Resveratrol restores glucocorticoid receptor and HDAC2 to overcome corticosteroid resistance in cigarette smoke-induced emphysema mice

Introduction

Chronic obstructive pulmonary disease (COPD), a major cause of mortality across the globe, is characterized by persistent chronic inflammation of the airways and non-structural damage, frequently accompanied by comorbidities such as cardiovascular disease and muscular atrophy (1). Long-term exposure to cigarette smoke (CS) is the main precursor for the onset of COPD (2,3). Currently, there is a paucity of effective therapeutic interventions that can halt the progression of the disease or address the cardiac remodeling and muscle atrophy associated with its comorbidities. In individuals diagnosed with COPD, the effectiveness of glucocorticoids is considerably diminished when compared to those with asthma, particularly in terms of managing chronic inflammation and enhancing lung function. Although inhaled and oral glucocorticoids can effectively control airway inflammation in asthma, their efficacy in treating COPD patients is relatively limited, even when administered at high doses (1). Clinical research indicates that the effectiveness of glucocorticoid monotherapy is limited in patients with COPD. However, this efficacy tends to increase when glucocorticoids are used in conjunction with long-acting bronchodilators (4). Nevertheless, a significant number of COPD patients continue to exhibit resistance to glucocorticoids, contributing to persistent inflammation, disease progression, a higher incidence of acute exacerbations, and an elevated risk of mortality (5). A previous study reveals that glucocorticoid-resistant patients are at greater risk of severe pulmonary inflammation and oxidative stress, ultimately leading to disease exacerbation and a diminished quality of life (3). Reports indicate that mice exposed to chronic CS exhibit an attenuated response to steroid therapy, rendering them a valuable model for studying steroid resistance in vivo (5).

The precise mechanism behind corticosteroid resistance in individuals with COPD is not yet fully comprehended. Recent studies suggest that major factors behind this resistance encompass the lowered expression of glucocorticoid receptors (GRs) and histone deacetylase 2 (HDAC2), alongside decreased activity and compromised functionality (6). Since GR serves as the primary target for the action of glucocorticoids, reduced levels have a direct impact on the effectiveness of these treatments. Within the pathological environment of COPD, both the levels of GR expression and its capacity for nuclear translocation are significantly diminished, which undermines its ability to effectively modulate the expression of genes related to inflammation (7). At the molecular level, the activation of kinase pathways, including p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun N-terminal kinase (JNK), can result in the phosphorylation of GRα at the Ser226 site, subsequently altering its biological function and obstructing the nuclear translocation process (8). Moreover, the upregulation of GRβ, an inhibitory isoform of GRα, further competes with GRα, intensifying the disruption of its function and contributing to glucocorticoid resistance (9). Additionally, HDAC2 is essential in mediating the anti-inflammatory properties of glucocorticoids by suppressing the transcription of inflammatory genes through the process of histone deacetylation (6). In individuals suffering from COPD, exposure to CS and oxidative stress significantly impairs both the expression levels and functionality of HDAC2, primarily through the activation of the phosphoinositide 3-kinase delta (PI3Kδ) signaling pathway (10). Moreover, oxidative stress not only directly influences HDAC2 but also enhances inflammatory responses and reduces the anti-inflammatory effectiveness of glucocorticoids by triggering inflammatory signaling pathways, such as PI3K-Akt and nuclear factor kappa B (NF-κB) (11). The mediators in the inflammatory response linked to COPD such as interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6) are of particular significance (12,13). Notably, there is no homologous gene corresponding to human IL-8 in mice. Consequently, chemokine (C-X-C motif) ligand 15 (CXCL15) is utilized as a surrogate for mouse IL-8 detection (14). The amplification of inflammation enhances the expression of myocardial hypertrophy markers, such as B-type natriuretic peptide (BNP) and β-myosin heavy chain (β-MHC) (13). Consequently, pharmacological agents designed to restore GR or HDAC2 levels and prevent the activation of NF-κB may enhance the anti-inflammatory properties of corticosteroids. However, the current state of research examining the GR protein expression within the lung tissues of COPD patients remains limited.

Resveratrol (RSV), a natural polyphenol, exhibits anti-inflammatory, antioxidant, anti-aging, and cardiovascular protective effects. The available research suggests that the continuous use of RSV may help to slow the progression of COPD (15,16). However, it should be noted that no atomizer formulations of RSV are currently available as therapeutic drugs. The combination of prednisolone and RSV has been shown to enhance the expression of silent information regulator 1 (SIRT1) in anti-steroid CD28nullCD8⁺ T cells derived from COPD patients, thereby reducing the production of inflammatory cytokines (17). Additionally, experimental evidence indicates that RSV exerts its anti-inflammatory effects by inhibiting the PI3K/Akt pathway, thereby lowering the pro-inflammatory cytokine levels (18). Our recent findings indicate that RSV can reduce emphysema by increasing HDAC2 expression, which is associated with skeletal muscle atrophy and aging (19). However, there is currently no literature examining whether RSV can restore steroid sensitivity in emphysema models, nor how RSV influences GR and HDAC2 levels in lung tissues.

This research explored the levels of GR and HDAC2 proteins in the lung tissues of COPD patients. Furthermore, a mouse model of emphysema, created through prolonged exposure to CS, was utilized to assess the impact of RSV in these animals. Our objective was to determine whether RSV could restore steroid sensitivity and assess its impact on GR and HDAC2 levels in the lungs, hearts, and gastrocnemius muscles. We present this article in accordance with the ARRIVE and MDAR reporting checklists (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-859/rc).

Methods

Human subjects

The participants in this research were categorized into two distinct categories: the normal lung function (N) category and the COPD category. Table 1 presents a comprehensive summary of data from all participants. The classification of the COPD patients was carried out according to Global Initiative for Chronic Obstructive Lung Disease (GOLD) criteria, ensuring that standardized measurements were utilized. All lung tissues were collected from a distance greater than 5 cm from the patients’ pulmonary nodules. Among the 10 subjects with normal lung function, five were current smokers, one had previously quit smoking, and four were non-smokers. In the COPD group, six patients were actively smoking, while four had stopped. None of the participants had a prior history of asthma or respiratory infections. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Ethics Committee of The First Affiliated Hospital of Guangxi Medical University (No. 2024-E327-01). Each subject signed a written informed consent form, indicating their understanding of the study and their willingness to take part.

Animals

Male C57BL/6 mice, aged 5 to 6 weeks and weighing approximately 14±2 g, were sourced from the Animal Experimental Center of Guangxi Medical University. Throughout the duration of the study, the mice were provided with unrestricted access to both food and water, allowing them to maintain a natural feeding behavior. Furthermore, the mice were kept under a controlled environment that simulated a 12-hour light-dark cycle, which is essential for maintaining their circadian rhythms and overall well-being during the experiment. Following the conclusion of the animal experiments, euthanasia was performed via intraperitoneal injection of lidocaine to minimize suffering.

CS-induced emphysema mouse model and treatment protocol

Five groups were formed by randomly assigning mice, with 8 mice in each group: a control group (Control), a CS exposure group (CS), a CS exposure group treated with RSV (CS + RSV), a CS exposure group treated with budesonide (CS + BUD), and a CS exposure group treated with both RSV and BUD (CS + RSV + BUD). Emphysema was induced as previously described in all groups except the control group, which was kept in a separate ventilated room and exposed to fresh air (20). RSV [200 mg/kg in dimethyl sulfoxide (DMSO)] (19) and BUD (2 mg/5 mL/cage) (21), along with vehicle control [10% DMSO in 0.5 mL phosphate-buffered saline (PBS)], were administered via gavage or nebulized inhalation from weeks 21 to 24, while CS exposure continued. The mice were sacrificed 24 hours following the final exposure to either CS or fresh air, and samples of lung, heart, and skeletal muscle were collected for analysis.

For the animal experiments above, only the experimenter knew the grouping. In addition, a protocol for animal experiments was prepared before the study with registration in the Guangxi Medical University Laboratory Animal Committee. All animal experiments were performed under a project license (No. 202304443) granted by the Guangxi Medical University Laboratory Animal Committee, in compliance with institutional guidelines for the care and use of animals.

Histology

Tissues from the right lung, right gastrocnemius muscle, and heart of the mouse were obtained and preserved in a 4% paraformaldehyde solution. Histopathological changes were observed through paraffin embedding and hematoxylin-eosin (HE) staining. For each tissue section, at least five random areas were selected for analysis. Based on prior research, the mean linear intercept (MLI) was determined for the alveolar space, which serves as an important metric in evaluating the structural characteristics of lung tissue. Additionally, the analysis included the measurement of cross-sectional areas for both myocardial and gastrocnemius tissues, providing further insight into their anatomical properties (19,22,23). Data analysis was performed using ImageJ software.

RNA extraction and real-time polymerase chain reaction (PCR)

Tissues sourced from the heart, lungs, and gastrocnemius muscle of mice were carefully processed through a homogenization procedure to prepare them for further analysis. Following this, RNA extraction was performed using the TRIzol reagent (Invitrogen, Carlsbad, USA) for isolating high-quality RNA. Prior to the synthesis of cDNA, the purity and concentration of the extracted RNA were rigorously assessed to ensure optimal conditions for downstream applications. The cDNA synthesis was conducted following the instructions provided with the Promega kit. Subsequently, the synthesized cDNA underwent PCR amplification to determine the expression levels of specific genes. This was accomplished using the SYBR^® Premix Ex Taq II kit from Takara, Japan. The expression levels of the target gene were analyzed via the 2^−ΔΔCT method, with β-actin serving as the internal reference control to normalize the data. The detailed information on the PCR primers employed is listed in Table 2.

Western blot

RIPA buffer (Solarbio, Beijing, China) was applied for total protein extraction from lung, heart, and gastrocnemius muscle tissues. The bicinchoninic acid (BCA) protein analysis kit (Pierce, Rockford, IL, USA) was then utilized for protein quantification. Subsequently, protein samples of 50 micrograms per well were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel discontinuous electrophoresis, after which the protein molecules on the gel were transferred to a polyvinylidene difluoride (PVDF) membrane provided by Millipore, USA. Following a 1-hour blocking step with 5% fat-free milk, the membranes were exposed to primary antibodies against HDAC2 (Abcam, Cambridge, UK), GR, β-MHC, and β-actin (Abclonal, Wuhan, China), IKBα and p-IKBα (Abmart, Shanghai, China), P65 and p-P65 (CST, Danvers, USA), BNP (Wanleibio, Shenyang, China), and GAPDH (Proteintech, Wuhan, China) at a dilution of 1:1,000 overnight at 4 ℃. Subsequent to this incubation, a secondary antibody labeled with fluorescent markers (1:10,000, CST, USA) was applied at room temperature for 1 hour. β-actin served as an internal control for lung tissue, while GAPDH was used for heart and skeletal muscle. ImageJ software was employed to measure the signal intensity.

Enzyme-linked immunosorbent assay (ELISA)

ELISA kits were utilized in the CXCL15 and TNF-α concentration quantification in mouse serum, following the manufacturer’s instructions (Shfksc, Shanghai, China).

Statistical analysis

The data analysis was performed utilizing the GraphPad Prism 8.0 software, and corresponding graphs were created. Results are presented as mean ± standard deviation (SD). To compare groups, simple comparisons between pairs of groups employed Student’s t-test, whereas more complex comparisons involving several groups were carried out using one-way analysis of variance (ANOVA) with subsequent post hoc Tukey-Kramer analysis. Statistical significance was set at P<0.05.

Results

GR and HDAC2 expression decrease in lung tissues of COPD patients

Our findings demonstrate a decrease in GR and HDAC2 protein levels in the lung of steroid-untreated COPD patients (Figure 1). These findings suggest a widespread resistance to steroids among COPD patients.

Figure 1 GR and HDAC2 expression in human lung tissues (n=10). ***, P<0.001. COPD, chronic obstructive pulmonary disease; GR, glucocorticoid receptor; N, normal lung function.

RSV restores corticosteroid sensitivity in CS-induced emphysema mice

An emphysema mouse model was developed, with mice receiving treatment of either RSV, BUD or a combination of both. The histological alterations in the lung tissue of these animal models are illustrated in Figure 2A. The MLI increased in mice subjected to CS exposure compared to Control mice (Figure 2B). Treatment with RSV alone reduced of MLI, while BUD alone did not prevent CS-induced emphysema. Notably, both RSV alone and the combination of RSV with BUD reduced MLI. Additionally, we assessed the grip strength of each group of mice (Figure 2C). The CS group exhibited diminished grip strength compared to the control group. Treatment with RSV alone improved grip strength, whereas BUD treatment alone did not yield significant enhancements. However, both RSV alone and the combination with BUD enhanced grip strength. These findings indicate that exposure to CS induces emphysema in mice and diminishes muscle strength, while treatment with RSV combined with BUD mitigates the detrimental impact of CS on lung tissue and skeletal muscle.

Figure 2 Resveratrol combined with BUD improves chronic cigarette exposure-induced emphysema and grip strength in mice. (A) HE results from lung tissues treated with RSV or BUD alone or in combination. Scale bars, 50 µm. (B,C) Comparison of mean alveolar lining intervals (B) and grip strength (C) in each group of mice (n=3–5). *, P<0.05; **, P<0.01; ***, P<0.001; ns, P>0.05. BUD, budesonide; CS, cigarette smoke; HE, hematoxylin-eosin; ns, not significant; RSV, resveratrol.

Furthermore, GR and HDAC2 protein levels in the lung tissues of mice within the CS group were lower compared to the Control group (Figure 3A). Notably, GR and HDAC2 levels could not be restored by BUD alone. However, treatment with RSV, either alone or in combination with BUD, improved the lung condition of these mice. Furthermore, the analysis of serum concentrations of CXCL15 (Figure 3B) and TNF-α (Figure 3C) in mice indicated that BUD did not reduce their levels. Conversely, RSV administration, whether used independently or alongside BUD, decreased the levels of these inflammatory markers. The findings indicate that mice exposed to CS display a blunted response to BUD therapy, as evidenced by reduced GR and HDAC2 in lung tissues, implying corticosteroid resistance. However, co-administration of RSV and BUD could potentially restore these protein levels, lower inflammatory factors levels, and overcome this resistance.

Figure 3 RSV combined with BUD restores corticosteroid sensitivity in CS-induced emphysema mice. (A) Protein expression of GR, and HDAC2 in lung tissue treated with RSV or BUD alone or in combination (n=5). (B,C) Concentration of CXCL15 (B) and TNF-α (C) in serum of mice (n=3). *, P<0.05; **, P<0.01; ***, P<0.001; ns, P>0.05. BUD, budesonide; CS, cigarette smoke; GR, glucocorticoid receptor; ns, not significant; RSV, resveratrol; TNF-α, tumor necrosis factor-alpha.

RSV suppresses NF-κB pathway and inflammation in emphysematous mouse lungs

Compared to the control group, the CS group’s lungs showed a significant rise in p-P65 and p-IKB-α protein levels (Figure 4A), coinciding with an upsurge in CXCL15 and TNF-α mRNA expression (Figure 4B). RSV gavage reduced the expression of p-P65 and p-IKB-α proteins and CXCL15 and TNF-α mRNA, suggesting that RSV may impede the NF-κB pathway and reduce lung inflammation in emphysema mouse models.

Figure 4 Gavage of RSV inhibits the NF-κB pathway and decreases the expression of inflammatory factors in lung tissue from CS-exposed mice. (A,B) Expressions of p-P65 and p-IKB-α protein (A) and CXCL15 and TNF-α mRNA (B) in lung tissues from CS-exposed mice treated with gavage RSV (n=5–6). *, P<0.05; **, P<0.01; ***, P<0.001. CS, cigarette smoke; NF-κB, nuclear factor kappa B; RSV, resveratrol; TNF-α, tumor necrosis factor-alpha.

RSV gavage reduces myocardial hypertrophy and modulates GR and HDAC2 expression in emphysema mice

Pathological analysis indicated that prolonged CS exposure enlarges the cross-sectional area of the mouse heart, suggesting myocardial hypertrophy, which RSV treatment notably reduced (Figure 5A). Additionally, GR and HDAC2 levels in the heart tissues of the CS group were downregulated, while RSV treatment elevated their expression (Figure 5B,5C). Furthermore, it was demonstrated that RSV therapy also suppressed the NF-κB signaling pathway, reducing P-P65 and P-IKB-α protein concentrations (Figure 5D), and downregulated IL-1β and IL-6 mRNA levels (Figure 5E). Concerning the myocardial hypertrophy index, the CS group mice showed elevated BNP and β-MHC expression in heart tissues, which decreased after RSV treatment (Figure 5F).

Figure 5 Gavage RSV upregulates GR and HDAC2 expression in cardiac tissues from CS-exposed mice, inhibits the NF-κB pathway, and downregulates the expression of indicators related to myocardial hypertrophy. (A) HE staining of the heart tissue from control, CS mice or treated with gavage RSV. Scale bars, 50 µm. Calculations were performed on the myocardium cross-sectional area (n=6). (B-F) Expressions of GR and HDAC2 protein (B) and mRNA (C), p-P65 and p-IKB-α protein (D), IL-1β and IL-6 mRNA (E), BNP and β-MHC protein (F) in heart tissues from CS-exposed mice treated with gavage RSV (n=5). (F) Expressions of BNP and β-MHC protein in heart tissue of mice (n=5–6). *, P<0.05; **, P<0.01; ***, P<0.001. β-MHC, β-myosin heavy chain; BNP, B-type natriuretic peptide; CS, cigarette smoke; GR, glucocorticoid receptor; IL, interleukin; NF-κB, nuclear factor kappa B; RSV, resveratrol.

RSV gavage enhances GR and HDAC2 expression of gastrocnemius muscle in mice with emphysema

Chronic exposure to CS led to gastrocnemius atrophy in mice, demonstrated by a decrease in gastrocnemius area in the CS group, which was subsequently restored following RSV gavage treatment (Figure 6A). Besides, in the gastrocnemius muscle of mice, there was a downward trend in GR and HDAC2 expression, while IL-1β and IL-6 mRNA levels were found to be elevated (Figure 6B,6C). Notably, RSV treatment was able to reverse these effects.

Figure 6 Gavage RSV upregulates GR and HDAC2 expression and decreases inflammatory factor expression in gastrocnemius muscle from CS-exposed mice. (A) HE staining of the gastrocnemius muscle from control, CS mice, or treated with gavage RSV. Scale bars, 50 µm. Calculations were performed on the gastrocnemius muscle cross-sectional area (n=6). Expressions of GR and HDAC2 protein (B) and IL-1β and IL-6 mRNA (C) in gastrocnemius muscle from CS-exposed mice treated with gavage RSV (n=6). *, P<0.05; **, P<0.01; ***, P<0.001. CS, cigarette smoke; GR, glucocorticoid receptor; HE, hematoxylin-eosin; IL, interleukin; RSV, resveratrol.

Discussion

COPD is marked by ongoing inflammation, primarily driven by pulmonary oxidative stress resulting from smoking, which can initiate an inflammatory cascade (24). Although COPD predominantly affects lung function, it is a complex disease that can lead to significant systemic inflammatory damage. Complications associated with COPD may arise at any stage of the disease (1). Resistance to corticosteroids complicates the treatment of COPD. This study used a smoking-induced emphysema model in male C57BL/6 mice, replicating the inflammation and pathogenesis observed in human patients (25). This model evaluated the efficacy of RSV in restoring glucocorticoid sensitivity and the expression of GR and HDAC2 in the lungs, heart, and gastrocnemius muscle of emphysema-affected mice.

Abnormal expression of GR and HDAC2 is recognized as a contributing factor to hormone resistance. Specifically, lung tissues of individuals with COPD and smokers have been observed to exhibit reduced HDAC2 expression (26). Moreover, GR expression levels in human bronchial epithelial cells are also affected by exposure to CS extract (27). However, there is currently limited research into GR protein expression within patient lung tissues. In this study, we evaluated GR and HDAC2 protein expression levels in lung tissue from steroid-untreated COPD patients and individuals with normal lung function. Our findings indicated that GR and HDAC2 levels were downregulated in the lung tissues of COPD patients. This further elucidates observations from Kahnert et al.’s study that COPD patients exhibit a limited response to the anti-inflammatory effects of corticosteroids, even at high inhalation or oral doses, suggesting a prevalent steroid resistance in this population (1).

Drugs such as theophylline, curcumin, erythromycin, and pericardial lactone have been shown to restore the activity and expression of HDAC2 in patients with COPD, thereby reinstating corticosteroid sensitivity (10,28,29). The literature indicates that RSV combined with prednisolone increases SIRT1 expression in CD28nullCD8⁺ T cells from COPD patients, leading to a reduction in inflammatory cytokines (17). However, there is a scarcity of studies investigating the ability of RSV to reverse steroid resistance, and it remains unclear whether RSV can elevate the expression of GR and HDAC2 in CS-induced mouse models. Our study demonstrates that in mice with CS-induced emphysema, aerosolized BUD does not effectively enhance GR and HDAC2 expression or reduce inflammation levels, indicating the presence of hormone resistance. Conversely, the combination of RSV and BUD successfully restores the expression levels of GR and HDAC2, significantly reduces serum levels of CXCL15 and TNF-α in emphysema-affected mice, alleviates emphysema, improves muscle strength, and reverses steroid resistance.

In addition to lung damage, patients with COPD also suffer from damage to the heart and skeletal muscles. Exposure to CS can induce myocardial hypertrophy in mice, lead to atrophy of skeletal muscles, and elevate levels of inflammatory factors. Research indicates that the downregulation of HDAC2 levels contributes to cardiac fibrosis and myocardial inflammation (30-32). HDAC2 expression was downregulated in the skeletal muscles of mice with emphysema in our previous study (19), but the GR expression in the heart and gastrocnemius of chronic CS-exposed mice and the effect of RSV treatment on GR expression was not clear. Observations were made that both GR and HDAC2 expression were suppressed in the heart and gastrocnemius of mice subjected to chronic tobacco smoke. However, these levels recovered following gastrointestinal treatment with RSV. In contrast to our experimental findings, some researchers suggested that enhanced GR signaling is critical in short-term hypoxia-induced muscle atrophy (33). It is hypothesized this discrepancy may arise from the chronic inflammatory stimulation of skeletal muscles in mice induced by chronic tobacco smoke, as well as differences between short-term hypoxia-induced skeletal muscle atrophy models, which may result in the downregulation of GR expression levels. Our experimental results demonstrate that RSV increases GR expression in the gastrocnemius muscles of emphysematous mice, enhances gastrocnemius cross-sectional area, and improves muscle strength.

The kBNF-κB signaling pathway is crucial in mediating glucocorticoid resistance and orchestrating both acute and chronic inflammatory responses (34-36). Our study demonstrates that RSV effectively inhibits the NF-κB pathway, with a consequent reduction in inflammatory factor levels in the lung, heart, and skeletal muscle. Additionally, some studies have suggested that RSV may alleviate left ventricular hypertrophy in elderly COPD rats (23). However, the effect of RSV on the expression of BNP and β-MHC as cardiac hypertrophy indices in mice exposed to chronic CS and emphysema remains unclear. Our findings indicate that BNP and β-MHC expression in heart tissue decreased, and the cross-sectional area of the myocardium also reduced following RSV treatment, which supports this perspective.

Based on our research findings, we propose that RSV may potentially alleviate steroid resistance. The PI3K/AKT signaling pathway can result in resistance to steroids by down-regulating the levels of GR and HDAC2 (10,28). When the JAK-STAT signaling pathway is activated, it can suppress the functionality of GR, thus reducing the anti-inflammatory properties of steroids (37). The stimulation of the p38 MAPK and ERK1/2 pathways decreases both the expression and function of GR, while also having an indirect effect on HDAC2 expression, which plays a role in steroid resistance (38,39). Moreover, NRF2 activation may indirectly influence HDAC2 expression by reducing oxidative stress (10). JNK/c-Jun is associated with corticosteroid sensitivity in peripheral blood mononuclear cells of COPD patients (40). However, due to the complexity of steroid resistance mechanisms, whether RSV acts on GR and HDAC2 through the aforementioned mechanisms or other mechanisms remains to be further investigated.

Conclusions

This study demonstrates that RSV can enhance GR and HDAC2 expression in lung tissue of mouse emphysema models, restore corticosteroid sensitivity in mouse models of emphysema. Additionally, our exploratory analyses suggest that RSV may have broader protective effects, including reducing myocardial hypertrophy and preventing skeletal muscle atrophy in emphysema mice.

However, the specific mechanisms by which RSV exerts these protective effects, particularly on myocardial hypertrophy, remain to be elucidated. Although we observed that RSV inhibited the activation of the NF-κB pathway and reduced inflammatory cytokine levels in cardiac tissues, the direct link between GR/HDAC2 upregulation and the inhibition of myocardial hypertrophy, as well as the potential involvement of other signaling pathways, requires further investigation. Similarly, the mechanisms underlying RSV’s protective effects on skeletal muscle atrophy also warrant further exploration. Future studies are needed to fully elucidate these mechanisms and their clinical implications.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the ARRIVE and MDAR reporting checklists. Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-859/rc

Data Sharing Statement: Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-859/dss

Peer Review File: Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-859/prf

Funding: This study was supported by the National Natural Science Foundation of China (No. 82260009), and Natural Science Foundation of Guangxi (No. 2023GXNSFBA026085).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-859/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 Ethics Committee of The First Affiliated Hospital of Guangxi Medical University (No. 2024-E327-01). Each subject signed a written informed consent form, indicating their understanding of the study and their willingness to take part. All animal experiments were performed under a project license (No. 202304443) granted by the Guangxi Medical University Laboratory Animal Committee, in compliance with institutional guidelines for the care and use of animals.

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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