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

Fibroblast growth factor 23 inhibition attenuates steroid-induced osteonecrosis of the femoral head through pyroptosis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Fibroblast growth factor 23 inhibition attenuates steroid-induced osteonecrosis of the femoral head through pyroptosis Lun Fang, Gang Zhang, Yadi Wu, Hao Li, Zhongzhe Li, Beilei Yu, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3897523/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 15 Jul, 2024 Read the published version in Scientific Reports → Version 1 posted 8 You are reading this latest preprint version Abstract Steroid-induced osteonecrosis of the femoral head (SONFH) is the predominant cause of non-traumatic osteonecrosis of the femoral head (ONFH). Impaired blood supply and reduced osteogenic activity of the femoral head are the key pathogenic mechanisms of SONFH. Fibroblast growth factor 23 (FGF23) levels are not only a biomarker for early vascular lesions caused by abnormal mineral metabolism, but can also act directly on the peripheral vascular system, leading to vascular pathology. The aim of this study was to observe the role of FGF23 on bone microarchitecture and vascular endothelium, and to investigate activation of pyroptosis in SONFH. Lipopolysaccharide (LPS) combined with methylprednisolone (MPS) was applied for SONFH mouse models, and adenovirus was used to increase or decrease the level of FGF23. Micro-CT and histopathological staining were used to observe the structure of the femoral head, and immunohistochemical staining was used to observe the vascular density. The cells were further cultured in vitro and placed in a hypoxic environment for 12h to simulate the microenvironment of vascular injury during SONFH. The effect of FGF23 on osteogenic differentiation was evaluated using alkaline phosphatase staining, alizarin red S staining and expression of bone formation-related proteins. Matrigel tube formation assay in vitro and immunofluorescence were used to detect the ability of FGF23 to affect endothelial cell angiogenesis. Steroids activated the pyroptosis signaling pathway, promoted the secretion of inflammatory factors in SONFH models, led to vascular endothelial dysfunction and damaged the femoral head structure. In addition, FGF23 inhibited the HUVECs angiogenesis and BMSCs osteogenic differentiation. FGF23 silencing attenuated steroid-induced osteonecrosis of the femoral head by inhibiting the pyroptosis signaling pathway, and promoting osteogenic differentiation of BMSCs and angiogenesis of HUVECs in vitro. Biological sciences/Cell biology Biological sciences/Molecular biology Osteonecrosis of the femoral head steroid FGF23 pyroptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Osteonecrosis of the femoral head (ONFH) is one of the common clinical diseases in orthopedics. The main clinical features are limited flexion and extension, claudication, severe pain in the hip and so on. It has a high prevalence in young and middle-aged people aged 20–50 years old, with a trend towards younger age [ 1 ]. There is a high disability rate in ONFH, which seriously affects patients' physical health and quality of life, and brings great psychological pressure to patients. ONFH is divided into non-traumatic and traumatic, and steroid-induced osteonecrosis of the femoral head (SONFH) is the main cause of non-traumatic osteonecrosis of the femoral head. The "vascular theory" plays a leading role in the pathophysiological mechanism of SONFH and is gradually being recognized [ 2 ]. The imbalance in the vascular homeostasis of the femoral head causes an impaired blood supply to the femoral head, resulting in hypoxia of the corresponding tissues, which can lead to programmed death and damage of osteoblasts and consequently to the collapse of the femoral head structure [ 3 ]. New research has shown that the treatment of ONFH by inhibiting inflammatory factors and protecting vascular endothelial cells from damage has been effective in animal models and in clinical practice [ 4 , 5 ]. Fibroblast growth factor 23 (FGF23) is a phosphophilic hormone produced by bone. Studies have shown that FGF23 is a physiological regulator of phosphate and vitamin D metabolism and is indispensable for maintaining serum phosphate levels [ 6 ]. FGF23 has been identified as a gene associated with autosomal dominant hypophosphatemic rickets (ADHR), which plays an important role in the development of bone diseases [ 7 , 8 ]. As research progressed, several reports identified different roles for FGF23 in metabolic regulation, rheumatism, and cardiac hypertrophy. In particular, FGF23 promotes reactive oxygen species (ROS) production by upregulating the expression of reductive coenzyme II oxidase 2 in coronary artery endothelial cells, and influences cell apoptosis and vascular injury by stimulating endothelial cell migration and proliferation [ 9 , 10 ]. In recent years, the study of SONFH has penetrated into the cellular biochemical level, which mainly includes apoptosis, autophagy [ 11 ], pyroptosis. Among them, pyroptosis is a new mode of cellular inflammatory and programmed death between apoptosis and necrosis, which is a programmed cellular death mediated by gasdermin (GSDMs) with the involvement of NOD-like receptor protein 3 (NLRP3) inflammatory vesicles and dependence on cysteinyl aspartate specific proteinase (caspase). Cells suffer from external stimuli through a series of immune responses to initiate pyroptosis, followed by swelling, rupture of the cell membrane and death, and at the same time release a large amount of inflammatory substances accumulated in the damaged area and thus inflammatory response occurs [ 12 ]. Cellular pyroptosis is widespread in eukaryotic organisms and is a form of cellular self-protection against external damage, but over-activation can lead to organismal damage. Steroid use can activate bone marrow mesenchymal stem cells (BMSCs), which is closely related to the development of SONFH. Therefore, this study was conducted to investigate the progression of ONFH in the case of FGF23 interference with avascular necrosis, with the intention of exploring the role of FGF23 in ONFH disease and related mechanisms, and providing a viable clinical treatment basis for the early prevention and treatment of SONFH. Materials and methods Animals and SONFH models. A total of 75 SPF-grade Sprague-Dawley (SD) male mice (7–8 weeks old, weight 250g ± 20g) were provided by the Experimental Animal Centre of Shandong First Medical University. The mice were randomly divided into the following five groups: blank control group (NC), model control group (con), LacZ group (LacZ), FGF23 overexpression group (ad-FGF23) and FGF23 silencing group (si-FGF23). Firstly, lipopolysaccharide (LPS, 10 µg/kg) was injected via the tail vein. 24 h later, methylprednisolone (MPS, 20 mg/kg) was injected intramusitoneally in three times with a 24 h interval [ 13 ]. While the blank control group was injected with an equal amount of saline. On the day before modeling, the mice in the LacZ group, ad-FGF23 group, and si-FGF23 group were injected with LacZ, FGF23 overexpression, and FGF23 silencing adenovirus via the tail vein to increase or decrease the corresponding protein levels (Hanheng Biological). The LacZ virus was used as a functional control for the adenovirus. This study has been approved by the Animal Ethics Committee of Shandong First Medical University (Shandong Academy of Medical Sciences), approval number: No.W202210070244. All experiments were conducted in accordance with the guidelines outlined in the Association for Research in Vision and Ophthalmology (ARVO) Statement. The study was carried out in compliance with the ARRIVE guidelines. Micro-computed tomography (micro-CT). After completion of the SONFH model, the femur was collected, the surrounding muscles and other soft tissues were removed, and the femoral specimen was fixed in 4% paraformaldehyde and scanned by micro-CT. Key trabeculae parameters were identified, including bone volume (BV, mm 3 ), bone volume fraction (BV/TV), number of trabeculae (Tb.N, 1/mm) and trabecular separation (Tb.Sp, mm). Histological staining. Femurs were fixed in 4% paraformaldehyde (cat.no.G1101; Servicebio), decalcified in 10% ethylenediaminetetraacetic acid (EDTA; cat.no.G1105; Servicebio) decalcifying solution for 4 weeks, and then embedded in paraffin. The tissue was sectioned longitudinally, cut into 5-µm-thick sections and stored at room temperature for use. Hematoxylin-Eosin (HE; cat.no.G1005; Servicebio) and Saffron O-Fast Green staining (cat.no.G1053; Servicebio) was performed. Finally, the sections were sealed with central gum, placed under a light microscope and photographed for analysis. Enzyme linked immunosorbent assay (ELISA). Serum of mice and cell culture medium from each group were collected. And ELISA kits were used to measure IL-1β (cat.no.ml003057; mlbio), IL-6 (cat.no.ml064292; mlbio) and TNF-α levels (cat.no.ml002859; mlbio). Immunohistochemical staining. Sections were dewaxed in xylene and hydrated in gradient ethanol for 5 min each, followed by antigen recovery. Sections were incubated in 3% H 2 O 2 at 37°C for 15 min, washed with phosphate buffer saline (PBS; cat.no.G4202-500ML; Servicebio) and blocked with 3% bovine serum albumin (BSA; cat.no.A8020; Solarbio) at room temperature for 30 min, incubated with anti-CD31 (1:1000; cat.no.3528; Cell Signaling Technology, Inc.), anti-VEGF (1:1000; cat.no.9698; Cell Signaling Technology, Inc.) antibodies overnight at 4°C. The sections were then incubated with secondary antibodies at room temperature for 1 h. They were developed with 3,3-diaminobenzidine tetrahydrochloride, then re-stained with hematoxylin for 3min, dehydrated and transparent, and sealed. Observation under the microscope. Isolation and culture of mouse bone marrow stromal cells (BMSCs). Male C57BL/6J mice of 3–4 weeks of age during the growth period were selected. The cervical vertebrae were dislocated and sterilized by immersion in 75% ethanol for about 5 min. The femur and tibia were separated and washed several times in sterilized 1× PBS containing 100 µg/ml streptomycin and 100 U/ml penicillin (Sigma-Aldrich, St Louis. MO, USA). The epiphyses of the two ends were cut to make the bone marrow cavity in an open state, The bone marrow cavity was rinsed with dulbecco's modified eagle medium (DMEM; Lot.8121218; Gibco) and the rinsate was collected, centrifuged at 1000 rpm/min for 5 min, the supernatant was discarded, and resuspended by adding DMEM complete culture medium, the cell suspension was inoculated in a constant temperature incubator at 37 ℃, with a volume fraction of 5% CO 2 , and the growth of the cells was observed under the microscope. Replace the complete culture medium after 2 days, wait until the cells are confluent to 80–90% for passaging, and then keep changing the medium 2–3 days and passaging culture in time. The third generation of cells was used for all subsequent experiments. Cell model. Human umbilical vein endothelial cells (HUVECs) were from American Type Culture Collection (ATCC, PCS-100-013). BMSCs and HUVECs were cultured in Oxoid AnaeroGen anaerobic tanks (cat. no. HBYY001; hopebio) with the aim of simulating the ischaemic-hypoxic microenvironment of vascular injury during SONFH in vitro. The anaerobic capsules in the sealed jars will rapidly absorb atmospheric oxygen and produce CO 2 , eventually bringing the oxygen concentration to less than 1% within 30 min [ 14 ]. Cell transfection and grouping. Logarithmically grown BMSCs and HUVECs were inoculated into 6-well plates at a cell density of 2×10 5 per well and transfected when cells were fused to 30%-50%. The adenovirus were synthesized by HANBIO (Hanheng Biological, Lot.xbd-001). LacZ adenovirus, ad-FGF23 adenovirus and si-FGF23 adenovirus were added dropwise to serum-free, antibiotic-free medium. Cells without treatment were also used as a control group. The medium was replaced with fresh complete medium after 6h of transfection. Cells were divided into the following five groups: normal control group (NC), hypoxia model control group (con), adenovirus functional control group (LacZ), FGF23 overexpression group (ad-FGF23) and FGF3 silencing group (si-FGF23). Alkaline Phosphatase (ALP) Staining and Alizarin Red Staining (ARS). BMSCs were seeded in 6-well plates at a cell density of 5×10 4 . When the cells were grown to 80%, the culture medium was replaced with osteogenic induction medium (complete medium containing 10 mM sodium β-glycerophosphate (cat.no.A56289; OKA) and 50 µg/ml ascorbic acid) to induce differentiation into mature osteoblasts [ 15 ]. After 7 days of induction, alkaline phosphatase activity was measured by ALP staining kit (cat.no.C3206; Beyotime Biotech Inc). After 21 days of induction, the mineralized nodule formation characteristics of BMSCs were measured by ARS (cat.no.G1452; Solarbio). Tube formation assay in vitro. Matrigel (cat.no.0827045; ABW) was diluted with DMEM at a ratio of 1:3 and seeded to a 96-well plate. 50µL matrigel was added to each well. HUVECs were inoculated at a density of 3×10 4 in the 96-well plate and continued to be cultured for 8 h. HUVECs were observed under a light microscope to see if they formed a tubular lumen-like structure [ 16 ]. Immunofluorescence. HUVECs were fixed in 4% paraformaldehyde solution (cat.no.P1110; Solarbio) for 30 min, permeabilised with 0.5% Triton X-100 (cat.no.T8200; Solarbio) for 10 min, and then blocked with 3% BSA (cat.no.A8020; Solarbio) at room temperature for 30 min. Subsequently, HUVECs were incubated with anti-VEGF (1:1000; cat.no.9698; Cell Signaling Technology, Inc.) antibody and BMSCs were incubated with anti-Runx2 (1: 500; cat.no.GB115631; Servisebio) and anti-α-Tubulin (1:500; cat.no.ab179484; Abcam) antibody overnight at 4°C and then incubated with secondary antibody at room temperature for 1 h. Cell nucleus were stained with DAPI (0.5 µg/mL; cat.no.C0060; Solarbio). Samples were observed under a fluorescent microscope. Hoechst 33342/PI fluorescent staining. Pyroptosis was assessed by double staining of cells with Hoechst 33342 and PI [ 17 ]. HUVECs were inoculated in 6-well plates in complete medium. After the indicated treatments, cells in each group were stained with staining solution Hoechst 33342 (cat.no.C1027; Beyotime) and 2 µg/mL PI (cat.no.C0080; Solarbio) for 20 min. The cells were then washed three times with PBS. Samples were photographed under a fluorescent microscope. Western blot. RIPA lysate (cat.no.R0010; Solarbio) was added to extract the total protein of femoral head and cells. BCA protein assay kit (cat.no.PC0020; Solarbio) was used to measure the protein concentration. 10% separation gel and 5% concentration gel were prepared. The samples were loaded sequentially at 30 µg protein per well. 5% concentrated gel was electrophoresed at 80 V for 30 min, then switched to 120 V for 10% separation gel for 1 h. The membranes were transferred at 100V for 1h in an ice bath. Block with 5% skimmed milk (cat.no.D8340; Solarbio) for 1 h. The primary antibodies were diluted with 5% BSA solution at a ratio of 1:1000. Remove the blocking solution and add the corresponding FGF23 (1:1000; Abcam; cat.no.ab56326), Runt-related transcription factor 2 (Runx2; 1: 1000; cat. no. 12556; Cell Signaling Technology, Inc.), Osteocalcin (OCN; 1: 1000; cat. no. ab93876; Abcam), ALP (1: 1000; cat. no. ab229126; Abcam), VEGF (1:1000; cat.no.9698; Cell Signaling Technology, Inc.), NOD-like receptor thermal protein domain associated protein 3 (NLRP3; 1:1000; cat.no.GB114320; Servicebio), caspase-1(1:1000; cat.no.GB11383; Servicebio), Gasdermin D (GSDMD; 1: 2000; cat.no.20770-1-AP; proteintech), β-actin (1: 5000; cat.no.AB0035; Abways Technology) primary antibody and incubated overnight at 4°C. The secondary antibody was incubated at room temperature for 1 h. The protein bands were observed using the ECL chemiluminescence kit (cat.no.P10200; New Cell & Molecular Biotech Co., Ltd). β-actin was used as a reference. Statistical analysis. The experiments were repeated three times and the data were statistically processed using GraphPad Prism 6.02 software. Student’s t -test was used to compare the two samples and one-way analysis of variance (ANOVA) was used to compare the repeated measures. Differences were considered statistically significant at P < 0.05. Results FGF23 exacerbates the destruction of the femoral head by steroids. Our previous study demonstrated that hypoxia led to upregulation of FGF23 expression in osteoblasts. To further verify the changes of FGF23 during the development of SONFH, FGF23 expression levels were found to be elevated in the femoral head necrosis region in SONFH modes by western blot (Fig. 1 A). To further investigate the effect of FGF23 on the bone microstructure of the femoral head in steroid-treated mice in vivo, we first overexpressed or silenced FGF23 by tail vein injection of LacZ, ad-FGF23 and si-FGF23 adenovirus. Analysis of the micro-CT results showed that the femoral head structure of the model control mice was distroyed (Fig. 1 B), the trabecular gap was enlarged, the bone density, BV, BV/TV, and Tb.N decreased (Fig. 1 C-F). This disruption was exacerbated by the overexpression of FGF23. FGF23 silencing resulted in an intact femoral head structure and improved BV, BV/TV, Tb.N and Tb.Sp. Histological staining showed the cartilage layer of the femoral head became thinner and bone trabeculae arranged disorderly after steroids treatment (Fig. 2 A-B). Further aggravation of femoral head destruction was observed in the ad-FGF23 group, which was partially reversed by FGF23 silencing. Furthermore, we extracted mice femoral head proteins and the results showed that FGF23 inhibited the expression levels of the bone formation marker genes ALP, Runx2 and OCN (Fig. 2 C). FGF23 promotes the secretion of inflammatory factors to damage blood vessels. In the early stage of SONFH, due to the disturbance of lipid metabolism, a large number of inflammatory factors are secreted, leading to vascular endothelial damage. Moreover, related studies have shown that FGF23 can act directly on the peripheral vascular system, leading to vascular lesions. In the present study, to verify the effect of FGF23 on angiogenesis in the femoral head, VEGF and CD31 were chosen for immunohistochemical staining. The results showed that VEGF and CD31 expression was reduced in the model group and the number of intact microvessels was less (Fig. 3 A). While VEGF and CD31-positive cells were increased, the microvascular structure was largely intact and the blood vessel density was increased in the si-FGF23 group. In addition, we found that the expression levels of inflammatory factors (IL-1β, IL-6, TNF-α) in the serum of the model group were significantly higher than those in the control group (Fig. 3 B-D). Moreover, we extracted femoral proteins and western blot results showed that the expression levels of NLRP3, caspase-1, and GSDMD were upregulated in the model group (Fig. 3 E). These results suggest that steroids increase the secretion of inflammatory factors in mice, which may activate the pyroptosis signaling pathway. FGF23 overexpression inhibited osteogenic differentiation in vitro. Since FGF23 overexpression was found to exacerbate steroids damage to the femoral head structure and to inhibit the expression of bone formation-related proteins, we further assessed whether FGF23 was associated with osteoblast differentiation in vitro. After induction of differentiation, ALP and ARS staining revealed reduced ALP activity and reduced mineralized nodule formation in hypoxia-treated BMSCs compared to the normal group (Fig. 4 A-B). ALP is a marker of early osteogenic differentiation and mineralized nodules are a marker of late osteogenic differentiation [ 18 ]. In addition, the protein expression levels of the typical osteogenic markers Runx2, ALP and OCN were reduced in the hypoxic group (Fig. 4 C). Overexpression of FGF23 further inhibited the osteogenic function of BMSCs. Silencing of FGF23 protected the ALP activity and mineralization properties of BMSCs and increased the expression of osteogenic-related genes. Immunofluorescence results also showed that Runx2 expression was up-regulated in the si-FGF23group (Fig. 4 D). These results suggest that FGF23 is essential for osteogenic differentiation. FGF23 silencing promotes angiogenesis in vitro. Experiments in vivo revealed that vascular injury was associated with FGF23 expression. Firstly, HUVECs were placed in a hypoxic environment to mimic the vascular injury microenvironment during SONFH. Western blot results showed that FGF23 expression in HUVECs was upregulated by hypoxia in a time-dependent manner, and the expression reached the highest level at 12h of hypoxia, so 12h of hypoxia was selected for subsequent experiments (Fig. 5 A). We used tube formation assay to further verify the effect of FGF23 on endothelial cell angiogenesis in vitro (Fig. 5 B). The results showed that vascular-like structures were incomplete or sparse in the ad-FGF23 group, whereas endothelial cells in the si-FGF23 group differentiated to form complete circular vessel-like structures. In addition, western blot results showed that FGF23 overexpression inhibited the expression level of VEGF in HUVECs, and FGF23 silencing reversed this result (Fig. 5 C). The results of immunofluorescence were consistent with those of western blot (Fig. 5 D). FGF23 silencing inhibits pyroptosis signaling pathway. FGF23 silencing protected the overall activity of HUVECs as revealed by MTT analysis. We then further evaluated the effect of FGF23 on pyroptosis in hypoxia-treated HUVECs after 12h (Fig. 6 A). In addition, FGF23 silencing significantly inhibited the release of IL-1β, IL-6, and TNF-α induced by hypoxia in HUVECs (Fig. 6 B-D). Hoechst 33342/PI fluorescence staining showed that FGF23 silencing significantly reduced the number of PI-positive cells in hypoxia-induced HUVECs (Fig. 6 E). Furthermore, FGF23 silencing reduced the protein expression of NLRP3, caspase-1 and GSDMD in hypoxia-induced HUVECs (Fig. 6 F). These results suggest that FGF23 silencing may partially protect HUVECs from hypoxia-induced endothelial pyroptosis. Discussion In the present study, we validated the role of FGF23 in SONFH. We found that steroids can upregulate FGF23 expression, increase the secretion of inflammatory factors and impair bone microarchitecture and angiogenesis. Our data also suggest that FGF23 silencing can promote osteogenic differentiation and reduce vascular endothelial damage in vitro, thereby preventing the development of ONFH. In this study, LPS combined with MPS was used to establish animal models of SONFH in mice. We found that the femoral head surface structure was incomplete and the trabecular structure of the subchondral bone area was severely damaged in the model group. The expression of FGF23 was associated with bone formation and bone resorption. In our study, the effect of FGF23 on the bone microarchitecture of SONFH in mice was first assessed by micro-CT and histopathological assays. It was found that FGF23 overexpression significantly reduced the estimation of the femoral head and disrupted the trabecular parameters, while FGF23 silencing improved this outcome. It was found that chronic steroids exposure, which resulted in upregulation of serum FGF23 and bone FGF23 expression in mice, partially reduced longitudinal bone growth, decreased mineral density and led to impaired bone growth [ 19 ]. Moreover, studies in vitro have shown that hypoxia inhibits osteoblast differentiation potential, while FGF23 silencing improved ALP activity, increases the number of calcified nodules and improved osteoblast differentiation. In addition, western blot results also showed that the expression of osteogenic markers Runx2, ALP and OCN was upregulated after FGF23 silencing. This finding is consistent with previous studies showing that interference with FGF23 expression can affect osteogenic differentiation, which in turn affects the generation of osteoblasts [ 20 , 21 ]. Therefore, these results suggest that interference with FGF23 expression can regulate osteoblast differentiation to influence bone formation and thus the development of ONFH. In the early stages of SONFH, much vascular damage occurs due to disruption of lipid metabolism, resulting in increased intravascular pressure and massive secretion of inflammatory factors. The results of the present study also revealed that reduced vascular density was found in the SONFH models, and immunohistochemical results showed the expression of key angiogenic proteins CD31 and VEGF decreased. Related studies have found that FGF23 plays an important role in regulating the secretion of inflammatory factors and damage to the vascular endothelium [ 22 ]. In the present study, we also confirmed that FGF23 overexpression inhibited the local angiogenesis of the femoral head in SONFH models, and the protein levels of CD31 and VEGF in bone tissue were significantly downregulated. In addition, we confirmed that FGF23 impairs the function of the vascular endothelium by further studies in vitro. Firstly, FGF23 reduced the overall activity of HUVECs as revealed by MTT analysis. Another report directly demonstrates that FGF23 causes vascular endothelial dysfunction. Tube-forming assays in vitro showed that FGF23 inhibited the differentiation of HUVECs into intact tube-like structures and impaired the angiogenic capacity of HUVECs. Recent studies have found that FGF23 impairs endothelial function by activating the NF-κB signaling pathway, increasing oxidative stress to interfere with NO bioavailability, promoting HUVECs apoptosis and attenuating HUVECs migration [ 23 ]. In addition, the results of this study showed that the expression levels of inflammatory factors in the serum of SONFH models were significantly higher than those in the normal group. Pyroptosis is a pro-inflammatory mode of programmed cell death, which is mediated by membrane porin (GSDM) and involves NOD-like receptor protein 3 (NLRP3) inflammasome and depends on cysteine aspartic acid (caspase) [ 24 ]. Also, NLRP3, GSDMD, and caspase-1 protein expression was found to be upregulated in our study, so we speculate that SONFH may activate the pyroptosis signaling pathway. The pyroptosis pathway in SONFH is a multifactorial and complex regulatory process. The current study found that NLRP3 inflammasomes mediated pyroptosis in BMSCs, thereby triggering a differentiation imbalance between osteoblasts and osteoclasts may play a key role in the development of SONFH [ 25 , 26 ]. NLRP3 inflammasomes activate capsase-1 to promote the maturation and secretion of IL-1β and IL-18, and Gasdermin D is cleaved to form peptides containing the N-terminal active domain of Gasdermin D, which enhances inflammation and mediates pyroptosis [ 27 ]. These results suggest inflammatory response and pyroptosis occur in SONFH. In addition, we also detected NLRP3, GSDMD, and caspase-1 activation in HUVECs, suggesting that inflammasomes are involved in the development of endothelial cell injury and exacerbate local ischemia and hypoxia in the necrotic femoral head to some extent, which is consistent with caspase-1-dependent inflammasomes activation in SONFH [ 28 ]. The results of this study also revealed that FGF23 silencing attenuated the inflammatory response of HUVECs and reduced the expression of NLRP3, GSDMD, and caspase-1. However, current studies on FGF23 in regulating pyroptosis signaling pathways are still limited. However, it has been suggested that inhibition of FGF23 improves inflammation in mice with chronic kidney disease in vivo and that blocking FGF23 activity could be a therapeutic target to reduce inflammation [ 29 ]. This study demonstrates that FGF23 can regulate the pyroptosis signaling pathway, increase the release of inflammatory factors in SONFH, damage vascular endothelium, and inhibit osteogenic differentiation, thus affecting the development of SONFH, which provides a theoretical basis for FGF23 as a potential therapeutic target for SONFH. Declarations Acknowledgements Not applicable. Funding The present study was supported by the Shandong Provincial Natural Science Foundation of China (grant no. ZR2019MH120). Availability of data and materials The datasets generated and analyzed during the present study are available from the corresponding author on reasonable request. Authors’ contributions L.F., G.Z. and L.Z. designed the study. L.F. and G.Z. performed the experiments, assisted the data analysis and manuscript preparation. W.-Y.D. and H.L. provided technical and material support and supervised the study. L.-Z.Z., Y.-B.L. and B.W. prepared the sample material. 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Pyroptosis: a new paradigm of cell death for fighting against cancer. J Exp Clin Cancer Res. 2021; 40(1): 153. Wang L, Chen K, Wan X, Wang F, Guo Z, Mo Z. NLRP3 inflammasome activation in mesenchymal stem cells inhibits osteogenic differentiation and enhances adipogenic differentiation. Biochem Biophys Res Commun. 2017; 484(4): 871–877. Chen Y, Qin X, An Q, Yi J, Feng F, Yin D, An N, Liu Z, Weng L, Chen S, Hu X, Yin W. Mesenchymal Stromal Cells Directly Promote Inflammation by Canonical NLRP3 and Non-canonical Caspase-11 Inflammasomes. EBioMedicine. 2018; 32: 31–42. Chen X, Liu G, Yuan Y, Wu G, Wang S, Yuan L. NEK7 interacts with NLRP3 to modulate the pyroptosis in inflammatory bowel disease via NF-κB signaling. Cell Death Dis. 2019; 10(12): 906. Li YF, Huang X, Li X, Gong R, Yin Y, Nelson J, Gao E, Zhang H, Hoffman NE, Houser SR, Madesh M, Tilley DG, Choi ET, Jiang X, Huang CX, Wang H, Yang XF. Caspase-1 mediates hyperlipidemia-weakened progenitor cell vessel repair. Front Biosci (Landmark Ed). 2016; 21(1): 178–91. Yan LJ. Folic acid-induced animal model of kidney disease. Animal Model Exp Med. 2021; 4(4): 329–342. Additional Declarations No competing interests reported. Supplementary Files WesternBlot.zip Cite Share Download PDF Status: Published Journal Publication published 15 Jul, 2024 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 18 Mar, 2024 Reviews received at journal 29 Feb, 2024 Reviewers agreed at journal 21 Feb, 2024 Reviewers invited by journal 20 Feb, 2024 Editor assigned by journal 20 Feb, 2024 Editor invited by journal 20 Feb, 2024 Submission checks completed at journal 20 Feb, 2024 First submitted to journal 25 Jan, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3897523","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":274111001,"identity":"37b179b0-c278-4f6d-80ba-5584f07344da","order_by":0,"name":"Lun Fang","email":"","orcid":"","institution":"Shandong First Medical University \u0026Shandong Academy Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Lun","middleName":"","lastName":"Fang","suffix":""},{"id":274111002,"identity":"8e4894c1-5397-49e1-bdde-66910c1e0aa4","order_by":1,"name":"Gang Zhang","email":"","orcid":"","institution":"The Second Affiliated Hospital of Shandong First Medical University","correspondingAuthor":false,"prefix":"","firstName":"Gang","middleName":"","lastName":"Zhang","suffix":""},{"id":274111003,"identity":"65ec1fed-3a71-4c8f-bd64-581692764d0d","order_by":2,"name":"Yadi Wu","email":"","orcid":"","institution":"Shandong First Medical University \u0026Shandong Academy Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Yadi","middleName":"","lastName":"Wu","suffix":""},{"id":274111004,"identity":"bf92ee74-3adf-4d18-a8a7-6a241dfa72ae","order_by":3,"name":"Hao Li","email":"","orcid":"","institution":"Shandong First Medical University \u0026 Shandong Academy of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Hao","middleName":"","lastName":"Li","suffix":""},{"id":274111005,"identity":"7c5be901-8513-479b-bff6-b001cff221da","order_by":4,"name":"Zhongzhe Li","email":"","orcid":"","institution":"Shandong First Medical University \u0026Shandong Academy Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Zhongzhe","middleName":"","lastName":"Li","suffix":""},{"id":274111006,"identity":"634d7967-9c56-4549-9794-8cdc21dd8d5a","order_by":5,"name":"Beilei Yu","email":"","orcid":"","institution":"Shandong First Medical University \u0026Shandong Academy Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Beilei","middleName":"","lastName":"Yu","suffix":""},{"id":274111007,"identity":"25bafabe-6358-41a5-bf52-b25d7aaa5de3","order_by":6,"name":"Bin Wang","email":"","orcid":"","institution":"Shandong First Medical University \u0026Shandong Academy Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Bin","middleName":"","lastName":"Wang","suffix":""},{"id":274111008,"identity":"a06983b5-e7f4-493c-8a98-d5b8fff8df52","order_by":7,"name":"Lu Zhou","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyklEQVRIiWNgGAWjYDACCSB+wGDDwHD4AIjLTKSWBIY0BoZjCaRpOUyCFvnZzcckEtvO2/Md406TYKiwTmxgP3sArxbGOcfSgFpuJ848xrtNguFMemIDT14CXi3MEjlmIC0JBvd7t0kwth1ObJDgMcCrhQ2i5Zy9AcgWxn9EaOGBaDnAuAGspYEILRISackWCeeSQX7ZbJFwLN24jScHvxb5GckHb3woswOGGO/GGx9qrGX72c/g14IKEkC+I0H9KBgFo2AUjAIcAAC+AkLPE31ZwQAAAABJRU5ErkJggg==","orcid":"","institution":"Shandong First Medical University \u0026Shandong Academy Medical Sciences","correspondingAuthor":true,"prefix":"","firstName":"Lu","middleName":"","lastName":"Zhou","suffix":""}],"badges":[],"createdAt":"2024-01-25 14:44:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3897523/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3897523/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-66799-z","type":"published","date":"2024-07-15T16:13:19+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":51465568,"identity":"cf9ab90b-96da-497e-a30d-f5039473efdd","added_by":"auto","created_at":"2024-02-22 06:08:47","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":90619,"visible":true,"origin":"","legend":"\u003cp\u003eFGF23 exacerbates the damage of steroids to the bone microstructure of the femoral head. (A) Western blot detection of FGF23 expression levels in the femoral head region of the SONFH model. (B) Micro-CT scan of the femoral head. (C-F) Bone trabecular parameters, including BV, BV/TV, Tb.N and Tb.Sp. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 versus NC group. #\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 versus LacZ group.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3897523/v1/ee286c279bafdd734aa500a2.jpg"},{"id":51465816,"identity":"a92d0698-78eb-4d92-b572-8bd159db96f8","added_by":"auto","created_at":"2024-02-22 06:16:47","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":185867,"visible":true,"origin":"","legend":"\u003cp\u003eInhibition of FGF23 reduces the damage of steroids to the structure of the femoral head. (A) Representative image of HE staining of the femoral head. (B) Representative images of the femoral head stained with Saffran O-Fast Green. (C) Western blot to detect the expression levels of bone formation-related proteins in the femoral head. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 versus NC group. #\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 versus LacZ group.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3897523/v1/b435ab8382fe58789132f7fb.jpg"},{"id":51465815,"identity":"04a19b3b-4f29-4ae4-9717-183837ef7cbd","added_by":"auto","created_at":"2024-02-22 06:16:47","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":125713,"visible":true,"origin":"","legend":"\u003cp\u003eFGF23 promotes the secretion of inflammatory factors that damage blood vessels. (A) Immunohistochemical staining of VEGF and CD31-related antigens and vascular density in the femoral head. (B-D) Levels of inflammatory factors including IL-1β, IL-6, TNF-α in serum of SONFH. (E) Western blot to detect the expression levels of proteins related to pyroptosis signaling pathway. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 versus NC group. #\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 versus LacZ group.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3897523/v1/d68405a39cafe7881b2b5b00.jpg"},{"id":51465566,"identity":"a8b7111f-81ce-436d-96ea-bb54ec0c1a11","added_by":"auto","created_at":"2024-02-22 06:08:47","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":120902,"visible":true,"origin":"","legend":"\u003cp\u003eInterference with FGF23 expression affects osteogenic differentiation in vitro. (A) ALP staining to detect the activity of ALP in BMSCs 7 days after induction of osteogenic differentiation (B) ARS staining to detect the number of mineralized nodules in BMSCs 21 days after induction of osteogenic differentiation. (C) Effect of FGF23 on the expression levels of bone formation-related proteins in BMSCs. (D) Immunofluorescence staining to detect the expression level of Runx2. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 versus NC group. #\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 versus LacZ group.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3897523/v1/532da4d482cb80317021a605.jpg"},{"id":51465569,"identity":"0e2e04ff-3c98-4358-a998-7386e72b8edc","added_by":"auto","created_at":"2024-02-22 06:08:47","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":86694,"visible":true,"origin":"","legend":"\u003cp\u003eFGF23 silencing promotes angiogenesis in vitro. (A) The expression level of FGF23 in HUVECs after hypoxia was detected by western blot. (B)Tube-forming assay in vitro to detect the effect of FGF23 on the formation of official lumen-like structures in HUVECs. (C) Effect of FGF23 on VEGF, an angiogenesis-related protein in HUVECs. (D) Immunofluorescence staining images of HUVECs after interference with FGF23. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 versus NC group. #\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 versus LacZ group.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3897523/v1/608a5054e3f1b6d8d9d4f10b.jpg"},{"id":51465571,"identity":"328bc430-a852-4cd0-a8bc-d10e6de46bee","added_by":"auto","created_at":"2024-02-22 06:08:47","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":79772,"visible":true,"origin":"","legend":"\u003cp\u003eFGF23 regulates the pyroptosis signaling pathway to influence the progression of SONFH. (A) MTT assay was used to examine the viability of HUVECs after hypoxia. (B-D) Levels of inflammatory factors including IL-1β, IL-6, TNF-α in HUVECs after interference with FGF23. (E) Hoechst 33342/PI fluorescence staining to detect activation of pyroptosis signaling pathway. (F) Western blot to detect expression levels of proteins related to pyroptosis signaling pathway. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 versus NC group. #\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 versus LacZ group.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3897523/v1/1575b980002ed5352d979cd4.jpg"},{"id":61595302,"identity":"d470d15f-d94d-4053-aee3-5a88008c179c","added_by":"auto","created_at":"2024-08-01 17:21:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1037011,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3897523/v1/65c016c0-2f16-4be1-b4b2-f2e3c8e2f64e.pdf"},{"id":51465573,"identity":"dde40a4f-ece4-499c-934d-703afb6d9ff8","added_by":"auto","created_at":"2024-02-22 06:08:47","extension":"zip","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":638265,"visible":true,"origin":"","legend":"","description":"","filename":"WesternBlot.zip","url":"https://assets-eu.researchsquare.com/files/rs-3897523/v1/90e13e37a8b7bf4f6a401f1e.zip"}],"financialInterests":"No competing interests reported.","formattedTitle":"Fibroblast growth factor 23 inhibition attenuates steroid-induced osteonecrosis of the femoral head through pyroptosis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOsteonecrosis of the femoral head (ONFH) is one of the common clinical diseases in orthopedics. The main clinical features are limited flexion and extension, claudication, severe pain in the hip and so on. It has a high prevalence in young and middle-aged people aged 20\u0026ndash;50 years old, with a trend towards younger age [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. There is a high disability rate in ONFH, which seriously affects patients' physical health and quality of life, and brings great psychological pressure to patients. ONFH is divided into non-traumatic and traumatic, and steroid-induced osteonecrosis of the femoral head (SONFH) is the main cause of non-traumatic osteonecrosis of the femoral head. The \"vascular theory\" plays a leading role in the pathophysiological mechanism of SONFH and is gradually being recognized [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The imbalance in the vascular homeostasis of the femoral head causes an impaired blood supply to the femoral head, resulting in hypoxia of the corresponding tissues, which can lead to programmed death and damage of osteoblasts and consequently to the collapse of the femoral head structure [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. New research has shown that the treatment of ONFH by inhibiting inflammatory factors and protecting vascular endothelial cells from damage has been effective in animal models and in clinical practice [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFibroblast growth factor 23 (FGF23) is a phosphophilic hormone produced by bone. Studies have shown that FGF23 is a physiological regulator of phosphate and vitamin D metabolism and is indispensable for maintaining serum phosphate levels [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. FGF23 has been identified as a gene associated with autosomal dominant hypophosphatemic rickets (ADHR), which plays an important role in the development of bone diseases [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. As research progressed, several reports identified different roles for FGF23 in metabolic regulation, rheumatism, and cardiac hypertrophy. In particular, FGF23 promotes reactive oxygen species (ROS) production by upregulating the expression of reductive coenzyme II oxidase 2 in coronary artery endothelial cells, and influences cell apoptosis and vascular injury by stimulating endothelial cell migration and proliferation [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn recent years, the study of SONFH has penetrated into the cellular biochemical level, which mainly includes apoptosis, autophagy [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], pyroptosis. Among them, pyroptosis is a new mode of cellular inflammatory and programmed death between apoptosis and necrosis, which is a programmed cellular death mediated by gasdermin (GSDMs) with the involvement of NOD-like receptor protein 3 (NLRP3) inflammatory vesicles and dependence on cysteinyl aspartate specific proteinase (caspase). Cells suffer from external stimuli through a series of immune responses to initiate pyroptosis, followed by swelling, rupture of the cell membrane and death, and at the same time release a large amount of inflammatory substances accumulated in the damaged area and thus inflammatory response occurs [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Cellular pyroptosis is widespread in eukaryotic organisms and is a form of cellular self-protection against external damage, but over-activation can lead to organismal damage. Steroid use can activate bone marrow mesenchymal stem cells (BMSCs), which is closely related to the development of SONFH.\u003c/p\u003e \u003cp\u003eTherefore, this study was conducted to investigate the progression of ONFH in the case of FGF23 interference with avascular necrosis, with the intention of exploring the role of FGF23 in ONFH disease and related mechanisms, and providing a viable clinical treatment basis for the early prevention and treatment of SONFH.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cem\u003eAnimals and SONFH models.\u003c/em\u003e A total of 75 SPF-grade Sprague-Dawley (SD) male mice (7\u0026ndash;8 weeks old, weight 250g\u0026thinsp;\u0026plusmn;\u0026thinsp;20g) were provided by the Experimental Animal Centre of Shandong First Medical University. The mice were randomly divided into the following five groups: blank control group (NC), model control group (con), LacZ group (LacZ), FGF23 overexpression group (ad-FGF23) and FGF23 silencing group (si-FGF23). Firstly, lipopolysaccharide (LPS, 10 \u0026micro;g/kg) was injected via the tail vein. 24 h later, methylprednisolone (MPS, 20 mg/kg) was injected intramusitoneally in three times with a 24 h interval [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. While the blank control group was injected with an equal amount of saline. On the day before modeling, the mice in the LacZ group, ad-FGF23 group, and si-FGF23 group were injected with LacZ, FGF23 overexpression, and FGF23 silencing adenovirus via the tail vein to increase or decrease the corresponding protein levels (Hanheng Biological). The LacZ virus was used as a functional control for the adenovirus. This study has been approved by the Animal Ethics Committee of Shandong First Medical University (Shandong Academy of Medical Sciences), approval number: No.W202210070244. All experiments were conducted in accordance with the guidelines outlined in the Association for Research in Vision and Ophthalmology (ARVO) Statement. The study was carried out in compliance with the ARRIVE guidelines.\u003c/p\u003e \u003cp\u003e \u003cem\u003eMicro-computed tomography (micro-CT).\u003c/em\u003e After completion of the SONFH model, the femur was collected, the surrounding muscles and other soft tissues were removed, and the femoral specimen was fixed in 4% paraformaldehyde and scanned by micro-CT. Key trabeculae parameters were identified, including bone volume (BV, mm\u003csup\u003e3\u003c/sup\u003e), bone volume fraction (BV/TV), number of trabeculae (Tb.N, 1/mm) and trabecular separation (Tb.Sp, mm).\u003c/p\u003e \u003cp\u003e \u003cem\u003eHistological staining.\u003c/em\u003e Femurs were fixed in 4% paraformaldehyde (cat.no.G1101; Servicebio), decalcified in 10% ethylenediaminetetraacetic acid (EDTA; cat.no.G1105; Servicebio) decalcifying solution for 4 weeks, and then embedded in paraffin. The tissue was sectioned longitudinally, cut into 5-\u0026micro;m-thick sections and stored at room temperature for use. Hematoxylin-Eosin (HE; cat.no.G1005; Servicebio) and Saffron O-Fast Green staining (cat.no.G1053; Servicebio) was performed. Finally, the sections were sealed with central gum, placed under a light microscope and photographed for analysis.\u003c/p\u003e \u003cp\u003e \u003cem\u003eEnzyme linked immunosorbent assay (ELISA).\u003c/em\u003e Serum of mice and cell culture medium from each group were collected. And ELISA kits were used to measure IL-1β (cat.no.ml003057; mlbio), IL-6 (cat.no.ml064292; mlbio) and TNF-α levels (cat.no.ml002859; mlbio).\u003c/p\u003e \u003cp\u003e \u003cem\u003eImmunohistochemical staining.\u003c/em\u003e Sections were dewaxed in xylene and hydrated in gradient ethanol for 5 min each, followed by antigen recovery. Sections were incubated in 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C for 15 min, washed with phosphate buffer saline (PBS; cat.no.G4202-500ML; Servicebio) and blocked with 3% bovine serum albumin (BSA; cat.no.A8020; Solarbio) at room temperature for 30 min, incubated with anti-CD31 (1:1000; cat.no.3528; Cell Signaling Technology, Inc.), anti-VEGF (1:1000; cat.no.9698; Cell Signaling Technology, Inc.) antibodies overnight at 4\u0026deg;C. The sections were then incubated with secondary antibodies at room temperature for 1 h. They were developed with 3,3-diaminobenzidine tetrahydrochloride, then re-stained with hematoxylin for 3min, dehydrated and transparent, and sealed. Observation under the microscope.\u003c/p\u003e \u003cp\u003e \u003cem\u003eIsolation and culture of mouse bone marrow stromal cells (BMSCs).\u003c/em\u003e Male C57BL/6J mice of 3\u0026ndash;4 weeks of age during the growth period were selected. The cervical vertebrae were dislocated and sterilized by immersion in 75% ethanol for about 5 min. The femur and tibia were separated and washed several times in sterilized 1\u0026times; PBS containing 100 \u0026micro;g/ml streptomycin and 100 U/ml penicillin (Sigma-Aldrich, St Louis. MO, USA). The epiphyses of the two ends were cut to make the bone marrow cavity in an open state, The bone marrow cavity was rinsed with dulbecco's modified eagle medium (DMEM; Lot.8121218; Gibco) and the rinsate was collected, centrifuged at 1000 rpm/min for 5 min, the supernatant was discarded, and resuspended by adding DMEM complete culture medium, the cell suspension was inoculated in a constant temperature incubator at 37 ℃, with a volume fraction of 5% CO\u003csub\u003e2\u003c/sub\u003e, and the growth of the cells was observed under the microscope. Replace the complete culture medium after 2 days, wait until the cells are confluent to 80\u0026ndash;90% for passaging, and then keep changing the medium 2\u0026ndash;3 days and passaging culture in time. The third generation of cells was used for all subsequent experiments.\u003c/p\u003e \u003cp\u003e \u003cem\u003eCell model.\u003c/em\u003e Human umbilical vein endothelial cells (HUVECs) were from American Type Culture Collection (ATCC, PCS-100-013). BMSCs and HUVECs were cultured in Oxoid AnaeroGen anaerobic tanks (cat. no. HBYY001; hopebio) with the aim of simulating the ischaemic-hypoxic microenvironment of vascular injury during SONFH in vitro. The anaerobic capsules in the sealed jars will rapidly absorb atmospheric oxygen and produce CO\u003csub\u003e2\u003c/sub\u003e, eventually bringing the oxygen concentration to less than 1% within 30 min [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eCell transfection and grouping.\u003c/em\u003e Logarithmically grown BMSCs and HUVECs were inoculated into 6-well plates at a cell density of 2\u0026times;10\u003csup\u003e5\u003c/sup\u003e per well and transfected when cells were fused to 30%-50%. The adenovirus were synthesized by HANBIO (Hanheng Biological, Lot.xbd-001). LacZ adenovirus, ad-FGF23 adenovirus and si-FGF23 adenovirus were added dropwise to serum-free, antibiotic-free medium. Cells without treatment were also used as a control group. The medium was replaced with fresh complete medium after 6h of transfection. Cells were divided into the following five groups: normal control group (NC), hypoxia model control group (con), adenovirus functional control group (LacZ), FGF23 overexpression group (ad-FGF23) and FGF3 silencing group (si-FGF23).\u003c/p\u003e \u003cp\u003e \u003cem\u003eAlkaline Phosphatase (ALP) Staining and Alizarin Red Staining (ARS).\u003c/em\u003e BMSCs were seeded in 6-well plates at a cell density of 5\u0026times;10\u003csup\u003e4\u003c/sup\u003e. When the cells were grown to 80%, the culture medium was replaced with osteogenic induction medium (complete medium containing 10 mM sodium β-glycerophosphate (cat.no.A56289; OKA) and 50 \u0026micro;g/ml ascorbic acid) to induce differentiation into mature osteoblasts [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. After 7 days of induction, alkaline phosphatase activity was measured by ALP staining kit (cat.no.C3206; Beyotime Biotech Inc). After 21 days of induction, the mineralized nodule formation characteristics of BMSCs were measured by ARS (cat.no.G1452; Solarbio).\u003c/p\u003e \u003cp\u003e \u003cem\u003eTube formation assay in vitro.\u003c/em\u003e Matrigel (cat.no.0827045; ABW) was diluted with DMEM at a ratio of 1:3 and seeded to a 96-well plate. 50\u0026micro;L matrigel was added to each well. HUVECs were inoculated at a density of 3\u0026times;10\u003csup\u003e4\u003c/sup\u003e in the 96-well plate and continued to be cultured for 8 h. HUVECs were observed under a light microscope to see if they formed a tubular lumen-like structure [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eImmunofluorescence.\u003c/em\u003e HUVECs were fixed in 4% paraformaldehyde solution (cat.no.P1110; Solarbio) for 30 min, permeabilised with 0.5% Triton X-100 (cat.no.T8200; Solarbio) for 10 min, and then blocked with 3% BSA (cat.no.A8020; Solarbio) at room temperature for 30 min. Subsequently, HUVECs were incubated with anti-VEGF (1:1000; cat.no.9698; Cell Signaling Technology, Inc.) antibody and BMSCs were incubated with anti-Runx2 (1: 500; cat.no.GB115631; Servisebio) and anti-α-Tubulin (1:500; cat.no.ab179484; Abcam) antibody overnight at 4\u0026deg;C and then incubated with secondary antibody at room temperature for 1 h. Cell nucleus were stained with DAPI (0.5 \u0026micro;g/mL; cat.no.C0060; Solarbio). Samples were observed under a fluorescent microscope.\u003c/p\u003e \u003cp\u003e \u003cem\u003eHoechst 33342/PI fluorescent staining.\u003c/em\u003e Pyroptosis was assessed by double staining of cells with Hoechst 33342 and PI [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. HUVECs were inoculated in 6-well plates in complete medium. After the indicated treatments, cells in each group were stained with staining solution Hoechst 33342 (cat.no.C1027; Beyotime) and 2 \u0026micro;g/mL PI (cat.no.C0080; Solarbio) for 20 min. The cells were then washed three times with PBS. Samples were photographed under a fluorescent microscope.\u003c/p\u003e \u003cp\u003e \u003cem\u003eWestern blot.\u003c/em\u003e RIPA lysate (cat.no.R0010; Solarbio) was added to extract the total protein of femoral head and cells. BCA protein assay kit (cat.no.PC0020; Solarbio) was used to measure the protein concentration. 10% separation gel and 5% concentration gel were prepared. The samples were loaded sequentially at 30 \u0026micro;g protein per well. 5% concentrated gel was electrophoresed at 80 V for 30 min, then switched to 120 V for 10% separation gel for 1 h. The membranes were transferred at 100V for 1h in an ice bath. Block with 5% skimmed milk (cat.no.D8340; Solarbio) for 1 h. The primary antibodies were diluted with 5% BSA solution at a ratio of 1:1000. Remove the blocking solution and add the corresponding FGF23 (1:1000; Abcam; cat.no.ab56326), Runt-related transcription factor 2 (Runx2; 1: 1000; cat. no. 12556; Cell Signaling Technology, Inc.), Osteocalcin (OCN; 1: 1000; cat. no. ab93876; Abcam), ALP (1: 1000; cat. no. ab229126; Abcam), VEGF (1:1000; cat.no.9698; Cell Signaling Technology, Inc.), NOD-like receptor thermal protein domain associated protein 3 (NLRP3; 1:1000; cat.no.GB114320; Servicebio), caspase-1(1:1000; cat.no.GB11383; Servicebio), Gasdermin D (GSDMD; 1: 2000; cat.no.20770-1-AP; proteintech), β-actin (1: 5000; cat.no.AB0035; Abways Technology) primary antibody and incubated overnight at 4\u0026deg;C. The secondary antibody was incubated at room temperature for 1 h. The protein bands were observed using the ECL chemiluminescence kit (cat.no.P10200; New Cell \u0026amp; Molecular Biotech Co., Ltd). β-actin was used as a reference.\u003c/p\u003e \u003cp\u003e \u003cem\u003eStatistical analysis.\u003c/em\u003e The experiments were repeated three times and the data were statistically processed using GraphPad Prism 6.02 software. Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test was used to compare the two samples and one-way analysis of variance (ANOVA) was used to compare the repeated measures. Differences were considered statistically significant at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cem\u003eFGF23 exacerbates the destruction of the femoral head by steroids.\u003c/em\u003e Our previous study demonstrated that hypoxia led to upregulation of FGF23 expression in osteoblasts. To further verify the changes of FGF23 during the development of SONFH, FGF23 expression levels were found to be elevated in the femoral head necrosis region in SONFH modes by western blot (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). To further investigate the effect of FGF23 on the bone microstructure of the femoral head in steroid-treated mice in vivo, we first overexpressed or silenced FGF23 by tail vein injection of LacZ, ad-FGF23 and si-FGF23 adenovirus. Analysis of the micro-CT results showed that the femoral head structure of the model control mice was distroyed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), the trabecular gap was enlarged, the bone density, BV, BV/TV, and Tb.N decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-F). This disruption was exacerbated by the overexpression of FGF23. FGF23 silencing resulted in an intact femoral head structure and improved BV, BV/TV, Tb.N and Tb.Sp. Histological staining showed the cartilage layer of the femoral head became thinner and bone trabeculae arranged disorderly after steroids treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B). Further aggravation of femoral head destruction was observed in the ad-FGF23 group, which was partially reversed by FGF23 silencing. Furthermore, we extracted mice femoral head proteins and the results showed that FGF23 inhibited the expression levels of the bone formation marker genes ALP, Runx2 and OCN (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eFGF23 promotes the secretion of inflammatory factors to damage blood vessels.\u003c/em\u003e In the early stage of SONFH, due to the disturbance of lipid metabolism, a large number of inflammatory factors are secreted, leading to vascular endothelial damage. Moreover, related studies have shown that FGF23 can act directly on the peripheral vascular system, leading to vascular lesions. In the present study, to verify the effect of FGF23 on angiogenesis in the femoral head, VEGF and CD31 were chosen for immunohistochemical staining. The results showed that VEGF and CD31 expression was reduced in the model group and the number of intact microvessels was less (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). While VEGF and CD31-positive cells were increased, the microvascular structure was largely intact and the blood vessel density was increased in the si-FGF23 group. In addition, we found that the expression levels of inflammatory factors (IL-1β, IL-6, TNF-α) in the serum of the model group were significantly higher than those in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-D). Moreover, we extracted femoral proteins and western blot results showed that the expression levels of NLRP3, caspase-1, and GSDMD were upregulated in the model group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). These results suggest that steroids increase the secretion of inflammatory factors in mice, which may activate the pyroptosis signaling pathway.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eFGF23 overexpression inhibited osteogenic differentiation in vitro.\u003c/em\u003e Since FGF23 overexpression was found to exacerbate steroids damage to the femoral head structure and to inhibit the expression of bone formation-related proteins, we further assessed whether FGF23 was associated with osteoblast differentiation in vitro. After induction of differentiation, ALP and ARS staining revealed reduced ALP activity and reduced mineralized nodule formation in hypoxia-treated BMSCs compared to the normal group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-B). ALP is a marker of early osteogenic differentiation and mineralized nodules are a marker of late osteogenic differentiation [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In addition, the protein expression levels of the typical osteogenic markers Runx2, ALP and OCN were reduced in the hypoxic group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Overexpression of FGF23 further inhibited the osteogenic function of BMSCs. Silencing of FGF23 protected the ALP activity and mineralization properties of BMSCs and increased the expression of osteogenic-related genes. Immunofluorescence results also showed that Runx2 expression was up-regulated in the si-FGF23group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). These results suggest that FGF23 is essential for osteogenic differentiation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eFGF23 silencing promotes angiogenesis in vitro.\u003c/em\u003e Experiments in vivo revealed that vascular injury was associated with FGF23 expression. Firstly, HUVECs were placed in a hypoxic environment to mimic the vascular injury microenvironment during SONFH. Western blot results showed that FGF23 expression in HUVECs was upregulated by hypoxia in a time-dependent manner, and the expression reached the highest level at 12h of hypoxia, so 12h of hypoxia was selected for subsequent experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). We used tube formation assay to further verify the effect of FGF23 on endothelial cell angiogenesis in vitro (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). The results showed that vascular-like structures were incomplete or sparse in the ad-FGF23 group, whereas endothelial cells in the si-FGF23 group differentiated to form complete circular vessel-like structures. In addition, western blot results showed that FGF23 overexpression inhibited the expression level of VEGF in HUVECs, and FGF23 silencing reversed this result (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). The results of immunofluorescence were consistent with those of western blot (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eFGF23 silencing inhibits pyroptosis signaling pathway.\u003c/em\u003e FGF23 silencing protected the overall activity of HUVECs as revealed by MTT analysis. We then further evaluated the effect of FGF23 on pyroptosis in hypoxia-treated HUVECs after 12h (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). In addition, FGF23 silencing significantly inhibited the release of IL-1β, IL-6, and TNF-α induced by hypoxia in HUVECs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB-D). Hoechst 33342/PI fluorescence staining showed that FGF23 silencing significantly reduced the number of PI-positive cells in hypoxia-induced HUVECs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). Furthermore, FGF23 silencing reduced the protein expression of NLRP3, caspase-1 and GSDMD in hypoxia-induced HUVECs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). These results suggest that FGF23 silencing may partially protect HUVECs from hypoxia-induced endothelial pyroptosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the present study, we validated the role of FGF23 in SONFH. We found that steroids can upregulate FGF23 expression, increase the secretion of inflammatory factors and impair bone microarchitecture and angiogenesis. Our data also suggest that FGF23 silencing can promote osteogenic differentiation and reduce vascular endothelial damage in vitro, thereby preventing the development of ONFH.\u003c/p\u003e \u003cp\u003eIn this study, LPS combined with MPS was used to establish animal models of SONFH in mice. We found that the femoral head surface structure was incomplete and the trabecular structure of the subchondral bone area was severely damaged in the model group. The expression of FGF23 was associated with bone formation and bone resorption. In our study, the effect of FGF23 on the bone microarchitecture of SONFH in mice was first assessed by micro-CT and histopathological assays. It was found that FGF23 overexpression significantly reduced the estimation of the femoral head and disrupted the trabecular parameters, while FGF23 silencing improved this outcome. It was found that chronic steroids exposure, which resulted in upregulation of serum FGF23 and bone FGF23 expression in mice, partially reduced longitudinal bone growth, decreased mineral density and led to impaired bone growth [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Moreover, studies in vitro have shown that hypoxia inhibits osteoblast differentiation potential, while FGF23 silencing improved ALP activity, increases the number of calcified nodules and improved osteoblast differentiation. In addition, western blot results also showed that the expression of osteogenic markers Runx2, ALP and OCN was upregulated after FGF23 silencing. This finding is consistent with previous studies showing that interference with FGF23 expression can affect osteogenic differentiation, which in turn affects the generation of osteoblasts [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Therefore, these results suggest that interference with FGF23 expression can regulate osteoblast differentiation to influence bone formation and thus the development of ONFH.\u003c/p\u003e \u003cp\u003eIn the early stages of SONFH, much vascular damage occurs due to disruption of lipid metabolism, resulting in increased intravascular pressure and massive secretion of inflammatory factors. The results of the present study also revealed that reduced vascular density was found in the SONFH models, and immunohistochemical results showed the expression of key angiogenic proteins CD31 and VEGF decreased. Related studies have found that FGF23 plays an important role in regulating the secretion of inflammatory factors and damage to the vascular endothelium [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In the present study, we also confirmed that FGF23 overexpression inhibited the local angiogenesis of the femoral head in SONFH models, and the protein levels of CD31 and VEGF in bone tissue were significantly downregulated. In addition, we confirmed that FGF23 impairs the function of the vascular endothelium by further studies in vitro. Firstly, FGF23 reduced the overall activity of HUVECs as revealed by MTT analysis. Another report directly demonstrates that FGF23 causes vascular endothelial dysfunction. Tube-forming assays in vitro showed that FGF23 inhibited the differentiation of HUVECs into intact tube-like structures and impaired the angiogenic capacity of HUVECs. Recent studies have found that FGF23 impairs endothelial function by activating the NF-κB signaling pathway, increasing oxidative stress to interfere with NO bioavailability, promoting HUVECs apoptosis and attenuating HUVECs migration [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn addition, the results of this study showed that the expression levels of inflammatory factors in the serum of SONFH models were significantly higher than those in the normal group. Pyroptosis is a pro-inflammatory mode of programmed cell death, which is mediated by membrane porin (GSDM) and involves NOD-like receptor protein 3 (NLRP3) inflammasome and depends on cysteine aspartic acid (caspase) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Also, NLRP3, GSDMD, and caspase-1 protein expression was found to be upregulated in our study, so we speculate that SONFH may activate the pyroptosis signaling pathway. The pyroptosis pathway in SONFH is a multifactorial and complex regulatory process. The current study found that NLRP3 inflammasomes mediated pyroptosis in BMSCs, thereby triggering a differentiation imbalance between osteoblasts and osteoclasts may play a key role in the development of SONFH [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. NLRP3 inflammasomes activate capsase-1 to promote the maturation and secretion of IL-1β and IL-18, and Gasdermin D is cleaved to form peptides containing the N-terminal active domain of Gasdermin D, which enhances inflammation and mediates pyroptosis [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. These results suggest inflammatory response and pyroptosis occur in SONFH. In addition, we also detected NLRP3, GSDMD, and caspase-1 activation in HUVECs, suggesting that inflammasomes are involved in the development of endothelial cell injury and exacerbate local ischemia and hypoxia in the necrotic femoral head to some extent, which is consistent with caspase-1-dependent inflammasomes activation in SONFH [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The results of this study also revealed that FGF23 silencing attenuated the inflammatory response of HUVECs and reduced the expression of NLRP3, GSDMD, and caspase-1. However, current studies on FGF23 in regulating pyroptosis signaling pathways are still limited. However, it has been suggested that inhibition of FGF23 improves inflammation in mice with chronic kidney disease in vivo and that blocking FGF23 activity could be a therapeutic target to reduce inflammation [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis study demonstrates that FGF23 can regulate the pyroptosis signaling pathway, increase the release of inflammatory factors in SONFH, damage vascular endothelium, and inhibit osteogenic differentiation, thus affecting the development of SONFH, which provides a theoretical basis for FGF23 as a potential therapeutic target for SONFH.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe present study was supported by the Shandong Provincial Natural Science Foundation of China (grant no. ZR2019MH120).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and analyzed during the present study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eL.F., G.Z. and L.Z. designed the study. L.F. and G.Z. performed the experiments, assisted the data analysis and manuscript preparation. W.-Y.D. and H.L. provided technical and material support and supervised the study. L.-Z.Z., Y.-B.L. and B.W. prepared the sample material. L.Z. reviewed the final manuscript. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Research Ethics Committee of Shandong First Medical University (Shandong Academy of Medical Sciences) (grant no. W202210070244).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there are no competing interests regarding the publication of this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePatient consent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eZhao D, Zhang F, Wang B, Liu B, Li L, Kim SY, Goodman SB, Hernigou P, Cui Q, Lineaweaver WC, Xu J, Drescher WR, Qin L. Guidelines for clinical diagnosis and treatment of osteonecrosis of the femoral head in adults (2019 version). J Orthop Translat. 2020; 21: 100\u0026ndash;110.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKerachian, M.A., C. Seguin, and E.J. Harvey, Glucocorticoids in osteonecrosis of the femoral head: a new understanding of the mechanisms of action. 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Animal Model Exp Med. 2021; 4(4): 329\u0026ndash;342.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Osteonecrosis of the femoral head, steroid, FGF23, pyroptosis","lastPublishedDoi":"10.21203/rs.3.rs-3897523/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3897523/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSteroid-induced osteonecrosis of the femoral head (SONFH) is the predominant cause of non-traumatic osteonecrosis of the femoral head (ONFH). Impaired blood supply and reduced osteogenic activity of the femoral head are the key pathogenic mechanisms of SONFH. Fibroblast growth factor 23 (FGF23) levels are not only a biomarker for early vascular lesions caused by abnormal mineral metabolism, but can also act directly on the peripheral vascular system, leading to vascular pathology. The aim of this study was to observe the role of FGF23 on bone microarchitecture and vascular endothelium, and to investigate activation of pyroptosis in SONFH. Lipopolysaccharide (LPS) combined with methylprednisolone (MPS) was applied for SONFH mouse models, and adenovirus was used to increase or decrease the level of FGF23. Micro-CT and histopathological staining were used to observe the structure of the femoral head, and immunohistochemical staining was used to observe the vascular density. The cells were further cultured in vitro and placed in a hypoxic environment for 12h to simulate the microenvironment of vascular injury during SONFH. The effect of FGF23 on osteogenic differentiation was evaluated using alkaline phosphatase staining, alizarin red S staining and expression of bone formation-related proteins. Matrigel tube formation assay in vitro and immunofluorescence were used to detect the ability of FGF23 to affect endothelial cell angiogenesis. Steroids activated the pyroptosis signaling pathway, promoted the secretion of inflammatory factors in SONFH models, led to vascular endothelial dysfunction and damaged the femoral head structure. In addition, FGF23 inhibited the HUVECs angiogenesis and BMSCs osteogenic differentiation. FGF23 silencing attenuated steroid-induced osteonecrosis of the femoral head by inhibiting the pyroptosis signaling pathway, and promoting osteogenic differentiation of BMSCs and angiogenesis of HUVECs in vitro.\u003c/p\u003e","manuscriptTitle":"Fibroblast growth factor 23 inhibition attenuates steroid-induced osteonecrosis of the femoral head through pyroptosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-22 06:08:42","doi":"10.21203/rs.3.rs-3897523/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-03-18T09:57:09+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-02-29T16:18:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"dddc9032-0d9b-4b6f-b26d-fadb5cb28441","date":"2024-02-21T14:07:35+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-02-20T15:45:55+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-02-20T15:41:07+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-02-20T15:26:13+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-02-20T15:24:54+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-01-25T14:30:11+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"aee8c1a0-6a83-4b3c-8aa3-344d8e5de89f","owner":[],"postedDate":"February 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":28892856,"name":"Biological sciences/Cell biology"},{"id":28892857,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2024-08-01T16:20:05+00:00","versionOfRecord":{"articleIdentity":"rs-3897523","link":"https://doi.org/10.1038/s41598-024-66799-z","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-07-15 16:13:19","publishedOnDateReadable":"July 15th, 2024"},"versionCreatedAt":"2024-02-22 06:08:42","video":"","vorDoi":"10.1038/s41598-024-66799-z","vorDoiUrl":"https://doi.org/10.1038/s41598-024-66799-z","workflowStages":[]},"version":"v1","identity":"rs-3897523","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3897523","identity":"rs-3897523","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}