Steroid-induced Avascular Necrosis Of The Femoral Head Research Articles

The IFT80/Hedgehog Pathway Regulates the Osteogenic-adipogenic Differentiation of Bone Marrow Mesenchymal Stem Cells.

Steroid-induced avascular necrosis of the femoral head (SANFH) is a typical refractory disease that often progresses irreversibly and has a high disability rate. Numerous studies have confirmed that abnormal osteogenic-adipogenic differentiation of bone marrow mesenchymal stem cells (BM-MSCs) is one of the major factors of SANFH. However, the mechanism remains to be elucidated. This study aimed to investigate the mechanism and effect of the IFT80/Hedgehog-mediated osteogenic-adipogenic differentiation of BM-MSCs in SANFH. Femoral head specimens of SANFH patients and femoral neck fractures (FNF) patients were collected to detect the expression of IFT80, Shh and osteogenic-adipogenic differentiation-related genes by immunohistochemistry (IHC), western blot (WB) and Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR). Based on the rabbit SANFH model, the mRNA expression and protein level of IFT80 and Shh were detected by RT-qPCR and WB. After the osteogenic/adipogenic differentiation based on rabbit BM-MSCs, the IFT80, Gli1, PPAR-γ, and Runx2 expression were detected. Differences in alkaline phosphodiesterase activity, calcium nodule, quantification/distribution of lipid droplets, expression of IFT80/Hedgehog axis, and the level of osteogenic- adipogenic associated factors were determined after IFT80 overexpression. RT-qPCR, WB and IHC revealed that IFT80 and Shh lowly expressed in the osteoblasts and intra-trabecular osteocytes at the edge of trabeculae and in the intercellular matrix of the bone marrow lumen in the SANFH specimens. The Runx2 expression was low, while the PPAR-γ expression was high in both human specimens and animal models of SANFH, suggesting that the balance of osteogenic-adipogenic differentiation was dysregulated. Rabbit BM-MSCs with stable overexpression of IFT80 showed increased alkaline phosphatase activity after induction of osteogenic differentiation, increased calcium nodule production, and decreased adipogenesis after induction of adipogenic differentiation. There is a dysregulation of the balance of osteogenic-adipogenic differentiation in SANFH. IFT80 may inhibit adipogenic differentiation while promoting osteogenic differentiation in rabbit BM-MSCs by activating the Hedgehog pathway.

Extracellular vesicles from human umbilical cord mesenchymal stem cells prevent steroid-induced avascular necrosis of the femoral head via the PI3K/AKT pathway.

Extracellular vesicles (EVs) secreted by human umbilical cord mesenchymal stem cells (hucMSC) have excellent therapeutic potential for many diseases. The aim of this study was to define the role of hucMSC-EVs in the prevention and treatment of steroid-induced avascular necrosis of the femoral head (SANFH). After establishing the SANFH rat model, the effects of hucMSC-EVs were assessed by measuring the microstructure of the femoral head using HE staining, micro-computed tomography (micro-CT), and TUNEL staining. The administration of hucMSC-EVs caused a significant reduction to glucocorticoids (GCs)-induced osteoblast apoptosis and empty lacuna of the femoral head, while effectively improving the microstructure. HucMSC-EVs rescued the deactivation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway induced by GCs, and reversed the proliferation and migration of osteoblasts inhibited by GCs. In addition, hucMSC-EVs attenuated the inhibitory effects of GCs on rat osteoblast osteogenesis, angiogenesis of endothelial cells, and prevented osteoblast apoptosis. However, the promoting effects of hucMSC-EVs were abolished following the blockade of PI3K/AKT on osteoblasts. hucMSC-EVs were found to prevent glucocorticoid-induced femoral head necrosis in rats through the PI3K/AKT pathway.

Houttuynia cordata Thunb repairs steroid-induced avascular necrosis of the femoral head through regulating NF-κB signaling pathway

To investigate the influences of Houttuynia cordata Thunb (HCT) in steroid-induced avascular necrosis of the femoral head (SANFH), we conducted a comprehensive study evaluating the effects of HCT on various aspects. Cell Counting Kit-8 assay was used to examine bone marrow stem cells (BMSCs) cell viability. Flow cytometry and lactate dehydrogenase detection assay were conducted to determine cell apoptosis. The levels of apoptosis-related proteins, osteogenic-related markers, inflammatory factors, and nuclear factor kappa B (NF-κB) pathway-associated proteins were determined via western blotting. Hematoxylin and eosin and terminal deoxynucleotidyl transferase dUTP nick-end labeling assays were utilized to verify the effects of HCT in SANFH rats. Our findings revealed that HCT could enhanced cell viability and arrested cell apoptosis in dexamethasone (Dex)-treated BMSCs. Dex increased the levels of cleaved caspase-3, Bcl2-associated X, interleukin (IL)-1β, IL-18, IL-6, p65, and inhibitor of NF-κB kinase β (IKKβ), while this promoting trend was weakened by HCT. Moreover, pyrrolidine dithiocarbamate (PDTC, an inhibitor of NF-κB signaling pathway) further increased the inhibitory role of apoptosis and the levels of IL-1β, IL-18, and IL-6 and the promotional effect of the levels of RUNX2 and ALP in Dex-treated BMSCs. The in-vivo assays showed that HCT decreased the percentage of empty lacunae, apoptosis, and the levels of IL-1β, IL-18, IL-6, p65, and IKKβ in SANFH rats. In conclusion, our study demonstrated that HCT relieved SANFH, which might be possibly achieved by NF-κB pathway.

LncAABR07053481 inhibits bone marrow mesenchymal stem cell apoptosis and promotes repair following steroid-induced avascular necrosis

The osteonecrotic area of steroid-induced avascular necrosis of the femoral head (SANFH) is a hypoxic microenvironment that leads to apoptosis of transplanted bone marrow mesenchymal stem cells (BMSCs). However, the underlying mechanism remains unclear. Here, we explore the mechanism of hypoxic-induced apoptosis of BMSCs, and use the mechanism to improve the transplantation efficacy of BMSCs. Our results show that the long non-coding RNA AABR07053481 (LncAABR07053481) is downregulated in BMSCs and closely related to the degree of hypoxia. Overexpression of LncAABR07053481 could increase the survival rate of BMSCs. Further exploration of the downstream target gene indicates that LncAABR07053481 acts as a molecular “sponge” of miR-664-2-5p to relieve the silencing effect of miR-664-2-5p on the target gene Notch1. Importantly, the survival rate of BMSCs overexpressing LncAABR07053481 is significantly improved after transplantation, and the repair effect of BMSCs in the osteonecrotic area is also improved. This study reveal the mechanism by which LncAABR07053481 inhibits hypoxia-induced apoptosis of BMSCs by regulating the miR-664-2-5p/Notch1 pathway and its therapeutic effect on SANFH.

Mechanism of vascular endothelial cell-derived exosomes modified with vascular endothelial growth factor in steroid-induced femoral head necrosis

Steroid-induced avascular necrosis of the femoral head (SANFH) is an intractable orthopedic disease. This study investigated the regulatory effect and molecular mechanism of vascular endothelial cell (VEC)-derived exosomes (Exos) modified with vascular endothelial growth factor (VEGF) in osteogenic and adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in SANFH. VECs were cultured in vitro and transfected with adenovirus Adv-VEGF plasmids. Exos were extracted and identified. In vitro/vivo SANFH models were established and treated with VEGF-modified VEC-Exos (VEGF-VEC-Exos). The internalization of Exos by BMSCs, proliferation and osteogenic and adipogenic differentiation of BMSCs were determined by the uptake test, cell counting kit-8 (CCK-8) assay, alizarin red staining, and oil red O staining. Meanwhile, the mRNA level of VEGF, the appearance of the femoral head, and histological analysis were assessed by reverse transcription quantitative polymerase chain reaction and hematoxylin-eosin staining. Moreover, the protein levels of VEGF, osteogenic markers, adipogenic markers, and mitogen-activated protein kinase (MAPK)/extracellular regulated protein kinases (ERK) pathway-related indicators were examined by Western blotting, along with evaluation of the VEGF levels in femur tissues by immunohistochemistry. Glucocorticoid (GC) induced adipogenic differentiation of BMSCs and inhibited osteogenic differentiation. VEGF-VEC-Exos accelerated the osteogenic differentiation of GC-induced BMSCs and inhibited adipogenic differentiation. VEGF-VEC-Exos activated the MAPK/ERK pathway in GC-induced BMSCs. VEGF-VEC-Exos promoted osteoblast differentiation and suppressed adipogenic differentiation of BMSCs by activating the MAPK/ERK pathway. VEGF-VEC-Exos accelerated bone formation and restrained adipogenesis in SANFH rats. VEGF-VEC-Exos carried VEGF into BMSCs and motivated the MAPK/ERK pathway, thereby promoting osteoblast differentiation of BMSCs in SANFH, inhibiting adipogenic differentiation, and alleviating SANFH.

Methylprednisolone Inhibits Autophagy of Vascular Endothelial Cells in Rat Femoral Head ViaPI3K/Akt/mTOR Pathway

ObjectiveTo study the relationship between vascular endothelial cells (VEC) and autophagy, and its regulatory mechanism in steroid‐induced avascular necrosis of the femoral head (SANFH).MethodsIn cell experiment, VEC were isolated and cultured from the femoral head of Sprague–Dawley rats and divided into three groups: blank control group (Ctrl), methylprednisolone group (MP), and methylprednisolone+mTOR‐shRNA group (MP + shmTOR). The autophagy formation was observed by transmission electron microscope. The mRNA expression of PI3K, Akt, mTOR, Beclin1 and MAP1LC3 was detected by RT‐PCR and the protein expression was detected by Western blot and immunofluorescence. Expression of the damage marker 6‐keto‐PGF1α was detected by the ELISA method. In vivo experiment, after establishing the model, the grouping method was the same as cell experiment. Autophagosomes were observed by same method, and the expression of related factors was detected by the same method in cell experiment.ResultsIn the cell experiment, autophagosomes in the MP group were significantly lower than in the Ctrl group, and the autophagosomes in the MP + shmTOR group were intermediate between two groups (P < 0.05). The mRNA expression levels of PI3K, Akt and mTOR in the MP group were significantly higher than in the Ctrl group, while the MP+ shmTOR group presented intermediate levels between these groups (average gray value were 3837.90, 2996.30, 3005.60, F = 428.64, P < 0.05). MRNA expression levels of Beclin1 and MAP1LC3 in the MP group were significantly lower than that in Ctrl group (P < 0.05). The content of 6‐keto‐PGF1α in the MP + shmTOR group was higher than in the Ctrl group and lower than in the MP group at the evaluated time intervals (average absorbance value were 104.98, 206.83, 145.91, F = 352.83, P < 0.01). In vivo experiment, the content of 6‐Keto‐PGF1α in the hormone group increased as time went on; the mTOR‐si group was higher than that in control group, but lower than that in the hormone group (P < 0.01). The mRNA expressions of Beclin1 and MAP1LC3 in the control group were higher than those in the hormone group, while the mRNA expressions of PI3K, Akt and mTOR were lower than those in the mTOR‐si group (P < 0.05).ConclusionThe steroid inhibited the physiological protective effect of autophagy on SANFH by increasing the expression of PI3K/Akt/mTOR signaling pathway related factors and decreasing the expression of Beclin1 and MAP1LC3 in the femoral head VEC.

C/EBPα regulates the fate of bone marrow mesenchymal stem cells and steroid-induced avascular necrosis of the femoral head by targeting the PPARγ signalling pathway

BackgroundThe imbalance of osteogenic/adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) is closely related to steroid-induced avascular necrosis of the femoral head (SANFH). We aimed to investigate the epigenetic mechanism of intramedullary fat accumulation and continuous osteonecrosis after glucocorticoid (GC) withdrawal in SANFH.MethodsAn SANFH model was established in SD rats, which received an intermittent high GC dose for the first 4 weeks followed by an additional 4 weeks without GC. We explored the synergistic effects and mechanisms of C/EBPα and PPARγ on the differentiation of BMSCs by lentivirus-mediated gene knockdown and overexpression assays. A chromatin immunoprecipitation assay was performed to identify epigenetic modification sites on PPARγ in vivo and in vitro.ResultsIn the SANFH model, intramedullary fat was significantly increased, and the transcription factors C/EBPα and PPARγ were upregulated simultaneously in the femoral head. In vitro, C/EBPα promoted adipogenic differentiation of BMSCs by targeting the PPARγ signalling pathway, while overexpression of C/EBPα significantly impaired osteogenic differentiation. Further studies demonstrated that histone H3K27 acetylation of PPARγ played an important role in the epigenetic mechanism underlying SANFH. C/EBPα upregulates the histone H3K27 acetylation level in the PPARγ promoter region by inhibiting HDAC1. Additionally, inhibiting the histone acetylation level of PPARγ effectively prevented adipogenic differentiation, thus slowing the progression of SANFH.ConclusionsOur results demonstrate the molecular mechanism by which C/EBPα regulates PPARγ expression by acetylating histones and revealed the epigenetic phenomenon in SANFH for the first time.Graphical abstract

Network-Based Pharmacology and Bioinformatics Study on the Mechanism of Action of Gujiansan in the Treatment of Steroid-Induced Avascular Necrosis of the Femoral Head.

Objective To investigate the main pharmacological basis and mechanism of action of Gujiansan in the treatment of steroid-induced avascular necrosis of the femoral head (SANFH). Methods The active constituents and targets of Gujiansan were screened by using TCMSP and other databases, and relevant disease targets were obtained by analyzing the microarray of SANFH in the GEO database. The intersection of the two was taken to obtain the potential targets of Gujiansan for the treatment of SANFH, and key active constituents were screened with the “active constituent-target” network constructed by the Cytoscape software; then, the STRING database was used to construct the protein interaction network to screen the key targets. The Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses of key targets were performed by the DAVID database, and the relationship between the “key active constituent-key target-key signaling pathway” was explored. Finally, the molecular docking between key active constituents and key targets was verified. In addition, qPCR detection technology was used to evaluate the preventive and therapeutic effects of key active constituents of Gujiansan in a rat osteoblast model of SANFH to verify the possible mechanism of the effect of Gujiansan in the treatment of SANFH. Results (1) 106 active constituents and 55 targets were obtained for the treatment of SANFH. (2) Quercetin, luteolin, kaempferol, cryptotanshinone, and naringenin were the key active constituents for the treatment of SANFH. (3) IL1B, STAT3, CAT, PTGS2, and MAPK3 were the key targets for the treatment of SANFH. (4) IL1B, STAT3, CAT, PTGS2, MAPK3, and HMOX1 are key targets in the protein interaction network. (5) DAVID enrichment analysis mainly covers the regulation of DNA-binding transcription factor activity, positive regulation of cytokine production, and response to oxidative stress and other biological processes, involving IL-17, AGE-RAGE, C-type lectin receptor, and other signaling pathways. (6) Gujiansan is a multitarget and multisignaling pathway for the treatment of SANFH. (7) Good binding activity exists between key active constituents and key targets. Conclusion This study analyzes the potential mechanism of action of Gujiansan in the treatment of SANFH with network pharmacology, which can provide a reference for the further study of its pharmacological basis and targets.

Oxidative Stress Induced Osteocyte Apoptosis in Steroid-Induced Femoral Head Necrosis.

ObjectiveTo investigate the effect and mechanism of Glucocorticoids (GCs) induced oxidative stress and apoptosis on necrosis of the femoral head in patients and rats.MethodsEight patients with steroid‐induced avascular necrosis of the femoral head (SINFH) and eight patients with developmental dysplasia of the hips (DDH) were enrolled in our study. In animal model, twenty male Sprague‐Dawley rats were randomly divided into two groups (SINFH group and NS group). The SINFH model group received the methylprednisolone (MPS) injection, while control group was injected with normal saline (NS). MRI was used to confirm SINFH rat model was established successfully. Then, the rats were sacrificed 4 weeks later and femoral head samples were harvested. Histopathological staining was preformed to evaluate osteonecrosis. TUNEL staining was performed with 8‐OHdG and DAPI immunofluorescence staining to evaluate oxidative injury and osteocyte apoptosis. Immunohistochemistry staining was used to detect Nox1, Nox2, and Nox4 protein expression.ResultsMRI showed signs of typical osteonecrosis of femoral head in SIHFH patients. Histopathological staining showed that the rate of empty lacunae in SINFH patients was significantly higher (56.88% ± 9.72% vs 19.92% ± 4.18%, T = −11.04, P < 0.001) than that in DDH patients. The immunofluorescence staining indicated that the TUNEL‐positive cell and 8‐OHdG‐positve cell in SINFH patients were significantly higher (49.32% ± 12.95% vs 8.00% ± 2.11%, T = −7.04, P = 0.002, 54.6% ± 23.8% vs 9.75% ± 3.31%, T = −4.17, P = 0.003) compared to the DDH patients. The immunohistochemistry staining showed that the protein expression of NOX1, NOX2 and NOX4 in SINFH patients were significantly increased (64.50% ± 7.57% vs 37.58% ± 9.23%, T = −3.88, P = 0.018, 90.84% ± 2.93% vs 49.56% ± 16.47%, T = −5.46, P = 0.001, 85.46% ± 9.3% vs 40.69% ± 6.77%, T = −8.03, P = 0.001) compared to the DDH patients. In animal model, MRI showed signs of edema of femoral head in MPS group, which represents SINFH rat model was established successfully. Histological evaluation showed the rate of empty lacunae in MPS group was significantly higher (25.85% ± 4.68% vs 9.35% ± 1.99%, T = −7.96, P < 0.001) than that in NS group. The immunofluorescence staining indicated that the TUNEL‐positive cell and 8‐OHdG‐positve cell (in MPS group were significantly increased (31.93% ± 1.01% vs 11.73% ± 1.16%, T = −32.26, P < 0.001, 47.59% ± 1.39% vs 22.07% ± 2.45%, T = −22.18, P < 0.001) compared to the NS group. The immunohistochemistry staining showed that the expression of NOX2 in MPS group was significantly increased (76.77% ± 8.34% vs 50.32% ± 10.84%, T = −4.74, P = 0.001) compare with NS group.ConclusionOur findings indicated that GC‐induced NOXs expression may be an important source of oxidative stress, which could lead to osteocyte apoptosis in the process of SINFH

Gujiansan Ameliorates Avascular Necrosis of the Femoral Head by Regulating Autophagy via the HIF-1α/BNIP3 Pathway.

Background Clinically, the traditional Chinese medicine compound Gujiansan has been widely used in the treatment of steroid-induced avascular necrosis of the femoral head (SANFH). The present study aimed to investigate the mechanisms underlying the therapeutic effect of Gujiansan. Methods A rat model of SANFH was established by the injection of dexamethasone (DEX) at a high dosage of 25 mg/kg/d. Then, Gujiansan was intragastrically administered for 2 weeks, 4 weeks, and 8 weeks, and histological examination of the femoral head was performed. The expression levels of related mRNAs and proteins were analyzed by qRT-PCR, Western blotting, and immunohistochemistry, and the levels of bone biochemical markers and cytokines were detected with ELISA kits. Results Gujiansan administration ameliorated SANFH and induced the expression of hypoxia-inducible factor-1α (HIF-1α), Bcl-2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3), LC3, and Beclin-1 in the rat model in a dose- and time-dependent manner, and Gujiansan promoted osteocalcin secretion at the femoral head. In addition, Gujiansan increased the levels of bone formation- and bone resorption-specific markers (osteocalcin (OC), bone-specific alkaline phosphatase (BAP), tartrate resistant acid phosphatase-5b (TRACP-5b), N-terminal telopeptides of type I collagen (NTX-1), and C-terminal telopeptide of type I collagen (CTX-1)) and decreased the levels of proinflammatory cytokines (TNF-α, IL-6, and CRP) in a dose- and time-dependent manner. Conclusions Gujiansan accelerates the formation of a new bone, promotes the absorption of the damaged bone, inhibits the inflammatory response, induces autophagy of the femoral head via the HIF-1α/BNIP3 pathway, and ultimately ameliorates SANFH.

Atorvastatin Upregulates microRNA-186 and Inhibits the TLR4-Mediated MAPKs/NF-κB Pathway to Relieve Steroid-Induced Avascular Necrosis of the Femoral Head.

Steroid-induced avascular necrosis of the femoral head (SANFH) is caused by the death of active components of the femoral head owing to hormone overdoses. The use of lipid-lowering drugs to prevent SANFH in animals inspired us to identify the mechanisms involving Atorvastatin (Ato) in SANFH. However, it is still not well understood how and to what extent Ato affects SANFH. This study aimed to figure out the efficacy of Ato in SANFH and the underlying molecular mechanisms. After establishment of the SANFH model, histological evaluation, lipid metabolism, inflammatory cytokines, oxidative stress, apoptosis, and autophagy of the femoral head were evaluated. The differentially expressed microRNAs (miRs) after Ato treatment were screened out using microarray analysis. The downstream gene and pathway of miR-186 were predicted and their involvement in SANFH rats was analyzed. OB-6 cells were selected to simulate SANFH in vitro. Cell viability, cell damage, inflammation responses, apoptosis, and autophagy were assessed. Ato alleviated SANFH, inhibited apoptosis, and promoted autophagy. miR-186 was significantly upregulated after Ato treatment. miR-186 targeted TLR4 and inactivated the MAPKs/NF-κB pathway. Inhibition of miR-186 reversed the protection of Ato on SANFH rats, while inhibition of TLR4 restored the protective effect of Ato. Ato reduced apoptosis and promoted autophagy of OB-6 cells by upregulating miR-186 and inhibiting the TLR4/MAPKs/NF-κB pathway. In conclusion, Ato reduced apoptosis and promoted autophagy, thus alleviating SANFH via miR-186 and the TLR4-mediated MAPKs/NF-κB pathway.

Total flavonoids of Rhizoma drynariae ameliorate steroid‑induced avascular necrosis of the femoral head via the PI3K/AKT pathway.

Steroid-induced avascular necrosis of the femoral head (SANFH) is a common orthopaedic disease that is difficult to treat. The present study investigated the effects of total flavonoids of Rhizoma drynariae (TFRD) on SANFH and explored its underlying mechanisms. The SANFH rat model was induced by intramuscular injection of lipopolysaccharides and methylprednisolone. Osteoblasts were isolated from the calvariae of neonatal rats and then cultured with dexamethasone (Dex). TFRD was used in vitro and in vivo, respectively. Haematoxylin and eosin staining was used to assess the pathological changes in the femoral head. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling assay and flow cytometry were conducted to detect apoptosis of osteoblasts. The 2,7-dichlorofluorescein-diacetate staining method was used to detect reactive oxygen species (ROS) levels in osteoblasts and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to detect osteoblast proliferation. The expression of caspase-3, Bax, Bcl-2, VEGF, runt-related transcription factor 2 (RUNX2), osteoprotegerin (OPG), osteocalcin (OCN), receptor activator of nuclear factor κB ligand (RANKL) and phosphoinositide 3-kinase (PI3K)/AKT pathway related-proteins were detected via western blotting. It was found that TFRD reduced the pathological changes, inhibited apoptosis, increased the expression of VEGF, RUNX2, OPG and OCN, decreased RANKL expression and activated the PI3K/AKT pathway in SANFH rats. TFRD promoted proliferation, inhibited apoptosis and reduced ROS levels by activating the PI3K/AKT pathway in osteoblasts. In conclusion, TFRD protected against SANFH in a rat model. In addition, TFRD protected osteoblasts from Dex-induced damage through the PI3K/AKT pathway. The findings of the present study may contribute to find an effective treatment for the management of SANFH.

Beware of Steroid-Induced Avascular Necrosis of the Femoral Head in the Treatment of COVID-19-Experience and Lessons from the SARS Epidemic.

SummaryThe recent outbreak of coronavirus disease 2019 (COVID-19) has become a global epidemic. Corticosteroids have been widely used in the treatment of severe acute respiratory syndrome (SARS), and the pathological findings seen in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are very similar to those observed in severe acute respiratory syndrome-related coronavirus (SARS-CoV) infection. However, the long-term use of corticosteroids (especially at high doses) is associated with potentially serious adverse events, particularly steroid-induced avascular necrosis of the femoral head (SANFH). In today’s global outbreak, whether corticosteroid therapy should be used, the dosage and duration of treatment, and ways for the prevention, early detection, and timely intervention of SANFH are some important issues that need to be addressed. This review aims to provide a reference for health care providers in COVID-19 endemic countries and regions.Article FocusHormones are a double-edged sword. This review aims to provide a reference for health care providers in coronavirus disease 2019 (COVID-19) endemic countries and regions, especially with respect to the pros and cons of corticosteroid use in the treatment of patients with COVID-19.Key MessagesIn today’s global outbreak, whether corticosteroid therapy should be used, the dosage and duration of treatment, and ways for the prevention, early detection, and timely intervention of SANFH are some important issues that need to be addressed.Strengths and LimitationsSince SARS was mainly prevalent in China at that time, many evidences in this paper came from the reports of Chinese scholars. There is a bias in the selection of data, which may ignore the differences in environment, race, living habits, medical level and so on. SANFH may be the result of multiple factors. Whether the virus itself is an independent risk factor for SANFH has not been confirmed. In this paper, through literature retrieval, some reference opinions on glucocorticoid usage, diagnosis and treatment of SANFH are given. However, due to the lack of large-scale research data support, it can not be used as the gold standard for the above problems.

Glucocorticoids induce femoral head necrosis in rats through the ROS/JNK/c-Jun pathway.

Osteonecrosis of the femoral head (ONFH) is a common clinical disease with a high disability rate. Apoptosis of osteoblasts caused by high-dose short-term or low-dose long-term glucocorticoid (GC) administration is the biological basis of steroid-induced avascular necrosis of the femoral head (SANFH). The pathogenesis of SANFH has not yet been fully elucidated, and there is currently a lack of effective clinical treatments. Here, we investigated the role of the reactive oxygen species (ROS)/JNK/c-Jun signaling pathway in SANFH. Dexamethasone (Dex) was used to induce apoptosis in osteoblasts, and this resulted in a significant increase in levels of p-JNK, p-c-Jun, Bax, caspase-3, caspase-9, cytochrome C, Beclin-1, and LC3, and a decrease in levels of P62 and Bcl-2. In addition, intracellular ROS levels were increased and mitochondrial membrane potential was decreased. Administration of 3-MA, an autophagy inhibitor, attenuated Dex-mediated changes in autophagy and apoptosis. A rat model of ONFH exhibited severe bone trabecular hollow bone pits along with a significant increase in femoral head cell apoptosis compared with the control group. Additionally, micro-CT analysis showed that both bone tissue content and femoral head integrity were significantly reduced in the ONFH group. Furthermore, 3-MA treatment decreased the effect of Dex on GC-induced ONFH and osteoblast apoptosis in rats and could counteract microstructure destruction due to femoral head necrosis. In summary, our data suggest that GC can induce osteoblast apoptosis and autophagy through the ROS/JNK/c-Jun signaling pathway, which contributes to ONFH.

Ghrelin attenuates avascular necrosis of the femoral head induced by steroids in rabbits

Purpose: Ghrelin is an endogenous ligand for growth hormone secretagogue receptor. The current study was aimed at examining the effect of ghrelin on avascular necrosis of the femoral head (ANFH) induced by steroids in a rabbit model and also exploring the underlying mechanism.&#x0D; Methods: Experimental rabbits were separated into three groups: Control, Vehicle and Ghrelin. We established a steroid-induced ANFH model in rabbits. Then, MRI scanning and hematoxylin-eosin staining (HE) were conducted to see ANFH. The mRNA levels of Vascular Endothelial Growth Factor (VEGF) and Bone Morphogenetic Protein 2 (BMP-2) were evaluated using real-time qRT-PCR.&#x0D; Results: Rabbits in the Vehicle group showed increased empty bone lacunae, reduced bone trabecula in femoral head; the number of hematopoietic cells in the bone marrow was reduced, whereas number of adipocytes increased with evident fusion phenomenon in comparison with the Control group. All of the changes induced in Vehicle group were attenuated in Ghrelin group. MRI scanning showed obvious necrosis of femoral head in the Vehicle group and less in the Ghrelin group. The mRNA levels of VEGF and BMP-2 were raised in Vehicle group and further enhanced in Ghrelin group.&#x0D; Conclusion: Ghrelin attenuates steroid-induced avascular necrosis in femoral head in rabbit model. A possible mechanism may be through VEGF/BMP-2 axis.&#x0D; Keywords: ANFH, BMP-2, Ghrelin, VEGF

Mechanism of Methylprednisolone-Induced Primary Cilia Formation Disorder and Autophagy in Osteoblasts.

ObjectiveTo study the role of primary cilia formation disorder and osteoblasts autophagy in the pathogenesis of steroid‐induced avascular necrosis of the femoral head (SANFH).MethodsOsteoblasts were isolated from rabbit bones and treated with 1 μM Methylprednisolone for 0, 12, 24, 48, and 72 h. The Beclin1, MAP1LC3, Atg‐5, Atg‐12, IFT20 and OFD1 mRNAs and proteins were detected by PCR and Western blotting, and their correlation was statistically analyzed. The lengths of osteoblast cilia were measured under a laser confocal microscope, and the autophagy flux was tracked by transfecting the osteoblasts with GFP‐RFP‐LC3 lentivirus.ResultsMethylprednisolone significantly upregulated Beclin1, MAP1LC3, Atg‐5, Atg‐12 and OFD1 mRNAs and proteins in a time‐dependent manner, and decreased that of IFT20 (P < 0.05). In addition, the autophagy flux in the osteoblasts also increased and the ciliary length decreased in a time‐dependent manner after Methylprednisolone treatment. The length of the cilia were 5.46 ± 0.11 um at 0 h, 4.08 ± 0.09 um at 12 h, 3.07 ± 0.07 um at 24 h, 2.31 ± 0.10 um at 48 h, and finally 1.15 ± 0.04 um at 72 h. Methylprednisolone treatment also affects primary cilium numbers in cultures, for 0 to 72 h. The autophagy regulatory genes, Beclin1, MAP1LC3, Atg‐5 and Atg‐12, were found to be negatively correlated with IFT20, with an average correlation coefficient of −0.81. A negative correlation was also found between OFD1 and IFT20, with an average correlation coefficient of −0.53.ConclusionMethylprednisolone inhibits primary cilia formation and promotes autophagy, which could be the pathological basis of SANFH. The exact regulatory mechanism needs to be further studied in vivo.

Protective effect of Agrimonia pilosa polysaccharides on dexamethasone-treated MC3T3-E1 cells via Wnt/β-Catenin pathway.

A water‐soluble polysaccharide (APP‐AW) was isolated from Agrimonia pilosa and prepared to three sulphated derivatives (S1, S2 and S3). The results showed that pre‐treatment with APP‐AW, S1, S2 and S3 each at the concentration of 50 μg/mL for 48 hours was able to prevent cytotoxicity induced by 1 μmol/L dexamethasone (Dex) in MC3T3‐E1 cells via inhibition of apoptosis, which is in line with the findings in flow cytometry analysis. Meanwhile, the decreased ALP activity, collagen content, mineralization, BMP2, Runx2, OSX and OCN protein expression in DEX‐treated MC3T3‐E1 cells were reversed by the addition of APP‐AW, S1, S2 and S3. Moreover, APP‐AW, S1, S2 and S3 rescued DEX‐induced increase of Bax, cytochrome c and caspase‐3 and decrease of Bcl‐2, Wnt3, β‐catenin and c‐Myc protein expression in MC3T3‐E1 cells. Our findings suggest that pre‐treatment with APP‐AW, S1, S2 and S3 could significantly protect MC3T3‐E1 cells against Dex‐induced cell injury via inhibiting apoptosis and activating Wnt/β‐Catenin signalling pathway, thus application of these polysaccharides may be a promising alternative strategy for steroid‐induced avascular necrosis of the femoral head (SANFH) therapy.

Structural characterization of a safflower polysaccharide and its promotion effect on steroid-induced osteonecrosis in vivo

A water-soluble polysaccharide (SPAW) was purified from Safflower and it was identified to be (1→3)-linked β-d-Glucan. The therapeutic effect and underlying mechanism of SPAW on steroid-induced avascular necrosis of the femoral head (SANFH) in a rabbit model was performed here. The abnormal histopathologic changes and apoptosis of femoral head in model group were significantly reverted after SPAW (25, 100 and 200 mg/kg) administration for 60 days, as evidenced by the a decline of empty lacunae rate, the average bone marrow fat cell size and the proportion of apoptotic cells. Furthermore, administration of SPAW significantly decreased the Bax and caspase-3 protein expression, but increased the protein expression of Bcl-2 when compared these in model rabbits. Meanwhile, increased hydroxyproline (HOP) and decreased serum hexosamine (HOM) concentration in rabbit serum were turned to the opposite way. The present study suggested that SPAW may provide an alternative treatment for the treatment of SANFH.

Influence of MicroRNA-141 on Inhibition of the Proliferation of Bone Marrow Mesenchymal Stem Cells in Steroid-Induced Osteonecrosis via SOX11.

ObjectiveTo investigate whether miR‐141 and the sex determination region of Y chromosome box 11 (SOX11) play roles in steroid‐induced avascular necrosis of the femoral head (SANFH), and to explore whether miR‐141 could target SOX11 to influence the proliferation of bone marrow mesenchymal stem cells (BMSC).MethodsBone marrow mesenchymal stem cells (BMSC) were isolated and cultured from 4‐week‐old Sprague Dawley rats. A flow cytometry assay was performed to identify BMSC. BMSC were divided into two groups: a control group and a dexamethasone (DEX) group. BMSC were transfected by miR‐141 mimic, miR‐141 inhibitor, and SOX11. Real‐time polymerase chain reaction (PCR) assay was performed to investigate the mRNA expression of miR‐141 and SOX11. The results were used to determine the effect of transfection and to verify the expression in each group and the association between miR‐141 and SOX11. Luciferase reporter assay revealed the targeted binding site between miR‐141 and the 3′‐untranslated region of SOX11 mRNA. MTT assays were performed to investigate the proliferation of BMSC in the miR‐141 mimic, miR‐141 inhibitor, and SOX11 groups.ResultThe results of the flow cytometry assay suggested that cells were positive for CD29 and CD90 while negative for CD45. This meant that the isolated and cultured cells were not hematopoietic stem cells. In addition, cell transfection was successful based on the expression of miR‐141 and SOX11. According to the results of real‐time PCR assay, the mRNA expression of miR‐141 in SANFH was upregulated (4.117 ± 0.042 vs 1 ± 0.027, P < 0.001), while SOX11 was downregulated (0.611 ± 0.055 vs 1 ± 0.027, P < 0.001) compared with the control group. Based on the results of the luciferase experiment, MiR‐141 could directly target the expression of SOX11. Inhibition of miR‐141 could upregulate the expression of SOX11 (2.623 ± 0.220 vs 1 ± 0.095, P < 0.001) according to the results of a real‐time PCR assay. MiR‐141 inhibited the proliferation of BMSC (0.618 ± 0.092 vs 1.004 ± 0.082, P < 0.001), while suppression of miR‐141 increased the proliferation of BMSC (0.960 ± 0.095 vs 0.742 ± 0.091, P < 0.001). Furthermore, according to the results of the MTT assay, SOX11 promoted the proliferation of BMSC (1.064 ± 0.093 vs 0.747 ± 0.090, P < 0.001).ConclusionMiR‐141 inhibited the proliferation of BMSC in SANFH by targeting SOX11. Inhibition of miR‐141 upregulated the expression of SOX11 and promoted the proliferation of BMSC. MiR‐141 and SOX11 could be new targets for investigating the mechanism of SANFH.

Allicin inhibits osteoblast apoptosis and steroid-induced necrosis of femoral head progression by activating the PI3K/AKT pathway.

Steroid-induced avascular necrosis of the femoral head (SANFH) is a major complication of long-term or excessive clinical use of glucocorticoids. Allicin is a classical ingredient extracted from garlic and has many functions such as anti-apoptosis and antibacterial effects. The purpose of this study was to investigate the effect and the mechanism of allicin on apoptosis of osteoblasts induced by dexamethasone (Dex) and SANFH in rats. In vitro, we performed CCK-8, western blotting, TUNEL and other experiments, and the results of these experiments showed that allicin could inhibit the Dex-induced abnormal expression of C-caspase3, C-caspase9, Bax, cytochrome C and Bcl-2 by activating the PI3K/AKT pathway. In vivo, the results of micro-CT, hematoxylin-eosin staining and immunohistochemical analysis suggested that allicin could effectively inhibit the progress of SANFH in rats. In summary, our experiments indicate that allicin is a potential drug for the treatment of SANFH.