M2 Macrophage-Derived Extracellular Vesicles Encapsulated in Hyaluronic Acid Alleviate Osteoarthritis by Modulating Macrophage Polarization.

An imbalance between M1 and M2 macrophage polarization is critical in osteoarthritis (OA) development. We investigated the effect of M2 macrophage-derived extracellular vesicles (M2-EVs) to reprogramme macrophages from the M1 to M2 phenotype for OA treatment. M1 macrophages and mouse OA models were treated with M2-EVs. Proteomic analysis was performed to evaluate macrophage polarization in vitro. The OA models were as follows: destabilization of the medial meniscus (DMM) surgery-induced OA and collagenase-induced OA (CIOA). Hyaluronic acid (HA) was used to deliver M2-EVs. M2-EVs decreased macrophage accumulation, repolarized macrophages from the M1 to M2 phenotype, mitigated synovitis, reduced cartilage degradation, alleviated subchondral bone damage, and improved gait abnormalities in the CIOA and DMM models. Moreover, HA increased the retention time of M2-EVs and enhanced the efficiency of M2-EVs in OA treatment. Furthermore, proteomic analysis demonstrated that M2-EVs exhibited a macrophage reprogramming ability similar to IL-4, and the pathways might be the NOD-like receptor (NLR), TNF, NF-κB, and Toll-like receptor (TLR) signaling pathways. M2-EVs reprogrammed macrophages from the M1 to M2 phenotype, which resulted in beneficial effects on cartilage and attenuation of OA severity. In summary, our study indicated that M2-EV-guided reprogramming of macrophages is a promising treatment strategy for OA.

NCF4 regulates antigen presentation of cysteine peptides by intracellular oxidative response and restricts activation of autoreactive and arthritogenic T cells.

Autoimmune diseases, such as rheumatoid arthritis (RA) and systemic lupus erythematous, are regulated by polymorphisms in genes contributing to the NOX2 complex. Mutations in both Ncf1 and Ncf4 affect development of arthritis in experimental models of RA, but the different regulatory pathways mediated by NOX2-derived reactive oxygen species (ROS) have not yet been clarified. Here we address the possibility that intracellular ROS, regulated by the NCF4 protein (earlier often denoted p40phox) which interacts with endosomal membranes, could play an important role in the oxidation of cysteine peptides in mononuclear phagocytic cells, thereby regulating antigen presentation and activation of arthritogenic T cells. To study the role of NCF4 we used mice with an amino acid replacing mutation (NCF4R58A), which is known to affect interaction with endosomal membranes, leading to decreased intracellular ROS production. To study the impact of NCF4 on T cell activation, we used the glucose phosphate isomerase peptide GPI325-339, which contains two cysteine residues (325-339c-c). Macrophages from mice with the NCF458A mutation efficiently presented the peptide when the two cysteines were intact and not crosslinked, leading to a strong arthritogenic T cell response. T cell priming occurred in the draining lymph nodes (LNs) within 8 days after immunization. Clodronate treatment, which depletes antigen-presenting mononuclear phagocytes, ameliorated arthritis severity, whereas treatment with FYT720, which traps activated T cells in LNs, prohibited arthritis. We conclude that NCF4-dependent intracellular ROS maintains cysteine peptides in an oxidized crosslinked state, which prevents presentation of peptides recognized by non-tolerized T cells and thereby protects against autoimmune arthritis.

Causal relationship between dementia and delirium: Insights from a bidirectional two-sample Mendelian randomization analysis.

BACKGROUND: Our previous study found dementia as a significant risk factor for delirium development in elderly patients with hip fracture. However, the causal relationship between dementia and delirium remains unclear. METHODS: To assess the causal relationship between delirium and dementia, we conducted a bidirectional Mendelian randomization (MR) analysis. Inversevariance weighted (IVW), weighted median, MR Egger, weighted mode, and simple mode were employed to conduct the MR analysis. Heterogeneity was assessed using the Cochran Q statistic in MR-Egger and IVW methods. Horizontal pleiotropy was examined via the MR pleiotropy residual sum and outliers (MR-PRESSO) and MR-Egger intercept tests. RESULTS: The forward MR analysis revealed a significant association between unclassified dementia (1.604 (1.326-1.941), p = 1.12 × 10-6), Alzheimer's disease (1.259 (1.128-1.405), p = 4.10 × 10-5), and dementia with Lewy bodies (1.121 (1.026-1.225), p = 0.011) with an increased risk of delirium. In the reverse MR analysis, delirium was also suggested to increase the risk of unclassified dementia (1.133 (1.066-1.204), p = 6.31 × 10-5) and vascular dementia (1.246 (1.075-1.444), p = 0.003). These significant results were further validated in the multivariable MR analysis. No evidence of heterogeneity or horizontal pleiotropy was observed (p > 0.05). LIMITATIONS: (1) Limited to European populations. (2) Sample population overlap between delirium and dementia. (3) Not all dementia subtypes were causally associated with delirium. CONCLUSIONS: This study provides genetic evidence supporting a causal relationship between dementia and delirium, indicating that dementia may influence the risk of delirium while delirium may also increase the risk of dementia.

Novel pyroptosis-related lncRNAs and ceRNAs predict osteosarcoma prognosis and indicate immune microenvironment signatures.

Objective: To study pyroptosis-related biomarkers that are associated with the prognosis and immune microenvironment characteristics of osteosarcoma (OS). The goal is to establish a foundation for the prognosis and treatment of OS. Methods: We retrieved transcriptome and clinical data from The Cancer Genome Atlas (TCGA) database for 88 OS patients. Using this data, we constructed a prognostic model to identify pyroptosis-related genes (PRGs) associated with OS prognosis. To further explore the biological function of these PRGs, we performed enrichment analysis. To identify pyroptosis-related long non-coding RNAs (PRLncs) associated with the prognosis of OS, we performed co-expression analysis. Subsequently, a risk prognostic model was constructed using these PRLncs to generate a risk score, termed as PRLncs-score, thereby obtaining PRLncs associated with the prognosis of OS. The accuracy of the prognostic model was verified through survival analysis, risk curve, independent prognostic analysis, receiver operating characteristic (ROC) curve, difference analysis between high- and low-risk groups, and clinical correlation analysis. And to determine whether PRLncs-score is independent prognostic factor for OS. In addition, we further conducted external and internal validation for the risk prognosis model. Further analyses of immune cell infiltration and tumor microenvironment were performed. A pyroptosis-related competitive endogenous RNA (PRceRNA) network was constructed to obtain PRceRNAs associated with the prognosis of OS and performed gene set enrichment analysis (GSEA) on PRceRNA genes. Results: We obtained five PRGs (CHMP4C, BAK1, GSDMA, CASP1, and CASP6) that predicted OS prognosis and seven PRLncs (AC090559.1, AP003119.2, CARD8-AS1, AL390728.4, SATB2-AS1, AL133215.2, and AC009495.3) and one PRceRNA (CARD8-AS1-hsa-miR-21-5p-IL1B) that predicted OS prognosis and indicated characteristics of the OS immune microenvironment. The PRLncs-score, in combination with other clinical features, was established as an independent prognostic factor for OS patients. Subsequent scrutiny of the tumor microenvironment and immune infiltration indicated that patients with low-PRLncs-scores were associated with reduced metastatic risk, improved survival rates, heightened levels of immune cells and stroma, and increased immune activity compared to those with high-PRLncs-scores. Conclusion: The study's findings offer insight into the prognosis of OS and its immune microenvironment, and hold promise for improving early diagnosis and immunotherapy.

The m6A/m1A/m5C-Related Methylation Modification Patterns and Immune Landscapes in Rheumatoid Arthritis and Osteoarthritis Revealed by Microarray and Single-Cell Transcriptome.

Purpose: The goal of this study was to explore the expression characteristics of RNA modification-related genes, reveal immune landscapes and identify novel potential diagnostic biomarkers in osteoarthritis (OA) and rheumatoid arthritis (RA) patients. Patients and Methods: RNA microarray and single-cell sequencing (scRNA-seq) data were downloaded from gene expression omnibus (GEO) database. Differentially expressed RNA modification-related genes were identified and then functionally annotated. Univariate logistic regression and lasso regression analysis were used to identify primary disease genes for OA and RA. Validation was done using scRNA-seq analysis and immunohistochemistry (IHC) in human knee synovial tissues and a murine destabilization of the medial meniscus (DMM) model. Through WGCNA analysis, genes associated with cell pyroptosis or autophagy in OA and RA were identified, which were then combined with differentially expressed RNA modification-related genes to construct a PPI interaction network. Furthermore, hub genes were selected for ceRNA interaction network analysis, correlation analysis with OA and RA molecular subtypes, as well as correlation analysis with 22 immune cells. Results: Six RNA modification-related genes (ADAMDEC1, IGHM, OGN, TNFRSF11B, SCARA3 and PTN) were identified as potential OA and RA pathogenesis biomarkers. Their expression was validated in human knee synovial tissues and a murine DMM model. Functional enrichment of differentially expressed RNA modification-related genes between RA and OA was analyzed using GO, KEGG, GSEA, and GSVA. Based on WGCNA and PPI analysis, the six hub genes related to pyroptosis and RNA modification (CXCL10, CXCL9, CCR7, CCL5, CXCL1, and CCR2) were identified as central nodes for ceRNA interaction, correlation with OA and RA molecular subtypes, and association with 22 immune cells. Conclusion: Our research revealed the significance of RNA modification-related genes in the development of OA and RA pathogenesis, thereby providing a novel research direction for understanding the mechanisms, diagnosis, and treatment of OA and RA.

Ferroptosis-related lncRNAs guiding osteosarcoma prognosis and immune microenvironment.

OBJECTIVE: To investigate the ferroptosis-related long non-coding RNAs (FRLncs) implicated in influencing the prognostic and immune microenvironment in osteosarcoma (OS), and to establish a foundational framework for informing clinical decision making pertaining to OS management. METHODS: Transcriptome data and clinical data pertaining to 86 cases of OS, the GSE19276, GSE16088 and GSE33382 datasets, and a list of ferroptosis-related genes (FRGs) were used to establish a risk prognostic model through comprehensive analysis. The identification of OS-related differentially expressed FRGs was achieved through an integrated analysis encompassing the aforementioned 86 OS transcriptome data and the GSE19276, GSE16088 and GSE33382 datasets. Concurrently, OS-related FRLncs were ascertained via co-expression analysis. To establish a risk prognostic model for OS, Univariate Cox regression analysis and Lasso Cox regression analysis were employed. Subsequently, a comprehensive evaluation was conducted, comprising risk curve analysis, survival analysis, receiver operating characteristic curve analysis and independent prognosis analysis. Model validation with distinct clinical subgroups was performed to assess the applicability of the risk prognostic model to diverse patient categories. Moreover, single sample gene set enrichment analysis (ssGSEA) was conducted to investigate variations in immune cell populations and immune functions within the context of the risk prognostic model. Furthermore, an analysis of immune checkpoint differentials yielded insights into immune checkpoint-related genes linked to OS prognosis. Finally, the risk prognosis model was verified by dividing the samples into train group and test group. RESULTS: We identified a set of seven FRLncs that exhibit potential as prognostic markers and influence factors of the immune microenvironment in the context of OS. This ensemble encompasses three high-risk FRLncs, denoted as APTR, AC105914.2 and AL139246.5, alongside four low-risk FRLncs, designated as DSCR8, LOH12CR2, AC027307.2 and AC025048.2. Furthermore, our analysis revealed notable down-regulation in the high-risk group across four distinct immune cell types, namely neutrophils, natural killer cells, plasmacytoid dendritic cells and tumor-infiltrating lymphocytes. This down-regulation was also reflected in four key immune functions, antigen-presenting cell (APC)-co-stimulation, checkpoint, cytolytic activity and T cell co-inhibition. Additionally, we identified seven immune checkpoint-associated genes with significant implications for OS prognosis, including CD200R1, HAVCR2, LGALS9, CD27, LAIR1, LAG3 and TNFSF4. CONCLUSION: The findings of this study have identified FRLncs capable of influencing OS prognosis and immune microenvironment, as well as immune checkpoint-related genes that are linked to OS prognosis. These discoveries establish a substantive foundation for further investigations into OS survival and offer valuable insights for informing clinical decision making in this context.

Identification of the potential regulatory interactions in rheumatoid arthritis through a comprehensive analysis of lncRNA-related ceRNA networks.

OBJECTIVE: This study aimed at constructing a network of competing endogenous RNA (ceRNA) in the synovial tissues of rheumatoid arthritis (RA). It seeks to discern potential biomarkers and explore the long non-coding RNA (lncRNA)-microRNA (miRNA)-messenger RNA (mRNA) axes that are intricately linked to the pathophysiological mechanisms underpinning RA, and providing a scientific basis for the pathogenesis and treatment of RA. METHODS: Microarray data pertaining to RA synovial tissue, GSE103578, GSE128813, and GSE83147, were acquired from the Gene Expression Omnibus (GEO) database ( http://www.ncbi.nlm.nih.gov/geo ). Conducted to discern both differentially expressed lncRNAs (DELncRNAs) and differentially expressed genes (DEGs). A ceRNA network was obtained through key lncRNAs, key miRNAs, and key genes. Further investigations involved co-expression analyses to uncover the lncRNA-miRNA-mRNA axes contributing to the pathogenesis of RA. To delineate the immune-relevant facets of this axis, we conducted an assessment of key genes, emphasizing those with the most substantial immunological correlations, employing the GeneCards database. Finally, gene set enrichment analysis (GSEA) was executed on the identified key lncRNAs to elucidate their functional implications in RA. RESULTS: The 2 key lncRNAs, 7 key miRNAs and 6 key genes related to the pathogenesis of RA were obtained, as well as 2 key lncRNA-miRNA-mRNA axes (KRTAP5-AS1-hsa-miR-30b-5p-PNN, XIST-hsa-miR-511-3p/hsa-miR-1277-5p-F2RL1). GSEA of two key lncRNAs obtained biological processes and signaling pathways related to RA synovial lesions. CONCLUSION: The findings of this investigation hold promise in furnishing a foundational framework and guiding future research endeavors aimed at comprehending the etiology and therapeutic interventions for RA.

Successful Transfection of MicroRNA Mimics or Inhibitors in a Regular Cell Line and in Primary Cells Derived from Patients with Rheumatoid Arthritis.

The transfection of microRNA (miRNA) mimics and inhibitors can lead to the gain and loss of intracellular miRNA function, helping us better understand the role of miRNA during gene expression regulation under specific physical conditions. Our previous research has confirmed the efficiency and convenience of using liposomes to transfect miRNA mimics or inhibitors. This work uses miR-424 as an example, to provide a detailed introduction for the transfection process of miRNA mimics and inhibitors in the regular SW982 cell line and primary rheumatoid arthritis synovial fibroblasts (RASF) cells from patients by using lipofection, which can also serve as a reference to miRNA transfection in other cell lines. Key features • MiRNA mimics and inhibitors transfection in regular SW982 cell line and primary RASF cells. • Treatment and culture of RASF primary cells before transfection. • Using liposomes for transfection purposes.

Total hip arthroplasty for posttraumatic osteoarthritis secondary to acetabular fracture: An evidence based on 1,284 patients from 1970 to 2018.

Background: Posttraumatic osteoarthritis (PTOA) can be a crippling sequela of acetabular fracture (AF), and total hip arthroplasty (THA) is often necessary to alleviate the clinical progression of symptoms. The purpose of this study was to summarize the existing clinical evidence concerning the surgical management of AF with THA through meta-analyses. Methods: Databases were searched for articles published between 1995 and January 2022 that contained the keywords "acetabular," "fracture," "arthroplasty," and "osteoarthritis." Our study was registered in PROSPERO under number CRD42022314997. Results: We screened 3,125 studies and included data from 31 studies with 1,284 patients. The median patient age at the time of THA was 52 years and ranged from 19 to 94 years. The pooled overall survival rate was 88% [86%-90%, 95% confidence interval (CI)] and could reach 83% at ≥15-year follow-up. For the Harris Hip Score, we pooled 22 studies with an overall mean difference of 43.25 (40.40-46.10, 95% CI; P < 0.001), indicating a large clinical effect. The pooled complications (incidence rates) across studies were: heterotopic ossification (22.53%), implant dislocation (4.66%), implant infection (3.44%), and iatrogenic nerve injury (1.07%). Conclusion: THA in patients with PTOA following AF leads to significant improvement in symptoms and function at ≥15-year follow-up. Survival rates of implants free from re-operation or revision after THA decreased with follow-up time and could still reach 83% at ≥15-year follow-up. THA might be an effective therapeutic method for patients with PTOA due to AF.

A novel signature to guide osteosarcoma prognosis and immune microenvironment: Cuproptosis-related lncRNA.

Objective: Osteosarcoma (OS) is a common bone malignancy with poor prognosis. We aimed to investigate the relationship between cuproptosis-related lncRNAs (CRLncs) and the survival outcomes of patients with OS. Methods: Transcriptome and clinical data of 86 patients with OS were downloaded from The Cancer Genome Atlas (TCGA). The GSE16088 dataset was downloaded from the Gene Expression Omnibus (GEO) database. The 10 cuproptosis-related genes (CRGs) were obtained from a recently published article on cuproptosis in Science. Combined analysis of OS transcriptome data and the GSE16088 dataset identified differentially expressed CRGs related to OS. Next, pathway enrichment analysis was performed. Co-expression analysis obtained CRLncs related to OS. Univariate COX regression analysis and least absolute shrinkage and selection operator (LASSO) regression analysis were used to construct the risk prognostic model of CRLncs. The samples were divided evenly into training and test groups to verify the accuracy of the model. Risk curve, survival, receiver operating characteristic (ROC) curve, and independent prognostic analyses were performed. Next, principal component analysis (PCA) and t-distributed stochastic neighbor embedding (t-SNE) analysis were performed. Single-sample gene set enrichment analysis (ssGSEA) was used to explore the correlation between the risk prognostic models and OS immune microenvironment. Drug sensitivity analysis identified drugs with potential efficacy in OS. Real-time quantitative PCR, Western blotting, and immunohistochemistry analyses verified the expression of CRGs in OS. Real-time quantitative PCR was used to verify the expression of CRLncs in OS. Results: Six CRLncs that can guide OS prognosis and immune microenvironment were obtained, including three high-risk CRLncs (AL645608.6, AL591767.1, and UNC5B-AS1) and three low-risk CRLncs (CARD8-AS1, AC098487.1, and AC005041.3). Immune cells such as B cells, macrophages, T-helper type 2 (Th2) cells, regulatory T cells (Treg), and immune functions such as APC co-inhibition, checkpoint, and T-cell co-inhibition were significantly downregulated in high-risk groups. In addition, we obtained four drugs with potential efficacy for OS: AUY922, bortezomib, lenalidomide, and Z.LLNle.CHO. The expression of LIPT1, DLAT, and FDX1 at both mRNA and protein levels was significantly elevated in OS cell lines compared with normal osteoblast hFOB1.19. The mRNA expression level of AL591767.1 was decreased in OS, and that of AL645608.6, CARD8-AS1, AC005041.3, AC098487.1, and UNC5B-AS1 was upregulated in OS. Conclusion: CRLncs that can guide OS prognosis and the immune microenvironment and drugs that may have a potential curative effect on OS obtained in this study provide a theoretical basis for OS survival research and clinical decision-making.

Development and Validation of a Nomogram for Predicting Postoperative Delirium in Patients With Elderly Hip Fracture Based on Data Collected on Admission.

Delirium is a common postoperative complication in elderly hip fracture patients that seriously affects patients' lives and health, and early delirium risk prediction, and targeted measures can significantly reduce the incidence of delirium. The purpose of this study was to develop and validate a nomogram for the prediction of postoperative delirium (POD) in elderly hip fracture patients. A total of 328 elderly patients with hip fractures enrolled retrospectively in department 1 of our hospital were randomly divided into the training set (n = 230) and the internal validation set (n = 98). The least absolute shrinkage and selection operator (LASSO) regression analysis was used for feature variable selection, and multivariate logistic regression with a backward stepwise method was used to construct a nomogram in the training set. The discrimination efficacy and calibration efficacy of the nomogram were evaluated through the receiver operating characteristic (ROC) curve and calibration curve, respectively. The clinical usefulness was estimated through decision curve analysis (DCA) and clinical impact curve (CIC) analysis. Another validation set from department 2 of our hospital, containing 76 elderly patients with hip fractures, was used for external validation of the nomogram. A total of 43 (13.1%) and 12 (15.8%) patients had POD in department 1 and department 2, respectively. The nomogram was constructed by three predictors, including dementia, chronic obstructive pulmonary disease (COPD), and albumin level. The nomogram showed good discrimination efficacy and calibration efficacy, with the AUC of 0.791 (95% CI, 0.708-0.873), 0.820 (95% CI, 0.676-0.964), and 0.841 (95% CI, 0.717-0.966) in the training set, the internal validation set, and the external validation set, respectively. Both DCA and CIC demonstrated that this nomogram has good clinical usefulness. The nomogram constructed by dementia, COPD, and albumin level can be conveniently used to predict POD in patients with elderly hip fractures.

Metformin Ameliorates Senescence of Adipose-Derived Mesenchymal Stem Cells and Attenuates Osteoarthritis Progression via the AMPK-Dependent Autophagy Pathway.

Osteoarthritis (OA) is one of the most serious age-related diseases worldwide that drastically affects the quality of life of patients. Despite advancements in the treatment of arthritis, especially with adipose-derived mesenchymal stem cells (ADSCs), senescence-induced alterations in ADSCs negatively affect the treatment outcomes. This study was aimed at mechanistically exploring whether metformin could ameliorate the senescence of ADSCs and at exploring the effect of metformin-preconditioned ADSCs in an experimental OA mouse model. In this study, an H2O2-induced mouse ADSC senescent model was established. Cell proliferation, senescence, and autophagy were investigated in vitro. Moreover, the effects of intra-articular injection of metformin-preconditioned ADSCs were investigated in vivo. Metformin could promote autophagy and activate the AMPK/mTOR pathway in ADSCs. The metformin-enhanced autophagy could improve the survival and reduce the senescence of ADSCs. The protective effects of metformin against senescence were partially blocked by 3-methyladenine and compound C. Injection of metformin-preconditioned ADSCs slowed OA progression and reduced OA pain in mice. The results suggest that metformin activates the AMPK/mTOR-dependent autophagy pathway in ADSCs against H2O2-induced senescence, while metformin-preconditioned ADSCs can potentially inhibit OA progression.

Bioinformatics Analysis Identified the Hub Genes, mRNA-miRNA-lncRNA Axis, and Signaling Pathways Involved in Rheumatoid Arthritis Pathogenesis.

Objective: Rheumatoid arthritis (RA) is a nonspecific, chronic, systemic autoimmune disease characterized by symmetric polyarticular synovitis. Bioinformatics analysis of potential biomarkers, mRNA-miRNA-lncRNA axes, and signaling pathways in the pathogenesis of RA provides potential targets and theoretical basis for further research on RA. Methods: The GSE1919 and GSE77298 datasets were downloaded from the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo). Perl was used to perform data merging, and R was used to perform batch correction. The "limma" package of R was used to screen differentially expressed genes, and the "clusterProfiler" package was used to perform enrichment analysis of the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes. Search Tool for the Retrieval of Interacting Genes/Proteins was used to construct the protein-protein interaction network, Cytoscape was used for module analysis, and R was used to screen for hub genes. GraphPad Prism was used to plot the receiver operating characteristic curve of the hub genes. Gene set enrichment analysis and competitive endogenous RNA network analysis were performed on hub genes with the greatest diagnostic values. The hub gene with the greatest diagnostic value was verified using immunohistochemical staining. Results: We obtained nine hub genes (ITGB2, VAMP8, HLA-A, PTAFR, SYK, FCER1G, HLA-DPB1, LCP2, and ACTR2) and four mRNA-miRNA-lncRNA axes (ITGB2-hsa-miR-486-3p-SNHG3, ITGB2-hsa-miR-338-5p-XIST, ITGB2-hsa-miR-5581-3p-XIST, and ITGB2-hsa-miR-1226-5p-XIST) related to the pathogenesis of RA. The nine hub genes were highly expressed, and ITGB2 had the highest diagnostic value for RA. We also identified signaling pathways related to the pathogenesis of RA: Fc epsilon Rl and chemokine signaling pathways. The immunohistochemical results showed that ITGB2 expression was significantly upregulated in RA. Conclusion: The hub genes, mRNA-miRNA-lncRNA axes, and signaling pathways related to RA pathogenesis identified in this study provide a new research direction for the mechanism, diagnosis, and treatment of RA.

Targeting intrinsically disordered regions facilitates discovery of calcium channels 3.2 inhibitory peptides for adeno-associated virus-mediated peripheral analgesia.

ABSTRACT: Ample data support a prominent role of peripheral T-type calcium channels 3.2 (Ca V 3.2) in generating pain states. Development of primary sensory neuron-specific inhibitors of Ca V 3.2 channels is an opportunity for achieving effective analgesic therapeutics, but success has been elusive. Small peptides, especially those derived from natural proteins as inhibitory peptide aptamers (iPAs), can produce highly effective and selective blockade of specific nociceptive molecular pathways to reduce pain with minimal off-target effects. In this study, we report the engineering of the potent and selective iPAs of Ca V 3.2 from the intrinsically disordered regions (IDRs) of Ca V 3.2 intracellular segments. Using established prediction algorithms, we localized the IDRs in Ca V 3.2 protein and identified several Ca V 3.2iPA candidates that significantly reduced Ca V 3.2 current in HEK293 cells stably expressing human wide-type Ca V 3.2. Two prototype Ca V 3.2iPAs (iPA1 and iPA2) derived from the IDRs of Ca V 3.2 intracellular loops 2 and 3, respectively, were expressed selectively in the primary sensory neurons of dorsal root ganglia in vivo using recombinant adeno-associated virus (AAV), which produced sustained inhibition of calcium current conducted by Ca V 3.2/T-type channels and significantly attenuated both evoked and spontaneous pain behavior in rats with neuropathic pain after tibial nerve injury. Recordings from dissociated sensory neurons showed that AAV-mediated Ca V 3.2iPA expression suppressed neuronal excitability, suggesting that Ca V 3.2iPA treatment attenuated pain by reversal of injury-induced neuronal hypersensitivity. Collectively, our results indicate that Ca V 3.2iPAs are promising analgesic leads that, combined with AAV-mediated delivery in anatomically targeted sensory ganglia, have the potential to be a selective peripheral Ca V 3.2-targeting strategy for clinical treatment of pain.

Peroxiredoxin 4 regulates tumor-cell-like characteristics of fibroblast-like synoviocytes in rheumatoid arthritis through PI3k/Akt signaling pathway.

Peroxiredoxin-4 (PRDX4), a member of PRDX family, which played an important role in scavenging reactive oxygen species (ROS). The up-regulation of PRDX4 in synovial tissue and synovial fluid from rheumatoid arthritis (RA) patients has been reported. However, the biological functions of PRDX4 in fibroblast-like synoviocytes (RA-FLS) remains unclear. In this research, we reveal that expression of PRDX4 was notably increased in RA synovial tissue, especially in hyperplastic synovial tissue. PRDX4 silencing significantly inhibited the tumor cell-like behaviors and mRNA expression of matrix metalloproteinases (MMPs) in RA-FLS. Furthermore, overexpression of PRDX4 markedly activated PI3K/Akt signaling pathway, which can be reverted by Akt inhibitor MK-2206. These observations identified elevated PRDX4 may regulates the tumor cell-like biological characteristic of RA-FLS via Pi3k/Akt pathway. Targeting PRDX4 and its downstream signaling pathway might provide a potential diagnostic markers and therapeutic target for RA.

Quantitative Proteomic Analysis of Synovial Tissue Reveals That Upregulated OLFM4 Aggravates Inflammation in Rheumatoid Arthritis.

Tandem mass tag (TMT)-coupled liquid chromatography coupled with tandem mass spectrometry is a powerful method to investigate synovial tissue protein profiles in patients with rheumatoid arthritis (RA) and osteoarthritis (OA). Protein was isolated from synovial tissue samples of 22 patients and labeled with a TMT kit. Over 500 proteins were identified as the differential expression protein on comparing RA and OA synovial tissue, including 239 upregulated and 271 downregulated proteins. Data are available via ProteomeXchange with identifier PXD027703. Gene ontology and Kyoto Encyclopedia of Genes and Genomes analysis showed that the majority participated in the developmental processes and protein processing in the endoplasmic reticulum. Olfactomedin 4 (OLFM4), a secreted glycoprotein, in joint inflammation of RA was explored. OLFM4 was upregulated in RA synovial tissue samples. In fibroblast-like synoviocytes (FLS), inflammation cytokines, TNF-α, interleukin (IL)-1ß, and LPS can upregulate OLFM4. After OLFM4 knockdown under TNF-α stimulation, RA FLS proliferation was inhibited and the expression of CXCL9, CXCL11, and MMP-1 was decreased. Overall, the RA synovial tissue protein expression profile by proteomic analysis shows some unique targets in RA pathophysiology, and OLFM4 in FLS plays an important role in RA joint inflammation. OLFM4 can be a promising therapeutic target in RA synovial tissue.

MicroRNA-497 Reduction and Increase of Its Family Member MicroRNA-424 Lead to Dysregulation of Multiple Inflammation Related Genes in Synovial Fibroblasts With Rheumatoid Arthritis.

Objectives: Mounting evidence has demonstrated that microRNAs (miRNAs) participate in rheumatoid arthritis (RA). The role of highly conserved miR-15/107 family in RA has not been clarified yet, and hence investigated in this study. Methods: Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to evaluate the expression of miRNAs and genes. Cell counting kit 8 (CCK-8) and FACS were used to detect proliferation and apoptosis. Protein expression was detected by using Western blotting. mRNA deep sequencing and cytokine antibody array were used to analyze differentially expressed genes, signaling pathways and cytokines. Results: The expression of miR-15a, miR-103, miR-497, and miR-646 was found decreased, while miR-424 increased in RA patients. MiR-424 and miR-497 were further investigated and the results showed that they could regulate the expression of multiple genes in rheumatoid arthritis synovial fibroblast (RASF) and affect signaling pathways. At the protein level, miR-497 mimic altered all the selected inflammation-related genes while miR-424 inhibitor only affected part of genes. MiR-497 mimic, rather than miR-424 inhibitor, had significant effects on proliferation and apoptosis of RASF. DICER1 was found to positively regulate the expression of miR-424 and miR-497, while DICER1 was also negatively regulated by miR-424. The increase of miR-424 could reduce miR-497 expression, thus forming a loop, which facilitated explaining the dysregulated miR-424 and miR-497 in RA. Conclusion: The miR-424 and miR-497 of miR-15/107 family affect cell proliferation and apoptosis in RA, and the proposed miR-424-DICER1-miR-497 feedback loop provides a novel insight into regulating miRNA expression and a candidate target for controlling RA.

Derlin-1, as a Potential Early Predictive Biomarker for Nonresponse to Infliximab Treatment in Rheumatoid Arthritis, Is Related to Autophagy.

Background: The goal of this study was to identify potential predictive biomarkers for the therapeutic effect of infliximab (IFX) in Rheumatoid arthritis (RA) and explore the potential molecular mechanism of nonresponse to IFX treatment to achieve individualized treatment of RA. Methods: Differential gene expression between IFX responders and nonresponders in the GSE58795 and GSE78068 datasets was identified. Coexpression analysis was used to identify the modules associated with nonresponse to IFX therapy for RA, and enrichment analysis was conducted on module genes. Least absolute shrink and selection operator (LASSO) regression was used to develop a gene signature for predicting the therapeutic effect of IFX in RA, and the area under the receiver operating characteristic curve (AUC) was used to evaluate the predictive value of the signature. Correlation analysis and single-sample gene set enrichment analysis (ssGSEA) were used to explore the potential role of the hub genes. Experimental validation was conducted in synovial tissue and RA fibroblast-like synoviocytes (RA-FLSs). Results: A total of 46 common genes were obtained among the two datasets. The yellow-green module was identified as the key module associated with nonresponse to IFX therapy for RA. We identified a 25-gene signature in GSE78068, and the AUC for the signature was 0.831 in the internal validation set and 0.924 in the GSE58795 dataset(external validation set). Derlin-1 (DERL1) was identified as the hub gene and demonstrated to be involved in the immune response and autophagy regulation. DERL1 expression was increased in RA synovial tissue compared with OA synovial tissue, and DERL1-siRNA partially inhibited autophagosome formation in RA-FLSs. Conclusion: The 25-gene signature may have potential predictive value for the therapeutic effect of IFX in RA at the beginning of IFX treatment, and autophagy may be involved in nonresponse to IFX treatment. In particular, DERL1 may be associated with the regulation of autophagy.

Enhanced T-type calcium channel 3.2 activity in sensory neurons contributes to neuropathic-like pain of monosodium iodoacetate-induced knee osteoarthritis.

The monosodium iodoacetate knee osteoarthritis model has been widely used for the evaluation of osteoarthritis pain, but the pathogenesis of associated chronic pain is not fully understood. The T-type calcium channel 3.2 (CaV3.2) is abundantly expressed in the primary sensory neurons, in which it regulates neuronal excitability at both the somata and peripheral terminals and facilitates spontaneous neurotransmitter release at the spinal terminals. In this study, we investigated the involvement of primary sensory neuron-CaV3.2 activation in monosodium iodoacetate osteoarthritis pain. Knee joint osteoarthritis pain was induced by intra-articular injection of monosodium iodoacetate (2 mg) in rats, and sensory behavior was evaluated for 35 days. At that time, knee joint structural histology, primary sensory neuron injury, and inflammatory gliosis in lumbar dorsal root ganglia, and spinal dorsal horn were examined. Primary sensory neuron-T-type calcium channel current by patch-clamp recording and CaV3.2 expression by immunohistochemistry and immunoblots were determined. In a subset of animals, pain relief by CaV3.2 inhibition after delivery of CaV3.2 inhibitor TTA-P2 into sciatic nerve was investigated. Knee injection of monosodium iodoacetate resulted in osteoarthritis histopathology, weight-bearing asymmetry, sensory hypersensitivity of the ipsilateral hindpaw, and inflammatory gliosis in the ipsilateral dorsal root ganglia, sciatic nerve, and spinal dorsal horn. Neuronal injury marker ATF-3 was extensively upregulated in primary sensory neurons, suggesting that neuronal damage was beyond merely knee-innervating primary sensory neurons. T-type current in dissociated primary sensory neurons from lumbar dorsal root ganglia of monosodium iodoacetate rats was significantly increased, and CaV3.2 protein levels in the dorsal root ganglia and spinal dorsal horn ipsilateral to monosodium iodoacetate by immunoblots were significantly increased, compared to controls. Perineural application of TTA-P2 into the ipsilateral sciatic nerve alleviated mechanical hypersensitivity and weight-bearing asymmetry in monosodium iodoacetate osteoarthritis rats. Overall, our findings demonstrate an elevated CaV3.2 expression and enhanced function of primary sensory neuron-T channels in the monosodium iodoacetate osteoarthritis pain. Further study is needed to delineate the importance of dysfunctional primary sensory neuron-CaV3.2 in osteoarthritis pain.

Clinical markers combined with HMGB1 polymorphisms to predict efficacy of conventional DMARDs in rheumatoid arthritis patients.

The efficacy of conventional synthetic disease-modifying antirheumatic drugs (csDMARDs) for rheumatoid arthritis (RA) patients was limited. However, there were no predictive markers for poor csDMARDs outcome. Clinical information of RA patients was collected and the high-mobility group box 1 (HMGB1) polymorphisms (rs4145277, rs2249825, rs1412125 and rs1045411) were examined. Among the 252 patients, 31.0% had no response of csDMARDs. Anti-citrullinated protein antibody (ACPA)-positive, C-reactive protein (CRP) and Disease Activity Score (DAS) 28- erythrocyte sedimentation rate (ESR) were the associated factors, which (DAC:DAS 28 > 4.7 and ACPA-positive and CRP > 7.1 mg/L) was used to predict poor csDMARDs outcome, the sensitivity and specificity was 87.2% and 60.9%, respectively. Among those DAC patients, the refractory RA rate in the rs2249825 GG genotype patients was 83.3%, the specificity was 98.5%. The clinical markers (DAC) and rs2249825 GG genotype can be used to predict poor csDMARDs outcome.