their underlying pathogenic roles may lead to discovering novel disease-modifying antirheumatic drugs. METHODS: Combining single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics, we sequenced six matched synovial tissue samples from three patients with relapse RA and three patients in remission. We analyzed the differences in the transcriptomes of the FLS subsets between the relapse and remitted phases. We validated several key signaling pathways using quantitative real-time PCR (qPCR) and multiplex immunohistochemistry (mIHC). We further targeted the critical signals in vitro and in vivo using the collagen-induced arthritis (CIA) model in rats. RESULTS: Lining and sublining FLS subsets were identified using scRNA-seq. Differential analyses indicated that the fibroblast growth factor (FGF) pathway was highly activated in the lining FLSs from patients with relapse RA for which mIHC confirmed the increased expression of FGF10. Although the type I interferon pathway was also activated in the lining FLSs, in vitro stimulation experiment suggested that it was independent of the FGF10 pathway. FGF10 knockdown by small interfering RNA in FLSs significantly reduced the expression of receptor activator of NF-κB ligand. Moreover, recombinant FGF10 protein enhanced bone erosion in the primary human-derived pannus cell culture, whereas the FGF receptor (FGFR) 1 inhibitor attenuated this process. Finally, administering an FGFR1 inhibitor displayed a therapeutic effect in a CIA rat model. CONCLUSION: The FGF pathway is a critical signaling pathway in relapse RA. Targeted tissue-specific inhibition of FGF10/FGFR1 may provide new opportunities to treat patients with relapse RA.

[Effect of Juanbi Qianggu Formula on biological behaviors of fibroblast-like synoviocytes in rheumatoid arthritis by regulating FGFR1 signaling pathway based on network pharmacology and cell function experiments].

This study aimed to explore the molecular mechanism of Juanbi Qianggu Formula(JBQGF), an empirical formula formulated by the prestigious doctor in traditional Chinese medicine, in the treatment of rheumatoid arthritis based on network pharmacology and cell function experiments. The main active components and targets of JBQGF were obtained through Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP) and Encyclopedia of Traditional Chinese Medicine(ETCM), and the core targets underwent functional enrichment analysis and signaling pathway analysis. Cytoscape 3.6.0 was used to construct a visualized "active component-target-signaling pathway" network of JBQGF. After screening, nine potential pathways of JBQGF were obtained, mainly including G protein-coupled receptor signaling pathway and tyrosine kinase receptor signaling pathway. As previously indicated, the fibroblast growth factor receptor 1(FGFR1) signaling pathway was highly activated in active fibroblast-like synoviocytes(FLS) in rheumatoid arthritis, and cell and animal experiments demonstrated that inhibition of the FGFR1 signaling pathway could significantly reduce joint inflammation and joint destruction in collagen-induced arthritis(CIA) rats. In terms of the tyrosine kinase receptor signal transduction pathway, the analysis of its target genes revealed that FGFR1 might be a potential target of JBQGF for rheumatoid arthritis treatment. The biological effect of JBQGF by inhibiting FGFR1 phosphorylation was preliminarily verified by Western blot, Transwell invasion assay, and pannus erosion assay, thereby inhibiting matrix metalloproteinase 2(MMP2) and receptor activator of nuclear factor-κB ligand(RANKL) and suppressing the invasion of fibroblasts in rheumatoid arthritis and erosive effect of pannus bone. This study provides ideas for searching potential targets of rheumatoid arthritis treatment and TCM drugs through network pharmacology.

The effect of JuanBiQiangGu granules in combination with methotrexate on joint inflammation in rheumatoid arthritis: a randomized controlled trial.

Background: Rheumatoid arthritis (RA) joint inflammation severely affects joint function and quality of life in patients and leads to joint deformities and limb disability. The non-steroidal anti-inflammatory drugs used in the treatment of RA do not fully control the progression of joint inflammation and bone destruction and have notable adverse reactions. Traditional Chinese medicine formula JuanBiQiangGu Granules (JBQG) are commonly used for the treatment of RA inflammation and delay of bone destruction, but has not been evaluated through high-quality clinical studies. There is a pressing need for well-designed, randomized, parallel, controlled clinical studies to evaluate the exact effect of JBQG on RA joint inflammation and improvement of patient quality of life. Methods: This is a randomized, parallel, controlled clinical study in which 144 patients with rheumatoid arthritis who met the inclusion criteria were randomly assigned to 2 groups in a 1:1 ratio. The JBQG group received methotrexate 7.5 mg qw and JBQG granules 8 mg tid, while the MTX group received methotrexate 7.5 mg qw. The endpoint was 12 weeks after treatment. Relevant indices at baseline, 4 weeks, 8 weeks, and 12 weeks after treatment were observed and recorded, and DAS28-ESR, HAQ-DI, and Sharp scores were recorded for each patient. Blood samples were collected to test for CRP, ESR, TNF-α, IL-1ß, IL-6, IL-17, and INF-γ, and adverse reactions and liver and kidney function (AST, ALT, Cr, BUN) were recorded for safety assessment. After 12 weeks of treatment, the effect of JBQG granules on disease activity, improvement in bone damage, and patient quality of life scores and safety in RA patients were evaluated. Results: A total of 144 subjects completed treatment (71 in the JBQG group and 73 in the MTX group) and were included in the analysis. At baseline, there were no significant differences between the groups in terms of the observed indicators (p > 0.05). After treatment, 76.06% of patients in the JBQG group had DAS28-ESR levels below or equal to Low, including 45.07% in Remission and 5.63% in High, compared to 53.1% in the MTX group below or equal to Low, 12.33% in Remission, and 17.81% in High. CRP was significantly reduced (8.54 ± 5.87 vs. 11.86 ± 7.92, p < 0.05, p = 0.005), ESR was significantly reduced (15.1 ± 6.11 vs. 21.96 ± 9.19, p < 0.0001), TNF-α was significantly reduced (1.44 ± 0.83 vs. 1.85 ± 1.07, p < 0.05, p = 0.011), IL-17 was significantly reduced (0.53 ± 0.33 vs. 0.71 ± 0.38, p < 0.05, p = 0.004), and INF-γ was significantly reduced (3.2 ± 1.51 vs. 3.89 ± 1.77, p < 0.05, p = 0.014). The median (IQR) OPG in the JBQG group was 2.54 (2.21-3.01), significantly higher than in the MTX group 2.06 (1.81-2.32), p < 0.0001), and the median (IQR) ß-CTX in the JBQG group was 0.4 (0.32-0.43), significantly lower than in the MTX group 0.55 (0.47-0.67), p < 0.0001). The median (IQR) VSA scores were 2 (1-3), a decrease from 3 (2-4) in the MTX group (p < 0.0001). The median (IQR) Sharp scores were 1 (1-2), a decrease from 2 (1-2) in the MTX group, but the difference was not statistically significant (p > 0.05, p = 0.28). The median (IQR) HAQ-DI scores were 11 (8-16), significantly lower than in the MTX group 26 (16-30) (p < 0.0001). The median (IQR) AST in the JBQG group was 16 (12-20), with a significant difference compared to the MTX group 19 (13-25) (p < 0.01, p = 0.004); the median (IQR) ALT in the JBQG group was 14 (10-18), with a significant difference compared to the MTX group 16 (11-22.5) (p < 0.05, p = 0.015). There were no statistically significant differences in Cr or BUN (p > 0.05). Conclusion: JuanBiQiangGu Granules can be used to treat patients with rheumatoid arthritis, alleviate joint inflammation, reduce the incidence of adverse reactions to methotrexate, and has good safety. Clinical Trial Registration: http://www.chinadrugtrials.org.cn/index.html; identifier: ChiCTR2100046373.

Identification of DNA methylation-regulated differentially expressed genes in RA by integrated analysis of DNA methylation and RNA-Seq data.

OBJECTIVE: To identify novel DNA methylation-regulated differentially expressed genes (MeDEGs) in RA by integrated analysis of DNA methylation and RNA-Seq data. METHODS: The transcription and DNA methylation profiles of 9 RA and 15 OA synovial tissue were generated by RNA-Seq and Illumina 850K DNA methylation BeadChip. Gene set enrichment analysis (GSEA) and Weighted gene co-expression network analysis (WGCNA) were used to analyze methylation-regulated expressed genes by R software. The differentially expressed genes (DEGs), differentially methylated probes (DMPs), differentially methylated genes (DMGs) were analyzed by DESeq and ChAMP R package. The functional correlation of MeDEGs was analyzed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). The protein-protein interaction (PPI) network of MeDEGs was constructed by STRING and Reactome FI Cytoscape Plugin. Correlation analysis between methylation level and mRNA expression was conducted with R software. RESULTS: A total of 17,736 genes, 25,578 methylated genes and 755,852 methylation probes were detected. A total of 16,421 methylation-regulated expressed genes were obtained. The GSEA showed that these genes are associated with activation of immune response, adaptive immune response, Inflammatory response in C5 (ontology gene sets). For KEGG analysis, these genes are associated with rheumatoid arthritis, NF-kappa B signaling pathway, T cell receptor signaling pathway. The WGCNA showed that the turquoise module exhibited the strongest correlation with RA (R = 0.78, P = 1.27 × 10- 05), 660 genes were screened in the turquoise module. A total of 707 MeDEGs were obtained. GO analysis showed that MeDEGs were enriched in signal transduction, cell adhesion for BP, enriched in plasma membrane, integral component of membrane for CC, and enriched in identical protein binding, calcium ion binding for MF. The KEGG pathway analysis showed that the MeDEGs were enriched in calcium signaling pathway, T cell receptor signaling pathway, NF-kappa B signaling pathway, Rheumatoid arthritis. The PPI network containing 706 nodes and 882 edges, and the enrichment p value < 1.0 × 10- 16. With Cytoscape, based on the range of more than 10 genes, a total of 8 modules were screened out. Spearman correlation analysis showed RGS1(cg10718027), RGS1(cg02586212), RGS1(cg10861751) were significantly correlated with RA. CONCLUSIONS: RGS1 can be used as novel methylated biomarkers for RA.

Expanded CD1c+CD163+ DC3 Population in Synovial Tissues Is Associated with Disease Progression of Osteoarthritis.

The mechanisms underlying osteoarthritis (OA) have recently been hypothesized to involve a dysfunctional immune system. In this study, we collected synovium, synovial fluid (SF), and peripheral blood from 21 patients. Mononuclear cells were characterized using FCM. H&E staining and mIHC histological assessment of synovium were performed. Cytokine levels in the SF were measured using ELISA. We observed similar frequencies of immune cells in the synovium and SF, which were enriched in DCs. Notably, CD1c+CD163+ DC3s were expanded in the synovium and SF. Furthermore, we found that DC3s were primarily located within the ectopic lymphoid-like structure (ELLS) in close proximity to CD8+ T cells. Finally, the level of TNF-α and IL12p70 in the SF correlated with the severity of OA. These data suggest that OA is an immune system-related disease and that DC3s may play an active role in OA progression by promoting ELLS formation and inflammatory responses.

RNA-seq and Network Analysis Reveal Unique Chemokine Activity Signatures in the Synovial Tissue of Patients With Rheumatoid Arthritis.

Purpose: This study aimed to provide a comprehensive understanding of the genome-wide expression patterns in the synovial tissue samples of patients with rheumatoid arthritis (RA) to investigate the potential mechanisms regulating RA occurrence and development. Methods: Transcription profiles of the synovial tissue samples from nine patients with RA and 15 patients with osteoarthritis (OA) (control) from the East Asian population were generated using RNA sequencing (RNA-seq). Gene set enrichment analysis (GSEA) was used to analyze all the detected genes and the differentially expressed genes (DEGs) were identified using DESeq. To further analyze the DEGs, the Gene Ontology (GO) functional enrichment and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed. The protein-protein interaction (PPI) network of the DEGs was constructed using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) and the hub genes were identified by topology clustering with the Molecular Complex Detection (MCODE)-Cytoscape. The most important hub genes were validated using quantitative real-time PCR (qRT-PCR). Results: Of the 17,736 genes detected, 851 genes were identified as the DEGs (474 upregulated and 377 downregulated genes) using the false discovery rate (FDR) approach. GSEA revealed that the significantly enriched gene sets that positively correlated with RA were CD40 signaling overactivation, Th1 cytotoxic module, overactivation of the immune response, adaptive immune response, effective vs. memory CD8+ T cells (upregulated), and naïve vs. effective CD8+ T cells (downregulated). Biological process enrichment analysis showed that the DEGs were significantly enriched for signal transduction (P = 3.01 × 10-6), immune response (P = 1.65 × 10-24), and inflammatory response (P = 5.76 × 10-10). Molecule function enrichment analysis revealed that the DEGs were enriched in calcium ion binding (P = 1.26 × 10-5), receptor binding (P = 1.26 × 10-5), and cytokine activity (P = 2.01 × 10-3). Cellular component enrichment analysis revealed that the DEGs were significantly enriched in the plasma membrane (P = 1.91 × 10-31), an integral component of the membrane (P = 7.39 × 10-13), and extracellular region (P = 7.63 × 10-11). The KEGG pathway analysis showed that the DEGs were enriched in the cytokine-cytokine receptor interaction (P = 3.05 × 10-17), chemokine signaling (P = 3.50 × 10-7), T-cell receptor signaling (P = 5.17 × 10-4), and RA (P = 5.17 × 10-4) pathways. We confirmed that RA was correlated with the upregulation of the PPI network hub genes, such as CXCL13, CXCL6, CCR5, CXCR5, CCR2, CXCL3, and CXCL10, and the downregulation of the PPI network hub gene such as SSTR1. Conclusion: This study identified and validated the DEGs in the synovial tissue samples of patients with RA, which highlighted the activity of a subset of chemokine genes, thereby providing novel insights into the molecular mechanisms of RA pathogenesis and identifying potential diagnostic and therapeutic targets for RA.

Cerebral mechanism of opposing needling for managing acute pain after unilateral total knee arthroplasty: study protocol for a randomized, sham-controlled clinical trial.

BACKGROUND: Opposing needling is a unique method used in acupuncture therapy to relieve pain, acting on the side contralateral to the pain. Although opposing needling has been used to treat pain in various diseases, it is not clear how opposing needling affects the activity of the central nervous system to relieve acute pain. We herein present the protocol for a randomized sham-controlled clinical trial aiming to explore the cerebral mechanism of opposing needling for managing acute pain after unilateral total knee arthroplasty (TKA). METHODS: This is a randomized sham-controlled single-blind clinical trial. Patients will be allocated randomly to two parallel groups (A: opposing electroacupuncture group; B: sham opposing electroacupuncture group). The Yinlingquan (SP9), Yanglingquan (GB34), Futu (ST32), and Zusanli (ST36) acupoints will be used as the opposing needling sites in both groups. In group A, the healthy lower limbs will receive electroacupuncture, while in group B, the healthy lower limbs will receive sham electroacupuncture. At 72 h after unilateral TKA, patients in both groups will begin treatment once per day for 3 days. Functional magnetic resonance imaging will be performed on all patients before the intervention, after unilateral TKA, and at the end of the intervention to detect changes in brain activity. Changes in pressure pain thresholds will be used as the main outcome for the improvement of knee joint pain. Secondary outcome indicators will include the visual analogue scale (including pain during rest and activity) and a 4-m walking test. Surface electromyography, additional analgesia use, the self-rating anxiety scale, and the self-rating depression scale will be used as additional outcome indices. DISCUSSION: The results will reveal the influence of opposing needling on cerebral activity in patients with acute pain after unilateral TKA and the possible relationship between cerebral activity changes and improvement of clinical variables, which may indicate the central mechanism of opposing needling in managing acute pain after unilateral TKA. TRIAL REGISTRATION: Study on the brain central mechanism of opposing needling analgesia after total kneearthroplasty based on multimodal MRI ChiCTR2100042429 . Registered on January 21, 2021.

DNA methylation change of HIPK3 in Chinese rheumatoid arthritis and its effect on inflammation.

Introduction: Homeodomain-interacting protein kinase 3 (HIPK3) plays an important role in cell proliferation, apoptosis, and inflammation. Over-expression of HIPK3 in immune cells in rheumatoid arthritis (RA) has been reported. In this study, we investigated blood methylation levels and clinical characteristics of RA in a Chinese population. Methods: A total of 235 patients with RA, 30 with osteoarthritis (OA), and 30 matched healthy controls were recruited. The methylation status of seven CpGs in the differentially methylated region of HIPK3 (cg05501357) was measured using targeted methylation-sequencing technology. The association between methylation haplotypes and the overall methylation status of HIPK3 with clinical characteristics was assessed using generalized linear regression. Results: All seven CpGs showed hypomethylation status in RA blood compared with OA and normal individuals (overall p= 1.143×10-8 and FDR= 2.799×10-7), which is consistent with the previously reported high expression of HIPK3 in RA immune cells. Among all seven CpGs, 33286785 showed the highest predictive power with an area under the curve (AUC) of 0.829; we received a higher AUC=0.864 when we combined HIPK3 with anti-citrullinated protein antibodies (ACPA -) and rheumatoid factor (RF +) in the prediction model, indicating that when a patient's ACPA is negative, HIPK3 can assist RF as a new clinical index for the diagnosis of RA. We also found that HIPK3 methylation levels were negatively correlated with C-reactive protein (CRP; r= -0.16, p= 0.01). Methylation haplotypes were analyzed, and the full methylation haplotype (FMH; r= 0.16, p= 0.01) and full non-methylation haplotype (FNH; r= 0.18, p= 0.0061) were negatively correlated with CRP. Conclusion: Circulating blood methylation levels in the protein region of HIPK3 can be utilized as a supportive diagnostic biomarker and CRP level indicator for RA.

Multiple intravenous tranexamic acid doses in total knee arthroplasty in patients with rheumatoid arthritis: a randomized controlled study.

BACKGROUND: We aimed to determine the efficacy and safety of multiple doses of intravenous tranexamic acid (IV-TXA) on perioperative blood loss in patients with rheumatoid arthritis (RA) who had undergone primary unilateral total knee arthroplasty (TKA). METHODS: For this single-center, single-blind randomized controlled clinical trial, 10 male and 87 female participants with RA, aged 50-75 years, who underwent unilateral primary TKA were recruited. The patients received one dose of 1 g IV-TXA 10 min before skin incision, followed by articular injection of 1.5 g tranexamic acid after cavity suture during the surgery. The patients were randomly assigned (1:1) into two groups and received an additional single dose of IV-TXA (1 g) for 3 h (group A) or three doses of IV-TXA (1 g) for 3, 6, and 12 h (group B) postoperatively. Primary outcomes were total blood loss (TBL), hidden blood loss (HBL), and maximum hemoglobin (Hb) level decrease. Secondary outcomes were transfusion rate and D-dimer levels. All parameters were measured postoperatively during inpatient hospital stay. RESULTS: The mean TBL, HBL, and maximum Hb level decrease in group B (506.1 ± 227.0 mL, 471.6 ± 224.0 mL, and 17.5 ± 7.7 g/L, respectively) were significantly lower than those in group A (608.8 ± 244.8 mL, P = 0.035; 574.0 ± 242.3 mL, P = 0.033; and 23.42 ± 9.2 g/L, P = 0.001, respectively). No episode of transfusion occurred. The D-dimer level was lower in group B than in group A on postoperative day 1 (P <  0.001), and the incidence of thromboembolic events was similar between the groups (P > 0.05). CONCLUSION: In patients with RA, three doses of postoperative IV-TXA further facilitated HBL and Hb level decrease without increasing the incidence of adverse events in a short period after TKA. TRIAL REGISTRATION: The trial was registered in the Chinese Clinical Trial Registry ( ChiCTR1900025013 ).

Effect of Multiple Doses of Intravenous Tranexamic Acid on Perioperative Blood Loss in Total Knee Arthroplasty: A Randomized Controlled Study.

OBJECTIVE: To identify the efficacy and safety of multiple doses of intravenous tranexamic acid (IV-TXA) following primary total knee arthroplasty (TKA) with a tourniquet. METHODS: This is a single-blind randomized controlled study that recruited osteoarthritis patients who had undergone primary unilateral TKA from May 2019 to May 2020 at our medical center. A total of 300 patients were randomly divided into three groups to receive: one dose (1 g) of IV-TXA before skin incision combined with one dose (1.5 g) of intra-articular tranexamic acid（IA-TXA) followed by a single dose of IV-TXA (1 g) for 3 h (group A); two doses of IV-TXA (1 g) for 3 and 6 h (group B); or three doses of IV-TXA (1 g) for 3, 6, and 12 h (group C) postoperatively. TKA with a tourniquet was performed by the same surgical team. The primary outcomes were total blood cell loss (TBL), hidden blood loss (HBL), maximum hemoglobin (Hb) drop, and transfusion rate. Secondary outcomes were levels of C-reactive protein (CRP) and D-dimer, and the incidence of postoperative complications. One-way analysis of variance, subgroup analysis, and multivariate correlation analysis were used to calculate the differences among the three groups. RESULTS: The study included 56 male and 244 female patients aged 60-80 years. The mean TBL, the mean HBL, and the maximum Hb drop in group C (471.2 ± 190.6 mL, 428.4 ± 190.3 mL, and 21.2 ± 3.8 g/L, respectively) were significantly lower than those in groups B (563.4 ± 224.6 mL, P = 0.030; 519.9 ± 226.4 mL, P = 0.033; and 23.2 ± 4.1 g/L, P = 0.001, respectively), and A (651.6 ± 254.1 mL, P < 0.001; 607.1 ± 254.3 mL, P < 0.001; and 25.1 ± 4.3 g/L, P < 0.001, respectively). No transfusions were required. The postoperative acute inflammatory reaction was less problematic for patients in Group C, and the incidence of thromboembolic events was similar among the groups (P > 0.05). In addition, there were positive correlations between the HBL and the tourniquet inflation time (r = 0.844, P < 0.001). Similarly, the level of CRP on POD1 (r = 0.393, P < 0.001) and POD3 (r = 0.149, P = 0.010), and the level of D-dimer on POD1 (r = 0.382, P < 0.001) were positively correlated with the HBL. CONCLUSION: Three doses of postoperative IV-TXA decreased blood loss and diminished the postoperative inflammatory and fibrinolytic response more than a single dose or two doses in elderly patients following TKA without increasing the incidence of adverse events.

Multiple-dose tranexamic acid for perioperative blood loss in total knee arthroplasty in patients with rheumatoid arthritis：a single-blinded, randomised, parallel-controlled study protocol in China.

INTRODUCTION: This clinical trial is designed to evaluate the effect of multiple-dose tranexamic acid (TXA) on perioperative blood loss in patients with rheumatoid arthritis (RA). METHODS AND ANALYSIS: A randomised, single-blinded, parallel-controlled study will be designed. Patients with RA (age 50-75 years) undergoing unilateral primary end-stage total knee arthroplasty will be randomly divided into group A or group B. Group A will be treated with one dose of TXA (1 g; intravenous injection 3 hours postsurgery) and group B with three doses (1 g; intravenous injection at 3, 6 and 12 hours postsurgery) after surgery. The primary outcomes will be evaluated with blood loss, maximum haemoglobin drop and transfusion rate. The secondary outcomes will be evaluated with knee function and complications. ETHICS AND DISSEMINATION: The Shanghai Guanghua Hospital of Integrated Traditional Chinese Medicine and Western Medicine Ethics Committee approved in this study in July 2019. Informed consent will be obtained from all participants. Results of the trial will be published in the Dryad and repository in a peer-reviewed journal. Additionally, deidentified data collected and analysed for this study will be available for review from the corresponding author on reasonable request. TRIAL REGISTRATION NUMBER: ChiCTR1900025013.

Using electroacupuncture to recover muscle strength in patients with knee osteoarthritis after total knee arthroplasty: a study protocol for a double-blinded, randomized, and placebo-controlled trial.

BACKGROUND: Total knee arthroplasty (TKA) is a gold standard for patients with terminal term gonarthrosis for reducing pain, correcting deformities, and regaining stability. However, post-TKA muscle strength recovery is often difficult. Although electroacupuncture (EA) enhances lower extremity muscle strength of the lower extremity, there is limited evidence regarding its effect on lower extremity muscle strength in post-TKA patients. Consequently, this trial intends to evaluate the efficacy of post-TKA EA on the recovery of lower extremity muscle strength, specifically, during the early post-TKA period. METHODS/DESIGN: This is a double-blinded, randomized, and controlled trial. It will be conducted between August 2020 and December 2020. Ninety-four participants with KOA who have undergone unilateral TKA will be randomized into a treatment (EA) group and a control (sham EA) group. The former and latter groups will receive EA and sham EA, respectively, at ST37, ST36, SP10, and SP9 acupoints. The participants will undergo ten treatment sessions over 2 weeks (5 sessions per week). The primary outcomes will include changes in muscle strength and the Hospital for Special Surgery score at the second week from baseline (pre-op 1 day or POD 3). The secondary outcomes will include a 4-m walk test, numerical rating scale score, the Hamilton Anxiety Scale score, and additional analgesia use. Additional outcomes will include the incidence of analgesia-related side effects and the participant satisfaction rate. Participant blinding will also be assessed where they will be asked to guess whether they received EA after the latest intervention. Adverse EA events will be documented and assessed throughout the trial. DISCUSSION: EA is helpful for post-TKA recovery and enhancement of lower limb muscle strength. TRIAL REGISTRATION: Chinese Clinical Trial Registry ChiCTR1900027806 . Registered on 29 November 2019.

Cyr61 participates in the pathogenesis of rheumatoid arthritis via promoting MMP-3 expression by fibroblast-like synoviocytes.

OBJECTIVES: The aim of this study was to investigate the effect and potential mechanism of Cysteine-rich 61 (Cyr61) on stimulating MMP-3 expression by fibroblast-like synoviocytes (FLS) from rheumatoid arthritis (RA) patients. METHODS: Primarily cultured RA FLS were treated with exogenous Cyr61 protein or Cyr61-siRNA, then, MMP-3 expression was analyzed by real-time PCR, western blotting and ELISA. Signal transduction pathways in Cyr61-induced MMP-3 production were examined by real-time PCR, western blotting, confocal microscopy, luciferase reporter assay. Mice with collagen-induced arthritis (CIA) were treated with anti-Cyr61 monoclonal antibodies (mAb), or IgG1 as control and MMP-3 in the joint was detected by IHC, real-time PCR and western blotting. RESULTS: High expressed MMP-3 and Cyr61 were positively correlated in RA ST; Cyr61 stimulated MMP-3 production in FLS of RA patients in an IL-1ß and TNF-α independent manner. Cyr61 induced MMP-3 could further enhance the invasive ability of RA FLS. Mechanistically, we found that Cyr61 promoted MMP-3 production via the P38, JNK-dependent AP-1 signaling pathway. Blockage of Cyr61 function with monoclonal antibody could decrease MMP-3 expression in the joints of CIA mice. CONCLUSION: This study provides new evidence that Cyr61 participates in RA pathogenesis not only as a pro-inflammatory factor but also plays a key role in bone erosion via promoting MMP-3 expression. We suggest that targeting of Cyr61 may represent a potential strategy in RA treatment.

Therapeutic Efficacy of E-64-d, a Selective Calpain Inhibitor, in Experimental Acute Spinal Cord Injury.

This study aims to investigate the therapeutic effect of calpain inhibitor E-64-d on SCI and to find a new approach to treat SCI. When an SCI rat model was established, it was immediately administered with E-64-d. RT-PCR and Western blotting were used to determine the protein and mRNA levels of calpain 1 and 68-kD NFP. TUNEL staining and NeuN labeling were performed to analyze neuronal apoptosis in the lesion. Immunohistochemistry assay was carried out to observe the expressions of calpain 1 and GFAP. Cyclooxygenase-2 activity was measured to show the immune response status. Locomotor function was evaluated by inclined plane test and Basso, Beattie, and Bresnahan locomotor rating scale. The results showed that calpain 1 was activated after SCI occurred. Treatment with E-64-d decreased expressions of calpain 1 and GFAP, alleviated neuronal apoptosis, inhibited cyclooxygenase-2 activity, and resulted in the promoted locomotor function. Furthermore, combination of E-64-d and MP had better efficacy than did E-64-d or MP alone. E-64-d is expected to be applied to treat SCI, and its alliance with MP may provide a valid strategy for SCI therapy.

Cyr61 participates in the pathogenesis of rheumatoid arthritis by promoting proIL-1ß production by fibroblast-like synoviocytes through an AKT-dependent NF-κB signaling pathway.

IL-1ß plays a major role in the development of rheumatoid arthritis (RA). We previously showed that Cyr61 participates in RA pathogenesis as a proinflammatory factor. Here, we found that the levels of IL-1ß and Cyr61 were higher in RA SF than in osteoarthritis (OA) SF. IL-1ß mRNA and proIL-1ß protein levels were remarkably increased in Cyr61-stimulated FLS; however, IL-1ß was hardly detectable in the supernatant. We also found that the level of adenosine triphosphate (ATP) in SF and ST was significantly increased in RA patients and that the level of IL-1ß in supernatants from Cyr61-activated FLS increased significantly when we added exogenous ATP to the culture. Mechanistically, Cyr61 induced proIL-1ß production in FLS via the AKT-dependent NF-κB signaling pathway, and ATP caused Cyr61-induced proIL-1ß to generate IL-1ß in a caspase-1-dependent manner. Our results reveal a novel role of Cyr61 in RA that involves the promotion of proIL-1ß production in FLS.

A novel p53/microRNA-22/Cyr61 axis in synovial cells regulates inflammation in rheumatoid arthritis.

OBJECTIVE: We previously showed that Cyr61 acts to promote fibroblast-like synoviocyte (FLS) proliferation and Th17 cell differentiation, suggesting that Cyr61 plays an important role in mediating the joint inflammation and damage in rheumatoid arthritis (RA). The aim of this study was to investigate whether Cyr61 expression is regulated at the posttranscription level, and if so, how this regulation connects to other etiologic factors in RA. METHODS: Expression of microRNA-22 (miR-22) in synovial tissue was detected by real-time polymerase chain reaction (PCR) using miRNA-specific TaqMan MGB probes. MicroRNA-22 promoter activity was analyzed using a Dual-Luciferase Reporter Assay. Cytokine expression was measured by enzyme-linked immunosorbent assay, and the expression of other factors was measured by real-time PCR or Western blotting. RESULTS: MicroRNA-22 directly targeted the 3'-untranslated region of Cyr61 messenger RNA and inhibited Cyr61 expression. Expression of miR-22 was down-regulated and was negatively correlated with Cyr61 expression in RA synovial tissue. Furthermore, wild-type p53 activated miR-22 transcription by binding to the promoter region of the miR-22 gene, while the mutant forms of p53 frequently found in RA synovial tissue were shown to have lost the ability to activate miR-22 expression. As a result, miR-22 was down-regulated, contributing to the overexpression of Cyr61 in RA FLS. CONCLUSION: Our results not only reveal a novel mechanism whereby p53 is involved in the posttranscriptional regulation of Cyr61 expression via miRNA-22, but also provide a molecular explanation for the role of somatic mutations of p53, which are frequently observed in RA synovial tissue, in the etiology of this autoimmune disease.

[Early efficacies of total knee arthroplasty for knees with severe lateral instability].

OBJECTIVE: To evaluate the outcomes of primary total knee arthroplasty (TKA) in the treatment of knee with severe lateral instability and summarize the essential points of operation and rehabilitation. METHODS: From February 2005 to August 2010, primary TKA was performed in 27 severe lateral unstable knees (25 cases), including 3 males (3 knees) and 22 females (24 knees). Their mean age was 57.8 (37-71) years. And their primary diseases included rheumatoid arthritis (22 knees in 21 cases) and osteoarthritis (5 knees in 4 cases). Thirteen lateral unstable knees were accompanied with 18.08° ± 5.96°(15-35°) varus deformity; in the rest 14 knees, there was medial instability with 20.71° ± 7.03° (15-35°) valgus deformity. Blood loss volume, operative duration and complications were recorded. During the follow-up period, HSS score, knee stability and varus/valgus status were recorded preoperatively, 1, 3, 6, 12 months and then annually postoperatively. RESULTS: AORI type I bone defect was found at the proximal tibia in 18 knees and distal lateral femoral condyle in 10 knees. All defects were reconstructed with cement or autograft. AORI type II bone defects at proximal tibia in 3 knees were reconstructed with metal augmentation. Blood loss during the first 24 hours were (438.9 ± 109.5) (400-700) ml and operative duration (91.1 ± 11.6) (70-110) min. The mean follow-up period was (41.6 ± 10.9) (27-60) months. At the final follow-up, the HSS score increased from (45.8 ± 5.4) to (85.4 ± 4.5) (t = 30.15, P < 0.01) .Five knees in 5 cases had mild postoperative instability. All cases were allowed to walk with knee orthosis for 4-6 weeks. At the end of follow-up, mild lateral instability of 2 knees persisted. One augmented knee had osteolysis beneath metal block. CONCLUSION: TKA for knees with severe lateral instability requires a deep understanding of causes and a rational treatment. Proper handling of bone defects and careful release of lateral soft tissue are two critical points for postoperative knee stability. Wearing knee orthosis during the early postoperative stage may be helpful or residual mild instability.

Cyr61 is involved in neutrophil infiltration in joints by inducing IL-8 production by fibroblast-like synoviocytes in rheumatoid arthritis.

INTRODUCTION: It is well known that neutrophils play very important roles in the development of rheumatoid arthritis (RA) and interleukin (IL)-8 is a critical chemokine in promoting neutrophil migration. We previously showed that increased production of Cyr61 by fibroblast-like synoviocytes (FLS) in RA promotes FLS proliferation and Th17 cell differentiation, thus Cyr61 is a pro-inflammatory factor in RA pathogenesis. In this study, we explored the role of Cyr61 in neutrophil migration to the joints of RA patients. METHODS: RA FLS were treated with Cyr61 and IL-8 expression was analyzed by real-time PCR and ELISA. The migration of neutrophils recruited by the culture supernatants was determined by the use of a chemotaxis assay. Mice with collagen-induced arthritis (CIA) were treated with anti-Cyr61 monoclonal antibodies (mAb), or IgG1 as a control. Arthritis severity was determined by visual examination of the paws and joint destruction was determined by hematoxylin-eosin (H&E) staining. Signal transduction pathways in Cyr61-induced IL-8 production were investigated by real-time PCR, western blotting, confocal microscopy, luciferase reporter assay or chromatin immunoprecipitation (ChIP) assay. RESULTS: We found that Cyr61 induced IL-8 production by RA FLS in an IL-1ß and TNF-α independent pathway. Moreover, we identified that Cyr61-induced IL-8-mediated neutrophil migration in vitro. Using a CIA animal model, we found that treatment with anti-Cyr61 mAb led to a reduction in MIP-2 (a counterpart of human IL-8) expression and decrease in neutrophil infiltration, which is consistent with an attenuation of inflammation in vivo. Mechanistically, we showed that Cyr61 induced IL-8 production in FLS via AKT, JNK and ERK1/2-dependent AP-1, C/EBPß and NF-κB signaling pathways. CONCLUSIONS: Our results here reveal a novel role of Cyr61 in the pathogenesis of RA. It promotes neutrophil infiltration via up-regulation of IL-8 production in FLS. Taken together with our previous work, this study provides further evidence that Cyr61 plays a key role in the vicious cycle formed by the interaction between infiltrating neutrophils, proliferated FLS and activated Th17 cells in the development of RA.

[Effects of Tuina and Chinese patent drug Shuxuetong injection on deep venous thrombosis and functional rehabilitation after total knee arthroplasty].

OBJECTIVE: To evaluate the efficacy of Tuina and Chinese patent drug Shuxuetong injection in preventing patients undergoing total knee arthroplasty from deep venous thrombosis and in functional rehabilitation. METHODS: A total of 120 patients with diagnosed rheumatoid arthritis in the Department of Orthopaedic Surgery, Shanghai Guanghua Hospital of Integrated Traditional Chinese and Western Medicine in China were enrolled for this study. The patients underwent total knee arthroplasty and were divided into treatment group (n=60) and control group (n=60) after surgery. Patients in the control group received conventional rehabilitation training, including using a continuous passive motion machine and training of muscle contractions of the lower limb. Patients in the treatment group were administered Shuxuetong injection and Tuina based on the conventional rehabilitation training. The course of treatment lasted for 2 weeks. Hospital for Special Surgery (HSS) knee score, rate of deep venous thrombosis and range of motion of the knee joint were evaluated before and after treatment. RESULTS: There was no significant difference in HSS knee score and range of motion as compared before and after treatment in two group (P>0.05). The rate of deep venous thrombosis of the treatment group was 13.33%, which was lower than 20% of the control group (P<0.05). CONCLUSION: Tuina combined with Shuxuetong injection treatment can prevent deep venous thrombosis in patients with rheumatoid arthritis after total knee arthroplasty.