Extracorporeal shock wave therapy alleviates glucocorticoid-induced injury and dysfunction of bone microvascular endothelial cells via the PI3K/AKT/FOXO1 pathway

Introduction

Osteonecrosis of the femoral head (ONFH) is a common orthopedic disorder with a high disability rate. Without timely intervention, it can lead to femoral head collapse, impaired hip joint function, and ultimately progress to osteoarthritis (1). High-dose glucocorticoid administration is recognized as the primary etiological factor in non-traumatic ONFH; however, its underlying pathogenic mechanisms have not been fully elucidated. Bone microvascular endothelial cells (BMECs), which line the inner surface of the microvasculature within bone tissue, exhibit specialized structural and functional properties that are essential for maintaining bone microcirculation and the homeostasis of the bone microenvironment (2). Accumulating evidence indicates that glucocorticoid-induced injury and dysfunction of BMECs in the femoral head are closely associated with the pathological development of ONFH (3,4). BMECs damage can induce a hypercoagulable state and promote microthrombus formation, leading to microcirculatory disturbances (5). Furthermore, various inflammatory mediators released by injured BMECs contribute to a persistent inflammatory response, which further aggravates endothelial damage and ultimately results in ischemic necrosis of the femoral head (6). Therefore, alleviating glucocorticoid-induced BMECs injury and dysfunction, as well as improving the local vascular supply to the femoral head, are of paramount importance for the prevention and early treatment of glucocorticoid-induced ONFH.

The early treatment of glucocorticoid-induced ONFH remains a clinical challenge. Compared to conventional surgical interventions, extracorporeal shock wave therapy (ESWT) offers several advantages, including its non-invasive nature, adjustable stimulation intensity, low procedural risk, and minimal complications. A growing body of clinical evidence has demonstrated that ESWT can alleviate pain, improve hip joint function, and delay the progression of ONFH (7,8). Moreover, as a preventive measure, ESWT may reduce the incidence of ONFH in high-risk populations (9). Despite its clinical efficacy, the underlying mechanisms by which ESWT exerts its therapeutic effects on ONFH remain poorly understood. Previous studies have confirmed that ESWT promotes angiogenesis and upregulates the expression of vascular endothelial growth factor (VEGF), thereby facilitating locomotor recovery after spinal cord injury (10). In addition, ESWT has been shown to enhance the proliferation of human umbilical vein endothelial cells (HUVECs) by modulating the Bach1/Wnt/β-catenin signaling pathway (11). However, it remains unclear whether ESWT can attenuate glucocorticoid-induced damage in BMECs and thereby delay the progression of ONFH.

The PI3K/AKT signaling pathway serves as a crucial transduction bridge connecting extracellular signals to intracellular responses and plays an essential role in regulating cell proliferation, migration, metabolism, and survival (12). Studies have shown that the PI3K/AKT pathway is closely associated with the pathogenesis of glucocorticoid-induced ONFH (13,14). Dex can inhibit the PI3K/AKT signaling pathway, leading to reduced phosphorylation of FOXO1, which subsequently increases the expression of pro-apoptotic factors such as cleaved caspase-3, cleaved caspase-9, and BAX, while decreasing the expression of the anti-apoptotic protein BCL-2, ultimately inducing apoptosis in MC3T3-E1 cells (15). In addition, the PI3K/AKT pathway mediates the biological effects of ESWT. ESWT has been shown to promote cell proliferation and differentiation via activation of this pathway (16), and in a rat model of post-infarction heart failure, ESWT alleviated fibrosis and improved ventricular function by upregulating phospho- (p-)PI3K and p-AKT expression (17). Our previous research (18) demonstrated that ESWT could protect vascular endothelial cells by modulating FOXO1 expression, but whether the PI3K/AKT signaling axis is involved remains unclear. Therefore, in this study, we established an in vitro model of glucocorticoid-induced injury in BMECs to investigate the effects of ESWT and its underlying mechanisms. Our findings will offer valuable insights into the potential application of ESWT for the treatment of femoral head necrosis. We present this article in accordance with the MDAR reporting checklist (available at https://aoj.amegroups.com/article/view/10.21037/aoj-25-36/rc).

Methods

The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Institutional Ethics Board of China-Japan Friendship Hospital (No. 2021-16-K08) and informed consent was taken from all the patients.

Isolation and identification of BMECs

According to previously established protocols (19), primary human BMECs were isolated from the cancellous bone of patients with femoral neck fractures undergoing total hip arthroplasty. Detailed patient information is provided in Table S1. To verify cell purity, immunofluorescence staining for von Willebrand factor (vWF) and CD31 was performed. BMECs at passages 2 to 4 were utilized for subsequent experiments.

Cell treatment

Dexamethasone (Dex; Beyotime, Shanghai, China) was dissolved in dimethyl sulfoxide (DMSO) at a stock concentration of 200 mM and subsequently diluted with endothelial cell medium (ECM; ScienCell, Carlsbad, CA, USA) to various concentrations for the establishment of a glucocorticoid-induced injury model in BMECs.

In this experiment, shock waves were generated using the Huikang SWT300 focused ESWT device. The shock wave frequency was set at 1 Hz. BMECs were collected and resuspended at a concentration of 1×10⁶/mL in sterile 15 mL centrifuge tubes. The tubes were fixed in a custom-designed water bath compatible with the shock wave device, ensuring that their position coincided with the focal point of the shock waves. The probe of the extracorporeal shock wave device was connected to the water bath through an elastic membrane to ensure effective energy transmission (Figure S1).

For studies investigating ESW-induced signal transduction, cells were pretreated with 25 µM LY294002 (Selleck, Shanghai, China), a PI3K pathway inhibitor, for 2 h. Following pretreatment, the cells were washed and resuspended, then exposed to the optimal intensity of ESW treatment as described above.

Cell viability assay

The viability of BMECs was determined using the Cell Counting Kit-8 (CCK-8) assay (Dojindo, Kumamoto, Japan). After treatment, 1×10⁴ BMECs were seeded in 96-well plates with 100 µL of medium in each well and cultured at 37 ℃ with 5% CO₂. At 24 and 48 h, 10 µL of the CCK-8 solution was added to each well, and the plates were incubated at 37 ℃ for 1 h. The absorbance was evaluated at 450 nm using a microplate reader.

Cell proliferation assay

Cell proliferation was assessed using the EdU-488 Cell Proliferation Kit (Ribobio, Shanghai, China) according to the manufacturer’s protocol. Briefly, cells were incubated with 5-ethynyl-2'-deoxyuridine (EdU) working solution for 2 h, followed by fixation and staining with Apollo reaction cocktail for 30 min. Fluorescent images were captured using a fluorescence inverted microscope and analyzed with ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Tube formation assay

To evaluate the angiogenic capacity of BMECs, a tube formation assay was conducted. Briefly, 50 µL of Matrigel (BD Biosciences, San Jose, CA, USA) was added to each well of a pre-cooled 96-well plate and incubated at 37 ℃ for 1 h to allow gelation. Subsequently, 4.0×10⁴ BMECs were seeded into each well and incubated at 37 ℃ with 5% CO₂ for 16 h. Tube-like structures were observed under an optical microscope, and quantitative analysis was performed with ImageJ software.

Migration assay

Cell migratory capacity was assessed using wound healing and Transwell assays. In the wound healing assay, a scratch was created in the confluent monolayer using a 200 µL pipette tip. Images were captured at 0 and 24 h to measure the width of the scratch, and the rate of wound closure was calculated.

For the Transwell assay, 1×10⁵ BMECs suspended in 200 µL of serum-free medium were seeded into the upper chamber of a 24-well Transwell insert. The lower chamber contained 800 µL of ECM supplemented with 10% fetal bovine serum as a chemoattractant. After 24 h of incubation, the inserts were stained with 0.1% crystal violet (Beyotime). Non-migrated cells on the upper surface of the membrane were gently removed using a cotton swab, and the migrated cells were observed and quantified under a light microscope.

Apoptosis assay

Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) assays (Beyotime) were used to assess cell apoptosis following the manufacturer’s instructions. The cells were incubated with Annexin V-FITC and PI reagent for 15 min in the dark. Following incubation, the cell apoptosis rate was evaluated by flow cytometry (FC).

Western blot analysis

BMECs from each group were collected, and total protein was extracted using a Total Protein Extraction Kit (Beyotime). Protein concentrations were measured using a BCA Protein Assay Kit (Beyotime). Equal amounts of protein (30 µg per sample) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% non-fat milk at room temperature for 2 h, followed by overnight incubation at 4 ℃ with the following primary antibodies: PI3K (1:1,000; cat. No. 4249; Cell Signaling Technology, Danvers, MA, USA), p-PI3K (1:1,000; cat. No. AB138364; Abcam, Cambridge, UK), AKT (1:1,000; cat. No. 4691; Cell Signaling Technology), p-AKT (1:1,000; cat. No. 4058; Cell Signaling Technology), FOXO1 (1:1,000; cat. No. 2880; Cell Signaling Technology), and p-FOXO1 (1:1,000; cat. No. 9461; Cell Signaling Technology). After washing with TBST for 10 min, the membranes were incubated with secondary antibodies at room temperature for 2 h. Protein bands were visualized using an electrochemiluminescence (ECL) detection system (Thermo Fisher Scientific, Waltham, MA, USA) and quantified using Image Lab software version 5.0 (Bio-Rad, Hercules, CA, USA).

Statistical analysis

All data are presented as mean ± standard error of the mean (SEM). Statistical significance between groups was determined using Student’s t-test or one-way analysis of variance (ANOVA) with a 95% confidence interval. No normality test was used cause the number of samples was too low. All statistical analyses were performed using GraphPad Prism software (version 8.0.1). A P value <0.05 was considered statistically significant.

Results

BMECs isolation and identification

We successfully isolated primary BMECs from the cancellous bone of the femoral head (Figure 1A,1B). After 1 week of culture, the cells exhibited a typical short spindle-shaped morphology (Figure 1B). Immunofluorescence staining showed high expression of endothelial cell markers vWF and CD31 (Figure 1C,1D), confirming their identity as BMECs.

Figure 1 BMECs identification and optimal parameters of ESWT. (A) Cancellous bone from the femoral head. (B) Morphology of BMECs. (C) BMECs identified by CD31 and vWF (immunofluorescence staining). (D) Ratio of CD31-positive and vWF-positive cells. (E) Viability of BMECs assessed by CCK-8 assay after treatment with different concentrations of Dex. (F) Viability of the BMECs injury model examined by CCK-8 assay following 1,000-pulse ESWT treatment at varying energy flux densities. (G) Viability of the BMECs injury model assessed by CCK-8 assay following ESWT treatment with varying pulse numbers at 0.06 mJ/mm². (H) CCK-8 results after Dex and/or ESWT treatment at 48 h. n=6; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. BMEC, bone microvascular endothelial cell; CCK-8, Cell Counting Kit-8; Dex, dexamethasone; ESWT, extracorporeal shock wave therapy; vWF, von Willebrand factor.

Optimal parameters of ESWT for a glucocorticoid-induced BMECs injury model

BMECs were treated with varying concentrations of Dex, and cell viability was assessed using the CCK-8 assay. Our results demonstrated that Dex inhibited cell viability in a dose-dependent manner (Figure 1E, Table S2). Based on these findings, a concentration of 200 µM Dex applied for 48 h was selected to establish a glucocorticoid-induced BMECs injury model.

To determine the optimal parameters for ESWT intervention, BMECs injury models were first subjected to 1,000 impulses of ESWT at varying energy flux densities. CCK-8 assay results showed that at 24 h post-treatment, ESWT at 0.06 mJ/mm² significantly enhanced cell viability (Figure 1F, Table S3). At 48 h, both 0.04 and 0.06 mJ/mm² treatments markedly increased BMECs viability, whereas 0.10 and 0.12 mJ/mm² resulted in notable cytotoxic effects at both 24 and 48 h (Figure 1F, Table S3). Subsequently, different impulse numbers at 0.06 mJ/mm² were tested. At both 24 and 48 h, 750 and 1,000 impulses significantly improved cell viability, while 1,500 impulses reduced it (Figure 1G, Table S4). Among all tested conditions, ESWT at 0.06 mJ/mm² with 1,000 impulses produced the most prominent improvement in cell viability at 48 h and was therefore identified as the optimal treatment parameter.

ESWT alleviated the Dex-induced reduction in BMECs viability and proliferation

BMECs viability and proliferation were evaluated using the CCK-8 assay and EdU assay, respectively. Results from the CCK-8 assay revealed that at 48 h, cell viability was significantly reduced in the Dex group compared to the Control group, while treatment with ESWT significantly restored cell viability in the Dex + ESWT group (Figure 1H, Table S5). Similarly, the EdU assay demonstrated that Dex treatment substantially suppressed BMECs proliferative capacity, which was partially rescued by ESWT (Figure 2A,2B, Table S6).

Figure 2 ESWT alleviated the Dex-induced reduction in BMECs proliferation and angiogenesis. (A,B) Cell proliferation of BMECs evaluated by EdU assay (stained with Apollo reaction cocktail). (C-E) Angiogenic capacity of BMECs detected by tube formation assay. n=3; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. BMEC, bone microvascular endothelial cell; DAPI, 4',6-diamidino-2-phenylindole; Dex, dexamethasone; EdU, 5-ethynyl-2'-deoxyuridine; ESWT, extracorporeal shock wave therapy.

ESWT alleviated the Dex-induced reduction in BMECs angiogenesis and migration

Tube formation assays were conducted to assess the effect of ESWT on angiogenic potential of BMECs. Quantitative analysis of total tube length and branch point numbers revealed that Dex exerted a pronounced anti-angiogenic effect compared to the control group, whereas ESWT treatment notably alleviated this inhibitory effect (Figure 2C-2E, Table S7). Furthermore, wound healing (Figure 3A,3B, Table S8) and Transwell assays (Figure 3C,3D, Table S9) were performed to evaluate migratory capacity. Dex treatment significantly impaired BMECs migration, while ESWT partially restored cell motility under these conditions.

Figure 3 ESWT mitigated Dex-induced impairment of BMECs migration and attenuated apoptosis. (A,B) Migration ability of BMECs evaluated by wound healing assay. (C,D) Migration ability of BMECs detected by Transwell assay (stained with 0.1% crystal violet). (E,F) Apoptosis of BMECs assessed through Annexin V-FITC/PI assay. n=3; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. BMEC, bone microvascular endothelial cell; Dex, dexamethasone; ESWT, extracorporeal shock wave therapy; FITC, fluorescein isothiocyanate; PI, propidium iodide.

ESWT alleviated the Dex-induced BMECs apoptosis

Cell apoptosis was observed using the Annexin V/PI assay. The results showed that the Dex group exhibited a significantly higher apoptosis rate compared to the control group. In contrast, ESWT treatment significantly inhibited Dex-induced apoptosis in BMECs (Figure 3E,3F, Table S10).

ESWT activated the PI3K/AKT/FOXO1 signaling pathway

Western blot analysis confirmed that ESWT activates the PI3K/AKT/FOXO1 signaling pathway. Dex treatment significantly downregulated the expression of p-PI3K/PI3K, p-AKT/AKT, and p-FOXO1/FOXO1, and this inhibitory effect was notably reversed by ESWT (Figure 4A-4D, Table S11).

Figure 4 ESWT activated the PI3K/AKT/FOXO1 signaling pathway. (A) Protein expression level was examined by the Western blot. (B-D) The relative protein level of p-PI3K/PI3K, p-AKT/AKT, and p-FOXO1/FOXO1. n=3; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. ESWT, extracorporeal shock wave therapy; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; p-, phospho-.

PI3K inhibition reversed the protective effect of ESWT on BMECs viability and proliferation

To investigate the role of PI3K in the ESWT-mediated enhancement of BMECs viability and proliferation, BMECs were pre-treated with the PI3K inhibitor LY294002. The CCK-8 assays revealed that cell viability in the Dex + ESWT + inhibitor group was significantly reduced at 48 h compared to the Dex + ESWT group (Figure 5A, Table S12). Similarly, the EdU assays demonstrated that PI3K inhibition reversed the proliferative effects of ESWT on BMECs (Figure 5B,5C, Table S13).

Figure 5 PI3K inhibition reversed the protective effect of ESWT on BMECs viability, proliferation, and angiogenesis. (A) Viability of BMECs assessed by CCK-8 assay. (B,C) Cell proliferation of BMECs evaluated by EdU assay (stained with Apollo reaction cocktail). (D-F) Angiogenic capacity of BMECs detected by tube formation assay. n=3; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. BMEC, bone microvascular endothelial cell; CCK-8, Cell Counting Kit-8; DAPI, 4',6-diamidino-2-phenylindole; EdU, 5-ethynyl-2'-deoxyuridine; ESWT, extracorporeal shock wave therapy.

PI3K inhibition reversed the protective effect of ESWT on BMECs angiogenesis and migration

We also observed that PI3K inhibition diminished the beneficial effects of ESWT on BMECs angiogenesis and migration. The tube length and number of branch points in the Dex + ESWT + inhibitor group were significantly reduced compared to the Dex + ESWT group (Figure 5D-5F, Table S14). Additionally, the PI3K inhibitor significantly impaired the migration of BMECs, as demonstrated by wound healing assay (Figure 6A,6B, Table S15) and Transwell assay (Figure 6C,6D, Table S16).

Figure 6 PI3K inhibition reversed the protective effect of ESWT on BMECs migration and apoptosis. (A,B) Migration ability of BMECs evaluated by wound healing assay. (C,D) Migration ability of BMECs detected by Transwell assay (stained with 0.1% crystal violet). (E,F) Apoptosis of BMECs assessed through Annexin V-FITC/PI assay. n=3; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. BMEC, bone microvascular endothelial cell; ESWT, extracorporeal shock wave therapy; FITC, fluorescein isothiocyanate; PI, propidium iodide.

PI3K inhibition reversed the protective effect of ESWT on BMECs apoptosis

The results of FC showed that the Dex + ESWT + inhibitor group exhibited a significantly higher apoptosis rate compared to the Dex + ESWT group (Figure 6E,6F, Table S17), indicating that ESWT attenuates BMECs apoptosis through activation of the PI3K signaling pathway.

The protective effects of ESWT on BMECs were mediated through the PI3K/AKT/FOXO1 signaling pathway

To further confirm that the protective effects of ESWT on BMECs are mediated through the PI3K/AKT/FOXO1 signaling pathway, the expression levels of p-PI3K, PI3K, p-AKT, AKT, p-FOXO1, and FOXO1 were assessed by Western blot. The results showed that, compared to the Dex + ESWT group, the ratios of p-PI3K/PI3K, p-AKT/AKT, and p-FOXO1/FOXO1 were significantly decreased in the Dex + ESWT + inhibitor group. These findings suggest that the beneficial effects of ESWT on BMECs are dependent on the activation of the PI3K/AKT/FOXO1 signaling pathway (Figure 7A-7D, Table S18).

Figure 7 The protective effects of ESWT on BMECs were mediated through the PI3K/AKT/FOXO1 signaling pathway. (A) Protein expression level was examined by the Western blot. (B-D) The relative protein level of p-PI3K/PI3K, p-AKT/AKT, and p-FOXO1/FOXO1. n=3; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. BMEC, bone microvascular endothelial cell; ESWT, extracorporeal shock wave therapy; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; p-, phospho-.

Discussion

A series of clinical studies has demonstrated the considerable potential of ESWT in the treatment of ONFH. ESWT has been shown to provide long-term pain relief, improve joint function, and enhance clinical outcomes in patients with ONFH (7,20). Moreover, several studies have reported that the therapeutic efficacy of ESWT is superior to that of core decompression or core decompression combined with bone grafting (21). Although previous studies suggest that ESWT may promote the expression of bone morphogenetic protein-2 (BMP-2) and VEGF, thereby facilitating angiogenesis and osteogenic repair (22), the precise mechanisms underlying its therapeutic effects remain incompletely understood. This study aims to investigate the effects and underlying mechanisms of ESWT in a glucocorticoid-induced injury model of bone BMECs.

Previous studies have demonstrated that the biological effects of ESWT on cells exhibit a significant dose-dependent pattern (23). Appropriate energy levels of ESWT can enhance cellular metabolism and improve cell function, whereas excessive energy levels may lead to cellular damage and apoptosis (24,25). In this study, the optimal ESWT parameters for the treatment of BMECs were determined. Based on previous experience, we initially applied a gradient of energy flux densities with 1,000 pulses. ESWT at 0.06 mJ/mm² significantly enhanced cell viability at 24 h post-treatment. By 48 h, both the 0.04 and 0.06 mJ/mm² groups showed significantly increased viability, while treatments at 0.10 and 0.12 mJ/mm² significantly suppressed cell viability at both time points. Additionally, when different pulse numbers were tested at a fixed energy level of 0.06 mJ/mm², 750 and 1,000 pulses significantly promoted cell viability, whereas 1,500 pulses led to a marked reduction at both time points. Notably, the 0.06 mJ/mm² group and the 1,000-pulse group exhibited the highest levels of cell viability at 48 h, suggesting that 0.06 mJ/mm² with 1,000 pulses may represent the optimal parameter for alleviating glucocorticoid-induced injury in BMECs.

Although the precise mechanisms underlying glucocorticoid-induced ONFH remain incompletely elucidated, accumulating evidence suggests that vascular endothelial cell injury and dysfunction play a central role in its pathogenesis (26). Glucocorticoids can induce vascular endothelial cell injury and apoptosis, leading to coagulopathy and sustained inflammatory responses. These pathological changes promote thrombosis and microcirculatory disturbances, ultimately resulting in ischemic ONFH (5). ESWT has been reported to promote the recruitment of circulating endothelial progenitor cells to renal tissue, thereby alleviating renal injury and facilitating repair by enhancing cell proliferation and angiogenesis (27). Moreover, ESWT has been shown to promote the migration and proliferation of HUVECs in vitro (28). Consistent with these findings, the present study demonstrates that ESWT alleviates glucocorticoid-induced injury and dysfunction in BMECs, enhancing their viability, proliferative capacity, migratory ability, and angiogenic potential, while simultaneously suppressing apoptosis.

The PI3K/AKT/FOXO1 signaling pathway is a key regulator of cellular processes, including survival, apoptosis, cell cycle progression, and metabolism (29). Dysregulation of this pathway has been implicated in various musculoskeletal disorders, such as osteoarthritis, osteoporosis, and osteosarcoma (30-32). Previous studies have indicated that this pathway plays a critical role in the development of glucocorticoid-induced ONFH. Glucocorticoids significantly reduce the expression of p-PI3K, p-AKT, and p-FOXO1 in osteoblasts, thereby inhibiting the pathway and promoting apoptosis by upregulating pro-apoptotic factors and downregulating anti-apoptotic factors (15). Consistent with these findings, our study revealed that Dex markedly suppressed the PI3K/AKT/FOXO1 signaling pathway in BMECs, leading to decreased cell viability, proliferation, migration, and angiogenic capacity, along with a notable increase in apoptosis.

In recent years, accumulating evidence has revealed the involvement of the PI3K/AKT signaling pathway in cellular mechanotransduction. ESWT has been shown to promote cell proliferation and differentiation by modulating the PI3K/AKT pathway (16). Recent studies have also demonstrated that ESWT enhances the levels of p-PI3K and p-AKT in extracellular vesicles derived from endothelial colony-forming cells, thereby reducing oxidative stress and apoptosis in cardiomyocytes and alleviating myocardial ischemia-reperfusion injury (33). Our previous study demonstrated that ESWT mitigates glucocorticoid-induced endothelial cell injury by modulating FOXO1 expression (18). However, it remains unclear whether the PI3K/AKT axis, as an upstream regulator of FOXO1, contributes to the protective role of ESWT in BMECs. In the present study, we observed that the ratios of p-PI3K/PI3K, p-AKT/AKT, and p-FOXO1/FOXO1 were significantly increased in the Dex + ESWT group compared to the Dex group, indicating activation of the PI3K/AKT/FOXO1 pathway by ESWT. Moreover, treatment with a selective PI3K inhibitor reversed the protective effects of ESWT on BMECs and concurrently suppressed the activation of the PI3K/AKT/FOXO1 signaling pathway, suggesting that ESWT alleviates glucocorticoid-induced BMECs injury by activating the PI3K/AKT/FOXO1 signaling pathway.

This study has several limitations that should be acknowledged. First, as the experiments were conducted in vitro, the findings require further validation in animal models. Second, only the central portion of the BMECs suspension was precisely aligned with the focal point of the shock wave device. Consequently, the entire sample may not have been uniformly exposed to the same stimulation intensity, potentially affecting the overall treatment effect. Another potential limitation of this study is the relatively small sample size. In addition, while our results indicate that ESWT alleviates glucocorticoid-induced injury and dysfunction in BMECs by activating the PI3K/AKT/FOXO1 signaling pathway, its effects on other cell types—such as osteoblasts, osteoclasts, and bone marrow mesenchymal stem cells—remain to be explored. Lastly, previous studies have shown that cellular responses to ESWT involve complex signaling networks. Therefore, further research is needed to investigate the potential multi-target mechanisms underlying the biological effects of ESWT.

Conclusions

In conclusion, our study demonstrates that ESWT activates the PI3K/AKT/FOXO1 signaling pathway, thereby alleviating glucocorticoid-induced injury in BMECs. ESWT enhances cell viability, proliferation, migration, and angiogenic capacity, while reducing apoptosis.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://aoj.amegroups.com/article/view/10.21037/aoj-25-36/rc

Data Sharing Statement: Available at https://aoj.amegroups.com/article/view/10.21037/aoj-25-36/dss

Peer Review File: Available at https://aoj.amegroups.com/article/view/10.21037/aoj-25-36/prf

Funding: This work was supported by grants from the National Natural Science Foundation of China (No. 82072524) and the Beijing Natural Science Foundation (No. 7242127).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://aoj.amegroups.com/article/view/10.21037/aoj-25-36/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Institutional Ethics Board of China-Japan Friendship Hospital (No. 2021-16-K08) and informed consent was taken from all the patients.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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