Integrative single-cell RNA and ATAC sequencing reveals that the FOXO1-PRDX2-TNF axis regulates tendinopathy.

Introduction: Tendinopathy, the most common form of chronic tendon disorder, leads to persistent tendon pain and loss of function. Profiling the heterogeneous cellular composition in the tendon microenvironment helps to elucidate rational molecular mechanisms of tendinopathy. Methods and results: In this study, through a multi-modal analysis, a single-cell RNA- and ATAC-seq integrated tendinopathy landscape was generated for the first time. We found that a specific cell subpopulation with low PRDX2 expression exhibited a higher level of inflammation, lower proliferation and migration ability, which not only promoted tendon injury but also led to microenvironment deterioration. Mechanistically, a motif enrichment analysis of chromatin accessibility showed that FOXO1 was an upstream regulator of PRDX2 transcription, and we confirmed that functional blockade of FOXO1 activity induced PRDX2 silencing. The TNF signaling pathway was significantly activated in the PRDX2-low group, and TNF inhibition effectively restored diseased cell degradation. Discussion: We revealed an essential role of diseased cells in tendinopathy and proposed the FOXO1-PRDX2-TNF axis is a potential regulatory mechanism for the treatment of tendinopathy.

N-Acetyl-L-cysteine facilitates tendon repair and promotes the tenogenic differentiation of tendon stem/progenitor cells by enhancing the integrin α5/ß1/PI3K/AKT signaling.

BACKGROUND: Tendon injury is associated with oxidative stress, leading to reactive oxygen species (ROS) production and inflammation. N-acetyl-L-cysteine (NAC) is a potent antioxidant. However, how NAC affects the biological functions of tendon stem/progenitor cells (TSPCs) and tendon repair has not been clarified.  METHOD: The impacts of NAC on the viability, ROS production, and differentiation of TSPCs were determined with the cell counting kit-8, fluorescence staining, Western blotting, and immunofluorescence. The effect of NAC on gene transcription in TSPCs was analyzed by transcriptomes and bioinformatics and validated by Western blotting. The potential therapeutic effect of NAC on tendon repair was tested in a rat model of Achilles tendon injury. RESULTS: Compared with the untreated control, treatment with 500 µM NAC greatly promoted the proliferation of TSPCs and significantly mitigated hydrogen peroxide-induced ROS production and cytotoxicity in vitro. NAC treatment significantly increased the relative protein expression of collagen type 1 alpha 1 (COL1A1), tenascin C (TNC), scleraxis (SCX), and tenomodulin (TNMD) in TPSCs. Bioinformatics analyses revealed that NAC modulated transcriptomes, particularly in the integrin-related phosphoinositide 3-kinase (PI3K)/AKT signaling, and Western blotting revealed that NAC enhanced integrin α5ß1 expression and PI3K/AKT activation in TSPCs. Finally, NAC treatment mitigated the tendon injury, but enhanced the protein expression of SCX, TNC, TNMD, and COLIA1 in the injured tissue regions of the rats. CONCLUSION: NAC treatment promoted the survival and differentiation of TSPCs to facilitate tendon repair after tendon injury in rats. Thus, NAC may be valuable for the treatment of tendon injury.

Micropattern Silk Fibroin Film Facilitates Tendon Repair In Vivo and Promotes Tenogenic Differentiation of Tendon Stem/Progenitor Cells through the α2ß1/FAK/PI3K/AKT Signaling Pathway In Vitro.

Background: Tendon injuries are common clinical disorders. Due to the limited regeneration ability of tendons, tissue engineering technology is often used as an adjuvant treatment. This study explored the molecular pathways underlying micropattern SF film-regulated TSPC propensity and their repairing effects to highlight the application value of micropattern SF films. Methods: First, we characterized the physical properties of the micropattern SF films and explored their repairing effects on the injured tendons in vivo. Then, we seeded TSPCs on SF films in vitro and determined the micropattern SF film-induced gene expression and activation of signaling pathways in TSPCs through high-throughput RNA sequencing and proteomics assays. Results: The results of in vivo studies suggested that micropattern SF films can promote remodeling of the injured tendon. In addition, immunohistochemistry (IHC) results showed that tendon marker genes were significantly increased in the micropattern SF film repair group. Transcriptomic and proteomic analyses demonstrated that micropattern SF film-induced genes and proteins in TSPCs were mainly enriched in the focal adhesion kinase (FAK)/actin and phosphoinositide 3-kinase (PI3K)/AKT pathways. Western blot analysis showed that the expression of integrins α2ß1, tenascin-C (TNC), and tenomodulin (TNMD) and the phosphorylation of AKT were significantly increased in the micropattern SF film group, which could be abrogated by applying PI3K/AKT inhibitors. Conclusion: Micropattern SF films modified by water annealing can promote remodeling of the injured tendon in vivo and regulate the tendon differentiation of TSPCs through the α2ß1/FAK/PI3K/AKT signaling pathway in vitro. Therefore, they have great medical value in tendon repair.

Single-cell analysis reveals that Jinwu Gutong capsule attenuates the inflammatory activity of synovial cells in osteoarthritis by inhibiting AKR1C3.

Jinwu Gutong capsule (JGC) is a traditional Chinese medicine formula for the treatment of osteoarthritis (OA). Synovitis is a typical pathological change in OA and promotes disease progression. Elucidating the therapeutic mechanism of JGC is crucial for the precise treatment of OA synovitis. In this study, we demonstrate that JGC effectively inhibits hyperproliferation, attenuates inflammation, and promotes apoptosis of synovial cells. Through scRNA-seq data analysis of OA synovitis, we dissected two distinct cell fates that influence disease progression (one fate led to recovery while the other fate resulted in deterioration), which illustrates the principles of fate determination. By intersecting JGC targets with synovitis hub genes and then mimicking picomolar affinity interactions between bioactive compounds and binding pockets, we found that the quercetin-AKR1C3 pair exhibited the best affinity, indicating that this pair constitutes the most promising molecular mechanism. In vitro experiments confirmed that the expression of AKR1C3 in synovial cells was reduced after JGC addition. Further overexpression of AKR1C3 significantly attenuated the therapeutic efficacy of JGC. Thus, we revealed that JGC effectively treats OA synovitis by inhibiting AKR1C3 expression.

Effect of Pore Size of Porous-Structured Titanium Implants on Tendon Ingrowth.

Purpose: The reconstruction of a tendon insertion on metal prostheses is a challenge in orthopedics. Of the available metal prostheses, porous metal prostheses have been shown to have better biocompatibility for tissue integration. Therefore, this study is aimed at identifying an appropriate porous structure for the reconstruction of a tendon insertion on metal prostheses. Methods: Ti6Al4V specimens with a diamond-like porous structure with triply periodic minimal surface pore sizes of 300, 500, and 700 µm and a porosity of 58% (designated Ti300, Ti500, and Ti700, respectively) were manufactured by selective laser melting and were characterized with micro-CT and scanning electron microscopy for their porosity, pore size, and surface topography. The porous specimens were implanted into the patellar tendon of rabbits. Tendon integration was evaluated after implantation into the tendon at 4, 8, and 12 weeks by histology, and the fixation strength was evaluated with a pull-out test at week 12. Results: The average pore sizes of the Ti300, Ti500, and Ti700 implants were 261, 480, and 668 µm, respectively. The Ti500 and Ti700 implants demonstrated better tissue growth than the Ti300 implant at weeks 4, 8, and 12. At week 12, the histological score of the Ti500 implant was 13.67 ± 0.58, and it had an area percentage of type I collagen of 63.90% ± 3.41%; both of these results were significantly higher than those for the Ti300 and Ti700 implants. The pull-out load at week 12 was also the highest in the Ti500 group. Conclusion: Ti6Al4V implants with a diamond-like porous structure with triply periodic minimal surface pore size of 500 µm are suitable for tendon integration.

The effect of acupuncture on the expression of inflammatory factors TNF-α, IL-6,IL-1 and CRP in cerebral infarction: A protocol of systematic review and meta-analysis.

BACKGROUND: The mechanisms of acupuncture on the treatment of cerebral infarction remain unclear, the aim of the present study was to provides a protocol of systematic review and meta-analysis, with which we will collect clinical evidence to verify whether acupuncture will have an effect on reducing the levels of tumor necrosis factor α (TNF-α), C-reactive protein (CRP), interleukin-1 (IL-1), and interleukin (IL-6) after cerebral infarction based on evidence-based studies. METHODS: Included studies will be retrieved according to inclusion and exclusion criteria from 5 English databases (the MEDLINE via PubMed, the Cochrane Library, Embase, the Web of Science, and Ovid database), and 4 Chinese databases (China Science and Technology Journal Database (VIP), Chinese Biomedical Literature Database (CBM), Wan-fang Database, China National Knowledge Infrastructure (CNKI)) from October 1990 to October 2017. The inflammatory factor levels of TNF-α and IL-1,IL-6,CRP will be marked as major outcomes. We will use RevMan V.5.3 software to calculate the data synthesis and will conduct meta-analysis based on the collected data. RESULTS: The inflammatory factor levels of TNF-α and IL-1,IL-6,CRP, mortality and adverse effects will be measured and comprehensively assessed to evaluate the adjunctive effect of XBP on CHF from this systematic review and meta-analysis with current clinical evidence. CONCLUSION: The systematic review and meta-analysis will assess the effect of acupuncture on the expression of inflammatory factors TNF-α, IL-6, IL-1 and CRP in cerebral infarction with up-to-date clinical evidence. PROSPERO REGISTRATION NUMBER: PROSPERO CRD42017078583.