Introduction

Hypertension remains one of the most significant public health challenges globally, affecting approximately one billion individuals and serving as the primary modifiable risk factor for cardiovascular and cerebrovascular diseases, including stroke, myocardial infarction, and heart failure. The global burden of hypertension is projected to escalate further, with estimates suggesting that by 2025, nearly 1.56 billion adults will be affected, imposing substantial economic and healthcare burdens on both developed and developing nations. The prevalence of hypertension is particularly concerning among elderly women, who experience age-related vascular stiffening, endothelial dysfunction, and altered autonomic regulation that collectively contribute to elevated blood pressure. Despite the availability of effective pharmacological interventions, a substantial proportion of patients fail to achieve adequate blood pressure control, highlighting the pressing need for adjunctive non-pharmacological strategies.

In recent years, the paradigm of hypertension management has expanded beyond traditional pharmacotherapy to embrace lifestyle modifications, particularly regular physical exercise. Aerobic exercise has emerged as a fundamental non-pharmacological intervention for blood pressure regulation, with accumulating evidence demonstrating its efficacy in reducing both systolic and diastolic blood pressure across diverse populations. The beneficial hemodynamic effects of exercise are multifaceted and include improved endothelial function, enhanced nitric oxide bioavailability, reduced sympathetic nervous system overactivity, and favorable modulation of the renin-angiotensin-aldosterone system. However, the precise molecular mechanisms underlying these adaptations, particularly regarding the role of epigenetic regulators, remain incompletely understood.

MicroRNAs constitute a class of small, non-coding RNA molecules, approximately 17 to 25 nucleotides in length, that function as potent post-transcriptional regulators of gene expression. These molecules negatively regulate gene expression by binding to complementary sequences in the 3' untranslated region of target messenger RNAs, leading to translational repression or mRNA degradation. The discovery that a single microRNA can regulate up to 200 target mRNAs, and that multiple microRNAs can converge on a single mRNA, underscores the remarkable regulatory capacity of these molecules. Dysregulation of microRNA expression has been increasingly implicated in the pathogenesis of hypertension, with specific microRNA signatures linked to endothelial dysfunction, vascular smooth muscle cell proliferation, inflammation, and activation of the renin-angiotensin-aldosterone system.

Emerging evidence suggests that exercise training can modulate the expression of numerous microRNAs in ways that often oppose the pathological patterns observed in hypertension and other cardiometabolic diseases. Specific microRNAs, including miR-21, miR-155, and miR-199a, have been identified as key regulators of endothelial function, vascular smooth muscle cell proliferation, and the nitric oxide pathway. However, the effects of aerobic exercise on these hypertension-associated microRNAs in elderly women remain inadequately characterized. Therefore, this study aimed to investigate the effects of twelve weeks of aerobic exercise training on the expression of miR-21, miR-155, and miR-199a in elderly women with hypertension.

Methods

This quasi-experimental study was conducted on 30 elderly women with hypertension, aged 50 to 65 years, who were purposefully selected from the community of Mashhad, Iran. Participants were diagnosed with hypertension, defined as a resting systolic blood pressure of 130 mmHg or higher or a resting diastolic blood pressure of 80 mmHg or higher, consistent with established clinical guidelines. Individuals with a history of myocardial infarction, uncontrolled cardiac arrhythmias, diabetic complications, regular physical activity, smoking, or specific therapeutic diets were excluded. All participants provided written informed consent after receiving a comprehensive explanation of the study's purpose, procedures, potential risks, and benefits.

Participants were randomly assigned using block randomization (block size of 4) to either an experimental group (n=15) or a control group (n=15). The experimental group performed progressive aerobic training three sessions per week for twelve weeks, with each session lasting 30 to 50 minutes, at an intensity of 50 to 74 percent of maximum heart rate calculated using the Karvonen formula. The intensity was progressively increased throughout the program. Each session began with 5 to 10 minutes of warm-up and ended with 5 to 10 minutes of cool-down. Blood samples (10 mL) were collected from the antecubital vein after a 12-hour overnight fast, both before and 48 hours after the final training session. Serum was separated by centrifugation at 1500 rpm for 10 minutes and stored at -80°C. Total RNA was extracted from serum samples using TRIzol LS Reagent (Invitrogen, Carlsbad, CA, USA). cDNA synthesis was performed using miRNA cDNA Synthesis Kit (Thermo Fisher Scientific, Catalog No: K1621). The relative expression of miR-21, miR-155, and miR-199a was quantified using quantitative real-time polymerase chain reaction (qRT-PCR) with the 2^(-ΔΔCt) method. miR-16 was used as an internal control, and its stability was confirmed using geNorm and NormFinder software. Data were analyzed using Mixed ANOVA, paired and independent t-tests, with a significance level set at 0.05. Effect sizes (Cohen's d) were calculated for all comparisons.

Results

The findings demonstrated significant changes in the experimental group following the twelve-week aerobic training program. The expression of miR-155 increased significantly from 4.10±0.98 to 6.55±0.44, representing a 59.8% increase (P<0.001, effect size: 2.34, 95%CI: 1.89-2.79). Similarly, miR-21 expression increased from 2.40±0.74 to 3.20±0.95, representing a 33.3% increase (P<0.001, effect size: 1.18, 95%CI: 0.72-1.64). In contrast, miR-199a expression decreased significantly from 6.80±1.52 to 3.20±0.96, representing a 52.9% decrease (P<0.001, effect size: 2.89, 95%CI: 2.41-3.37). In the control group, no significant changes in microRNA expression were observed for any of the three target microRNAs.

The between-group comparisons using independent t-tests revealed significant differences in the magnitude of changes for miR-155 (t=4.12, P<0.001), miR-199a (t=17.4, P<0.001), and miR-21 (t=3.42, P=0.002), confirming that the observed changes were attributable to the exercise intervention. Mixed ANOVA results showed significant time×group interaction effects for miR-155 (F(1,28)=18.45, P<0.001), miR-199a (F(1,28)=22.87, P<0.001), and miR-21 (F(1,28)=8.92, P=0.006).

Additionally, significant reductions in systolic blood pressure (from 142.3±8.5 to 131.7±7.2 mmHg, P<0.001) and diastolic blood pressure (from 89.6±5.3 to 82.4±4.8 mmHg, P<0.001) were observed in the experimental group, while no significant changes were observed in the control group (P>0.05).

Discussion

The findings of this study provide compelling evidence that twelve weeks of aerobic exercise training significantly modulates the expression of hypertension-associated microRNAs in elderly women with hypertension. The significant increase in miR-21 expression observed in our experimental group aligns with previous research indicating that exercise training can reverse the pathological downregulation of specific miRNAs associated with hypertension. This finding is particularly noteworthy given that miR-21 has been implicated in the regulation of endothelial nitric oxide synthase activity and nitric oxide production. Studies have demonstrated that miR-21 targets phosphatase and tensin homolog and Bcl-2, thereby influencing apoptosis and nitric oxide bioavailability. The increased expression of miR-21 following aerobic training may therefore contribute to improved endothelial function through enhanced nitric oxide production.

The elevation of miR-155 levels observed in our training group is consistent with its established role in the regulation of endothelial nitric oxide synthase expression and nitric oxide production. miR-155 has been shown to directly target endothelial nitric oxide synthase, and its expression is modulated by inflammatory stimuli and shear stress. Our findings suggest that aerobic exercise may enhance miR-155 expression, potentially through increased hemodynamic forces and reduced systemic inflammation. This is particularly relevant given that aging is associated with reduced miR-155 levels in vascular tissues, contributing to age-related vasoconstriction and blood pressure elevation.

The most striking finding of our study was the substantial reduction in miR-199a expression following aerobic exercise training. This observation is consistent with recent evidence demonstrating that miR-199a is differentially regulated during physiological and pathological cardiac hypertrophy. The mechanistic importance of miR-199a repression is underscored by its role in regulating key metabolic and structural pathways, including the nitric oxide pathway in endothelial cells. The exercise-induced reduction in miR-199a expression may therefore enhance nitric oxide bioavailability and improve endothelial function, contributing to reduced peripheral vascular resistance and blood pressure.

The significant reductions in systolic and diastolic blood pressure observed in the experimental group are consistent with previous studies demonstrating the antihypertensive effects of aerobic exercise. These blood pressure reductions may be mediated through multiple mechanisms, including improved endothelial function, enhanced nitric oxide bioavailability, reduced sympathetic nervous system overactivity, and favorable modulation of the renin-angiotensin-aldosterone system. However, it should be noted that this study only measured microRNA expression and did not directly assess eNOS, NO, or endothelial function. The proposed mechanisms are therefore hypotheses for future research rather than definitive conclusions.

 Conclusion

In conclusion, this study demonstrates that twelve weeks of aerobic exercise training significantly modulates the expression of hypertension-associated microRNAs in elderly women with hypertension. We observed a significant increase in miR-155 and miR-21 expression and a marked reduction in miR-199a expression following the intervention. These changes are consistent with the established roles of these microRNAs in regulating endothelial function, nitric oxide bioavailability, and vascular health. The coordinated regulation of miR-21, miR-155, and miR-199a expression suggests that exercise acts through a complex network of molecular pathways to promote cardiovascular health. The finding that exercise induces opposite regulation of miR-199a compared to pathological stimuli underscores its potential as a therapeutic target and biomarker for cardiovascular health. Our results support the use of aerobic exercise as an effective non-pharmacological strategy for promoting molecular adaptations that may contribute to blood pressure control and improved cardiovascular outcomes in elderly women. Future studies with larger sample sizes and longer follow-up periods are warranted to validate these findings and to explore the clinical utility of monitoring microRNA expression as a marker of exercise adherence and efficacy. The identification of exercise-modulated microRNAs may ultimately contribute to the development of novel therapeutic approaches and personalized strategies for managing hypertension in elderly populations.

Footnote

Ethics Approval

This study was reviewed and approved by the Ethics Committee of the University of Qom IR.QOM.REC.1405.057. Written informed consent was obtained from all participants, and all research procedures were conducted in accordance with the principles of the Declaration of Helsinki.

 Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. All costs related to the implementation of this study, including the provision of equipment, laboratory kits, blood sampling, and other necessities, were covered by the researchers.

 Authors' Contributions

Z. R. contributed to the conceptualization, methodology, investigation, data curation, formal analysis, and writing of the original draft. M. A. B. was responsible for supervision, project administration, methodology, validation, and writing—review and editing. M. F. contributed to the conceptualization, resources, validation, and writing—review and editing. All authors have read and agreed to the published version of the manuscript. All authors have read and approved the final version of the manuscript.

 Conflict of Interest

The authors declare that they have no competing interests, whether financial or personal, that could have influenced the conduct of this research, the analysis of data, the writing of the manuscript, or the decision to publish the results.

 Declaration of Generative AI and AI-Assisted Technologies in the Writing Process

During the preparation of this work, the authors used ChatGPT-4 CoPilot for editing the manuscript. After using this tool, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication.

 Acknowledgments

The authors wish to express their sincere gratitude and appreciation to all participants who patiently cooperated in the training sessions and blood sampling procedures. We are also grateful to all the respected professors and experts who provided valuable cooperation and guidance at various stages of this study, including the design of the exercise protocol, conducting the tests, and data analysis. We would like to express our appreciation to the authorities of the University of Qom, the Department of Sports Sciences, and all centers that facilitated the implementation of this research.