A static magnetic field enhances the repair of osteoarthritic cartilage by promoting the migration of stem cells and chondrogenesis.

Objective: To investigate the therapeutic effects of static magnetic field (SMF) and its regulatory mechanism in the repair of osteoarthritic cartilage. Methods: Fourteen-week-old female C57BL/6 mice were randomly divided into the sham operation group and the osteoarthritis (OA) groups with and without SMF application. SMF was applied at 200 â€‹mT for two consecutive weeks. Changes in knee cartilage were examined by histomorphometry, and the chondrogenesis and migration of endogenous stem cells were assessed. The expression of SRY-related protein 9 (SOX9), Collagen type II (COL2), matrix metallopeptidase 13 (MMP13), stromal cell-derived factor 1/C-X-C chemokine receptor type 4 (SDF-1/CXCR4), Piezo1 and other genes was evaluated, and the mechanism of SMF's action was tested using the CXCR4 inhibitor, AMD3100, and Piezo1 siRNA. Results: SMF significantly decreased the OARSI scores after induction of OA. SMF was beneficial to chondrogenesis by elevating SOX9. In the OA mouse model, an increase in MMP13 with a decrease in COL2 led to the destruction of the cartilage extracellular matrix, which was suppressed by SMF. SMF promoted the migration of cartilage-derived stem/progenitor cells and bone marrow-derived mesenchymal stem cells (MSCs). It increased SDF-1 and CXCR4, while the CXCR4 inhibitor significantly suppressed the beneficial effects of SMF. The application of Piezo1 siRNA inhibited the SMF-induced increase of CXCR4. Conclusion: SMF enhanced chondrogenesis and improved cartilage extracellular matrices. It activated the Piezo1-mediated SDF-1/CXCR4 regulatory axis and promoted the migration of endogenous stem cells. Collectively, it attenuated the pathological progression of cartilage destruction in OA mice. The Translational potential of this article: The findings in this study provided convincing evidence that SMF could enhance cartilage repair and improve OA symptoms, suggesting that SMF could have clinical value in the treatment of OA.

METTL3 promotes SMSCs chondrogenic differentiation by targeting the MMP3, MMP13, and GATA3.

Objective: Synovium-derived mesenchymal stem cells (SMSCs) are multipotential non-hematopoietic progenitor cells that can differentiate into various mesenchymal lineages in adipose and bone tissue, especially in chondrogenesis. Post-transcriptional methylation modifications are relative to the various biological development procedures. N6-methyladenosine (m6A) methylation has been identified as one of the abundant widespread post-transcriptional modifications. However, the connection between the SMSCs differentiation and m6A methylation remains unknown and needs further exploration. Methods: SMSCs were derived from synovial tissues of the knee joint of male Sprague-Dawley (SD) rats. In the chondrogenesis of SMSCs, m6A regulators were detected by quantitative real-time PCR (RT-PCR) and Western blot (WB). We observed the situation that the knockdown of m6A "writer" protein methyltransferase-like (METTL)3 in the chondrogenesis of SMSCs. We also mapped the transcript-wide m6A landscape in chondrogenic differentiation of SMSCs and combined RNA-seq and MeRIP-seq in SMSCs by the interference of METTL3. Results: The expression of m6A regulators were regulated in the chondrogenesis of SMSCs, only METTL3 is the most significant factor. In addition, after the knockdown of METTL3, MeRIP-seq and RNA-seq technology were applied to analyze the transcriptome level in SMSCs. 832 DEGs displayed significant changes, consisting of 438 upregulated genes and 394 downregulated genes. DEGs were enriched in signaling pathways regulating the glycosaminoglycan biosynthesis-chondroitin sulfate/dermatan sulfate and ECM-receptor interaction via Kyoto Encyclopedia of genes and genomes (KEGG) pathway enrichment analysis. The findings of this study indicate a difference in transcripts of MMP3, MMP13, and GATA3 containing consensus m6A motifs required for methylation by METTL3. Further, the reduction of METTL3 decreased the expression of MMP3, MMP13, and GATA3. Conclusion: These findings confirm the molecular mechanisms of METTL3-mediated m6A post-transcriptional change in the modulation of SMSCs differentiating into chondrocytes, thus highlighting the potential therapeutic effect of SMSCs for cartilage regeneration.

Teriparatide ameliorates articular cartilage degradation and aberrant subchondral bone remodeling in DMM mice.

Objective: Knee osteoarthritis (KOA) is a highly prevalent musculoskeletal disorder characterized by degeneration of cartilage and abnormal remodeling of subchondral bone (SCB). Teriparatide (PTH (1-34)) is an effective anabolic drug for osteoporosis (OP) and regulates osteoprotegerin (OPG)/receptor activator of nuclear factor ligand (RANKL)/RANK signaling, which also has a therapeutic effect on KOA by ameliorating cartilage degradation and inhibiting aberrant remodeling of SCB. However, the mechanisms of PTH (1-34) in treating KOA are still uncertain and remain to be explored. Therefore, we compared the effect of PTH (1-34) on the post-traumatic KOA mouse model to explore the potential therapeutic effect and mechanisms. Methods: In vivo study, eight-week-old male mice including wild-type (WT) (n â€‹= â€‹54) and OPG-/- (n â€‹= â€‹54) were investigated and compared. Post-traumatic KOA model was created by destabilization of medial meniscus (DMM). WT mice were randomly assigned into three groups: the sham group (WT-sham; n â€‹= â€‹18), the DMM group (WT-DMM; n â€‹= â€‹18), and the PTH (1-34)-treated group (WT-DMM â€‹+ â€‹PTH (1-34); n â€‹= â€‹18). Similarly, the OPG-/- mice were randomly allocated into three groups as well. The designed mice were executed at the 4th, 8th, and 12th weeks to evaluate KOA progression. To further explore the chondro-protective of PTH (1-34), the ATDC5 chondrocytes were stimulated with different concentrations of PTH (1-34) in vitro. Results: Compared with the WT-sham mice, significant wear of cartilage in terms of reduced cartilage thickness and glycosaminoglycan (GAG) loss was detected in the WT-DMM mice. PTH (1-34) exhibited cartilage-protective by alleviating wear, retaining the thickness and GAG contents. Moreover, the deterioration of the SCB was alleviated and the expression of PTH1R/OPG/RANKL/RANK were found to increase after PTH (1-34) treatment. Among the OPG-/- mice, the cartilage of the DMM mice displayed typical KOA change with higher OARSI score and thinner cartilage. The damage of the cartilage was alleviated but the abnormal remodeling of SCB didn't show any response to the PTH (1-34) treatment. Compared with the WT-DMM mice, the OPG-/--DMM mice caught more aggressive KOA with thinner cartilage, sever cartilage damage, and more abnormal remodeling of SCB. Moreover, both the damaged cartilage from the WT-DMM mice and the OPG-/--DMM mice were alleviated but only the deterioration of SCB in WT-DMM mice was alleviated after the administration of PTH (1-34). In vitro study, PTH (1-34) could promote the viability of chondrocytes, enhance the synthesis of extracellular matrix (ECM) (AGC, COLII, and SOX9) at the mRNA and protein level, but inhibit the secretion of inflammatory cytokines (TNF-α and IL-6). Conclusion: Both wear of the cartilage was alleviated and aberrant remodeling of the SCB was inhibited in the WT mice, but only the cartilage-protective effect was observed in the OPG-/- mice. PTH (1-34) exhibited chondro-protective effect by decelerating cartilage degeneration in vivo as well as by promoting the proliferation and enhancing ECM synthesis of chondrocytes in vitro. The current investigation implied that the rescue of the disturbed SCB is dependent on the regulation of OPG while the chondro-protective effect is independent of modulation of OPG, which provides proof for the treatment of KOA. The translational potential of this article: Systemic administration of PTH (1-34) could exert a therapeutic effect on both cartilage and SCB in different mechanisms to alleviate KOA progression, which might be a novel therapy for KOA.

Integrative RNA profiling of TBEV-infected neurons and astrocytes reveals potential pathogenic effectors.

Tick-borne encephalitis virus (TBEV), the most medically relevant tick-transmitted flavivirus in Eurasia, targets the host central nervous system and frequently causes severe encephalitis. The severity of TBEV-induced neuropathogenesis is highly cell-type specific and the exact mechanism responsible for such differences has not been fully described yet. Thus, we performed a comprehensive analysis of alterations in host poly-(A)/miRNA/lncRNA expression upon TBEV infection in vitro in human primary neurons (high cytopathic effect) and astrocytes (low cytopathic effect). Infection with severe but not mild TBEV strain resulted in a high neuronal death rate. In comparison, infection with either of TBEV strains in human astrocytes did not. Differential expression and splicing analyses with an in silico prediction of miRNA/mRNA/lncRNA/vd-sRNA networks found significant changes in inflammatory and immune response pathways, nervous system development and regulation of mitosis in TBEV Hypr-infected neurons. Candidate mechanisms responsible for the aforementioned phenomena include specific regulation of host mRNA levels via differentially expressed miRNAs/lncRNAs or vd-sRNAs mimicking endogenous miRNAs and virus-driven modulation of host pre-mRNA splicing. We suggest that these factors are responsible for the observed differences in the virulence manifestation of both TBEV strains in different cell lines. This work brings the first complex overview of alterations in the transcriptome of human astrocytes and neurons during the infection by two TBEV strains of different virulence. The resulting data could serve as a starting point for further studies dealing with the mechanism of TBEV-host interactions and the related processes of TBEV pathogenesis.

Long non-coding RNA HCAR promotes endochondral bone repair by upregulating VEGF and MMP13 in hypertrophic chondrocyte through sponging miR-15b-5p.

Endochondral bone formation is an important route for bone repair. Although emerging evidence has revealed the functions of long non-coding RNAs (lncRNAs) in bone and cartilage development, the effect of lncRNAs in endochondral bone repair is still largely unknown. Here, we identified a lncRNA, named Hypertrophic Chondrocyte Angiogenesis-related lncRNA (HCAR), and proved it to promote the endochondral bone repair by upregulating the expression of matrix metallopeptidase 13 (Mmp13) and vascular endothelial growth factor α (Vegfa) in hypertrophic chondrocytes. Lnc-HCAR knockdown in hypertrophic chondrocytes restrained the cartilage matrix remodeling and decrease the CD31hiEmcnhi vessels number in a bone repair model. Mechanistically, we proved that lnc-HCAR was mainly enriched in the cytoplasm using fluorescence in situ hybridization (FISH) assay, and it acted as a molecular sponge for miR-15b-5p. Further, in hypertrophic chondrocytes, lnc-HCAR competitively bound to miR-15b-5p to increase Vegfa and Mmp13 expression. Our results proved that lncRNA is deeply involved in endochondral bone repair, which will provide a new theoretical basis for future strategies for promoting fracture healing.

AncPhore: A versatile tool for anchor pharmacophore steered drug discovery with applications in discovery of new inhibitors targeting metallo-ß-lactamases and indoleamine/tryptophan 2,3-dioxygenases.

We herein describe AncPhore, a versatile tool for drug discovery, which is characterized by pharmacophore feature analysis and anchor pharmacophore (i.e., most important pharmacophore features) steered molecular fitting and virtual screening. Comparative analyses of numerous protein-ligand complexes using AncPhore revealed that anchor pharmacophore features are biologically important, commonly associated with protein conservative characteristics, and have significant contributions to the binding affinity. Performance evaluation of AncPhore showed that it had substantially improved prediction ability on different types of target proteins including metalloenzymes by considering the specific contributions and diversity of anchor pharmacophore features. To demonstrate the practicability of AncPhore, we screened commercially available chemical compounds and discovered a set of structurally diverse inhibitors for clinically relevant metallo-ß-lactamases (MBLs); of them, 4 and 6 manifested potent inhibitory activity to VIM-2, NDM-1 and IMP-1 MBLs. Crystallographic analyses of VIM-2:4 complex revealed the precise inhibition mode of 4 with VIM-2, highly consistent with the defined anchor pharmacophore features. Besides, we also identified new hit compounds by using AncPhore for indoleamine/tryptophan 2,3-dioxygenases (IDO/TDO), another class of clinically relevant metalloenzymes. This work reveals anchor pharmacophore as a valuable concept for target-centered drug discovery and illustrates the potential of AncPhore to efficiently identify new inhibitors for different types of protein targets.