Apocynin exerts cytoprotective effects on dexamethasone‐induced osteoblasts by inhibiting oxidative stress through the Nrf2 signalling pathway

Abstract Steroid‐induced femoral head necrosis (SIFHN) is a serious clinical complication that is caused by prolonged or excessive use of glucocorticoids (GCs). Osteoblast apoptosis and osteogenic differentiation dysfunction caused by GC‐induced oxidative stress and mitochondrial impairment are strongly implicated in SIFHN. Apocynin (APO) is a kind of acetophenone extracted from an herb. In recent years, APO has received much attention for its antiapoptotic and antioxidant properties. This study aimed to investigate whether APO could protect against SIFHN and explore the mechanism. In our study, low‐dose APO had no toxic effects on osteoblasts and restored dexamethasone (Dex)‐treated osteoblasts by improving survival, inhibiting OS and restoring mitochondrial dysfunction. Mechanistically, APO alleviated Dex‐induced osteoblast injury by activating the Nrf2 pathway, and the use of ML385 to block Nrf2 significantly eliminated the protective effect of APO. In addition, APO could reduce the formation of empty lacunae, restore bone mass and promote the expression of Nrf2 in SIFHN rats. In conclusion, APO protects osteoblasts from Dex‐induced oxidative stress and mitochondrial dysfunction through activation of the Nrf2 pathway and may be a beneficial drug for the treatment of SIFHN.

oxygen species (ROS) in osteoblasts and inhibit antioxidant enzymes, leading directly to oxidative damage and dysfunction in cells and ultimately to apoptosis. 5,9Mitochondria are the primary organelles that generate ROS, 10 and mitochondrial impairment is a major contributor to apoptosis. 11GCs have been reported to induce apoptosis by altering the mitochondrial membrane potential (MMP) of osteoblasts and producing excessive ROS. 11Therefore, the key to treating SIFHN is to prevent intracellular OS and restore mitochondrial function in osteoblasts.
Nuclear factor erythroid 2-related factor 2 (Nrf2) maintains cellular redox homeostasis by promoting the expression of downstream haem oxygenase-1 (HO-1) and antioxidant enzymes and plays an important protective role by reducing OS damage and inhibiting apoptosis. 12,13The Nrf2 pathway also protects mitochondrial function through the restoration of MMP and reducing mitochondrial ROS production. 14,15It has been demonstrated that the Nrf2 pathway is significantly downregulated in GC-treated osteoblasts, and its activation reduces ROS production, restores mitochondrial function and inhibits apoptosis in osteoblasts. 16Thus, this pathway might be a promising therapeutic target for SIFHN.
Recently, natural compounds have shown great promise in the treatment of SIFHN due to their powerful antioxidant properties.

Apocynin (APO) is a naturally occurring acetophenone that has been
shown to alleviate various diseases such as diabetes, stroke, hypertension and acute pancreatitis. 17[20] However, it is not yet clear whether APO is protects against SIFHN.
Consequently, the objective of the current study was to study apoptosis and OS physiology and its signalling system and identify the effects of APO on dexamethasone (Dex)-induced apoptosis in osteoblasts.
To establish the SIFHN cell model, osteoblasts were cultured in medium containing 1 μM dexamethasone for 24 h.For APO treatment, osteoblasts were exposed to 1, 10 and 100 μM APO for 24 h.
In the Dex + APO group, osteoblasts were pretreated with diverse concentrations of APO (1, 10, 100 μM) for 2 h and then conditioned with Dex for 24 h.In the Dex + APO + ML385 group, osteoblasts were pretreated with ML385 (5 μM) for 24 h, followed by APO (100 μM) treatment for 2 h, and then stimulation with Dex for 24 h.

| Proliferation assay
The CCK-8 kit was used to investigate the cytoprotective effects of APO on osteoblasts.When the cells reached 80% confluence, the osteoblasts were treated with different concentrations of APO (0.01, 0.1, 1, 10, 100, 1000 μM) for 24 h.To explore the cytoprotective effect of APO on osteoblasts, MC3T3-E1 cells were pretreated with various concentrations of APO (1, 10 and 100 μM) for 2 h with or without 1 μM Dex treatment.Subsequently, CCK-8 (10 μL) working solution was added dropwise to each well and incubated for 4 h.Finally, the optical density (OD, 450 nm) was recorded with a microplate reader.

| Mitochondrial function assay
The MMP and the levels of mitochondrial superoxide (Mito-Sox) in each group of MC3T3-E1 cells were measured using a JC-1 Assay Kit and Mito-Sox Red fluorescence assay probe.

| Detection of osteoid differentiation and mineralization
The activity of alkaline phosphatase (ALP) was determined to evaluate whether APO could reduce Dex-mediated osteoblast damage.Cells (5 × 104 each well) were inoculated in 12-well plates and supplemented with osteogenic induction medium after the different drug interventions.After 1 week, the cells were lysed with 100 μL of working solution, and ALP reagent kits (Beyotime, China) were used to measure ALP activity.
Alizarin Red S staining was employed to detect calcium deposition in osteoblasts by complexing calcium ions with Alizarin Red S. After osteogenic induction in the different treatment groups, the cells were stained with ARS on Day 21 (Cyagen, China).

| Drug targets identified by bioinformatics analysis
To further explore the mechanism by which APO attenuates OS in osteonecrosis of the femoral head, we used bioinformatics tools to search for therapeutic targets of APO.First, genes associated with femoral head necrosis were collected from the CTD database (https://ctdba se.org/), and then the Amigo2 database (http://geneo ntolo gy.org/) was searched for genes involved in OS using the words 'oxidative stress'.Finally, we searched for potential targets of APO in the SuperPRED database (https://predi ction.charite.de/).The intersection was determined by constructing Venn diagrams and was used to identify potential targets by which APO regulates OS in femoral head necrosis.To understand the biological process (BP), cell component (CC), molecular function (MF) and pathways in which these targets were involved, enrichment analysis of these targets was performed using the R package clusterProfiler with the minimum gene set to 5 and maximum gene set to 5000.A p-value of <0.05 and a false discovery rate (FDR) of <0.1 were considered statistically significant.Protein-protein interaction (PPI) network maps of the targets were built from the STRING database (https://cn.string-db. org/).The results were imported into Cytoscape software, and the significant nodes in the PPI were calculated using the BottleNeck algorithm of the cytoHubba plugin.

| Animal model and groupings
Twenty-four 12-week-old Sprague-Dawley rats (male, 380 ± 50 g) were obtained from the Tianjin Medical University Experimental Animal Center and maintained under specific pathogen-free (SPF) conditions at 23°C.According to the experimental requirements, after 1 week of adaptation, the animals were grouped as follows: negative control (NC) group, Dex treatment group (Dex) and APO pretreatment group (Dex + APO), n = 8/per group).The Dex group received daily intramuscular injections of 21 mg/kg Dex for 28 days following to the previous protocol. 21The Dex + APO group received 100 mg/kg APO daily by gavage 1 h before Dex treatment.For the NC group, rats were administered normal saline daily.
Four weeks later, the rats were sacrificed under isoflurane anaesthesia, and the femurs were extracted and preserved in 4% paraformaldehyde for 48 h.

| MRI
MRI was performed on bilateral femurs using a 9.3 T small-animal scanner (Bruker, Germany) after isoflurane euthanasia, and image analysis was conducted using ImageJ software as previously described. 22The image signal intensity of the femoral head was averaged over the region of interest, which was manually drawn on the original image and normalized to the average image intensity of adjacent muscle regions calculated over the same size region of interest.

| Histology and immunofluorescence staining
First, the fixed femoral head was decalcified with EDTA for 6 weeks, followed by ethanol gradient dehydration and xylene transparency.Subsequently, paraffin embedding was performed, and the femoral head was cut into 5μm-thick sections.Routine HE staining was executed to observe the pathological changes in subchondral bone within the femoral head.For immunofluorescence staining, after deparaffinization and rehydration, the sections were incubated with primary antibodies against Nrf2, HO-1,4-HNE, cleaved caspase 3 and 8-OHdG overnight and then incubated with rabbit anti-rat secondary antibodies for 120 min in the dark.Five randomly selected fields per section and three sections per rat were analysed to determine the percentage of positive cells per field.

| TUNEL assay
Cell and tissue apoptosis were detected by a TUNEL Assay Kit (Beyotime, China).To examine apoptosis, 1 × 104 cells were seeded on coverslips for 24 h before the various treatments.Then, the cells were subjected to fixation and permeabilization.After dewaxing and rehydration, the paraffin sections were incubated in TUNEL working solution, and 4′,6-diamidino-2-phenylindole (DAPI) was added to counterstain the cell nuclei.Images were acquired using a fluorescence microscope.

Tianjin Medical University General Hospital's Animal Research
Ethics Committee authorized all animal experimentation protocols in accordance with the US National Institutes of Health's guidelines for the care and use of animals.

| Statistical analysis
All experiments were repeated at least three times.The data are expressed as the mean ± standard deviation (SD).Comparisons between the two groups were assessed by Student's t-test for mean differences or one-way analysis of variance with a Bonferroni/Dunn post hoc test for multiple comparisons.Statistical analysis was conducted using GraphPad Prism 8.0.2 (United States) (*p < 0.05, **p < 0.01, ***p < 0.001).

| APO alleviated Dex-induced injury in MC3T3-E1 cells
The molecular formula of APO is HOC 6 H 3 (OCH 3 )COCH 3 , and its chemical structure is illustrated in Figure 1A.Firstly, to investigate the toxicity of APO on osteoblasts, we treated osteoblasts with different concentrations of APO for 24 h.The CCK-8 results showed that 100 μM was the maximum safe concentration (Figure 1B).Therefore, we selected APO concentrations of 1, 10 and 100 μM for subsequent experiments.In addition, Dex (1 μM) significantly reduced osteoblast activity, and APO mitigated this effect of Dex on osteoblasts (Figure 1C).The TUNEL staining (Figure 1D,E

| APO attenuated Dex-induced osteogenic differentiation dysfunction in MC3T3-E1 osteoblasts
The osteogenic differentiation and mineralization abilities of osteoblasts were examined.Figure 2A,B shows that Dex treatment significantly decreased the number of calcified nodules in osteoblasts, while APO pretreatment significantly restored the mineralization capacity of osteoblasts compared with that in the Dex group.In addition, Dex significantly suppressed ALP activity in osteoblasts, while APO pretreatment markedly enhanced ALP activity (Figure 2C,D).We also discovered Dex downregulated the levels of ALP, OPG and RUNX2, which indicated that Dex impaired osteogenic differentiation (Figure 2E-H).However, APO pretreatment upregulated the protein expression of ALP, OPG and RUNX2.These findings suggested that Dex inhibited the osteogenic differentiation and mineralization potential of osteoblasts, and APO ameliorated osteogenic differentiation dysfunction.

| APO protected osteoblasts by inhibiting the production of ROS
Many studies have revealed that ROS are critical in the proapoptotic signalling cascade 23,24 ; therefore, we used flow cytometry and immunofluorescence staining to explore the effect of APO on However, APO pretreatment significantly inhibited the production of these OS products.OS is an impairment of redox homeostasis caused by excessive ROS and decreased antioxidant enzyme activity in an organism.APO inhibited Dex-induced ROS production and reduced OS damage in osteoblasts.

| APO relieved Dex-induced mitochondrial dysfunction in osteoblasts in vitro
Mitochondria are the main sites of ROS production, and mitochondrial dysfunction is a major cause of apoptosis.Therefore, we examined the effects of APO on MMP and mitochondrial ROS in Dex-treated osteoblasts using the probes JC-1 (Figure 4A) and Mito-Sox (Figure 4C), respectively.MMP was assessed by determining the ratio between red (aggregated JC-1) and green (monomeric JC-1) fluorescence.Compared with control cells, Dex-treated osteoblasts had a significantly lower MMP and higher levels of ROS, and APO significantly restored the MMP and decreased mitochondrial superoxide anion levels in osteoblasts (Figure 4B,D).In summary, APO restored the MMP and inhibited mitochondrial ROS generation in osteoblasts.

| Nrf2 might be a therapeutic target if APO according to bioinformatics analysis
To further explore the mechanism by which APO attenuates OS in osteoblasts, we identified therapeutic targets of APO by using bioinformatics tools.The Venn diagrams showed six intersecting genes related to osteonecrosis of the femoral head, OS and APO targets: NFE2L2 (Nrf2), ERN1, PDGFRB, PDGFRA, KEAP1 and CDK1 (Figure 5A).PPI network analysis showed that Nrf2 was the most important target (Figure 5B).These genes are involved in a variety of signalling pathways, including the Nrf2 pathway, OS pathway and osteoblast pathway (Figure 5C).Gene

| Nrf2 activation was involved in the osteoprotective effect of APO
Nrf2 plays a critical role in cellular sensing of ROS and antioxidant defence. 25To further confirm whether the antioxidant effect of

| APO pretreatment alleviated disease progression in the SIFHN rat model
To investigate the therapeutic effects of APO, we established a rat model of SIFHN.After 4 weeks of treatment, we used MRI, histological and micro-CT evaluations to assess the protective effect of APO on bone.The T2-weighted MRI (T2WI) results showed that the femoral head oedema signal was markedly higher in Dextreated rats than in the NC group, and the rats in the Dex + APO group had significantly reduced oedema signals in the femoral head (Figure 8A,E).In the NC group, the surface of the femoral head appeared porcelain white with a smooth surface, whereas the articular cartilage surface appeared focally congested and oedematous with the cartilage exhibiting a greyish colour in the Dex group.APO treatment significantly suppressed the congestion and oedema in cartilage and exhibited a colour between that in the NC group and Dex group (Figure 8B).HE staining indicated that empty lacunae and necrotic haematopoietic cells in the subchondral region were apparently more abundant in the Dex group than

| APO restored the activity of antioxidant enzymes and decreased oxidative stress products by upregulating Nrf2 in a rat of SIFHN
We investigated the effect of APO treatment on the expression of Nrf2, HO-1 and antioxidant enzymes in a rat model of SIFHN.As shown in Figure 9A,B, compared with those in the Dex group, APO pretreatment significantly elevated the levels of Nrf2 and HO-1 and decreased the levels of GSH, CAT, GPx and SOD (Figure 9E-H) in rat serum in the Dex + APO group.APO also decreased the levels of the lipid peroxidation products 4-HNE (Figure 9C) and 8-OhdG (Figure 9D).Our findings indicated that APO inhibited Dex-induced OS by activating Nrf2 to promote the expression of downstream antioxidant enzymes, thereby alleviating the progression of SIFHN (Figure 10).

| DISCUSS ION
The incidence of SIFHN is increasing year by year and accounts for the largest proportion of non-traumatic femoral head necrosis.Cumulative or high-dose shock therapy with GCs is the most important risk factor for SIFHN. 26Since 2020, when COVID-19 spread worldwide, patients who were severely infected with coronaviruses have received high-dose, pulsed treatment with GCs, which may cause these patients to progressively develop SIFHN during the following years.However, 67 per cent of patients with SIFHN are asymptomatic in the early stages, 27 and Shimuzu et al. 28 reported that the progres- APO can alleviate a wide variety of conditions, including diabetesrelated complications, neurodegenerative disorders, cardiovascular diseases, cancer and ischaemia-reperfusion injury. 17Stéphane M. Camus et al. 29 proved that APO reduced ROS generation and inhibited endothelial cell apoptosis in sickle cell disease.In addition, APO protected osteoblasts from antimycin A-induced mitochondrial dysfunction and apoptosis. 30Targeted delivery of mitoapocynin effectively alleviated ROS production in mitochondria, showing promising therapeutic potential. 31Consistent with previous findings, we showed that APO promoted cell survival and reversed changes in C-caspase3, BAX and BCL-2 expression in Dex-induced osteoblasts.
OS and mitochondrial dysfunction-mediated apoptosis in osteoblasts play a pivotal role in GC-induced bone metabolism abnormalities. 53][34] Excessive ROS attack macromolecules, including proteins, DNA and lipids, and trigger pathological mitochondrial clearance and cell apoptosis via ROS-induced ROS release. 10,35GCs influence mitochondria by modifying the expression of mitochondrial and nuclear genes and reducing the MMP by binding to their receptors on the mitochondrial membrane. 36,37The loss of MMP, which is an early pathologic event in apoptosis, promotes the release of intracellular cytochrome C, which activates C-caspase3 and ultimately triggers the intrinsic mitochondrial apoptotic pathway. 35 The Nrf2 pathway plays a crucial antioxidant role in cells. 40Nrf2 translocates from the cytoplasm into the nucleus and promotes the transcription of downstream antioxidant enzyme. 41,42Geng et al. 43 showed that GCs suppress the Nrf2 signalling pathway and the antioxidant capacity of tissues.GSH bind with lipid peroxide and free radicals to resist the destruction of sulfhydryl groups by oxidants. 44 our study, APO promoted the expression of Nrf2 and HO-1.In addition, APO enhanced antioxidant enzyme activity in cells and animal serum.These results suggested that APO attenuated oxidative stress-induced damage in Dex-induced osteoblasts through Nrf2 pathway activation.
In the present study, APO treatment inhibited osteoblast apoptosis induced by OS and mitochondrial dysfunction via the Nrf2 signalling pathway.However, the in-depth mechanism needs to be further

Flow
cytometry was used to estimate the apoptosis rate and reactive oxygen levels in osteoblasts in each group.The cells were harvested after centrifugation and resuspension.Subsequently, the cells were stained with 5 μL of Annexin-V and 7-AAD solution and incubated for 20 min at room temperature.Then, we performed flow cytometry (BD Biosciences) to detect apoptosis.To detect intracellular ROS levels, different interventions were implemented when the cells reached 80% confluence.Osteoblasts were labelled with DCFH-DA and incubated for 20 min at 37°C.Then, ROS levels were detected by flow cytometry (BD Biosciences, USA).
) and flow cytometry (Figure 1F,G) results showed that Dex significantly increased the osteoblast apoptosis ratio, and APO exerted a significant cytoprotective effect.To investigate whether the mitochondrial pathway was responsible for the antiapoptotic effect of APO, western blotting and immunofluorescent staining were performed to measure the expression of proteins related to the mitochondrial pathway.The expression of BAX and the mean fluorescence intensity of C-caspase3 were increased significantly, while BCL-2 protein expression was considerably decreased after Dex treatment (Figure 1H-L).APO pretreatment dramatically prevented this change in the experimental group.In conclusion, APO significantly increased cell survival and inhibited apoptosis in osteoblasts induced by Dex.
ROS generation in MC3T3-E1 cells.Our results demonstrated that Dex promoted the formation of ROS compared with those in the NC group, and APO significantly suppressed ROS generation (Figure 3A,B).4-HNE and 8-OHdG are the end products of OS, and the intracellular levels of these products can be used to assess the state of OS.As shown in Figure 3C-F, Dex promoted the intracellular levels of 4-HNE and 8-OHdG compared with those in the NC group.F I G U R E 1 Cytoprotective effect of APO pretreatment on the viability of Dex-induced osteoblasts.(A) The chemical structure of APO.(B) Osteoblasts were incubated with various concentrations of APO for 24 h, and viability was examined with CCK-8 assays.(C) Osteoblasts were pretreated with the indicated concentration of APO for 2 h and then exposed to Dex for 24 h.Then, viability was analysed by CCK-8 assays.(D-G) Osteoblast apoptosis was analysed by TUNEL staining and flow cytometry.(H, I) Quantitative analysis of the MFI after C-caspase3 staining.(J-L) Western blot of the expression of BAX and BCL-2.Scale bar, 100 μm.
annotations and classifications based on BP, CC and MF are shown in Figure 5D-F.
APO involved Nrf2 signalling pathway activation, we measured the protein levels of Nrf2, HO-1 and downstream antioxidant enzymes in vitro.As shown in Figure 6A-D, Dex prominently reduced the fluorescence intensities of Nrf2 and HO-1, which indicated that the expression of Nrf2 and HO-1 was inhibited.Consistent with the immunofluorescence staining results, western blot analysis of Nrf2 and HO-1 showed that Dex markedly downregulated Nrf2 and HO-1 protein expression, while APO dramatically upregulated their expression (Figure 6E-G).Moreover, as shown in Figure 6H-K, APO pretreatment increased the activity of downstream antioxidant enzymes (GSH, CAT, GPX, SOD) by activating the Nrf2 pathway.These data suggested that the cytoprotective effect of APO antagonized OS damage and cell apoptosis by activating the Nrf2 pathway.

3. 7 |
Nrf2 activation improved cell viability and osteogenic differentiation by inhibiting OS and restoring mitochondrial function We used ML385 (an inhibitor of Nrf2) to block the Nrf2 pathway to examine the role of Nrf2 in the protective effect of APO against oxidative stress.Compared with those in the Dex + APO group, the levels of Nrf2 and HO-1 were significantly inhibited in the Dex + APO + ML385 group after ML385 (5 μM) treatment (Figure 7A ,D-H).In the Dex + APO + ML385 group, blocking the Nrf2 pathway downregulated the expression of osteogenesisrelated markers (ALP, OPG, RUNX2) and the apoptosis-related marker BCL-2 and upregulated the expression of BAX (Figure 7A-C).In addition, ML385 treatment impaired the ability of APO to antagonize OS damage and protected mitochondrial function.Therefore, the expression of the oxidative stress-related markers 4-HNE and 8-OHdG (Figure 7I-L) was significantly increased, and mitochondrial function (Figure 7M-P) was notably impaired.As shown in Figure 7Q-T, blocking the Nrf2 pathway markedly increased the fluorescence intensities of TUNEL and C-caspase3, which suggested that ML385 increased apoptosis in the Dex + APO + ML385 group.ML385 also exerted an inhibitory effect on ALP activity and mineralization in osteoblasts induced by Dex (Figure 7U-X).

F
I G U R E 2 APO restored osteogenic differentiation and mineralization in osteoblasts induced by Dex.After APO (100 μM) pretreatment for 2 h, osteoblasts were exposed to Dex (1 μM) for 24 h in the Dex + APO group.(A) ARS staining measured the mineralization potential of osteoblasts after 21 days of osteogenic induction.(B) Quantitative analysis of the ARS-positive area.(C, D) ALP activity in osteoblasts was examined after 7 days of osteogenic induction.(E-H) Western blot of the expression of ALP, OPG and RUNX2.Scale bar, 50 μm.that in the NC group (Figure 8C,F).In addition, TUNEL staining demonstrated that pretreatment with APO significantly reduced the MFI in the femoral head compared with that in the Dex group (Figure 8D).Micro-CT revealed that compared to the NC group, Dex significantly damaged the subchondral bone and resulted in marked declines in BV/TV, Tb.Th, Tb. S and BMD.However, APO F I G U R E 3 APO inhibited Dex-induced ROS generation in MC3T3-E1 cells.(A) Intracellular ROS levels in osteoblasts were determined by flow cytometry.(B) Quantitative analysis of the ROS levels in the different groups.(C) Representative 8-OHdG immunofluorescence staining image.(D) Quantitative analysis of the MFI after 8-OHdG staining.(E) Representative 4-HNE immunofluorescence staining image.(F) The MFI of 4-HNE immunofluorescence staining was analysed.Scale bar, 20 μm.significantly reversed the toxic effects of Dex on bone reconstruction (Figure 8H-M).
sion from ARCO Stage I to Stage IV takes an average of 49 months.Therefore, to effectively treat SIFHN, early and prompt intervention, the identification of prospective targets and the development of effective medications are essential and urgent.In the current work, we assessed the effect of APO on Dex-mediated osteoblast apoptosis.Our findings suggested that APO protected osteoblasts from Dexmediated OS and mitochondrial functional impairment.Mechanistically, APO suppressed intracellular ROS generation and restored F I G U R E 4 APO restored mitochondrial function in Dex-treated osteoblasts.Osteoblasts were incubated with 1 μM Dex with or without APO (1, 10, and 100 μM) for 24 h.(A) Representative images of the MMP after staining with JC-1.Red indicates normal mitochondria, and green indicates MMP depolarization.(B) Fluorescence intensities (ratio of red/green JC-1 fluorescence) of MC3T3-E1 cells.(C) Representative images of mitochondrial ROS levels after staining with Mito-Sox red.(D) MFI of Mito-Sox red fluorescence.Scale bar, 20 μm.F I G U R E 5 Analysis of potential therapeutic targets of APO, as determined by network pharmacology.(A) Venn diagram showing APO target prediction of OS in osteonecrosis of the femoral head (ONFH).(B) PPI network of 6 targets.(C) Pathways of the potential targets.(D-F) GO enrichment analysis of the targets.Biological process (D), cell component (E) and molecular function (F).BP, biological process; CC, cell component; GO, Gene Ontology; MF, molecular function; PPI, protein-protein interaction.F I G U R E 6 APO activated the Nrf2 pathway and upregulated the expression of antioxidant enzymes.(A, B) Immunofluorescence was conducted to examine the levels of Nrf2 and HO-1 after 24 h of APO treatment (100 μM).(C, D) The MFI of Nrf2 and HO-1 in the three groups.(E-G) Western blotting was performed to measure Nrf2 and HO-1 expression.(H-K) The levels of GSH and the activities of CAT, GPx and SOD.Scale bar, 20 μm.CAT, catalase; GPx, glutathione peroxidase; GSH, glutathione; MDA, malondialdehyde; SOD, superoxidase dismutase.F I G U R E 7 Blocking the Nrf2 pathway neutralized the cytoprotective effect of APO on Dex-induced osteoblasts.(A-G) Western blotting was used to detect the protein expression of Nrf2, HO-1, ALP, OPG, RUNX2, BAX and BCL-2.(I-L) 4-HNE and 8-OHdG immunofluorescence staining was used to detect OS levels in MC3T3-E1 cells.(M-P) Mito-Sox and JC-1 staining were performed to assess ROS production and the membrane potential polarization status in mitochondria.(Q-T) C-caspase3 and TUNEL staining were used to measure apoptosis in MC3T3-E1 cells.(U-X) ALP activity assays and ARS staining were used to evaluate the osteogenic differentiation of osteoblasts.Scale bar, 50 μm.mitochondrial function through activation of the Nrf2 pathway.In vitro, APO inhibited apoptosis and attenuated osteonecrosis by increasing the expression of Nrf2 in the rat femoral head.APO is a naturally occurring acetophenone found in rooibos and black walnut roots.Initially, APO was primarily reported to be a NOX inhibitor.However, it has now been demonstrated that F I G U R E 8 APO relieved the progression of SIFHN.(A, E) Representative MRI images of the rat femoral head (T2WI) and relative MRI signal intensity.(B) Representative macroscopic images of the femoral head in rats.(C, F) Representative images of HE staining.(D, G) Representative image of TUNEL staining and the MFI of TUNEL staining.(H-M) Representative images of micro-CT scanning and quantitative analysis of BMD (I), Tb.N (J), Tb. S (K), BV/TV (L) and Tb.Th (M).The red rectangle indicates the region of interest (ROI).n.s., not significant.Scale bar, 100 μm.
Our results suggested Dex markedly raised the levels of ROS and aggravated OS injury, thus disrupting the MMP.These changes lead to apoptosis.Encouragingly, APO significantly reduced ROS levels in cells, restored mitochondrial function and inhibited the activation of C-caspase3.Weakened mechanical strength in bone trabeculae and reduced osteogenic differentiation in the subchondral bone region F I G U R E 9 APO promoted Nrf2 expression and suppressed oxidative stress levels in the femoral head.Representative immunofluorescence pictures and quantitative analysis of the MFI of Nrf2 (A), HO-1 (B), 4-HNE (C) and 8-OHdG (D) in the different treatment groups.(E-H) The contents of antioxidant enzymes (GSH, CAT, GPx, SOD) in rat plasma.Scale bar (A, B) = 200 μm, scale bar (C, D) = 100 μm. of the femoral head are important factors in the development of collapse during SIFHN. 5,38Eric E. Beier et al. 39 demonstrated that GCs significantly accelerated bone loss and prevented osteoblasts from forming mineralized nodules, which resulted in microfractures of the trabeculae and a decline in trabeculae-related parameters, such as trabecular volume (TV), Tb.N and Tb.Th, as well as a corresponding decline in BMD.However, APO rescued the deterioration of mechanical strength and the mineralization properties of bone trabeculae in the subchondral bone region.This may be another crucial function of APO in delaying the collapse and progression of SIFHN.
explored.The potential mechanism of Nrf2 activation by APO may be as follows: (1) APO treatment may weaken the binding ability of GCs to GC receptors (GRs) and inhibit downstream gene transcription.(2) APO may attenuate GR activity.(3) In the physiological state, the binding of Keap1 and Nrf2 is the main mediator of Nrf2 ubiquitination and degradation.APO inhibits the degradation of Nrf2 by facilitating the dissociation of KEAP1 and Nrf2. 45(4) The interaction between Keap1 and Nrf2 can be altered by the phosphorylation of Nrf2, and APO may regulate Nrf2 phosphorylation through protein kinases.In addition, studies have revealed that osteoclast overactivation plays a critical role in the progression of SIFHN, while APO may alleviate SIFHN by inhibiting osteoclastogenesis. 465 | CON CLUS ION In summary, APO inhibits apoptosis in Dex-treated osteoblasts.APO protects osteoblasts from Dex-mediated oxidative stress and mitochondrial impairment by activating Nrf2 signalling.In addition, APO mitigates the occurrence and progression of SIFHN in rats.These results provide theoretical support for the pharmacological treatment of SIFHN.APO may be a promising targeted medicine for the management of steroid-induced necrosis of the femoral head.AUTH O R CO NTR I B UTI O N S Xinglong Zhang: Conceptualization (equal); methodology (equal); writing -original draft (equal).Ran Pang: Methodology (supporting).Kai Zhang: Methodology (supporting); writing -original draft (supporting).Qian Xu: Writing -review and editing (equal).Chunlei Xu: Methodology (supporting).Wei Shi: Data curation (equal).Xinyu Liang: Methodology (equal).Dong Li: Data curation (equal).Wenhao Cui: Writing -review and editing (equal).Shucai Bai: Data F I G U R E 1 0 Schematic model of the osteoprotective effect of APO.APO promotes the activation of Nrf2 pathway to reduce cellular ROS and restore the MMP.