The novel HSP90 monoclonal antibody 9B8 ameliorates articular cartilage degeneration by inhibiting glycolysis via the HIF-1 signaling pathway.

Osteoarthritis (OA) is a prevalent chronic degenerative disease that affects the bones and joints, particularly in middle-aged and elderly individuals. It is characterized by progressive joint pain, swelling, stiffness, and deformity. Notably, treatment with a heat shock protein 90 (HSP90) inhibitor has significantly curtailed cartilage destruction in a rat model of OA. Although the monoclonal antibody 9B8 against HSP90 is recognized for its anti-tumor properties, its potential therapeutic impact on OA remains uncertain. This study investigated the effects of 9B8 on OA and its associated signaling pathways in interleukin-1ß (IL-1ß)-stimulated human chondrocytes and a rat anterior cruciate ligament transection (ACLT) model. A specific concentration of 9B8 preserved cell viability against IL-1ß-induced reduction. In vitro, 9B8 significantly reduced the expression of extracellular matrix-degrading enzyme such as disintegrin and metallopeptidase-4 (ADAMTS4) of thrombospondin motifs, matrix metalloproteinase-13 (MMP-13), as well as cellular inflammatory factors such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), which were upregulated by IL-1ß. In vivo, 9B8 effectively protected the articular cartilage and subchondral bone of the rat tibial plateau from ACLT-induced damage. Additionally, gene microarray analysis revealed that IL-1ß substantially increased the expression of SLC2A1, PFKP, and ENO2 within the HIF-1 signaling pathway, whereas 9B8 suppressed the expression of these genes. Thus, 9B8 effectively mitigates ACLT-induced osteoarthritis in rats by modulating the HIF-1 signaling pathway, thereby inhibiting overexpression involved in glycolysis. These results collectively indicate that 9B8 is a promising novel drug for the prevention and treatment of OA.

Identification and verification of four candidate biomarkers for early diagnosis of osteoarthritis by machine learning.

Background: Osteoarthritis (OA) is a common chronic joint disease. This study aimed to investigate possible OA diagnostic biomarkers and to verify their significance in clinical samples. Methods: We exploited three datasets from the Gene Expression Omnibus (GEO) database, serving as the training set. We first determined differentially expressed genes and screened candidate diagnostic biomarkers by applying three machine learning algorithms (Random Forest, Least Absolute Shrinkage and Selection Operator logistic regression, Support Vector Machine-Recursive Feature Elimination). Another GEO dataset was used as the validation set. The test set consisted of RNA-sequenced peripheral blood samples collected from patients and healthy donors. Blood samples and chondrocytes were collected for quantitative real-time PCR to confirm expression levels. Receiver operating characteristic curves were generated for individual and combined biomarkers. Results: In total, 251 DEGs were screened, where B3GALNT1, SCRG1 and ZNF423 were screened by all three algorithms. The area under the curve (AUC) of various biomarkers in our test set did not reach as high as that in public datasets. GRB10 exhibited highest AUC of 0.947 in the training set but 0.691 in our test set, while the favorable combined model comprising B3GALNT1, GRB10, KLF9 and SCRG1 demonstrated an AUC of 0.986 in the training set, 1.000 in the validation set and 0.836 in our test set. Conclusion: We identified a combined model for early diagnosis of OA that includes B3GALNT1, GRB10, KLF9 and SCRG1. This finding offers new avenues for further exploration of mechanisms underlying OA.

[Analysis of genetic variants and molecular pathogenesis in a Chinese pedigree affected with Multiple epiphyseal dysplasia].

OBJECTIVE: To analyze the genetic variant and molecular pathogenesis in a Chinese pedigree affected with Multiple epiphyseal dysplasia (MED). METHODS: A MED pedigree which had presented at the Beijing Jishuitan Hospital Affiliated to Capital Medical University on September 13, 2020 was selected as the study subject. Clinical data of the pedigree were collected. Peripheral blood samples were drawn from pedigree members for the extraction of genomic DNA. Whole exome sequencing (WES) was carried out for the pedigree. Candidate variant was verified by Sanger sequencing. Wild type and mutant SLC26A2 expression plasmids were constructed and transfected into human primary chondrocytes. The effect of the variants on the protein localization and cell proliferation was determined by immunofluorescence and CCK8 assays. RESULTS: WES and Sanger sequencing revealed that the proband has harbored compound heterozygous variants of the SLC26A2 gene, including a paternally derived c.484G>T (p.Val162Leu) missense variant and a maternally derived c.485_486delTG (p.Val162Glyfs*12) frameshifting variant. The SLC26A2WT and its mutant SLC26A2Val162Leu and SLC26A2Val162Glyfs*12 expression plasmids were distributed in the nuclei and cytoplasm of human primary chondrocytes. Compared with SLC26A2WT, the expressions of SLC26A2Val162Leu and SLC26A2Val162Glyfs*12 were decreased, along with reduced proliferation of human primary chondrocytes. CONCLUSION: The c.484G>T and c.485_486delTG compound heterozygous variants of the SLC26A2 gene may affect the proliferation of human primary chondrocytes and underlay the pathogenesis of MED in this pedigree.

Puerarin modulates proliferation, inflammation and ECM metabolism in human nucleus pulposus mesenchymal stem cells via the lncRNA LINC01535.

Background: Intervertebral disc degeneration (IVDD) is a highly prevalent musculoskeletal disorder characterized by progressive destruction of the intervertebral disc, leading to chronic low back pain and disability. Emerging evidence suggests that dysregulation of ferroptosis, a recently discovered form of regulated cell death, participates in IVDD pathogenesis. Puerarin, a natural flavonoid compound from Pueraria lobata, has shown promise in modulating ferroptosis in various diseases. Methods: Human nucleus pulposus-derived mesenchymal stem cells (NPMSCs) were isolated and identified by flow cytometry. We investigated the effects of puerarin on human NPMSCs and examined the underlying molecular mechanisms. Results: Puerarin significantly promoted human NPMSC proliferation, as evidenced by the increased cell viability and colony formation ability. Furthermore, puerarin suppressed the expression of cyclooxygenase-2 and the proinflammatory cytokine interleukin-6 in NPMSCs, demonstrating the anti-inflammatory properties of the compound. Notably, puerarin attenuated ECM breakdown by downregulating the ECM-degrading enzymes MMP3, MMP13 and ADAMTS5, and it increased ECM component synthesis, including collagen type II and aggrecan, by NPMSCs. Moreover, puerarin inhibited ferroptosis in NPMSCs by modulating the expression of key ferroptosis-related genes, including ACSL4, PTGS2 and GPX4. Depletion of LINC01535 abolished the effects of puerarin on proliferation, inflammation and ECM metabolism, suggesting a key role of this lncRNA in mediating the effects of puerarin. Conclusion: Our findings show that puerarin promotes the proliferation of human NPMSCs and ECM synthesis by these cells. Furthermore, puerarin inhibits inflammation and ECM degradation by suppressing ferroptosis via LINC01535. These results provide insights into the molecular mechanisms underlying the therapeutic effects of puerarin in IVDD. Targeting ferroptosis and its regulatory factors, such as LINC01535, may have therapeutic potential for the treatment of IDD and other degenerative disorders of the intervertebral disc. Further studies are needed to uncover the translational potential of puerarin and its downstream targets in preclinical and clinical applications.

Biallelic variants in SLC26A2 cause multiple epiphyseal dysplasia-4 by disturbing chondrocyte homeostasis.

BACKGROUND: Multiple epiphyseal dysplasia-4 (MED-4, MIM 226900) is a rare autosomal recessive disease characterized by disproportionate height and early onset osteoarthritis of the lower limbs. MED-4 is caused by homozygous or compound heterozygous pathogenic variants in the SLC26A2 gene. However, the underlying pathogenic mechanisms in chondrocytes remains unknown. This study aimed to identify the pathogenic variants within a MED-4 family and explore the molecular etiology of this condition in human primary chondrocyte cells. METHODS: Clinical data were recorded and peripheral blood samples were collected for analysis. Whole exome sequencing (WES) and bioinformatic analyses were performed to determine causative variants. Wild-type SLC26A2 and corresponding mutant expression plasmids were constructed and transfected into human primary chondrocytes. The expression and subcellular distribution of SLC26A2 protein in chondrocytes were detected by immunoblotting and immunofluorescence. Effects of these variants on chondrocytes viability and apoptosis were measured by Cell Counting Kit-8 (CCK-8) assay. Expression of genes related to cartilage homeostasis was subsequently analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: We identified two compound heterozygous variants c.1020_1022delTGT(p.Val341del) and c.1262 T > C(p.Ile421Thr) in the SLC26A2 gene in the patients. Mutant SLC26A2Val341del and SLC26A2Ile421Thr proteins were distributed in relatively few cells and were observed only within the nucleus. The viability of chondrocytes with the SLC26A2 variant group was similar to the wild-type (WT) group. However, the protein expressions of SLC26A2Val341del and SLC26A2Ile421Thr were decreased compared with SLC26A2WT. Expression levels of matrix metallopeptidase 13 (MMP13), α-1 chain of type X collagen (COL10A1), and Runt-related transcription factor 2 (RUNX2) were significantly decreased in the variant group. However, aggrecan (ACAN) expression was higher in the variant group than the WT group. CONCLUSIONS: Overall, our data demonstrate that the variants p.Val341del and p.Ile421Thr in SLC26A2 cause MED-4 and that these two variants promote chondrocyte proliferation while inhibiting chondrocyte differentiation.

Identification and validation of ferroptosis-related lncRNA signature in intervertebral disc degeneration.

Low back pain influences people of every age and is one of the major contributors to the global cost of illness. Intervertebral disc degeneration (IVDD) is a major contributor to low back pain, but its pathogenesis is unknown. Recently, ferroptosis has been shown to have a substantial role in modulating IVDD progression. However, the function of ferroptosis-related long non-coding RNAs (lncRNAs) has rarely been reported in IVDD. Consequently, the research was conducted to explore the ferroptosis-related lncRNA signature in the IVDD occurrence and development. We analyzed two datasets (GSE167199 and GSE167931) archived in the NCBI Gene Expression Omnibus (GEO) public database. We screened differentially expressed genes (DEGs) and differentially expressed lncRNAs (DELncs) in these datasets using the limma package. Ferroptosis-related genes (FRGs) were derived from the FerrDb V2 website and the intersection of DEGs and FRGs was considered as differentially expressed ferroptosis-related genes (DFGs). These genes were then subjected to Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis. Correlations between DFGs and DELncs were shown by Pearson test to determine differential expression of ferroptosis-related lncRNAs. The Pearson test showed that CPEB1-HTR2A-AS1 and ACSL3-DNAJC27-AS1 pairs had correlation coefficients over 0.9. Twenty ferroptosis-related lncRNAs were identified and validated in IVDD. Eight of these lncRNAs were upregulated in IVDD nucleus pulposus cells, including HTR2A-AS1, MIF-AS1, SLC8A1-AS1, LINC00942, DUXAP8, LINC00161, LUCAT1 and LINC01615. Twelve were downregulated in IVDD nucleus pulposus cells, including DNAJC27-AS1, H19, LINC01588, LINC02015, FLNC1, CARMN, PRKG1-AS1, APCDD1L-DT, LINC00839, LINC00536, LINC00710 and LINC01535. Eighteen of the 20 lncRNAs (excluding H19 and LUCAT1) were identified as ferroptosis-related lncRNAs for the first time and verified in IVDD. We have identified a ferroptosis-related lncRNA signature involved in IVDD and revealed a close relationship between CPEB1 and HTR2A-AS1, and between ACSL3 and DNAJC27-AS1. Our findings indicate that ferroptosis-related lncRNAs are a new target set for the early detection and therapy of IVDD.

Integration analysis of lncRNA and mRNA expression data identifies DOCK4 as a potential biomarker for elderly osteoporosis.

BACKGROUND: We aimed to identify some potential biomarkers for elderly osteoporosis (OP) by integral analysis of lncRNA and mRNA expression data. METHODS: A total of 8 OP cases and 5 healthy participants were included in the study. Fasting peripheral venous blood samples were collected from individuals, and total RNA was extracted. RNA-seq library was prepared and sequenced on the Illumina HiSeq platform. Differential gene expression analysis was performed using "DESeq2" package in R language. Functional enrichment analysis was conducted using the "clusterProfiler" package, and the cis- and trans-regulatory relationships between lncRNA and target mRNA were analyzed by the lncTar software. A protein-protein interaction (PPI) network was constructed using the STRING database, and hub genes were identified through the MCODE plugin in Cytoscape. RESULTS: We identified 897 differentially expressed lncRNAs (DELs) and 1366 differentially expressed genes (DEGs) between normal and OP samples. After co-expression network analysis and cis-trans regulatory genes analysis, we identified 69 candidate genes regulated by lncRNAs. Then we further screened 7 genes after PPI analysis. The target gene DOCK4, trans-regulated by two lncRNAs, was found to be significantly upregulated in OP samples. Additionally, 4 miRNAs were identified as potential regulators of DOCK4. The potential diagnostic value of DOCK4 and its two trans-regulatory lncRNAs was supported by ROC analysis, indicating their potential as biomarkers for OP. CONCLUSION: DOCK4 is a potential biomarker for elderly osteoporosis diagnostic. It is identified to be regulated by two lncRNAs and four miRNAs.

Characterization and role exploration of ferroptosis-related genes in osteoarthritis.

Osteoarthritis (OA), viewing as a degenerative aseptic inflammatory disease, is characterized by joint pain and inflammation that significantly affects the quality of patients' life, especially for the elder. Although rapid progress has been achieved in elucidating the underlying mechanisms of OA occurrence and progression, there is still a lack of effective clinical therapeutics for OA patients. Currently the most common treatments including drug therapy and surgical operations are not very satisfactory in majority of cases, so it is worthy to explore new remedies. During the past few decades, a number of novel forms of regulated cell death have been reported widely, typified by ferroptosis, with its prominent features including reactive oxygen species (ROS) elevation, lipid peroxidation, iron accumulation and glutathione deprivation. Our study was designed to identify the functional roles of differentially expressed ferroptosis-related genes in OA, which were screened out by referring to GEO database via bioinformatics analyses. Human chondrocytes were applied to validate the above findings in the scenario of ferroptosis inhibitors administration. Results partially proved the consistency with bioinformatics analyses that ATF3 and TFRC were highly expressed in interleukin-1ß (IL-1ß)-stimulated chondrocytes whereas CXCL2 and JUN were downregulated. Besides, TFRC was firstly validated to be upregulated in IL-1ß-stimulated chondrocytes, which could be reversed by ferroptosis inhibitors. In conclusion, our study reported two prominent ferroptosis-related genes, ATF3 and TFRC are upregulated in IL-1ß-stimulated chondrocytes while CXCL2 and JUN are downregulated. And preliminary results demonstrated that TFRC might serve as an accomplice of ferroptosis process in IL-1ß-stimulated chondrocytes and ferroptosis inhibitors have the potential to inhibit ROS in IL-1ß-stimulated chondrocytes.