Rat Femoral Head Research Articles

Tension-Free Weight-Bearing Model of Steroid-Induced Osteonecrosis of Femoral Head in Rats.

Unlike humans, rats are animals that walk on all fours, while humans are bipedal animals that stand, and the hips are subjected to tremendous pressure when walking and standing. In rat steroid-induced femoral head necrosis models, the biomechanical characteristics of the human hip under higher pressure often need to be simulated. Some scholars try to emulate the state of human hip pressure by making rats bear a certain weight, but fixing the weight-bearing object on the rat is tough. Rats can easily break free of immobilization, and sticking the weight on the rats with adhesive tape will cause the rats to suffocate or die from intestinal obstruction. Our research group used elastic therapeutic tape to perform tension-free immobilization of weight-bearing objects in rats so that the rats could breathe freely and not break away from the immobilization under weight-bearing conditions. Compared to the usual steroid-induced femoral head necrosis rat model, we found that this weight-bearing intervention can aggravate the progression of femoral head necrosis in rats.

Effect of genetically engineered drugs in aseptic necrosis of the femoral head in rats

Bone tissue is a dynamic structure with a metabolic function. The maintenance of bone homeostasis is carried out due to the continuous process of its renewal, remodeling. At the same time, a number of pathological processes, such as ischemic catastrophe, can lead to a violation of the balance of maintaining the constancy of the bone structure. One of these diseases is aseptic necrosis of the femoral head. The presented study analyzes the dynamics of expression of genes involved in maintaining bone tissue homeostasis, changes in the histological picture during the development of aseptic necrosis of the femoral head in laboratory rats that did not receive genetically engineered drugs and against the background of the use of inhibitors of biological action IL-6, TNF-α. After induction of aseptic necrosis in the proximal epiphysis of the femur, the histological picture in animals of different groups was not the same. More preserved bone architectonics and a larger volume of bone plates were recorded in rats receiving genetically engineered drugs compared to animals without the introduction of biological agents. The latter also had the most vivid picture of osteodestruction with increased expression of proinflammatory cytokine genes. In animals, against the background of the use of drugs of inhibitors of the biological action of IL-6, TNF-α, from the second week after induction of aseptic necrosis of the mRNA, the profile of the spongiose bone of the proximal epiphysis of the femur tended to increase the expression of osteoreparation genes. At the same time, the greatest inhibition of osteoclastogenesis gene expression was obtained in rats after injection of a monoclonal antibody to the IL-6 receptor.

The effect of the IL-6 monoclonal blocker on the course of aseptic femoral head necrosis in the experiment (pilot study)

Background There is currently no pathogenetically based treatment for aseptic necrosis of the femoral head. One of the most promising areas of possible targeted therapy is the use of genetically engineered drugs, including monoclonal blockers of proinflammatory cytokines, aimed at inhibiting inflammation and indirectly reducing the activity of osteodestruction. The aim of the work is to evaluate the effectiveness of the use of the IL-6 monoclonal blocker in the course of aseptic necrosis of the femoral head in an experiment.Purpose Evaluate the preliminary results of the use of the IL-6 monoclonal blocker in the course of aseptic necrosis of the femoral head in an experiment.Materials and methods Surgical induction of aseptic necrosis of the femoral head was performed in 18 male Wistar rats. The animals were divided into two groups of 9 individuals each. The first group did not receive any treatment, the second received therapy with a monoclonal IL-6 receptor blocker, starting from the second week of the experiment, one injection once every two weeks. All animals were removed from the experiment at 4, 6 and 8 weeks after the induction of aseptic necrosis, 3 rats from each group at a time. Total RNA was isolated from the femoral head on the aseptic necrosis side and the conditionally healthy side as a control. The expression of genes of regulatory proteins of osteogenesis was studied by PCR. To study the features of osteodestructive processes, histological examination of femoral head preparations in all animals was conducted.Results Histological preparations of femoral heads of the second group animals were characterized by less pronounced osteodestructive, chondrodestructive processes compared to the animals that did not receive therapy. The mRNA profile of the rats of the second group displayed an increase in the expression of genes encoding proteins involved in osteoreparation at all stages of the experiment. At the same time, the activity of genes encoding proteins of proinflammatory cytokines, regulatory molecules of osteoclastogenesis was reduced relative to the first group.Discussion The data obtained indicate an important role of inflammation in the regulation of osteodestruction. Inhibition of the biological action of IL-6 contributed to inhibition of the expression of osteoclastogenesis genes, increased activity of bone metabolism genes, and caused a decrease in the intensity of osteodestruction and activation of osteoreparation. Conclusion Preliminary results of the use of a monoclonal blocker of the proinflammatory cytokine IL-6 indicate the inhibition of osteodestructive and strengthening of osteoreparative processes due to the correction of the expression of bone metabolism genes during the progression of aseptic necrosis of the femoral head in rats in an experimental model.

Palliative effect of rotating magnetic field on glucocorticoid-induced osteonecrosis of the femoral head in rats by regulating osteoblast differentiation

With the substantial increase in the overuse of glucocorticoids (GCs) in clinical medicine, the prevalence of glucocorticoid-induced osteonecrosis of the femoral head (GC-ONFH) continues to rise in recent years. However, the optimal treatment for GC-ONFH remains elusive. Rotating magnetic field (RMF), considered as a non-invasive, safe and effective approach, has been proved to have multiple beneficial biological effects including improving bone diseases. To verify the effects of RMF on GC-ONFH, a lipopolysaccharide (LPS) and methylprednisolone (MPS)-induced invivo rat model, and an MPS-induced invitro cell model have been employed. The results demonstrate that RMF alleviated bone mineral loss and femoral head collapse in GC-ONFH rats. Meanwhile, RMF reduced serum lipid levels, attenuated cystic lesions, raised the expression of anti-apoptotic proteins and osteoprotegerin (OPG), while suppressed the expression of pro-apoptotic proteins and nuclear factor receptor activator-κB (RANK) in GC-ONFH rats. Besides, RMF also facilitated the generation of ALP, attenuated apoptosis and inhibits the expression of pro-apoptotic proteins, facilitated the expression of OPG, and inhibited the expression of RANK in MPS-stimulated MC3T3-E1 cells. Thus, this study indicates that RMF can improve GC-ONFH in rat and cell models, suggesting that RMF have the potential in the treatment of clinical GC-ONFH.

Astaxanthin-mediated Nrf2 activation ameliorates glucocorticoid-induced oxidative stress and mitochondrial dysfunction and impaired bone formation of glucocorticoid-induced osteonecrosis of the femoral head in rats

BackgroundOsteonecrosis of the femoral head caused by glucocorticoids (GIONFH) is a significant issue resulting from prolonged or excessive clinical glucocorticoid use. Astaxanthin, an orange-red carotenoid present in marine organisms, has been the focus of this study to explore its impact and mechanism on osteoblast apoptosis induced by dexamethasone (Dex) and GIONFH.MethodsIn this experiment, bioinformatic prediction, molecular docking and dynamics simulation, cytotoxicity assay, osteogenic differentiation, qRT-PCR analysis, terminal uridine nickend labeling (TUNEL) assay, determination of intracellular ROS, mitochondrial function assay, immunofluorescence, GIONFH rat model construction, micro-computed tomography (micro-CT) scans were performed.ResultsOur research demonstrated that a low dose of astaxanthin was non-toxic to healthy osteoblasts and restored the osteogenic function of Dex-treated osteoblasts by reducing oxidative stress, mitochondrial dysfunction, and apoptosis. Furthermore, astaxanthin rescued the dysfunction in poor bone quality, bone metabolism and angiogenesis of GIONFH rats. The mechanism behind this involves astaxanthin counteracting Dex-induced osteogenic damage by activating the Nrf2 pathway.ConclusionAstaxanthin shields osteoblasts from glucocorticoid-induced oxidative stress and mitochondrial dysfunction via Nrf2 pathway activation, making it a potential therapeutic agent for GIONFH treatment.

Jintiange capsule ameliorates glucocorticoid-induced osteonecrosis of the femoral head in rats by regulating the activity and differentiation of BMSCs

Background and aimA surplus of glucocorticoids (GC) is a main cause of non-traumatic osteonecrosis of the femoral head (ONFH), and Jintiange (JTG), as one of the traditional Chinese medicines (TCM), also plays an instrumental role in the alleviation of bone loss simultaneously. Therefore, JTG was thought to be able to reverse GC-induced ONFH (GC-ONFH) to a certain extent. Experimental procedureIn vivo, the effect of JTG on trabeculae in the subchondral bone of the femoral head was investigated using micro-computed tomography (micro-CT), TdT-mediated dUTP nick end labeling (TUNEL) and histological staining; in vitro, proliferation, viability, apoptosis, and senescence of purified bone mesenchymal stem cells (BMSCs) were examined to demonstrate the direct impact of JTG on these cells. Meanwhile after using a series of interventions, the function of JTG on BMSC differentiation could be assessed by measuring of osteogenic and adipogenic markers at levels of protein and mRNA. ResultsOur final results demonstrated that with the involvement of Wnt/β-catenin pathway, JTG was able to significantly promote osteogenesis, restrain adipogenesis, delay senescence in BMSCs, reduce osteoclast number, weaken apoptosis, and enhance proliferation of osteocytes, all of which could mitigate the progression of subchondral osteonecrosis. ConclusionAccording to the results of experiments in vitro and vivo, JTG was deemed to relieve the early GC-ONFH using the prevention of destruction of subchondral bone, which was contributed to regulating the differentiation of BMSCs and the number of osteoclasts.

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.

Echinacoside alleviates glucocorticoid induce osteonecrosis of femoral head in rats through PI3K/AKT/FOXO1 pathway

Steroid-induced osteonecrosis of the femoral head (SONFH), caused by glucocorticoid (GC) administration, is known to exhibit a high incidence worldwide. Although osteoblast apoptosis has been reported as an important cytological basis of SONFH, the precise mechanism remains elusive. Echinacoside (Ech), a natural phenylethanoid glycoside, exerts multiple beneficial effects, such as facilitation of cell proliferation and anti-inflammatory and anticancer activities. Herein, we aimed to explore the regulatory mechanism underlying glucocorticoid-induced osteoblast apoptosis and determine the protective efficacy of Ech against SONFH. We comprehensively surveyed multiple public databases to identify SONFH-related genes. Using bioinformatics analysis, we identified that the PI3K/AKT/FOXO1 signaling pathway was most strongly associated with SONFH. We examined the protective effect of Ech against SONFH using in vivo and in vitro experiments. Specifically, dexamethasone (Dex) decreased p-PI3K and p-AKT levels, which were reversed following Ech addition. Validation of the PI3K inhibitor (LY294002) and molecular docking of Ech and PI3K/AKT further indicated that Ech could directly enhance PI3K/AKT activity to alleviate Dex-induced inhibition. Interestingly, Dex upregulated the expression of FOXO1, Bax, cleaved-caspase-9, and cleaved-caspase-3 and enhanced MC3T3-E1 apoptosis; application of Ech and siRNA-FOXO1 reversed these effects. In vitro, Ech decreased the number of empty osteocytic lacunae, reduced TUNEL and FOXO1 positive cells, and improved bone microarchitecture. Our results provide robust evidence that PI3K/AKT/FOXO1 plays a crucial role in the development of SONFH. Moreover, Ech may be a promising candidate drug for the treatment of SONFH.

Analysis of the Potential Angiogenic Mechanisms of BuShenHuoXue Decoction against Osteonecrosis of the Femoral Head Based on Network Pharmacology and Experimental Validation.

Osteonecrosis of the femoral head (ONFH) is a common orthopedic disease with a high disability rate. The clinical effect of BuShenHuoXue decoction (BSHX) for ONFH is satisfactory. We aimed to elucidate the potential angiogenic mechanisms of BSHX in a rat femoral osteonecrosis model and bone marrow mesenchymal stem cells (BMSCs). With in vivo experiments, we established the steroid-induced osteonecrosis of the femoral head (SONFH) model using Sprague-Dawley (SD) rats (8-week-old). The rats were randomly divided into five group of 12 rats each and given the corresponding interventions: control, model (gavaged with 0.9% saline), BSHX low-, medium- and high-dose groups (0.132 3, 0.264 6, and 0.529 2 g/mL BSHX solution by gavage). After 12 weeks, haematoxylin and eosin (H&E) staining was preformed to evaluate rat osteonecrosis. the expression of angiogenic factors (CD31, VEGFA, KDR, VWF) in rat femoral head was detected by immunohistochemistry, qPCR and western blotting. In cell experiment, BMSCs were isolated and cultured in the femoral bone marrow cavity of 4-week-old SD rats. BMSCs were randomly divided into eight groups and intervened with different doses of BSHX-containing serum and glucocorticoids: control group (CG); BSHX low-, medium-, and high-dose groups (CG + 0.661 5, 1.323, and 2.646 g/kg BSHX gavage rat serum); dexamethasone (Dex) group; and Dex + BSHX low-, medium-, and high-dose groups (Dex + 0.661 5, 1.323, and 2.646 g/kg BSHX gavaged rat serum), the effects of BSHX-containing serum on the angiogenic capacity of BMSCs were examined by qPCR and Western blotting. A co-culture system of rat aortic endothelial cells (RAOECs) and BMSCs was then established. Migration and angiogenesis of RAOECs were observed using angiogenesis and transwell assay. Identification of potential targets of BSHX against ONFH was obtained using network pharmacology. BSHX upregulated the expression of CD31, VEGFA, KDR, and VWF in rat femoral head samples and BMSCs (p < 0.05, vs. control group or model group). Different concentrations of BSHX-containing serum significantly ameliorated the inhibition of CD31, VEGFA, KDR and VWF expression by high concentrations of Dex. BSHX-containing serum-induced BMSCs promoted the migration and angiogenesis of RAOECs, reversed to some extent the adverse effect of Dex on microangiogenesis in RAOECs, and increased the number of microangiogenic vessels. Furthermore, we identified VEGFA, COL1A1, COL3A1, and SPP1 as important targets of BSHX against ONFH. BSHX upregulated the expression of angiogenic factors in the femoral head tissue of ONFH model rats and promoted the angiogenic capacity of rat RAOECs and BMSCs. This study provides an important basis for the use of BSHX for ONFH prevention and treatment.

Development of triple-functionalized calcium phosphate nanoparticles as an advanced drug delivery system for bone tissue repair

IntroductionDuring tissue repair or regeneration, several bioactive molecules are released and interact with each other and act as complex additives or inhibitors for tissue reconstruction. In this study, the bone-healing effects of the combination treatment with tumor necrosis factor-α (TNF-α) inhibition, vascular endothelial growth factor A (VEGF-A) and bone morphogenetic protein-7 (BMP-7) release by gene silencing, and gene transfection with calcium phosphate nanoparticles (CaP) in the rat femoral head was histologically, morphologically, and biochemically evaluated. MethodsA triple-functionalized paste of CaP carrying plasmid DNA encoding for BMP-7 and for VEGF), and siRNA against TNF-α was developed and denoted as CaP3mix. To compare the effects of 3mixCaP, CaP with plasmid DNA encoding BMP-7, VEGF, or siRNA encoding TNF-α was prepared and denoted as CaP/PEI/pBMP-7/SiO2, CaP/PEI/pVEGF/SiO2, or CaP/PEI/siRNA-TNF-α/SiO2, respectively. The bone healing in bone defects in the rat femoral head was investigated after 10 and 21 days of implantation. ResultsThe levels of bone formation-related markers OCN, Runx2, and SP7 increased at the protein and gene levels in 3mixCaP after 10 days, and 3mixCaP significantly accelerated bone healing compared with the other treatments after 21 days of implantation. ConclusionThe triple-functionalized CaP paste loading plasmid DNA encoding BMP-7 and VEGF and siRNA encoding TNF-α is a promising bioactive material for bone tissue repair.

Erythropoietin suppresses osteoblast apoptosis and ameliorates steroid-induced necrosis of the femoral head in rats by inhibition of STAT1-caspase 3 signaling pathway

BackgroundSteroid-induced avascular necrosis of the femoral head (SANFH) is characterized by osteoblast apoptosis, leading to a loss of bone structure and impaired hip joint function. It has been demonstrated that erythropoietin (EPO) performs a number of biological roles.ObjectiveWe examined the effects of EPO on SANFH and its regulation of the STAT1-caspase 3 signaling pathway.MethodIn vitro, osteoblasts were treated with dexamethasone (Dex) or EPO. We identified the cytotoxicity of EPO by CCK-8, the protein expression of P-STAT1, cleaved-caspase9, cleaved-caspase3, Bcl-2, BAX, and cytochrome c by Western blotting, and evaluated the apoptosis of osteoblasts by flow cytometry. In vivo, we analyzed the protective effect of EPO against SANFH by hematoxylin and eosin (H&E), Immunohistochemical staining, and Micro-computed tomography (CT).ResultsIn vitro, EPO had no apparent toxic effect on osteoblasts. In Dex-stimulated cells, EPO therapy lowered the protein expression of BAX, cytochrome c, p-STAT1, cleaved-caspase9, and cleaved-caspase3 while increasing the expression of Bcl-2. EPO can alleviate the apoptosis induced by Dex. In vivo, EPO can lower the percentage of empty bone lacunae in SANFH rats.ConclusionThe present study shows that EPO conferred beneficial effects in rats with SANFH by inhibiting STAT1-caspase 3 signaling, suggesting that EPO may be developed as a treatment for SANFH.

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.

Cyasterone has a protective effect on steroid-induced Osteonecrosis of the femoral head.

Cyasterone alleviated the apoptosis of BMSCs induced by Dexamethasone via the PI3K/AKT signaling pathway. In addition, Cyasterone had a protective effect on SIONFH model rats by reducing the percentage of empty bone lacunae. To study the effect of Cyasterone on apoptosis of rat BMSCs and its function on the SIONFH rat model. Rat BMSCs were cultured and divided into Control, DXM and Cyasterone (DXM+Cyasterone) groups. The apoptosis of each group was detected by flow cytometry, the expressions of Caspase-3 and Caspase-9 were detected by immunofluorescence staining, and the mRNA and protein expressions of AKT, BAX, P53, P85, Bcl-2 and Cytochrome C were detected by qPCR and WB. In animal experiments, the femoral head of rats were subjected to HE staining and Micro-CT to observe the necrosis and repair conditions. The apoptosis rate of DXM and Cyasterone groups increased compared with Control group, and the apoptosis rate of Cyasterone group decreased compared with DXM group. Compared with DXM group, the mRNA expression of BAX, P53, P85 and Cytochrome C in Cyasterone group were increased, while the protein expression of AKT and Bcl-2 decreased. The histopathological and morphological analysis showed that Cyasterone promoted the trabecular bone structure in rat, which evenly benefit for the repair of SIONFH. Cyasterone can reduce the apoptosis of rat BMSCs induced by Dexamethasone, and help promoting the bone repair in SIONFH rats.

Effects of Thymoquinone on Urotensin-II and TGF-β1 Levels in Model of Osteonecrosis in Rats.

Objectives: We aimed to investigate the therapeutic effects of thymoquinone (TMQ) treatment in osteonecrotic rats by evaluating protein levels, osteonecrosis (ON) levels, fatty acid degeneration, oxidative status, and plasma levels of Urotensin-II (U-II) and transforming growth factor-beta (TGF-β1). Materials and Methods: 40 weight-matched adult male Wistar rats were grouped as control (n = 10), methylprednisolone acetate (MPA) (n = 10), thymoquinone (TMQ) (n = 10), and MPA + TMQ (n = 10). To induce ON, 15-week-old animals were subcutaneously injected with MPA at a dose of 15 mg/kg twice weekly for 2 weeks. TMQ was injected into 15-week-old rats via gastric gavage at a dose of 80 mg/kg per day for 4 weeks. The rats in the MPA + TMQ group were administered TMQ 2 weeks before the MPA injection. At the end of the treatments, cardiac blood samples and femur samples were collected for biochemical and histological evaluations. Results: In the control and TMQ groups, no ON pattern was observed. However, in tissues exposed to MPA, TMQ treatment resulted in significantly decreased ON levels compared to the MPA group. The number of cells that were positive for 8-OHdG and 4-HNE was significantly lower in the MPA + TMQ group than in the MPA group (p < 0.05). In terms of TGF-β1 and U-II levels, we observed that both TGF-β1 (367.40 ± 23.01 pg/mL vs. 248.9 ± 20.12 pg/mL) and U-II protein levels (259.5 ± 6.0 ng/mL vs. 168.20 ± 7.90 ng/mL) increased significantly in the MPA group compared to the control group (p < 0.001). Furthermore, TGF-β1 (293.50 ± 14.18 pg/mL) and U-II (174.80 ± 4.2 ng/mL) protein levels were significantly decreased in the MPA + TMQ group compared to the MPA group (p < 0.05 and p < 0.01, respectively). There was a statistically positive correlation (p < 0.05) between the TGF-β1 and U-II protein levels in all groups (p = 0.002, rcontrol = 0.890; p = 0.02, rTMQ = 0.861; p = 0.024, rMPA+TMQ = 0.868) except for the MPA group (p < 0.03, rMedrol = -0.870). Conclusions: As far as we know, this is the first study to demonstrate the curative functions of TMQ on ON by causing a correlated decrease in the expression of U-II and TGF-β1 in the femoral heads of rats.

LncRNA DGCR5-encoded polypeptide RIP aggravates SONFH by repressing nuclear localization of β-catenin in BMSCs

The differentiation fate of bone marrow mesenchymal stem cells (BMSCs) affects the progression of steroid-induced osteonecrosis of the femoral head (SONFH). We find that lncRNA DGCR5 encodes a 102-amino acid polypeptide, RIP (Rac1 inactivated peptide), which promotes the adipogenic differentiation of BMSCs and aggravates the progression of SONFH. RIP, instead of lncRNA DGCR5, binds to the N-terminal motif of RAC1, and inactivates the RAC1/PAK1 cascade, resulting in decreased Ser675 phosphorylation of β-catenin. Ultimately, the nuclear localization of β-catenin decreases, and the differentiation balance of BMSCs tilts toward the adipogenesis lineage. In the femoral head of rats, overexpression of RIP causes trabecular bone disorder and adipocyte accumulation, which can be rescued by overexpressing RAC1. This finding expands the regulatory role of lncRNAs in BMSCs and suggests RIP as a potential therapeutic target.

Administration of necrostatin-1 ameliorates glucocorticoid-induced osteonecrosis of the femoral head in rats.

Glucocorticoid (GC)-induced osteonecrosis of the femoral head (ONFH) is a serious complication of glucocorticoid treatment and is characterized by dysfunctional bone reconstruction at necrotic sites. Our previous study confirmed the protective potential of necrostatin-1, a selective blocker of necroptosis, in glucocorticoid-induced osteoporosis. In this study, rat models of GC-induced ONFH were established to evaluate the effects of necrostatin-1 on osteonecrotic changes and repair processes. Osteonecrosis was verified by histopathological staining. An analysis of trabecular bone architecture was performed to evaluate osteogenesis in the osteonecrotic zone. Then, necroptotic signaling molecules such as RIP1 and RIP3 were examined by immunohistochemistry. Histopathological observations indicated that necrostatin-1 administration reduced the incidence of osteonecrosis and the osteogenic response in subchondral areas. Additionally, bone histomorphometry demonstrated that necrostatin-1 intervention could restore bone reconstruction in the necrotic zone. The protective mechanism of necrostatin-1 was related to the inhibition of RIP1 and RIP3. Necrostatin-1 administration alleviated GC-induced ONFH in rats by attenuating the formation of necrotic lesions, recovering the function of osteogenesis, and suppressing glucocorticoid-induced osteocytic necroptosis by inhibiting the expression of RIP1 and RIP3.

Proanthocyanidins Inhibit Osteoblast Apoptosis via the PI3K/AKT/Bcl-xL Pathway in the Treatment of Steroid-Induced Osteonecrosis of the Femoral Head in Rats.

Steroid-induced osteonecrosis of the femoral head (SONFH) is a common clinical disease caused by massive or prolonged use of steroids. Its pathogenesis is unclear, but its incidence is increasing annually. It is characterized by an insidious and rapid onset, and high disability rate, causing a great burden on patients' daily life. Therefore, clarifying its pathogenesis and providing early and effective treatment for steroid osteonecrosis is important. In vivo, we used methylprednisolone (MPS) to construct a SONFH rat model and employed Mirco-ct, Hematoxylin and eosin (H&E) staining, and TdT-mediated dUTP nick end labeling (TUNEL) staining analysis to evaluate the therapeutic effects of proanthocyanidins (PACs). Network pharmacology analysis was conducted to mine targets associated with femoral head necrosis, and PACs analyzed possible molecular mechanisms. In vitro, PACs were added at different doses after treatment of cells with dexamethasone (DEX), and human osteoblast-like sarcoma(MG-63) cell apoptosis was determined by Annexin V-FITC-PI. The mechanisms by which PACs regulate bone metabolism via the Phosphoinositide 3-kinase(PI3K)/protein kinase B(AKT)/Recombinant Human B-Cell Leukemia/Lymphoma 2 XL(Bcl-xL) axis were explored by Western blotting. In vivo studies showed that PACs prevented SONFH in rat model. The PI3K/AKT/Bcl-xL signaling pathway was selected by network pharmacology approach; in vitro studies showed that proanthocyanidin-activated AKT and Bcl-xL inhibited osteoblast apoptosis. PACs can inhibit excessive osteoblast apoptosis in SONFH via the PI3K/AKT/Bcl-xL signaling axis and have potential therapeutic effects.

Therapeutic effect of human umbilical cord mesenchymal stem cells in early traumatic osteonecrosis of the femoral head.

BackgroundOsteonecrosis of the femoral head (ONFH) is a refractory disease due to its unclear pathomechanism. Therapies during the early stage of ONFH have not achieved satisfactory results. Therefore, this study aims to explore the available evidence for the therapeutic effect of human umbilical cord mesenchymal stem cells (HUCMSCs) on early-stage traumatic ONFH.MethodsEarly-stage traumatic ONFH was established. The femoral heads of rats were then locally administered HUCMSCs. Four weeks and eight weeks after surgery, bone repair of the necrotic area in the femoral head was analyzed to evaluate the therapeutic effect of HUCMSCs using micro-CT, histopathological staining, immunofluorescence staining, Luminex.ResultsHUCMSCs were still present in the femoral head four weeks later, and the morphological, micro-CT and histopathological outcomes in the 4-week HUCMSC-treated group were better than those in the model, NS and 8-week HUCMSC-treated groups. Local transplantation of HUCMSCs promoted bone repair and prevented bone loss in the necrotic area of the femoral head.ConclusionsHUCMSCs can survive and positively affect the femoral head through local transplantation in early-stage traumatic ONFH. The conclusions of this study can provide a treatment option for patients who have ONFH and can serve as basic research on the advanced development of this disease.The Translational potential of this articleThe study indicated that the positive effect of exogenous HUCMSCs in the treatment of early-stage traumatic ONFH provides the solid basis and guidance for the clinical application of HUCMSCs.

SIRT6 Prevents Glucocorticoid-Induced Osteonecrosis of the Femoral Head in Rats.

Objective Glucocorticoid-induced osteonecrosis of the femoral head is one of the most common causes of nontraumatic osteonecrosis of the femoral head, but its exact pathogenesis remains unclear. The aim of this study was to investigate the role of SIRT6 in the maintenance of bone tissue morphology and structure, intravascular lipid metabolism, and its potential molecular mechanism in glucocorticoid-induced osteonecrosis of the femoral head. Methods SIRT6 adenovirus was transfected into GIONFH in rats. The microstructure of rat bone was observed by micro-CT and histological staining, and the expression of bone formation-related proteins and angiogenesis-related factors was determined through western blot and immunohistochemistry. Alkaline phosphatase activity, alizarin red staining, and the expression levels of Runx2 and osteocalcin were used to evaluate the osteogenic potential. And in vitro tube formation assay and immunofluorescence were used to detect the ability of endothelial cell angiogenesis. Results Dexamethasone significantly inhibited osteoblast differentiation, affected bone formation, and destroyed microvessel formation, increased the intracellular Fe2+ and ROS levels and induced the occurrence of ferroptosis. SIRT6 can inhibit ferroptosis and restore the ability of bone formation and angiogenesis. Conclusion SIRT6 can inhibit the occurrence of ferroptosis, reduce the damage of vascular endothelium, and promote osteogenic differentiation, so as to prevent the occurrence of osteonecrosis of the femoral head.

Glucocorticoids induce osteonecrosis of the femoral head in rats via PI3K/AKT/FOXO1 signaling pathway.

BackgroundSteroid-induced osteonecrosis of the femoral head (SONFH) is a disorder that causes severe disability in patients and has a high incidence worldwide. Although glucocorticoid (GC)-induced apoptosis of osteoblasts is an important cytological basis of SONFH, the detailed mechanism underlying SONFH pathogenesis remains elusive. PI3K/AKT signaling pathway was reported to involve in cell survival and apoptosis.ObjectiveWe explored the role of PI3K/AKT/FOXO1 signaling pathway and its downstream targets during glucocorticoid -induced osteonecrosis of the femoral head.MethodsWe obtained gene expression profile of osteoblasts subjected to dexamethasone (Dex) treatment from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) were screened out and functional enrichment analysis were conducted by bioinformatics analysis. In vitro, we analyzed Dex-induced apoptosis in MC3T3-E1 cells and explored the role of PI3K/AKT/FOXO1 signaling pathway in this phenomenon by employing siRNA-FOXO1 and IGF-1(PI3K/AKT agonist). Finally, we verified our results in a rat model of SONFH.ResultsIn Dex-treated osteoblasts, DEGs were mainly enriched in the FOXO signaling pathway. Dex inhibited MC3T3-E1 cell viability in a dose-dependent effect and induced apoptosis by increasing the expression levels of FOXO1, Bax, cleaved-Caspase-3, and cleaved-Caspase-9, while reducing the expression of Bcl-2. Notably, these results were reversed by siRNA-FOXO1 treatment. Dex inhibited PI3K/AKT signaling pathway, upregulated FOXO1 expression and increased FOXO1 nuclear translocation, which were reversed by IGF-1. Compared to normal rats, the femoral head of SONFH showed increased expression of FOXO1, increased number of apoptotic cells, and empty osteocytic lacunas, as well as decreased bone tissue content and femoral head integrity. Significantly, the effects of GC-induced SONFH were alleviated following IGF-1 treatment.ConclusionDex induces osteoblast apoptosis via the PI3K/AKT/FOXO1 signaling pathway. Our research offers new insights into the underlying molecular mechanisms of glucocorticoid-induced osteonecrosis in SONFH and proposes FOXO1 as a therapeutic target for this disease.