GC-MS based comparative metabolomic analysis of human cancellous bone reveals the critical role of linoleic acid metabolism in femur head necrosis.

INTRODUCTION: Femur head necrosis (FHN) is a challenging clinical disease with unclear underlying mechanism, which pathologically is associated with disordered metabolism. However, the disordered metabolism in cancellous bone of FHN was never analyzed by gas chromatography-mass spectrometry (GC-MS). OBJECTIVES: To elucidate altered metabolism pathways in FHN and identify putative biomarkers for the detection of FHN. METHODS: We recruited 26 patients with femur head necrosis and 22 patients with femur neck fracture in this study. Cancellous bone tissues from the femoral heads were collected after the surgery and were analyzed by GC-MS based untargeted metabolomics approach. The resulting data were analyzed via uni- and multivariate statistical approaches. The changed metabolites were used for the pathway analysis and potential biomarker identification. RESULTS: Thirty-seven metabolites distinctly changed in FHN group were identified. Among them, 32 metabolites were upregulated and 5 were downregulated in FHN. The pathway analysis showed that linoleic acid metabolism were the most relevant to FHN pathology. On the basis of metabolites network, L-lysine, L-glutamine and L-serine were deemed as the junctions of the whole metabolites. Finally, 9,12-octadecadienoic acid, inosine, L-proline and octadecanoic acid were considered as the potential biomarkers of FHN. CONCLUSION: This study provides a new insight into the pathogenesis of FHN and confirms linoleic acid metabolism as the core.

MiR-654-3p, reduced by the excessive ALKBH5, Alleviated the Inflammation in OA by targeting TNFRSF9, the trigger of the NF-κB pathway.

MicroRNA (miRNA) is one of the most potent therapeutic targets for osteoarthritis (OA). We identified that miR-654-3p protected the phenotype of chondrocytes. We demonstrated that TNF receptor superfamily member 9 (TNFRSF9) was the target of miR-654-3p by binding to its 3'UTR regions, based on a dual-luciferase reporter assay and an RNA binding protein immunoprecipitation (RIP) assay. In addition, further experiments proved that TNFRSF9, as a trigger of the NF-κB pathway, correlated with the inflammation in chondrocytes. MiR-654-3p overexpressed in the knee of mice alleviated the OA in vivo. Moreover, we examined the m6A enzyme level in OA, proving that the abnormal expression of α-ketoglutarate-dependent dioxygenase alkB homolog 5 (ALKBH5) contributed to the miR-654-3p decrease. Our research illustrated the significant role of miR-654-3p in OA, including its maturation and the mechanism in protecting the phenotype of chondrocytes, which could be a new treatment target for OA.

LncRNA HOXA11-AS intercepts the POU2F2-mediated downregulation of SLC3A2 in osteoarthritis to suppress ferroptosis.

BACKGROUND: Osteoarthritis (OA) is a prevalent ailment characterized by the gradual degradation of joints, resulting in discomfort and restricted movement. The recently proposed mechanism of ferroptosis is intricately associated with the initiation and progression of OA. Our study found that the long non-coding RNA HOXA11-AS reduces ferroptosis by increasing the expression of SLC3A2 through the transcription factor POU2F2. MATERIALS AND METHODS: HOXA11-AS was identified through lncRNA microarray analysis, and its impact on chondrocytes and extracellular matrix was assessed using real-time quantitative PCR, western blotting, and CCK8 assays. Subsequently, overexpression of HOXA11-AS in the knee joints of mice confirmed its protective efficacy on chondrocyte phenotype in the OA model. The involvement of HOXA11-AS in regulating ferroptosis via SLC3A2 was further validated through RNA sequencing analysis of mouse cartilage and the assessment of malondialdehyde levels and glutathione peroxidase activity. Finally, a combination of RNA sequencing, pull-down assays, mass spectrometry (MS), and chromatin immunoprecipitation (ChIP) techniques was employed to identify POU2F2 as the crucial transcription factor responsible for repressing the expression of SLC3A2, which can be effectively inhibited by HOXA11-AS. RESULTS: Our study demonstrated that HOXA11-AS effectively enhanced the metabolic homeostasis of chondrocytes, and alleviated the progression of OA in vitro and in vivo experiments. Furthermore, HOXA11-AS was found to enhance SLC3A2 expression, a key regulator of ferroptosis, by interacting with the transcriptional repressor POU2F2. CONCLUSIONS: HOXA11-AS promotes SLC3A2 expression and inhibits chondrocyte ferroptosis, by binding to the transcriptional repressor POU2F2, offering a promising and innovative therapeutic approach for OA.

Role of immune cell infiltration and small molecule drugs in adhesive capsulitis: Novel exploration based on bioinformatics analyses.

Background: Adhesive capsulitis (AC) is a type of arthritis that causes shoulder joint pain, stiffness, and limited mobility. The pathogenesis of AC is still controversial. This study aims to explore the role of immune related factors in the occurrence and development of AC. Methods: The AC dataset was downloaded from Gene Expression Omnibus (GEO) data repository. Differentially expressed immune-related genes (DEIRGs) were obtained based on R package "DESeq2" and Immport database. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were performed to explore the functional correlation of DEIRGs. MCC method and Least Absolute Shrinkage and Selection Operator (LASSO) regression were conducted to identify the hub genes. The immune cell infiltration in shoulder joint capsule between AC and control was evaluated by CIBERSORTx, and the relationship between hub genes and infiltrating immune cells was analyzed by Spearman's rank correlation. Finally, potential small molecule drugs for AC were screened by the Connectivity Map database (CMap) and further verified by molecular docking. Results: A total of 137 DEIRGs and eight significantly different types of infiltrating immune cells (M0 macrophages, M1 macrophages, regulatory T cells, Tfh cells, monocytes, activated NK cells, memory resting CD4+T cells and resting dendritic cells) were screened between AC and control tissues. MMP9, FOS, SOCS3, and EGF were identified as potential targets for AC. MMP9 was negatively correlated with memory resting CD4+T cells and activated NK cells, but positively correlated with M0 macrophages. SOCS3 was positively correlated with M1 macrophages. FOS was positively correlated with M1 macrophages. EGF was positively correlated with monocytes. Additionally, dactolisib (ranked first) was identified as a potential small-molecule drug for the targeted therapy of AC. Conclusions: This is the first study on immune cell infiltration analysis in AC, and these findings may provide a new idea for the diagnosis and treatment of AC.

Identification of SLC3A2 as a Potential Therapeutic Target of Osteoarthritis Involved in Ferroptosis by Integrating Bioinformatics, Clinical Factors and Experiments.

Osteoarthritis (OA) is a type of arthritis that causes joint pain and limited mobility. In recent years, some studies have shown that the pathological process of OA chondrocytes is related to ferroptosis. Our study aims to identify and validate differentially expressed ferroptosis-related genes (DEFRGs) in OA chondrocytes and to investigate the potential molecular mechanisms. RNA-sequencing and microarray datasets were downloaded from Gene Expression Omnibus (GEO) data repository. Differentially expressed genes (DEGs) were screened by four methods: limma-voom, edgeR, DESeq2, and Wilcoxon rank-sum test. Weighted correlation network analysis (WGCNA), protein-protein interactions (PPI), and cytoHubba of Cytoscape were applied to identify hub genes. Clinical OA cartilage specimens were collected for quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis, western blotting (WB), histological staining, transmission electron microscopy (TEM), and transfection. Sankey diagram was used to visualize the relationships between the expression level of SLC3A2 in the damaged area and clinical factors. Based on bioinformatics analysis, clinical factors, and experiment validation, SLC3A2 was identified as a hub gene. It was down-regulated in OA cartilage compared to normal cartilage (p < 0.05). Functional enrichment analysis revealed that SLC3A2 was associated with ferroptosis-related functions. Spearman correlation analysis showed that the expression level of SLC3A2 in the OA cartilage-damaged area was closely related to BMI, obesity grade, and Kellgren-Lawrence grade. Furthermore, in vitro experiments validated that SLC3A2 inhibited ferroptosis and suppressed cartilage degeneration in OA. In summary, we demonstrated that SLC3A2 inhibited ferroptosis and suppressed cartilage degeneration in OA. These findings provide a new idea for the study of the pathogenesis of OA, thus providing new means for the clinical diagnosis and targeted therapy of OA.

tRNA-derived fragment TRF365 regulates the metabolism of anterior cruciate ligament cells by targeting IKBKB.

tRNA-derived fragments (tRFs) are new noncoding RNAs, and recent studies have shown that tRNAs and tRFs have important functions in cell metabolism via posttranscriptional regulation of gene expression. However, whether tRFs regulate cellular metabolism of the anterior cruciate ligament (ACL) remains elusive. The aim of this study was to investigate the role and action mechanism of tRFs in ACL cell metabolism. A tRF array was used to determine tRF expression profiles in different human ACL cells, and quantitative real-time polymerase chain reaction and fluorescence in situ hybridisation were used to determine TRF365 expression. ACL cells were transfected with a TRF365 mimic or a TRF365 inhibitor to determine whether TRF365 regulates IKBKB expression. A rescue experiment and dual-luciferase reporter assay were conducted to determine whether the 3'-untranslated region (UTR) of IKBKB has a TRF365-binding site. TRF365 was weakly expressed in osteoarthritis (OA) ACL and interleukin-1ß-treated ACL cells. IKBKB was highly expressed in OA ACL and interleukin-1ß-treated ACL cells; transfection with the TRF365 mimic suppressed IKBKB expression, whereas transfection with the TRF365 inhibitor had the opposite effect. A dual-luciferase reporter assay showed that TRF365 silenced the expression of IKBKB by binding to its 3'-UTR. Thus, TRF365 regulates the metabolism of ACL cells by targeting IKBKB. In summary, TRF365 may provide a new direction for the study of ACL degeneration and on the pathophysiological process of OA.

The plane of the distal femur anterior cortex is a useful index for femoral component rotation in total knee arthroplasty.

OBJECTIVE: To investigate the reference role of the plane of the distal femur anterior cortex in determining the lateral rotation angle of femoral component in total knee arthroplasty (TKA). METHODS: Computed tomography (CT) scan was performed for full length of both lower limbs in 27 patients, and a total of 53 legs were examined by radiological department of the First Affiliated Hospital of Sun Yat-sen University. Case inclusion criteria were as follows: no obvious congenital bone deformity on the lower limbs, no knee purulent infection, and no severe knee deformity after surgery. 3D reconstruction was performed by Mimics 16.0 software for the scan images to calculate the angle between the plane of the distal femur anterior cortex and the Posterior Condylar Line (PCL) (defined as FPA), and the angle between the plane of the distal femur anterior cortex and the Clinical Epicondylar Axis (CEA) (defined as ACA), as well as the angle between the CEA and PCL, known as the condylar twist angle (CTA). Finally, Kolmogorov-Smirnov test and Student's t test were used to analyze the measured value. RESULTS: The average value of FPA was -4.70 ± 3.77°, that of CTA, +5.04 ± 2.36°, and that of ACA, -9.17 ± 5.78°. Each angle measured above has no significant differences in the gender (P > 0.05). CONCLUSION: ACA was measured to be 9.17 ± 5.78° on the direction of medial femoral rotation, proving that the plane of distal femur anterior cortex plays a relatively accurate reference role in determining the lateral rotation angle of the femoral component in TKA.

Cementless acetabular component with or without upward placement in dysplasia hip: Early results from a prospective, randomised study.

BACKGROUND: Total hip arthroplasty (THA) in developmental dysplasia of hip (DDH) is difficult for the abnormal acetabulum. The purpose of this study was to evaluate the difference of anatomic and upward placement of acetabular component during early stage. METHODS: From April 2014 through June 2015, forty DDH patients (Crowe I to III, 42 hips) were prospectively randomized to either anatomic or upward group. Patient recorded diaries were collected. Radiographs were reviewed. WOMAC and Harris scores were tabulated from pre-operation to 12 months after surgery. RESULTS: The patients' characteristics including age and body mass index (BMI) had no significant difference (P > 0.05). There were no statistically differences between two groups for surgery time, intraoperative blood loss, hemoglobin (Hb), blood transfusion, albumin decrease, length of stay-day, but surgery time and blood loss in patients with structural bone graft was much higher in anatomic group. The postoperative limb-length discrepancy (LLD) was also no difference, but limb lengthening was better in anatomic group (P = 0.042). The total hospital costs in the anatomic group were higher, but no significant differences. With regard to Harris and WOMAC score, there were significant improved after surgery in both groups, and the anatomic group was better in the value, but these differences were no statistically significant. CONCLUSIONS: Acetabular reconstruction for DDH subluxation should be reconstructed as close to the actual acetabular location as possible, but an appropriate (<20 mm) upward placement that can achieve at least 70% native bone coverage of the acetabular implant is a valuable technique for early faster recovery.

[Effect of chondrogenesis related miR-4287 on expression of aggrecanase-1 in human chondrocytes].

Objective: To investigate the effect and mechanism of miR-4287, a chondrogenesis associated microRNA, regulated the expression of aggrecanase-1 (a disintegrin and metalloproteinase with thrombospondin motif 4, ADAMTS4) in human chondrocytes. Methods: First, the voluntarily donated normal and osteoarthritic knee articular cartilages were used to detect the expressions of miR-4287 and ADAMTS4 mRNA by real-time fluorescence quantitative PCR. Then, chondrocytes were isolated from knee articular cartilages. The effect of IL-1ß on the expression of miR-4287 and ADAMTS4 mRNA was validated by the first generation of osteoarthritic chondrocytes. To confirm the influence of IL-1ß signal pathways on the expression of miR-4287 and ADAMTS4 mRNA, osteoarthritic chondrocytes were pretreated with MAPK signal pathway inhibitor SP600125, NF-κB pathway inhibitor SN50, and finally stimulated with IL-1ß. Chondro cytes were transfected with miR-4287 mimics and mimics negative control, inhibitors and inhibitors negative control respectively to value the effect of miR-4287 on ADAMTS4 expression. Luciferase reporter assay was used to verify the direct interaction between miR-4287 and putative site in the 3-untranslated region (3'UTR) of ADAMTS4 mRNA. Results: Compared with normal knee articular cartilages, the miR-4287 expression was markedly diminished and conversely ADAMTS4 mRNA expression was raised in osteoarthritis cartilages ( P<0.05). Stimulation with IL-1ß led to a reduction in miR-4287 expression and upregulation in ADAMTS4 mRNA expression, showing significant difference when compared with the untreated groups ( P<0.05). Pretreatment with IL-1ß signal pathway inhibitors induced miR-4287 expression and attenuated ADAMTS4 mRNA expression in human chondrocytes, which were significantly different from that of unstimulated cells ( P<0.05). ADAMTS4 mRNA and protein were suppressed by transfection with miR-4287 mimics ( P<0.05) and elevated by transfection with miR-4287 inhibitors ( P<0.05). As luciferase reporter assay showed, overexpression miR-4287 failed to alter the luciferase activity of a reporter construct containing either wild or mutant 3'UTR of ADAMTS4 mRNA ( P>0.05). Conclusion: miR-4287, a chondrogenesis associated microRNA, may play an important role in cartilage degeneration. miRNA-4287 is able to regulate ADAMTS4 expression in human chondrocytes, but not by means of directly targeted the ADAMTS4 mRNA 3'UTR. The exact mechanisms need to be further addressed.

[[INFLUENCES OF PREOPERATIVE SEVERE LOWER LIMB DEFORMITY ON PROSTHESIS INSTALLATION AND ALIGNMENT RESTORATION IN TOTAL KNEE ARTHROPLASTY].

OBJECTIVE: To investigate the effect of preoperative valgus or varus deformity on the prosthesis installation and alignment restoration in total knee arthroplasty (TKA). METHODS: Between January 2012 and December 2013, 198 patients (245 knees) with osteoarthritis underwent primary TKA, and the clinical data were retrospectively analyzed. There were 23 males and 175 females, with the average age of 67 years (range, 43-90 years). Single knee and double knees were involved in 151 and 47 cases respectively. The disease duration was from 1 month to 30 years (mean, 8.99 years). The anteroposterior X-ray films of whole lower limbs were taken, and the femorotibial angle (FT) was measured before operation and at 1 week after operation; the mechanical femoral angle (MF) and the anatomical tibial angle (AT) at 1 week after operation were measured. The correlation analysis was made for pre- and post-operative FT, MF, and AT. According to the valgus or varus deformity before operation, all patients were divided into 5 groups: ≥ 20 degrees varus (group A), 10-20 degrees varus (group B), ≤ 10 degrees varus (group C), < 10 degrees valgus (group D), and ≥ 10 degrees valgus (group E), and the above indicators were compared between groups. And the rate of the good limb alignment was recorded after operation. RESULTS: The pre- and post-operative FT were (171.53 ± 9.12) and (177.38 ± 3.57)degrees respectively, and postoperative MF and AT were (89.00 ± 2.68) and (88.62 ± 2.16) respectively. Preoperative FT was associated with postoperative FT and MF (r = 0.375, P = 0.000; r = 0.386, P = 0.000), but it was not correlated with AT (r = 0.024, P = 0.710). Postoperative FT was associated with MF and AT (r = 0.707, P = 0.000; r = 0.582, P = 0.000). Postoperative FT was significantly increased when compared with preoperative FT in each group (P < 0.05). There were significant differences in preoperative FT between groups (P < 0.05). There were significant differences in postoperative FT when compared group A with groups B, C, D, and E (P < 0.05), and when compared groups B and C with groups D and E (P<0.05), but there was no significant difference between groups B and C, and between groups D and E (P > 0.05). The rate of good alignment was 70.2% (172/245); it was 27.8% (5/18), 66.0% (62/94), 74.4% (67/90), 88.9% (32/36), and 85.7% (6/7) in groups A, B, C, D, and E respectively, showing significant differences between groups (P < 0.05). There was no significant difference in postoperative AT between groups (P > 0.05). Except for between group D and group E (P > 0.05), significant difference in MF was shown between the other groups (P < 0.05). CONCLUSION: The more severe deformity of lower limb before TKA, the higher risk of deviation for prosthesis installation and poor alignment in TKA.