Steroid-induced Osteonecrosis Of The Femoral Head Research Articles

Predicting steroid-induced osteonecrosis of the femoral head: role of lipid metabolism biomarkers and radiomics in young and middle-aged adults.

Femoral head necrosis is a common orthopedic disease that results in significant physical disability in patients. Early prediction and diagnosis of steroid-induced osteonecrosis of the femoral head (SONFH) are crucial for the prevention and treatment of this condition. In this study, initial CT images and clinical data of patients with SONFH, admitted from January 2019 to December 2022, were collected. Patients were grouped as follows: (1) those diagnosed with SONFH at the initial diagnosis (control group), and (2) those with high-risk factors but no symptoms at first diagnosis, who developed SONFH two years later (experimental group). CT imaging histological features, clinical characteristics, and transcriptome screening for differentially expressed genes, pathway enrichment, and immune infiltration analyses were performed. Significant differences were found in triglyceride (TG) levels between the training and validation groups. Age, sex, alkaline phosphatase (ALP), and hemoglobin levels differed between the training and internal validation groups, while HDL and red blood cell counts varied between the training and external validation groups. Univariate analysis showed that age, TG, HDL, and Radiomics scores influenced SONFH, while multivariate analysis revealed TG, HDL, and Radiomics scores were closely related to SONFH. Transcriptomic analysis showed associations with sphingolipid and adipocyte signaling pathways, along with immune cell involvement, linking SONFH to lipid metabolism and atherosclerosis. These findings indicate a significant association between steroid-induced osteonecrosis of the femoral head and age, with TG and HDL serving as indicators of lipid metabolism closely correlated with the occurrence of SONFH. Radiomics scores were also found to correlate with SONFH occurrence, supported by transcriptomic and CT imaging findings. However, this study has limitations, including its retrospective design and a relatively limited sample size, which may impact the generalizability of the results. Further prospective studies with larger, more diverse populations are needed to validate and enhance the predictive model.

Mitochondrial maintenance as a novel target for treating steroid-induced osteonecrosis of femoral head: a narrative review.

The pathogenesis of steroid-induced osteonecrosis of the femoral head (SONFH) remains unclear; however, emerging evidence suggests that mitochondrial injury plays a significant role. This review aims to elucidate the involvement of mitochondrial dysfunction in SONFH and explore potential therapeutic targets. A comprehensive literature search was conducted in PubMed, Web of Science, and Elsevier ScienceDirect, focusing on mitochondrial homeostasis, including mitophagy, mitochondrial biogenesis, mitochondrial dynamics, and oxidative stress in SONFH. Ultimately, we included and analyzed a total of 16 studies. Glucocorticoids initially promote but later inhibit mitochondrial biogenesis in osteoblasts, leading to excessive ROS production and mitochondrial dysfunction. This dysfunction impairs osteoblast survival and bone formation, contributing to SONFH progression. Key proteins such as mitochondrial transcription factor A (TFAM) and peroxisome proliferator-activated receptor γ coactivator 1-α (PGC1α) are potential therapeutic targets for promoting mitochondrial biogenesis and reducing ROS-induced damage. Enhancing mitochondrial function and reducing oxidative stress in osteoblasts may prevent or slow the progression of SONFH. Future research should focus on developing these strategies.

Plasma SQSTM1/p62 act as a biomarker for steroid-induced osteonecrosis of the femoral head

Autophagy is closely associated with the onset and progression of steroid-induced osteonecrosis of the femoral head (SIONFH). SQSTM1/p62 is an important indicator of autophagic activity. The aim of this study was to investigate the role of SQSTM1/p62 in the development of SIONFH. From May 2021 through November 2021, 36 patients diagnosed with SIONFH and 36 healthy controls were recruited for this study. Evaluations included imaging and pathologic assessment of clinical bone tissue, location and level of SQSTM1/p62 expression, plasma SQSTM1/p62 levels, and receiver operating characteristic (ROC) curves. We observed that the expression level of SQSTM1/p62 in bone samples decreased with the Association Research Circulation Osseous (ARCO) phase. Plasma SQSTM1/p62 levels were significantly higher in the SIONFH group compared to healthy controls. Plasma SQSTM1/p62 levels were higher in pre-crash patients than in post-crash patients, and lower plasma SQSTM1/p62 levels were associated with elevated ARCO stage. Plasma SQSTM1/p62 may represent a potential biomarker for different stages during SIONFH. Lower plasma SQSTM1/p62 levels indicate an advanced stage of SIONFH. This study provides new clues for early diagnosis of SIONFH.

Identification and validation of endoplasmic reticulum stress-related genes in patients with steroid-induced osteonecrosis of the femoral head

Steroid-induced osteonecrosis of the femoral head (SONFH) is a debilitating condition caused by long-term corticosteroid use, leading to impaired blood flow and bone cell death. The disruption of cellular processes and promotion of apoptosis by endoplasmic reticulum stress (ERS) is implicated in the pathogenesis of SONFH. We identified ERS-associated genes in SONFH and investigated their potential as therapeutic targets. We analysed the GSE123568 GEO dataset to identify differentially expressed genes (DEGs) related to ERS in SONFH. We conducted Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses, identified hub genes by protein-protein interaction (PPI) analyses, and evaluated their functions by gene set enrichment analysis (GSEA). We constructed mRNA–miRNA networks, identified potential therapeutics, and assessed immune cell infiltration. We performed cross-validation using the GEO dataset GSE74089, qRT-PCR on clinical samples from patients with SONFH and controls, and a receiver operating characteristic (ROC) curve analysis to assess the diagnostic performance of the hub genes. We identified 195 ERS-related genes in SONFH, which were primarily involved in oxidative stress, immune responses, and metabolic pathways. The PPI network suggested CXCL8, STAT3, IL1B, TLR4, PTGS2, TLR2, CASP1, CYBB, CAT, and HOMX1 to be key hub genes, which were shown by GSEA to be involved in biological pathways related to metabolism, immune modulation, and cellular integrity. We also identified 261 microRNAs (miRNAs) as well as drugs such as dibenziodolium and N-acetyl-L-cysteine that modulated inflammatory responses in SONFH. Twenty-two immune cell subtypes showed significant correlations, such as a positive correlation between activated mast cells and Tregs, and patients with SONFH had fewer dendritic cells than controls. The hub genes CYBB and TLR4 showed significant correlations with M1 macrophages and CD8 T cells, respectively. Cross-validation and qRT-PCR confirmed the upregulation of STAT3, IL1B, TLR2, and CASP1 in patients with SONFH, validating the bioinformatics findings. An ROC curve analysis confirmed the diagnostic potential of the hub genes. The top 10 hub genes show promise as ERS-related diagnostic biomarkers for SONFH. We discovered that 261 miRNAs, including hsa-miR-23, influence these genes and identified potential therapeutics such as dibenziodolium and simvastatin. Immune profiling indicated altered immune functions in SONFH, with significant correlations among immune cell types. Validation confirmed the upregulation of STAT3, IL1B, TLR2 and CASP1, which had diagnostic potential. The findings suggest potential diagnostic markers and therapeutic targets for SONFH.

Integrated lipidomics and network pharmacology analysis to determine how Gu Fu Sheng Capsule improves lipid metabolism in rats with steroid-induced osteonecrosis of the femoral head

BackgroundSteroid-induced osteonecrosis of the femoral head (SONFH) is a prevalent refractory condition in orthopedics, often causing high disability rates. Gu Fu Sheng Capsule (GFSC) is commonly used to treat SONFH during the early and intermediate stages. However, the precise underlying mechanism of action of GFSC remains elusive. ObjectiveTo elucidate the mechanism through which GFSC improves lipid metabolism in rats with SONFH. MethodsA rat model of SONFH was established by administering methylprednisolone and lipopolysaccharide. Micro-computed tomography assessed femoral head effects, while hematoxylin–eosin staining examined femoral head tissues. Pathological changes were evaluated using an automated biochemical analyzer to measure changes in serum lipid levels. Besides, quantitative lipidomics and network pharmacology were used to determine how GFSC impacts SONFH. qRT-PCR and Western blotting were performed to assess mRNA and protein expression levels of key targets. ResultsGFSC can enhance bone density and prevents surface collapse of the femoral head, improve hollow bone lacuna density, reduce adipocyte infiltration, minimize osteocyte cavities, regulate the serum lipid levels and enhance lipid metabolism in SONFH rats. Moreover, integrated lipidomics and network pharmacology suggested that GFSC affects lipid metabolism-related genes by regulating nine targets, thereby influencing four metabolites and two metabolic pathways, which may mainly be due to components in GFSC such as quercetin, tanshinone iia, berberine. Western blotting and qRT-PCR data highlighted that GFSC improves lipid metabolism by regulating TP53, HSP90AA1, and ESR1. ConclusionGFSC effectively retards SONFH progression, potentially attributed to its ability to regulate lipid metabolism.

Genetic Analysis of Potential Biomarkers and Therapeutic Targets in Ferroptosis from Steroid-Induced Osteonecrosis of the Femoral Head Based on Machine Learning.

To locate the candidate therapeutic target genes involved in ferroptosis in steroid-induced osteonecrosis of the femoral head (SONFH). Bioinformatics analysis study. Place and Duration of the Study: Department of Orthopaedic Surgery, Zhuhai Hospital of Integrated Traditional Chinese and Western Medicine, Guangdong, China, from March to July 2023. After processing the gene expression omnibus (GEO) data with the R programming language, differentially expressed ferroptosis-related genes in SONFH were identified. To pinpoint the genes most strongly linked to SONFH in association with ferroptosis, least absolute shrinkage and selection operator (LASSO) regression and support vector machine-recursive feature elimination (SVM-RFE) were employed. Subsequently, the screened essential genes were analysed to investigate immune cell infiltration, and competing endogenous RNA (ceRNA) networks involving these marker genes were constructed. The machine learning algorithms identified three genes i.e., SOCS1 (suppressor of cytokine signalling1), MYCN (N-myc proto-oncogene protein), and KLF2 (Kruppel-like factor 2) as diagnostic feature biomarkers associated with ferroptosis. Additionally, CIBERSORT analysis revealed that alterations in the immune microenvironment, such as Macrophages M1, Monocytes, and T cells CD4 naive, could be linked to SOCS1, MYCN, and KLF2. Moreover, the competing endogenous RNA (ceRNA) network exposed a complex regulatory relationship based on marker genes. SOCS1, MYCN, and KLF2 are potential biomarkers associated with ferroptosis in SONFH, pending confirmation in future studies. Steroid-induced osteonecrosis of the femoral head, Ferroptosis, Machine learning, Genetic analysis.

Fibroblast growth factor 23 inhibition attenuates steroid-induced osteonecrosis of the femoral head through pyroptosis

Steroid-induced osteonecrosis of the femoral head (SONFH) is the predominant cause of non-traumatic osteonecrosis of the femoral head (ONFH). Impaired blood supply and reduced osteogenic activity of the femoral head are the key pathogenic mechanisms of SONFH. Fibroblast growth factor 23 (FGF23) levels are not only a biomarker for early vascular lesions caused by abnormal mineral metabolism, but can also act directly on the peripheral vascular system, leading to vascular pathology. The aim of this study was to observe the role of FGF23 on bone microarchitecture and vascular endothelium, and to investigate activation of pyroptosis in SONFH. Lipopolysaccharide (LPS) combined with methylprednisolone (MPS) was applied for SONFH mouse models, and adenovirus was used to increase or decrease the level of FGF23. Micro-CT and histopathological staining were used to observe the structure of the femoral head, and immunohistochemical staining was used to observe the vascular density. The cells were further cultured in vitro and placed in a hypoxic environment for 12 h to simulate the microenvironment of vascular injury during SONFH. The effect of FGF23 on osteogenic differentiation was evaluated using alkaline phosphatase staining, alizarin red S staining and expression of bone formation-related proteins. Matrigel tube formation assay in vitro and immunofluorescence were used to detect the ability of FGF23 to affect endothelial cell angiogenesis. Steroids activated the pyroptosis signaling pathway, promoted the secretion of inflammatory factors in SONFH models, led to vascular endothelial dysfunction and damaged the femoral head structure. In addition, FGF23 inhibited the HUVECs angiogenesis and BMSCs osteogenic differentiation. FGF23 silencing attenuated steroid-induced osteonecrosis of the femoral head by inhibiting the pyroptosis signaling pathway, and promoting osteogenic differentiation of BMSCs and angiogenesis of HUVECs in vitro.

Atorvastatin exerts a preventive effect against steroid-induced necrosis of the femoral head by modulating Wnt5a release.

Steroid-induced osteonecrosis of the femoral head (SONFH) is a prevalent form of osteonecrosis in young individuals. More efficacious clinical strategies must be used to prevent and treat this condition. One of the mechanisms through which SONFH operates is the disruption of normal differentiation in bone marrow adipocytes and osteoblasts due to prolonged and extensive use of glucocorticoids (GCs). In vitro, it was observed that atorvastatin (ATO) effectively suppressed the impact of dexamethasone (DEX) on bone marrow mesenchymal stem cells (BMSCs), specifically by augmenting their lipogenic differentiation while impeding their osteogenic differentiation. To investigate the underlying mechanisms further, we conducted transcriptome sequencing of BMSCs subjected to different treatments, leading to the identification of Wnt5a as a crucial gene regulated by ATO. The analyses showed that ATO exhibited the ability to enhance the expression of Wnt5a and modulate the MAPK pathway while regulating the Wnt canonical signaling pathway via the WNT5A/LRP5 pathway. Our experimental findings provide further evidence that the combined treatment of ATO and DEX effectively mitigates the effects of DEX, resulting in the upregulation of osteogenic genes (Runx2, Alpl, Tnfrsf11b, Ctnnb1, Col1a) and the downregulation of adipogenic genes (Pparg, Cebpb, Lpl), meanwhile leading to the upregulation of Wnt5a expression. So, this study offers valuable insights into the potential mechanism by which ATO can be utilized in the prevention of SONFH, thereby holding significant implications for the prevention and treatment of SONFH in clinical settings.

EGFL6 activates the ERK signaling to improve angiogenesis and osteogenesis of BMSCs in patients with steroid-induced osteonecrosis of the femoral head.

Recently, epidermal growth factor-like domain protein 6 (EGFL6) was proposed as a candidate gene for coupling angiogenesis to osteogenesis during bone repair; however, the exact role and underlying mechanism are largely unknown. Here, using immunohistochemical and Western blotting analyses, we found that EGFL6 was downregulated in the femoral head tissue of patients with steroid-induced osteonecrosis of the femoral head (SONFH) compared to patients with traumatic femoral neck fracture (FNF), accompanied by significantly downregulation of osteogenic and angiogenic marker genes. Then, bone marrow mesenchymal stem cells (BMSCs) were isolated from the FNF and the SONFH patients, respectively, and after identification by immunofluorescence staining surface markers, the effect of EGFL6 on their abilities of osteogenic differentiation and angiogenesis was evaluated. Our results of alizarin red staining and tubular formation experiment revealed that BMSCs from the SONFH patients (SONFH-BMSCs) displayed an obviously weaker ability of osteogenesis than FNF-BMSCs, and EGFL6 overexpression improved the abilities of osteogenic differentiation and angiogenesis of SONFH-BMSCs. Moreover, EGFL6 overexpression activated extracellular signal-regulated kinases 1/2 (ERK1/2). ERK1/2 inhibitor U0126 reversed the promoting effect of EGFL6 overexpression on the expression of osteogenesis and angiogenesis-related genes in the SONFH femoral head. In conclusion, EGFL6 plays a protective role in SONFH, it promotes osteogenesis and angiogenesis of BMSCs, and its effect is likely to be related to ERK1/2 activation.

Epidemiological investigation and diagnostic analysis of osteonecrosis of the femoral head in three northeastern provinces of China

BackgroundIn this retrospective case investigation, we analysed the data of patients with osteonecrosis of the femoral head (ONFH) to reveal demographic and clinical diagnostic features of ONFH in three northeastern provinces of China and provide a reference for its prevention, diagnosis, and treatment.MethodsWe collected data from patients in Beijing Orthopaedic Hospital of Liaoning, focusing on the aetiology and diagnosis of ONFH. Medical records and self-designed questionnaires were used to collect information for statistical analysis, including age, aetiology, reason for glucocorticoid use, hospital level at first visit, and diagnosis.ResultsIn total, 906 patients with complete medical records were included in the analysis. The mean patient age was 47.65 ± 12.12 years. The peak age distribution was in the 40s for men and the 50s for women. Among the total cohort, 72 patients (7.95%; 40 men and 32 women) had traumatic ONFH, 198 (21.85%; 131 men and 67 women) had steroid-induced ONFH, 230 (25.39%; 121 men and 109 women) had idiopathic ONFH, and 406 (44.81%; 397 men and 9 women) had alcohol-induced ONFH. Six hundred and twenty patients were diagnosed with ONFH at the first visit, while 286 patients were misdiagnosed, with a diagnosis rate of 68.43%. The diagnosis rate at the first visit in tertiary hospitals was 76.14%. The diagnosis rate at the first visit in second-class hospitals was 52.07%.ONFH was most likely to be misdiagnosed as lumbar disc herniation.ConclusionsMost patients with ONFH in three northeastern provinces of China were middle-aged, male, and had alcohol-induced ONFH. The misdiagnosis rate of ONFH at the first visit was very high, especially for misdiagnosis of lumbar disc herniation, indicating that the diagnosis of ONFH requires further improvement.

Bioinformatics analysis of PANoptosis regulators in the diagnosis and subtyping of steroid-induced osteonecrosis of the femoral head.

In this study, we aimed to investigate the involvement of PANoptosis, a form of regulated cell death, in the development of steroid-induced osteonecrosis of the femoral head (SONFH). The underlying pathogenesis of PANoptosis in SONFH remains unclear. To address this, we employed bioinformatics approaches to analyze the key genes associated with PANoptosis. Our analysis was based on the GSE123568 dataset, allowing us to investigate both the expression profiles of PANoptosis-related genes (PRGs) and the immune profiles in SONFHallowing us to investigate the expression profiles of PRGs as well as the immune profiles in SONFH. We conducted cluster classification based on PRGs and assessed immune cell infiltration. Additionally, we used the weighted gene co-expression network analysis (WGCNA) algorithm to identify cluster-specific hub genes. Furthermore, we developed an optimal machine learning model to identify the key predictive genes responsible for SONFH progression. We also constructed a nomogram model with high predictive accuracy for assessing risk factors in SONFH patients, and validated the model using external data (area under the curve; AUC = 1.000). Furthermore, we identified potential drug targets for SONFH through the Coremine medical database. Using the optimal machine learning model, we found that 2 PRGs, CASP1 and MLKL, were significantly correlated with the key predictive genes and exhibited higher expression levels in SONFH. Our analysis revealed the existence of 2 distinct PANoptosis molecular subtypes (C1 and C2) within SONFH. Importantly, we observed significant variations in the distribution of immune cells across these subtypes, with C2 displaying higher levels of immune cell infiltration. Gene set variation analysis indicated that C2 was closely associated with multiple immune responses. In conclusion, our study sheds light on the intricate relationship between PANoptosis and SONFH. We successfully developed a risk predictive model for SONFH patients and different SONFH subtypes. These findings enhance our understanding of the pathogenesis of SONFH and offer potential insights into therapeutic strategies.

METTL3 inhibits BMSC apoptosis and facilitates osteonecrosis repair via an m6A-IGF2BP2-dependent mechanism

Hypoxia-induced apoptosis of bone marrow mesenchymal stem cells (BMSCs) limits the efficacy of their transplantation for steroid-induced osteonecrosis of the femoral head (SONFH). As apoptosis and RNA methylation are closely related, exploring the role and mechanism of RNA methylation in hypoxic apoptosis of BMSCs is expected to identify new targets for transplantation of BMSCs for SONFH and enhance transplantation efficacy. We performed methylated RNA immunoprecipitation sequencing (MeRIP-seq) combined with RNA-seq on a hypoxia-induced apoptosis BMSC model and found that the RNA methyltransferase-like 3 (METTL3) is involved in hypoxia-induced BMSC apoptosis. The expression of METTL3 was downregulated in BMSCs after hypoxia and in BMSCs implanted in osteonecrosis areas. Knockdown of METLL3 under normoxic conditions promoted apoptosis of BMSCs. In contrast, overexpression of METTL3 promoted the survival of BMSCs under hypoxic conditions, and overexpression of METTL3 promoted the survival of BMSCs in the osteonecrosis area and the repair of the osteonecrosis area. Regarding the mechanism, the m6A levels of the mRNAs of anti-apoptotic genes Bcl-2, Mcl-1, and BIRC5 were significantly increased upon the overexpression of METTL3 under hypoxic conditions, which promoted the binding of Bcl-2, Mcl-1, and BIRC5 mRNAs to IGF2BP2, enhanced the mRNA stability, and increased the protein expression of the three anti-apoptotic genes. In conclusion, overexpression of METTL3 promoted m6A modification of mRNAs of Bcl-2, Mcl-1, and BIRC5, promoted the binding of IGF2BP2 to the above-mentioned mRNAs, enhanced mRNA stability, inhibited hypoxia-induced BMSC apoptosis, and promoted repair of SONFH, thereby providing novel targets for transplantation of BMSCs for SONFH.

Human umbilical cord mesenchymal stem cells promote steroid-induced osteonecrosis of the femoral head repair by improving microvascular endothelial cell function.

Recently, there has been growing interest in using cell therapy through core decompression (CD) to treat osteonecrosis of the femoral head (ONFH). Our study aimed to investigate the effectiveness and mechanism of human umbilical cord mesenchymal stem cells (hUCMSCs) in treating steroid-induced ONFH. We constructed a steroid-induced ONFH rabbit model as well as dexamethasone (Dex)-treated bone microvascular endothelial cells (BMECs) model of human femoral head. We injected hUCMSCs into the rabbit femoral head via CD. The effects of hUCMSCs on steroid-induced ONFH rabbit model and Dex-treated BMECs were evaluated via micro-CT, microangiography, histology, immunohistochemistry, wound healing, tube formation, and western blotting assay. Furthermore, we conducted single-cell RNA sequencing (scRNA-seq) to examine the characteristics of endothelial cells, the activation of signaling pathways, and inter-cellular communication in ONFH. Our data reveal that hUCMSCs improved the femoral head microstructure and bone repair and promoted angiogenesis in the steroid-induced ONFH rabbit model. Importantly, hUCMSCs improved the migration ability and angioplasty of Dex-treated BMECs by secreting COL6A2 to activate FAK/PI3K/AKT signaling pathway via integrin α1β1.

Exosomes derived from M2 macrophages prevent steroid-induced osteonecrosis of the femoral head by modulating inflammation, promoting bone formation and inhibiting bone resorption

Inflammatory reactions are involved in the development of steroid-induced osteonecrosis of the femoral head(ONFH). Studies have explored the therapeutic efficacy of inhibiting inflammatory reactions in steroid-induced ONFH and revealed that inhibiting inflammation may be a new strategy for preventing the development of steroid-induced ONFH. Exosomes derived from M2 macrophages(M2-Exos) display anti-inflammatory properties. This study aimed to examine the preventive effect of M2-Exos on early-stage steroid-induced ONFH and explore the underlying mechanisms involved. In vitro, we explored the effect of M2-Exos on the proliferation and osteogenic differentiation of bone marrow-derived mesenchymal stem cells(BMMSCs). In vivo, we investigated the role of M2-Exos on inflammation, osteoclastogenesis, osteogenesis and angiogenesis in an early-stage rat model of steroid-induced ONFH. We found that M2-Exos promoted the proliferation and osteogenic differentiation of BMMSCs. Additionally, M2-Exos effectively attenuated the osteonecrotic changes, inhibited the expression of proinflammatory mediators, promoted osteogenesis and angiogenesis, reduced osteoclastogenesis, and regulated the polarization of M1/M2 macrophages in steroid-induced ONFH. Taken together, our data suggest that M2-Exos are effective at preventing steroid-induced ONFH. These findings may be helpful for providing a potential strategy to prevent the development of steroid-induced ONFH.

TRIM21 silencing inhibits the apoptosis and expedites the osteogenic differentiation of dexamethasone‑induced MC3T3‑E1 cells by activating the Keap1/Nrf2 pathway.

Steroid-induced osteonecrosis of the femoral head (ONFH) is a serious complication caused by long-term or excessive use of glucocorticoids. The present study aimed to ascertain the effects of tripartite motif-containing protein 21 (TRIM21) on the process of steroid-induced ONFH and its hidden action mechanism. TRIM21 expression in dexamethasone (Dex)-treated mouse MC3T3-E1 preosteoblast cells was examined using reverse transcription-quantitative PCR and western blotting. The Cell Counting Kit-8 (CCK-8) method and lactate dehydrogenase release assay were used to respectively measure cell viability and injury. Flow cytometry analysis was used to assay cell apoptosis. Caspase 3 activity was evaluated using a specific assay, while alkaline phosphatase and Alizarin red S staining were used to evaluate osteogenesis. 2,7-dichloro-dihydrofluorescein diacetate fluorescence probe was used to estimate reactive oxygen species generation. Specific assay kits were used to appraise oxidative stress levels. In addition, the expression of apoptosis-, osteogenic differentiation- and Kelch-like ECH-associated protein 1 (Keap1)/nuclear factor erythroid 2-related factor 2 (Nrf2) signaling-associated proteins was assessed using western blotting. In Nrf2 inhibitor (ML385)-pretreated MC3T3-E1 cells exposed to Dex, cell apoptosis, osteogenesis and oxidative stress were detected again as aforementioned. Results revealed that TRIM21 expression was raised in Dex-induced MC3T3-E1 cells and TRIM21 deletion improved the viability and osteogenic differentiation, whereas it hampered the oxidative stress and apoptosis in MC3T3-E1 cells with Dex induction. In addition, silencing of TRIM21 activated Keap1/Nrf2 signaling. Moreover, ML385 partially abrogated the effects of TRIM21 depletion on the oxidative stress, apoptosis and osteogenic differentiation in MC3T3-E1 cells exposed to Dex. In conclusion, TRIM21 silencing might activate Keap1/Nrf2 signaling to protect against steroid-induced ONFH.

Elucidating the role of the GC/GR/GLUT1 axis in steroid-induced osteonecrosis of the femoral head: A proteomic approach

BackgroundSteroid-induced osteonecrosis of the femoral head (SONFH) is a prevalent and incapacitating condition that affects the hip joint. Unfortunately, early diagnostic and treatment measures are limited. MethodsOur study employed Tandem Mass Tag (TMT) labeling mass spectrometry (MS)-based quantitative proteome to compare the proteins of femoral head tissues in patients with SONFH with those of patients who sustained femoral neck fracture (FNF). We investigated the level and effects of glucose transporter member 1 (GLUT1) in SONFH patients and MC3T3-E1 cells and examined the function and molecular mechanism of GLUT1 in the context of SONFH using in vivo and in vitro approaches. ResultsThe SONFH group exhibited significant changes in protein expression levels compared to the fracture group. Specifically, we observed the up-regulation of 86 proteins and the down-regulation of 138 proteins in the SONFH group. Among the differentially expressed proteins, GLUT1 was down-regulated and associated with glucose metabolic processes in the SONFH group. Further analysis using Parallel Reaction Monitoring (PRM), WB, and PCR confirmed that the protein was significantly down-regulated in both femoral head tissue samples from SONFH patients and dexamethasone-treated MC3T3-E1 cells. Moreover, overexpression of GLUT1 effectively reduced glucocorticoid (GC)-induced apoptosis and the suppression of osteoblast proliferation and osteogenic differentiation in MC3T3-E1 cells, as well as GC-induced femoral head destruction in GC-induced ONFH rat models. Additionally, our research demonstrated that GC down-regulated GLUT1 transcription via glucocorticoid receptors in MC3T3-E1 cells. ConclusionsGLUT1 was down-regulated in patients with SONFH; furthermore, down-regulated GLUT1 promoted apoptosis and inhibited osteoblast ossification in dexamethasone-induced MC3T3-E1 cells and contributed to GC-induced femoral head destruction in a SONFH rat model. Glucocorticoids inhibited the transcriptional activity of GLUT1, leading to a reduction in the amount and activity of GLUT1 in the cells and ultimately promoting apoptosis and inhibiting osteoblast ossification via the GC/GR/GLUT1 axis in SONFH.

Gene expression profiling of peripheral blood in patients with steroid-induced osteonecrosis of the femoral head.

Aim: Steroid-induced osteonecrosis of the femoral head (SONFH) is a severe complication following glucocorticoid therapy. This study aimed to identify the differential mRNA expression and investigate the molecular mechanisms of SONFH. Materials & methods: RNA sequencing was performed in eight SONFH patients, five non-SONFH patients and five healthy individuals. Results: Atotal of 1555, 3997 and 5276 differentially expressed mRNAs existed between the following combinations: SONFH versus non-SONFH, SONFH versus healthy subjects and non-SONFH versus healthy subjects. Increased ISM1 expression might contribute to a high risk of SONFH through antiangiogenesis. Decreased FOLR3 expression might affect the metabolism of homocysteine, leading to avascular necrosis of the femoral head. KCNJ2, which plays a pivotal role in regulating bone development, was also deregulated. Conclusion: ISM1, FOLR3 and KCNJ2 might be related to the occurrence of SONFH.

HIF-1α in cartilage homeostasis, apoptosis, and glycolysis in mice with steroid-induced osteonecrosis of the femoral head.

With the prevalence of coronavirus disease 2019, the administration of glucocorticoids (GCs) has become more widespread. Treatment with high-dose GCs leads to a variety of problems, of which steroid-induced osteonecrosis of the femoral head (SONFH) is the most concerning. Since hypoxia-inducible factor 1α (HIF-1α) is a key factor in cartilage development and homeostasis, it may play an important role in the development of SONFH. In this study, SONFH models were established using methylprednisolone (MPS) in mouse and its proliferating chondrocytes to investigate the role of HIF-1α in cartilage differentiation, extracellular matrix (ECM) homeostasis, apoptosis and glycolysis in SONFH mice. The results showed that MPS successfully induced SONFH in vivo and vitro, and MPS-treated cartilage and chondrocytes demonstrated disturbed ECM homeostasis, significantly increased chondrocyte apoptosis rate and glycolysis level. However, compared with normal mice, not only the expression of genes related to collagens and glycolysis, but also chondrocyte apoptosis did not demonstrate significant differences in mice co-treated with MPS and HIF-1α inhibitor. And the effects observed in HIF-1α activator-treated chondrocytes were similar to those induced by MPS. And HIF-1α degraded collagens in cartilage by upregulating its downstream target genes matrix metalloproteinases. The results of activator/inhibitor of endoplasmic reticulum stress (ERS) pathway revealed that the high apoptosis rate induced by MPS was related to the ERS pathway, which was also affected by HIF-1α. Furthermore, HIF-1α affected glucose metabolism in cartilage by increasing the expression of glycolysis-related genes. In conclusion, HIF-1α plays a vital role in the pathogenesis of SONFH by regulating ECM homeostasis, chondrocyte apoptosis, and glycolysis.

Transcriptome data mining combined with clinical and animal experiments for identification of syndrome element marker genes during entire course of steroid-induced osteonecrosis of femoral head

A research strategy combining transcriptome data mining and experimental verification was adopted to identify the marker genes characterizing the syndrome elements of phlegm, stasis, and deficiency in steroid-induced osteonecrosis of the femoral head(SONFH). Firstly, the common differentially expressed gene sets of SONFH with the syndromes of phlegm-stasis obstructing collaterals, vessel obstruction, and liver-kidney deficiency were obtained from the clinical transcriptomic analysis of a previous study. The differential expression trend analysis and functional gene mining were then employed to predict the candidate marker gene sets representing phlegm, stasis, and deficiency. The whole blood samples from SONFH patients, whole blood samples from SONFH rats, and affected femoral head tissue samples were collected for qPCR, which aimed to determine the expression levels of the candidate marker genes mentioned above. Furthermore, the receiver operating characteristic curve(ROC) was established to objectively evaluate the syndrome differentiation effectiveness of the candidate marker genes mentioned above. The transcriptome data analysis results showed that the candidate marker genes for phlegm was ELOVL fatty acid elongase 6(ELOVL6), and those for stasis were ankyrin 1(ANK1), glycophorin A/B(GYPA/B), and Rh-associated glycoprotein(RHAG). The candidate marker genes for deficiency were solute carrier family 2 member 1(SLC2A1) and stomatin(STOM). The qPCR results showed that compared with that in the non-SONFH group, ELOVL6 had the lowest expression level in the peripheral blood of the SONFH patients with the syndrome of phlegm-stasis obstructing collaterals(P<0.05). Compared with that in the normal control group, ELOVL6 had the lowest expression level in the peripheral blood and affected femoral head tissue of SONFH rats modeled for 4 weeks(P<0.01), and it showed better syndrome differentiation effectiveness of rats modeled for 4 weeks(AUC=0.850, P=0.006) than at other modeling time points(8, 12, 16, and 21 weeks, AUC of 0.689, 0.766, 0.588, and 0.662, respectively). Compared with that in the non-SONFH group, the expression levels of ANK1, GYPA, and RHAG were the lowest in the peripheral blood of SONFH patients with the vessel obstruction syndrome(P<0.05). The expression levels of the three genes were the lowest in the peripheral blood and affected femoral head tissue of SONFH rats modeled for 12 weeks(P<0.05, P<0.01), and their syndrome differentiation effectiveness in the rats modeled for 12 weeks(GYPA: AUC=0.861, P=0.012; ANK1: AUC=0.855, P=0.006; RHAG: AUC=0.854, P=0.009) was superior to that for 4, 8, 16, and 21 weeks(GYPA: AUC=0.646, 0.573, 0.691, and 0.617, respectively; ANK: AUC1=0.630, 0.658, 0.657, and 0.585, respectively; RHAG: AUC=0.592, 0.511, 0.515, and 0.536, respectively). Compared with the non-SONFH group, both SLC2A1 and STOM had the lowest expression levels in the peripheral blood of patients with the syndrome of liver and kidney deficiency(P<0.05). Compared with the normal control group, their expression levels were the lowest in the peripheral blood and affected femoral head tissue of SONFH rats modeled for 21 weeks(P<0.05, except STOM in the peripheral blood of rats). Moreover, the syndrome differentiation effectiveness of SLC2A1 in the rats modeled for 21 weeks(AUC=0.806, P=0.009) was superior to that for 4, 8, 12, and 16 weeks(AUC=0.520, 0.580, 0.741, 0.774, respectively), and STOM was meaningless in syndrome differentiation. In summary, the candidate marker gene for phlegm in SONFH is ELOVL6; the candidate marker genes for stasis are GYPA, RHAG, and ANK1; the candidate marker gene for deficiency is SLC2A1. The results help to reveal the biological connotations of phlegm, stasis, and deficiency in SONFH at the genetic level.

Mechanism of KDM5A-mediated H3K4me3 modification in the osteogenic differentiation of mesenchymal stem cells in steroid-induced osteonecrosis of the femoral head.

Steroid-induced osteonecrosis of the femoral head (SONFH) is characterized by impaired osteogenesis in bone marrow mesenchymal stem cells (BMSCs). This study investigates the role of lysine-specific demethylase 5A (KDM5A) in SONFH to identify potential therapeutic targets. Human BMSCs were isolated and characterized for cell surface markers and differentiation capacity. A SONFH cell model was established using dexamethasone treatment. BMSCs were transfected with KDM5A overexpression vectors or si-KDM5A, and the expression of KDM5A, miR-107, runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), and osteopontin (OPN) was assessed. Alizarin red staining was used to observe mineralization nodules, while alkaline phosphatase activity and cell viability were measured. The enrichment of KDM5A and histone 3 lysine 4 trimethylation (H3K4me3) on the promoters of RUNX2, OCN, and OPN was analyzed. The binding between miR-107 and KDM5A 3'UTR was validated, and the combined effect of miR-107 overexpression and KDM5A overexpression on BMSC osteogenic differentiation was evaluated. KDM5A was upregulated in BMSCs from SONFH. Inhibition of KDM5A promoted osteogenic differentiation of BMSCs, associated with increased RUNX2, OCN, and OPN promoters. KDM5A bound to the promoters of RUNX2, OCN, and OPN, leading to reduced H3K4me3 levels and downregulation of their expression. Overexpression of miR-107 inhibited KDM5A and enhanced BMSC osteogenic differentiation. KDM5A negatively regulates BMSC osteogenic differentiation by modulating H3K4me3 levels on the promoters of key osteogenic genes. miR-107 overexpression counteracts the inhibitory effect of KDM5A on osteogenic differentiation. These findings highlight the potential of targeting the KDM5A/miR-107 axis for SONFH therapy.