Identification and analysis of mitochondria-related central genes in steroid-induced osteonecrosis of the femoral head, along with drug prediction.

Purpose: Steroid-induced osteonecrosis of the femoral head (SONFH) is a refractory orthopedic hip joint disease that primarily affects middle-aged and young individuals. SONFH may be caused by ischemia and hypoxia of the femoral head, where mitochondria play a crucial role in oxidative reactions. Currently, there is limited literature on whether mitochondria are involved in the progression of SONFH. Here, we aim to identify and validate key potential mitochondrial-related genes in SONFH through bioinformatics analysis. This study aims to provide initial evidence that mitochondria play a role in the progression of SONFH and further elucidate the mechanisms of mitochondria in SONFH. Methods: The GSE123568 mRNA expression profile dataset includes 10 non-SONFH (non-steroid-induced osteonecrosis of the femoral head) samples and 30 SONFH samples. The GSE74089 mRNA expression profile dataset includes 4 healthy samples and 4 samples with ischemic necrosis of the femoral head. Both datasets were downloaded from the Gene Expression Omnibus (GEO) database. The mitochondrial-related genes are derived from MitoCarta3.0, which includes data for all 1136 human genes with high confidence in mitochondrial localization based on integrated proteomics, computational, and microscopy approaches. By intersecting the GSE123568 and GSE74089 datasets with a set of mitochondrial-related genes, we screened for mitochondrial-related genes involved in SONFH. Subsequently, we used the good Samples Genes method in R language to remove outlier genes and samples in the GSE123568 dataset. We further used WGCNA to construct a scale-free co-expression network and selected the hub gene set with the highest connectivity. We then intersected this gene set with the previously identified mitochondrial-related genes to select the genes with the highest correlation. A total of 7 mitochondrial-related genes were selected. Next, we performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis on the selected mitochondrial-related genes using R software. Furthermore, we performed protein network analysis on the differentially expressed proteins encoded by the mitochondrial genes using STRING. We used the GSEA software to group the genes within the gene set in the GSE123568 dataset based on their coordinated changes and evaluate their impact on phenotype changes. Subsequently, we grouped the samples based on the 7 selected mitochondrial-related genes using R software and observed the differences in immune cell infiltration between the groups. Finally, we evaluated the prognostic significance of these features in the two datasets, consisting of a total of 48 samples, by integrating disease status and the 7 gene features using the cox method in the survival R package. We performed ROC analysis using the roc function in the pROC package and evaluated the AUC and confidence intervals using the ci function to obtain the final AUC results. Results: Identification and analysis of 7 intersecting DEGs (differentially expressed genes) were obtained among peripheral blood, cartilage samples, hub genes, and mitochondrial-related genes. These 7 DEGs include FTH1, LACTB, PDK3, RAB5IF, SOD2, and SQOR, all of which are upregulated genes with no intersection in the downregulated gene set. Subsequently, GO and KEGG pathway enrichment analysis revealed that the upregulated DEGs are primarily involved in processes such as oxidative stress, release of cytochrome C from mitochondria, negative regulation of intrinsic apoptotic signaling pathway, cell apoptosis, mitochondrial metabolism, p53 signaling pathway, and NK cell-mediated cytotoxicity. GSEA also revealed enriched pathways associated with hub genes. Finally, the diagnostic value of these key genes for hormone-related ischemic necrosis of the femoral head (SONFH) was confirmed using ROC curves. Conclusion: BID, FTH1, LACTB, PDK3, RAB5IF, SOD2, and SQOR may serve as potential diagnostic mitochondrial-related biomarkers for SONFH. Additionally, they hold research value in investigating the involvement of mitochondria in the pathogenesis of ischemic necrosis of the femoral head.

Neohesperidin promotes the proliferation and osteogenic differentiation of BMSCs via BMP2-Wnt/ß-catenin pathway.

The present study aimed to investigate the role of neohesperidin (NH) in mice with steroid-induced femoral head necrosis (SONFH) and in bone marrow stromal cells (BMSCs). The SONFH model was established. The effects of NH on SONFH mice were detected by hematoxylin-eosin (HE) staining and micro-CT, while those on proliferation, osteogenic differentiation and associated pathways of BMSCs were detected by molecular experiments. Besides, the effects of NH on ß-catenin nuclear translocation and the H3K27me3 abundance on the transcriptional start site of Bone Morphogenetic Protein 2 (BMP2) were also determined by immunofluorescence staining and Chromatin Immunoprecipitation. Results indicated that NH not only reduced histopathological changes and improved the structures of the femoral heads of the SONFH mice but also promoted the proliferation and osteogenic differentiation of mouse BMSCs, enhanced alkaline phosphatase (ALP) activity, and upregulated expressions of osteoblast markers in a dose-dependent manner. Moreover, NH was also confirmed to upregulate the expressions of genes related to osteogenesis and Wnt/ß-catenin pathway of BMSCs, which, however, were all noticeably downregulated by Noggin and DKK1. Additionally, Noggin and DKK1 in combination further promoted the suppressive effect on genes related to osteogenesis and Wnt/ß-catenin pathway than alone. Besides, NH induced nuclear translocation of ß-catenin in BMSCs and further reduced H3K27me3-triggered enrichment of BMP2. In conclusion, NH could promote proliferation and osteogenic differentiation of BMSCs via BMP2-Wnt/ß-catenin pathway.

Early detection of steroid-induced femoral head necrosis using 99mTc-Cys-Annexin V-based apoptosis imaging in a rabbit model.

BACKGROUND: At present, the early diagnosis of femoral head necrosis mainly relies on Magnetic resonance imaging (MRI), and most early patients are difficult to make an accurate diagnosis. Therefore, to investigate the early diagnostic value of 99mTc-Cys-Annexin V Single-photon emission computed tomography (SPECT) imaging were compared with MRI in rabbit models of steroid-induced femoral head necrosis. METHODS: The animal model of steroid-induced femoral head necrosis (SIFHN) was established in 5-month-old healthy New Zealand white rabbits by injecting horse serum into ear vein and methylprednisolone into gluteal muscle, the purpose of modeling is to simulate the actual clinical situation of SIFNH. 99mTc-Cys-Annexin V SPECT imaging and MRI were performed at 2nd week, 4th week, and 6th week after modeling. After that, histopathology was used to verify the success of modeling. Apoptosis was detected by transmission electron microscopy (TEM) and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL). RESULTS: At 2 weeks after the injection of hormone, 99mTc-Cys-Annexin V SPECT image showed abnormal radioactive uptake in the bilateral femoral head. And over time, the radioactivity concentration was more obvious, and the ratio of T/NT (target tissue/non-target tissues, which is the ratio of femoral head and the ipsilateral femoral shaft) was gradually increased. In the 99mTc-Cys-Annexin V SPECT imaging at each time point, T/NT ratio of the model group was significantly higher than that of the control group (P < 0.01); at 4 weeks after the injection of hormone, MRI showed an abnormal signal of osteonecrosis. At 2, 4, and 6 weeks after hormone injection, apoptosis was observed by TUNEL and TEM. CONCLUSIONS: 99mTc-Cys-Annexin V SPECT imaging can diagnose steroid-induced femoral head necrosis earlier than MRI, and has potential application value for non-invasively detecting early and even ultra-early stage of femoral head necrosis.

Osteogenic Effect of tsRNA-10277-Loaded Exosome Derived from Bone Mesenchymal Stem Cells on Steroid-Induced Osteonecrosis of the Femoral Head.

PURPOSE: Steroids are known to inhibit osteogenic differentiation and subsequent bone formation in bone mesenchymal stem cells (BMSCs). However, little is known about the role of BMSC exosomes (Exos) and tRNA-derived small RNAs (tsRNAs) in steroid-induced osteonecrosis of the femoral head (SONFH). The objective of this study was to characterize the tsRNA expression profiles of plasma Exos collected from SONFH patients and healthy individuals using small RNA sequencing and further explore the effect of BMSC Exos carrying specific tsRNAs on osteogenic differentiation. MATERIALS AND METHODS: Based on insights from small RNA sequencing, five differentially expressed (DE) tsRNAs were selected for quantitative real-time polymerase chain reaction (qRT-PCR). The regulatory networks associated with interactions of the tsRNAs-mRNA-pathways were reconstructed. The osteogenesis and adipogenesis in BMSCs were detected via ALP and oil red O staining methods, respectively. RESULTS: A total of 345 DE small RNAs were screened, including 223 DE tsRNAs. The DE tsRNAs were enriched in Wnt signaling pathway and osteogenic differentiation. We identified five DE tsRNAs, among which tsRNA-10277 was significantly downregulated in plasma Exos of SONFH patients compared to that in healthy individuals. Dexamethasone-induced BMSCs were associated with an increased fraction of lipid droplets and decreased osteogenic differentiation, whereas BMSC Exos restored the osteogenic differentiation of that. After treatment of tsRNA-10277-loaded BMSC Exos, the lipid droplets and osteogenic differentiation ability were found to be decreased and enhanced in dexamethasone-induced BMSCs, respectively. CONCLUSION: An altered tsRNA profile might be involved in the pathophysiology of SONFH. tsRNA-10277-loaded BMSC Exos enhanced osteogenic differentiation ability of dexamethasone-induced BMSCs. Our results provide novel insights into the osteogenic effect of BMSC Exos carrying specific tsRNAs on SONFH.

Osteogenic effect of bone marrow mesenchymal stem cell-derived exosomes on steroid-induced osteonecrosis of the femoral head.

BACKGROUND: Animal studies have demonstrated the therapeutic effect of mesenchymal stem cells (MSCs) on osteogenesis, but little is known about the functions of exosomes (Exos) released by bone MSCs (BMSCs). Here, we investigated the effect of BMSC Exos on steroid-induced femoral head necrosis (SFHN) and explored the vital genes involved in this process. MATERIALS AND METHODS: BMSCs were isolated from healthy and SFHN rats. BMSC Exos were isolated using the Exosome Precipitation Kit and characterized by transmission electron microscopy and Western blotting. SFHN BMSCs were incubated with Exos from healthy BMSCs. Osteogenic ability was assessed by oil red O staining and alizarine red staining. Differentially expressed genes (DEGs) induced by Exos were screened using the Osteogenesis RT2 Profiler PCR Array. The effect of upregulated Sox9 was examined using lentivirus-mediated siRNA. RESULTS: The results revealed that BMSC Exos were 100-150 nm in size and expressed CD63. Moreover, BMSC Exo-treated SFHN cells exhibited suppressed adipogenesis compared to model cells. PCR array showed that eleven and nine genes were upregulated and downregulated, respectively, in the BMSC Exo-treated SFHN cells compared to the model group. Among the DEGs, osteogenesis-related genes, including Bmp2, Bmp6, Bmpr1b, Mmp9, and Sox9, may play important roles in SFHN. Furthermore, the DEGs were mainly involved in immune response, osteoblast differentiation, and in the transforming growth factor-ß/bone morphogenetic protein signaling pathway. The level of the SOX9 protein was upregulated by Exos, and Sox9 silencing significantly decreased the osteogenic effect of BMSC Exos. CONCLUSION: Our data suggest that Exos derived from BMSCs mainly affect SFHN osteogenesis, and this finding can be further investigated to develop a novel therapeutic agent for SFHN.

Comparative experimental study of different administration methods for steroid-induced femoral head nec-rosis in rabbits / 医学研究生学报

Objective The method of administration is one of the important factors influencing steroid-induced femoral head necrosis ( FHN) .From the perspective of administration compliance, time and persons needed, and pharmacodynamics, this study compared different administration methods for steroid-induced FHN in rabbits aiming to provide some experimental evidence for selec-ting correct methods of administration. Methods The steroid-induced femoral head necrosis rabbits ( New Zealand, male) were ran-domly divided into five groups according to the different administration methods of intervention:deep oral administration group (n=10), in-tragastric gavage administration group ( n =10 ) , free-drinking drug group (n=10) and model control group (n=5), blank control group (n=5), the administration compliance, administration time, mortal-ity, pharmacodynamic index of lipid content and empty lacunae rate were compared among deep oral administration group, intragastric ga-vage administration group, free-drinking drug group. Results Compliance effect size of deep oral administration group ( 1.78 ± 0.64) lower than intragastric gavage administration group (4.04 ±0.87) and free-drinking drug group (8.94 ±1.05) (P<0.01). Administration time among deep oral administration group ([0.94 ±0.02]min), intragastric gavage administration group ([9.47 ± 0.31]min) and free-drinking drug group ([889.50 ±235.38]min) overall comparison gradually increased (P=0.000), the differ-ence between the two groups was statistically significant (P<0.05).At the 2 and 4 weeks, cholesterol, triglycerides and low-density lipoprotein of deep oral administration group, intragastric gavage administration group, free-drinking drug group compared with model control group and blank control group the difference was statistically significant ( P<0.05) , and deep oral administration group com-pared with free-drinking drug group, the difference was also statistically significant ( P<0.05) .At the 2 weeks, empty lacunae rate of deep oral administration group ([15.44 ±2.68]%), intragastric gavage administration group ([15.02 ±3.34])%), free-drinking drug group ([16.72 ±4.06]%) compared with model control group ([18.59 ±3.12]%) and blank control group ([10.82 ± 2.76]%, the difference was statistically significant (P<0.05).At the 4 weeks, empty lacunae rate of deep oral administration group ([18.53 ±3.26]%), intragastric gavage administration group ([18.85 ±3.17]%), free-drinking drug group ([20.41 ±4.18]%) compared with model control group ([24.66 ±3.74]%) and blank control group ([11.37 ±2.23]%), the difference was also sta-tistically significant ( P<0.05 ) . Conclusion Compared with traditional methods of administration, deep oral administration has better compliance, shorter administration time, and similar to intragastric gavage administration in pharmacodynamics, but more effec-tive than free-drinking drug administration, and it is a new and effective method of administration.