Steroid-induced Osteonecrosis Of Femoral Head Research Articles

Endothelial Cell-Derived Exosomes Inhibit Osteoblast Apoptosis and Steroid-Induced Necrosis of Femoral Head Progression by Activating the PI3K/Akt/Bcl-2 Pathway

The aim of the study was to investigate the therapeutic potential of exosomes secreted by endothelial cells (EC-exos) on steroid-induced osteonecrosis of femoral head (SNFH). First, we successfully obtained EC-exos through differential centrifugation. Then, the effects of EC-exos on mouse embryo osteoblast precursor (MC3T3-E1) cells under high concentration of dexamethasone (Dex) were analysed in vitro, which included cell migration, viability, and apoptosis. In vivo, a SNFH rat model was successfully established and treated with EC-exos. Micro-computed tomography (micro-CT) and haematoxylin and eosin (H&E) were used to observe femoral trabeculae. Our in vitro results showed that EC-exos improved cell viability and migration of osteoblasts and reduced the apoptotic effect of high concentration of Dex on osteoblasts in vitro. Phosphoinositide 3-kinase (PI3K)/Akt/Bcl-2 signalling pathway was activated in MC3T3-E1 cells under the response to EC-exos. In vivo, increased bone volume per tissue volume (BV/TV) p=0.031, trabecular thickness (Tb.Th) p=0.020, and decreased separation (Tb.Sp) p=0.040 were observed in SNFH rats treated with EC-exos. H&E staining revealed fewer empty lacunae and pyknotic osteocytes in trabeculae. The expression of Bcl-2 and Akt in EC-exos group was significantly increased in trabeculae tissue. Overall, our finding indicated that EC-exos could attenuate SNFH by inhibiting osteoblast apoptosis via the PI3K/Akt/Bcl-2 pathway.

[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.

Dexamethasone Induced Osteocyte Apoptosis in Steroid-Induced Femoral Head Osteonecrosis through ROS-Mediated Oxidative Stress.

Glucocorticoid (GC) overuse is strongly associated with steroid-induced osteonecrosis of the femoral head (SINFH). However, the underlying mechanism of SINFH remains unclear. This study aims to investigate the effect of dexamethasone (Dex)-induced oxidative stress on osteocyte apoptosis and the underlying mechanisms. Ten patients with SINFH and 10 patients with developmental dysplasia of the hips (DDH) were enrolled in our study. Sixty rats were randomly assigned to the Control, Dex, Dex + N-Acetyl-L-cysteine (NAC), Dex + Dibenziodolium chloride (DPI), NAC, and DPI groups. Magnetic resonance imaging (MRI) was used to examine edema in the femoral head of rats. Histopathological staining was performed to assess osteonecrosis. Immunofluorescence staining with TUNEL and 8-OHdG was conducted to evaluate osteocyte apoptosis and oxidative damage. Immunohistochemical staining was carried out to detect the expression of NOX1, NOX2, and NOX4. Viability and apoptosis of MLO-Y4 cells were measured using the CCK-8 assay and TUNEL staining. 8-OHdG staining was conducted to detect oxidative stress. 2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) staining was performed to measure reactive oxygen species (ROS). The expression of NOX1, NOX2, and NOX4 in MLO-Y4 cells was analyzed by Western blotting. Multiple comparisons were performed using one-way analysis of variance (ANOVA). In patients and the rat model, hematoxylin-eosin (HE) staining revealed a significantly higher rate of empty lacunae in the SINFH group than in the DDH group. Immunofluorescence staining indicated a significant increase in TUNEL-positive cells and 8-OHdG-positive cells in the SINFH group compared to the DDH group. Immunohistochemical staining demonstrated a significant increase in the expression of NOX1, NOX2, and NOX4 proteins in SINFH patients compared to DDH patients. Moreover, immunohistochemical staining showed a significant increase in the proportion of NOX2-positive cells compared to the Control group in the femoral head of rats. In vitro, Dex significantly inhibited the viability of osteocyte cells and induced apoptosis. After Dex treatment, the intracellular ROS level increased. However, Dex treatment did not alter the expression of NOX proteins in vitro. Additionally, NAC and DPI inhibited the generation of intracellular ROS and partially alleviated osteocyte apoptosis in vivo and in vitro. This study demonstrates that GC promotes apoptosis of osteocyte cells through ROS-induced oxidative stress. Furthermore, we found that the increased expression of NOXs induced by GC serves as an important source of ROS generation.

Modified osteochondral autograft transplantation for steroid-induced osteonecrosis of femoral head in idiopathic thrombocytopenic purpura: a case report and literature

Osteochondral autograft transplantation (OAT) has been commonly applied in the knee and ankle while the technique has not yet been a popularity in the femoral head. In this article, we present a 28-year-old female patient, who has a history of 1-year-use of glucocorticoid in the treatment of idiopathic thrombocytopenic purpura, with steroid-induced osteonecrosis of the femoral head (SONFH). She underwent surgical hip dislocation, osteochondroplasty, OAT, and internal fixation. Her Harris Hip Score improved from 64 to 82 in 36 months to follow-up. The case is valuable considering that a single, instead of several, 1.5 cm autograft was harvested from the non-bearing part of the same femoral head. This modification dispensed with the need of surgery for harvesting autograft from knee or ankle and reduced the structural vulnerability brought by the multihole donor part of the femoral head.

The protective role of vitamins C and E in steroid-induced femoral head osteonecrosis: An experimental study in rats.

This study aimed to determine whether vitamin C (VC) and vitamin E (VE) can effectively protect the femoral head and reduce the risk of developing osteonecrosis in rats that have been treated with steroids. The study was conducted on 30 young adult male Sprague-Dawley rats (mean weight: 356±18 g; range, 330 to 375 g), which were randomly assigned to one of five groups. The control group received saline solution, while the other groups were given lipopolysaccharide/methylprednisolone (LPS/MPS) to induce osteonecrosis. Three groups in which osteonecrosis was induced were also intraperitoneally administered either VC, VE, or both once a day for four weeks. Intracardiac blood samples were taken at the end of the fourth week for biochemical examination, and the rats were then sacrificed under general anesthesia. After sacrification, right femurs were removed for histopathological, immunohistochemical, and radiologic examinations. The results showed that the mean trabecular number increased significantly in the VC+VE group. There was a substantial decrease observed in the mean trabecular separation within the LPS/MPS group compared to the control group, although trabecular separation decreased in all three vitamin groups compared to the LPS/MPS group. The surface area/bone volume was significantly increased in the VC+VE group compared to the LPS/MPS group. Histological, immunohistochemical, and radiological examinations showed that the administration of VC and VE significantly reduced oxidative stress, inflammation, and microvascular dysfunction in rats with steroid-induced femoral head osteonecrosis. This study suggests that VC, VE, and particularly VC+VE have a protective effect on the femoral head in rats with steroid-induced femoral head osteonecrosis. These findings may lead to new treatment options for patients.

Natural drug asiaticoside inhibits osteoclast differentiation and promotes repair of steroid-induced osteonecrosis of femoral head via RANKL pathway

Asiaticoside is one of the most classic traditional Chinese medicines. The interaction between Receptor activator of nuclear factor κB (RANK) and Receptor Activator of Nuclear Factor-κ B Ligand (RANKL) activates a series of intracellular signaling pathways. Our research explored the mechanism of asiaticoside inhibiting osteoclast differentiation and repair of steroid-induced osteonecrosis of femoral head (SIONFH) through RANKL pathway. Asiaticoside’s effect on osteoclasts was determined by Microneedle Therapy System (MTS) method, and the number of Tartrate resistant acid phosphatase (TRAcP) multinucleated cells (3 nuclei) was quantitatively analyzed. Under different dose treatments, the changes of bone function were detected by eroded area and TRAcP staining on hydroxyapatite-coated plate, and the expression of different osteocyte formation and osteogenic differentiation were detected. Asiaticoside weakened the osteoclast function induced by RANKL. When the concentration of asiaticoside was 10 μmol/L, osteoclast formation regulatory genes (NFATc1 and Acp5) and osteoclast cell function-related genes (AP-1 and P65) were significantly downregulated in the presence of RANKL. Asiaticoside (10 μmol/L) significantly decreased calcium oscillations induced by RANKL via preventing RANKL-mediated NF-κB activation and Ca2+ oscillation.

Dragon blood resin ameliorates steroid-induced osteonecrosis of femoral head through osteoclastic pathways.

Objective: Dragon's Blood resin (DBR) is a traditional medicinal substance renowned for its diverse pharmacological effects, which consists of potent anti-inflammatory, antioxidant and angiogenic properties. This study aimed to elucidate its therapeutic mechanism in alleviating steroid-induced osteonecrosis of the femoral head (SIONFH). Methods: Techniques such as SPR and LC-MS were employed to identify and analyze the target proteins of DBR in bone marrow macrophages (BMMs). In vitro, BMMs were treated with RANKL and DBR, and TRAcP staining and actin belt staining were utilized to assess osteoclast activity. The inhibitory effects and underlying mechanisms of DBR on osteoclastogenesis and reactive oxygen species (ROS) generation were determined using real-time PCR, western blotting and immunofluorescence staining. An in vivo SIONFH rat model was set up to assess the curative impacts of DBR using micro-CT scanning and pathological staining. Results: Bioinformatic tools revealed a pivotal role of osteoclast differentiation in SIONFH. Proteomic analysis identified 164 proteins binding in BMMs. In vitro assessments demonstrated that DBR hindered osteoclastogenesis by modulating the expression of specific genes and proteins, along with antioxidant proteins including TRX1 and Glutathione Reductase. Notably, the resin effectively inhibited the expression of crucial proteins, such as the phosphorylation of JNK and the nuclear localization of p65 within the TRAF6/JNK and NFκB signaling pathways. In vivo experiments further confirmed that DBR mitigated the onset of SIONFH in rats by curbing osteoclast and ROS activities. Conclusion: These findings underscore the potential of Dragon's Blood as an effective administration for early-stage SIONFH, shedding light on its therapeutic influence on ROS-mediated osteoclastic signaling pathways.

Effects of Simvastatin on Cartilage Homeostasis in Steroid-Induced Osteonecrosis of Femoral Head by Inhibiting Glucocorticoid Receptor.

Steroid-induced osteonecrosis of femoral head (SONFH) is one of the most common bone disorders in humans. Statin treatment is beneficial in preventing the development of SONFH through anti-inflammation effects and inhibition of the glucocorticoid receptor (GR). However, potential mechanisms of statin action remain to be determined. In this study, pulse methylprednisolone (MP) treatment was used to induce SONFH in broilers, and then MP-treated birds were administrated with simvastatin simultaneously to investigate the changes in cartilage homeostasis. Meanwhile, chondrocytes were isolated, cultured, and treated with MP, simvastatin, or GR inhibitor in vitro. The changes in serum homeostasis factors, cell viability, and expression of GR were analyzed. The results showed that the morbidity of SONFH in the MP-treated group increased significantly compared with the simvastatin-treated and control group. Furthermore, MP treatment induced apoptosis and high-level catabolism and low-level anabolism in vitro and vivo, while simvastatin significantly decreased catabolism and slightly recovered anabolism via inhibiting GR and the hypoxia-inducible factor (HIF) pathway. The GR inhibitor or its siRNA mainly affected the catabolism of cartilage homeostasis in vitro. In conclusion, the occurrence of SONFH in broilers was related to the activation of GR and HIF pathway, and imbalance of cartilage homeostasis. Simvastatin and GR inhibitor maintained cartilage homeostasis via GR and the HIF pathway.

MiR-145 inhibits the differentiation and proliferation of bone marrow stromal mesenchymal stem cells by GABARAPL1 in steroid-induced femoral head necrosis

Steroid-induced osteonecrosis of femoral head (SANFH) involves impaired differentiation of bone marrow mesenchymal stem cells (BMSC), the mechanism of which is regulated by multiple microRNAs. Studies have shown that miR-145 is a key regulatory molecule of BMSC cells, but its mechanism in steroid-induced femur head necrosis remains unclear. The present study mainly explored the specific mechanism of miR-145 involved in SANFH. In this study dexamethasone, a typical glucocorticoid, was used to induce osteogenic differentiation of BMSC cells. Western blot, qPCR, CCK8 and flow cytometry were used to investigate the effects of miR-145 on the proliferation and differentiation of BMSC. The relationship between miR-145 and GABA Type A Receptor Associated Protein Like 1(GABARAPL1) was identified using dual luciferase reports and the effects of the two molecules on BMSC were investigated in vitro. The results showed that miR-145 was up-regulated in SANFH patients, while GABARAPL1 was down-regulated. Inhibition of miR-145 can improve apoptosis and promote proliferation and activation of BMSC. GABARAPL1 is a downstream target gene of miR-145 and is negatively regulated by miR-145. In conclusion, miR-145 regulates the proliferation and differentiation of glucocorticoid-induced BMSC cells through GABARAPL1 and pharmacologically inhibit targeting miR-145 may provide new aspect for the treatment of SANFH.

The effects of thymoquinone on steroid-induced femoral head osteonecrosis: An experimental study in rats.

In this study, we aimed to evaluate the antioxidant, anti-apoptotic, osteoblastic and hypolipidemic effects of thymoquinone (TQ) treatment on the steroid-induced osteonecrosis of femoral head (ONFH) model in rats. A total of 24 rats were randomly divided into four groups: the control group administered saline; the TQ group administered 10 mg/kg/day TQ orally; lipopolysaccharide/methylprednisolone (LPS/MPS) group administered 20 µg/kg intraperitoneally LPS and 40 mg/kg intramuscularly MPS to establish ONFH model; and the LPS/ MPS+TQ group administered both LPS/MPS and, then, TQ once daily for four weeks. All rats were sacrificed after intracardiac blood collection and their right femurs were removed. Micro-computed tomography showed a higher bone mineral density and lower porosity, Tr. Sp and Tr. Sep data were detected in the LPS/MPS+TQ group. In histopathology, osteonecrosis increased significantly in the LPS/MPS group and osteonecrosis decreased in the LPS/MPS+TQ group compared to the LPS/MPS group (p=0.0077). Histomorphometric examination revealed that the percentage of BV/TV in the LPS/MPS group was significantly lower compared to control and other groups (p<0.01 and p<0.05, respectively), while it reached normal rates in the LPS/MPS+TQ group. Immunohistochemically, antioxidant, anti-apoptotic, and angiogenesis indicators (8-hydroxy-20- deoxyguanosine [8-OHdG], malondialdehyde [MDA], B-cell lymphoma [Bcl-2], caspase-3, vascular endothelial growth factor [VEGF]) were significantly improved in tissue and serum with TQ. Furthermore, TQ significantly reduced low- and high-density lipoprotein cholesterol ratio and carboxy-terminal type 1 collagen crosslink (CTX) in serum. Vascular and hematopoietic cell damages that occur due to steroid-induced deoxyribonucleic acid (DNA) oxidative and lipid peroxidative damages in an ONFH model can be successfully ameliorated by TQ administration. This antioxidant and anti-apoptotic effects of TQ may be a promising treatment option for early stage of osteonecrosis.

Effects of angiotensin II combined with asparaginase and dexamethasone on the femoral head in mice: A model of steroid-induced femoral head osteonecrosis.

Background: To study the pathogenesis of steroid-induced femoral head osteonecrosis, an ideal animal model is very important. As experimental animals, mice are beneficial for studying the pathogenesis of disease. However, there are currently few mouse models of steroid-induced femoral head osteonecrosis, and there are many questions that require further exploration and research. Purposes: The purpose of this study was to establish a new model of osteonecrosis in mice using angiotensin II (Ang II) combined with asparaginase (ASP) and dexamethasone (DEX) and to study the effects of this drug combination on femoral head osteonecrosis in mice. Methods: Male BALB/c mice (n = 60) were randomly divided into three groups. Group A (normal control, NC) was treated with physiological saline and given a normal diet. Group B (DEX + ASP, DA) was given free access to food and water (containing 2 mg/L DEX) and subjected to intraperitoneal injection of ASP (1200 IU/kg twice/week for 8 weeks). Group C (DEX + ASP + Ang II, DAA) was treated the same as group B, it was also given free access to food and water (containing 2 mg/L DEX) and subjected to intraperitoneal injection of ASP (1200 IU/kg twice/week for 8 weeks), but in the 4th and 8th weeks, subcutaneous implantation of a capsule osmotic pump (0.28 mg/kg/day Ang II) was performed. The mice were sacrificed in the 4th and 8th weeks, and the model success rate, mouse mortality rate, body weight, blood lipids, coagulation factors, histopathology, and number of local vessels in the femoral head were evaluated. Results: DAA increased the model success rate [4th week, 30% (DA) vs. 40% (DAA) vs. 0% (NC); 8th week, 40% (DA) vs. 70% (DAA) vs. 0% (NC)]. There was no significant difference in mortality rate between the groups [4th week, 0% (DA) vs. 0% (DAA) vs. 0% (NC); 8th week, 5% (DA) vs. 10% (DAA) vs. 0% (NC)]. DAA affected mouse body weight and significantly affected blood lipids and blood coagulation factors. DAA reduces the number of blood vessels in the femoral head and destroys the local blood supply. Conclusion: Angiotensin II combined with asparaginase and dexamethasone can obviously promote the necrosis of femoral head and provide a new idea for the model and treatment of osteonecrosis.

MIR31HG polymorphisms are related to steroid-induced osteonecrosis of femoral head among Chinese Han population

BackgroundsMIR31 host gene (MIR31HG) polymorphisms play important roles in the occurrence of osteonecrosis. However, the association of MIR31HG polymorphisms with the risk of steroid-induced osteonecrosis of the femoral head (SONFH) remains unclear. In this study, we aimed to investigate the correlation between MIR31HG polymorphisms and SONFH susceptibility in the Chinese Han population.MethodsA total of 708 volunteers were recruited to detect the effect of seven single nucleotide polymorphisms (SNPs) in the MIR31HG gene on SONFH risk in the Chinese Han population. Genotyping of MIR31HG polymorphisms was performed using the Agena MassARRAY platform. The odds ratio (OR) and 95% confidence interval (95% CI) were used to evaluate the correlation between MIR31HG polymorphisms and SONFH risk using logistic regression model.ResultsAccording to the results of genetic model, rs10965059 in MIR31HG was significantly correlated with the susceptibility to SONFH (OR = 0.56, p = 0.002). Interestingly, the stratified analysis showed that rs10965059 was associated with the reduced risk of SONFH in subjects aged > 40 years (OR = 0.30, p < 0.001) and male populations (OR = 0.35, p < 0 .001). Moreover, rs10965059 was associated with the reduced risk of bilateral SONFH (OR = 0.50, p = 0.002). Finally, multi-factor dimension reduction (MDR) results showed that the combination of rs1332184, rs72703442, rs2025327, rs55683539, rs2181559, rs10965059 and rs10965064 was the best model for predicting SONFH occurrence (p < 0.0001).ConclusionThe study indicated that rs10965059 could be involved in SONFH occurrence in the Chinese Han population, which might provide clues for investigating the role of MIR31HG in the pathogenesis of SONFH.

Huogu injection alleviates SONFH by regulating adipogenic differentiation of BMSCs via targeting the miR-34c-5p/MDM4 pathway

Although the precise pathogenesis of steroid-induced osteonecrosis of femoral head (SONFH) is not yet fully understood, evidence shows miRNAs-mediated posttranscription control directs the adipogenesis of bone marrow mesenchymal stem cells (BMSCs) and plays a pivotal role in the SONFH processes. Huogu injection formulated according to traditional Chinese medicine (TCM) theory has been used to treat SONFH by intra-articular injection. In this study, we asked whether the therapeutic effects of Huogu injection might depend on the inhibition of adipogenic differentiation of BMSCs, and if so, the pathway might be a therapeutic target to promote bone repair. Consequently, miR-34c-5p was upregulated in the dexamethasone (DEX)-treated BMSCs and might participate in the adipogenesis of BMSCs. TargetScan database and the luciferase reporter assay showed miR-34c-5p targeted on the MDM4 and negatively regulated its expression. Huogu injection in vitro inhibited the adipogenesis in the DEX-treated BMSCs by inhibiting the expression levels of PPARγ and C/EBPα, as well as reducing miR-34c-5p to prevent the degradation of MDM4. Moreover, miR-34c-5p mimic or MDM4 knockdown using shRNA neutralized the anti-adipogenesis of Huogu injection in BMSCs. In vivo, the results of X-ray imaging confirmed that Huogu injection alleviated the bone loss in rat SONFH. Consistent with results in vitro, Huogu injection reduced the lipid accumulation, removed the suppression of MDM4 by downregulating the expression of miR-34c-5p, and inhibited the expression of C/EBPα and PPARγ in bone tissues. When the lentivirus encoding miR-34c-5p was conducted by intra-articular injection, the overexpression of miR-34c-5p antagonized the therapeutic effects of Huogu injection. Our results underline the critical importance of the miR-34c-5p/MDM4 pathway in regulating the adipogenic outcome of BMSCs, suggesting the miR-34c-5p as a potentially effective therapeutic target in SONFH. These results further reinforce the potential of Huogu injection as an alternative approach in SONFH.

Hsa_circ_0058122 knockdown prevents steroid-induced osteonecrosis of the femoral head by inhibiting human umbilical vein endothelial cells apoptosis via the miR-7974/IGFBP5 axis.

BackgroundSteroid‐induced osteonecrosis of femoral head (SONFH) is a serious complication of glucocorticoid overused. Recent evidence has demonstrated that circRNAs exert key pathophysiological roles in a variety of disease processes. However, the role of circRNA in SONFH remains largely unknown. The current study sought to evaluate how hsa_circ_0058122 affects SONFH in dexamethasone (DEX) treated human umbilical vein endothelial cells (HUVECs) model.MethodsRT‐PCR was used to demonstrate the hsa_circ_0058122 expression level in Dex‐treated HUVECs cells. The effects of hsa_circ_0058122 on HUVECs apoptosis were evaluated via overexpression plasmid and siRNA. Using dual‐luciferase and fluorescence in situ hybridization assays, we demonstrated that hsa_circ_0058122 binds to miR‐7974 thereby facilitating HUVECs apoptosis. Bioinformatics analysis and western blot were performed to confirm target genes of hsa‐miR‐7974.ResultsIn our previous work, we revealed the top 20 elevated circRNAs in SONFH patients were hsa_circ_0010027, hsa_circ_0058115, hsa_circ_0010026, hsa_circ_0058839, hsa_circ_0056886, hsa_circ_0056885, hsa_circ_0058146, hsa_circ_0058105, hsa_circ_0058112, hsa_circ_0058143, hsa_circ_0058102, hsa_circ_0058090, hsa_circ_0075353, hsa_circ_0058126, hsa_circ_0058130, hsa_circ_0058140, hsa_circ_0058122, hsa_circ_0058123, hsa_circ_0058103, and hsa_circ_0058121. Among these, hsa_circ_0058122 was finally selected for further investigation. We found hsa_circ_0058122 expression was markedly elevated in Dex‐treated HUVECs cells, and the Dex‐mediated HUVEC apoptosis was impaired in hsa_circ_0058122‐silenced cells and increased in hsa_circ_0058122‐overexpressing cells. hsa_circ_0058122 competitively binds to hsa‐miR‐7974, which in turn interacts with insulin‐like growth factor binding protein 5 (IGFBP5).Conclusionshsa_circ_0058122/miR‐7974/IGFBP5 was proposed to be a key regulatory pathway for SONFH. DEX treatment upregulated hsa_circ_0058122 expression in HUVECs, which sponged miR‐7974, thereby increasing IGFBP5 expression, the hsa_circ_0058122/miR‐7974/IGFBP5 axis contributed to the Dex‐mediated apoptosis. These findings may identify novel targets for SONFH molecular therapy.

LINC00473-modified bone marrow mesenchymal stem cells incorporated thermosensitive PLGA hydrogel transplantation for steroid-induced osteonecrosis of femoral head: A detailed mechanistic study and validity evaluation.

The pathogenesis of steroid‐induced osteonecrosis of the femoral head (SONFH) involves a glucocorticoid‐induced imbalance of osteogenic and adipogenic differentiation, and apoptosis of bone marrow mesenchymal stem cells (BMSCs). An increasing number of genes, especially noncoding RNAs, have been implicated in the function of BMSCs. Our previous studies have confirmed the key role of LINC00473 and miR‐23a‐3p on the osteogenic, adipogenic differentiation, and apoptosis of BMSCs. However, the underlying mechanism of this process is still unclear. Based on bioinformatics analysis, here we investigated the effects of LINC00473 on the LRP5/Wnt/β‐catenin signaling pathway in the osteogenesis and adipogenesis of BMSCs, as well as the PEBP1/Akt/Bad/Bcl‐2 signaling pathway in dexamethasone‐ (Dex‐) induced apoptosis of BMSCs. Our data showed that LINC00473 could promote osteogenesis and suppress the adipogenesis of BMSCs through the activation of the miR‐23a‐3p/LRP5/Wnt/β‐catenin signaling pathway axis, while rescuing BMSCs from Dex‐induced apoptosis by activating the miR‐23a‐3p/PEBP1/Akt/Bad/Bcl‐2 signaling pathway axis. Notably, we observed that LINC00473 interacted with miR‐23a‐3p in an Argonaute 2 (AGO2)‐dependent manner based on dual‐luciferase reporter assay, AGO2‐related RNA immunoprecipitation, and RNA antisense purification assay. Furthermore, injectable thermosensitive polylactic‐co‐glycolic acid (PLGA) hydrogel loaded with rat‐derived BMSCs (rBMSCs) modified by LINC00473 were used for the treatment of SONFH in a rat model. Our results demonstrated that PLGA hydrogels provided a suitable environment for harboring rBMSCs. Besides, transplantation of PLGA hydrogels loaded with rBMSCs modified by LINC00473 could significantly promote the bone repair and reconstruction of the necrotic area at the femoral head in our SONFH rat model. Surprisingly, compared with the transplantation of BMSCs alone, the transplanted rBMSCs encapsulated within the PLGA hydrogel could migrate from the medullary cavity to the femoral head. In summary, LINC00473 promoted osteogenesis, inhibited adipogenesis, and antagonized Dex‐induced apoptosis of BMSCs. Therefore, LINC00473 could provide a new strategy for the treatment of SONFH.

Risk factors and pathogenesis of steroid-induced osteonecrosis of femoral head - A scoping review

Steroid induced osteonecrosis of the femoral head is commonly seen in clinical practice, but yet not fully understood. It is intriguing why only some cases develop and others escape from it. We did an extensive and up to date literature review on it, with the aim to identify its incidence, associated risk factor and pathogenesis. We found that the incidence ranged from 03 to 40%, and was more with associated risk factors. Several risk factors were identified, which included higher dose and prolonged duration of steroid consumption, and underlying disease for which the steroids were given. Pathogenesis of this disease is complex and not yet fully understood. Awareness about this condition and associated risk factors should help the clinicians in identifying the cases who are prone to develop osteonecrosis with the use of steroids.

Astragalus polysaccharide ameliorates steroid-induced osteonecrosis of femoral head through miR-206/HIF-1α/BNIP3 axis.

Declining autophagy and rising apoptosis are the main factors driving the development of steroid-induced osteonecrosis of the femoral head (SONFH). Here, we showed that astragalus polysaccharide (APS) improved femoral head necrosis via regulation of cell autophagy and apoptosis through microRNA (miR)-206/hypoxia inducible factor-1 (HIF-1α)/BCL2 interacting protein 3 (BNIP3) axis. The expression of miR-206, HIF-1α, and BNIP3 in SONFH specimens and cell model were measured using qPCR. SONFH cell model was treated with APS. Cell autophagy was evaluated using LC3-immunofluorescence assays. Flow cytometry was conducted to assess cell apoptosis. Apoptosis-related proteins and autophagy-related proteins were determined using western blot. Besides, dual-luciferase reporter assay was employed to investigate the relationship between miR-206 and HIF-1α. Here we showed that miR-206 expression was upregulated in SONFH tissues and cell model. APS promoted autophagy and inhibited apoptosis in SONFH cell model via downregulating miR-206. What is more, HIF-1α was the target of miR-206. Knockdown of HIF-1α reversed the recovery effect of miR-206 inhibitor on SONFH cell model. Furthermore, BNIP3 was the target of HIF-1α. HIF-1α overexpression promoted autophagy and inhibited apoptosis, and knockdown of BNIP3 abolished the recovery effect of HIF-1α overexpression in SONFH cell model. These results provided evidence that APS reduced miR-206 expression, and the downregulated miR-206 increased BNIP3 expression by targeting HIF-1α to promote autophagy and inhibit bone cell apoptosis. Our research proved that APS effectively improved SONFH by regulating cell autophagy and apoptosis.

Research on the Mechanism of Asiaticoside Inhibiting Osteoclast Differentiation and Promoting the Recovery of Steroid-Induced Osteonecrosis of the Femoral Head (SIONFH)

Our study aims to assess whether asiaticoside promotes the recovery of SINOFH by inhibiting bone marrow stem cells (BMSCs) differentiation into osteoclasts (OC). BMMs were induced to form OC system by dexamethasone in vitro and ELISA detected the expression of OC-related genes formation by asiaticoside. BMSCs were cultured followed by analysis of BMSCs morphology under microscope, gene expression by qRT-PCR. TRACP and c-Src level by western blot, RANKL, OPG and TRACP5b level by ELISA. Asiaticoside inhibited the expression of OC formation in SIONFH. The expression of OC-related genes increased with the induction days. With the increasing of induction days, asiaticoside level in culture fluid was decreased. While after asiaticoside interference, OCrelated genes and proteins levels were significantly down-regulated. Aasiaticoside can significantly increase the RANKL signaling protein expression. In conclusion, asiaticoside promotes the recovery of SINOFH by inhibiting BMSCs differentiation into OC.

Novel MRI technique for the quantification of biochemical deterioration in steroid-induced osteonecrosis of femoral head: a prospective diagnostic trial.

To explore the novel magnetic resonance imaging techniques, IVIM-DWI and IDEAL-IQ in detecting bone marrow fat and microcirculation in steroid-induced osteonecrosis of the femoral head (SIONFH). In this prospective study, 49 patients (80 hips) with SIONFH taking glucocorticoids and 24 healthy volunteers (48 hips) were recruited and assessed by T1WI, T2WI/fs, IDEAL-IQ and IVIM-DWI. The affected hips, contralateral asymptomatic hips and normal hips, as well as normal, penumbra and necrotic areas in the affected hips, were classified and evaluated. Imaging results were compared with histologic bone sections obtained from SIONFH patients undergoing surgery. The fat fraction (FF) and perfusion fraction (f) differences between groups were analyzed using analysis of variance, the LSD t-test, Pearson correlation analysis and ROC curve analysis. Our results demonstrate that IDEAL-IQ (FF) and IVIM-DWI (f) enable the classification of SIONFH at different ARCO stages. The FF was positively associated with the progression of the disease (r = 0.72), in contrast to f (r = −0.17). The FF and f were significantly different among the necrotic, penumbra and normal areas, and they were negatively correlated with each other (r = −0.37). The diagnostic sensitivity and specificity of IDEAL-IQ were 96.9% and 86.7%, and those of IVIM-DWI were 72.34% and 58.33%, respectively. The FF in contralateral asymptomatic hips was significantly higher than in normal hips, but no difference was found for f. IDEAL-IQ, and not IVIM-DWI, was identified to successfully detect bone marrow fat, which is beneficial to the diagnosis of the severity of SIONFH.

HEMGN and SLC2A1 might be potential diagnostic biomarkers of steroid-induced osteonecrosis of femoral head: study based on WGCNA and DEGs screening

BackgroundSteroid-induced osteonecrosis of the femoral head (SONFH) is a chronic and crippling bone disease. This study aims to reveal novel diagnostic biomarkers of SONFH.MethodsThe GSE123568 dataset based on peripheral blood samples from 10 healthy individuals and 30 SONFH patients was used for weighted gene co-expression network analysis (WGCNA) and differentially expressed genes (DEGs) screening. The genes in the module related to SONFH and the DEGs were extracted for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Genes with |gene significance| > 0.7 and |module membership| > 0.8 were selected as hub genes in modules. The DEGs with the degree of connectivity ≥5 were chosen as hub genes in DEGs. Subsequently, the overlapping genes of hub genes in modules and hub genes in DEGs were selected as key genes for SONFH. And then, the key genes were verified in another dataset, and the diagnostic value of key genes was evaluated by receiver operating characteristic (ROC) curve.ResultsNine gene co-expression modules were constructed via WGCNA. The brown module with 1258 genes was most significantly correlated with SONFH and was identified as the key module for SONFH. The results of functional enrichment analysis showed that the genes in the key module were mainly enriched in the inflammatory response, apoptotic process and osteoclast differentiation. A total of 91 genes were identified as hub genes in the key module. Besides, 145 DEGs were identified by DEGs screening and 26 genes were identified as hub genes of DEGs. Overlapping genes of hub genes in the key module and hub genes in DEGs, including RHAG, RNF14, HEMGN, and SLC2A1, were further selected as key genes for SONFH. The diagnostic value of these key genes for SONFH was confirmed by ROC curve. The validation results of these key genes in GSE26316 dataset showed that only HEMGN and SLC2A1 were downregulated in the SONFH group, suggesting that they were more likely to be diagnostic biomarkers of SOFNH than RHAG and RNF14.ConclusionsOur study identified that two key genes, HEMGN and SLC2A1, might be potential diagnostic biomarkers of SONFH.