The results of the present study demonstrate that sacubitril/valsartan administration effectively reduces blood pressure and heart rate in DOCA-salt-treated rats. This therapeutic efficacy was observed both when administered after the establishment of hypertension (therapeutic group) and when initiated concurrently with DOCA-salt exposure (preventive group). Notably, early initiation of sacubitril/valsartan had a more pronounced effect in mitigating elevations in systemic systolic and diastolic blood pressure, suggesting potential utility for early intervention in this experimental model. A primary focus of this study was evaluating how early versus late sacubitril/valsartan intervention influences diurnal hemodynamic variations. It must be explicitly noted as a study limitation that systemic BP and HR were recorded at only three specific time points (09:00, 15:00, and 21:00). Consequently, these data reflect discrete time-of-day variations rather than a full, continuous 24-hour circadian profile. They cannot be used to mathematically define definitive circadian parameters such as nocturnal dipping, amplitude, acrophase, or phase shifts.

​

In our experimental model, the control group showed steady fluctuations across the measured hours. Conversely, the untreated hypertensive group exhibited an altered pattern, with significantly elevated blood pressure values persisting across the morning and afternoon points. While these findings indicate a clear disruption in normal time-of-day blood pressure variations under DOCA-salt exposure, interpreting these patterns requires caution. Because rats are nocturnal, their physiological rest/activity cycles are reversed compared to humans'. In our facility, the 12 h light/12 h dark cycle was strictly controlled, with lights on at 07:00 and lights off at 19:00. Thus, the 09:00 and 15:00 measurements correspond to the animals' early- to mid-quiescent (sleep) phase.

​

In contrast, the 21:00 measurement occurs during their active (dark) phase. The high blood pressure values observed during the resting phases in Group II align with a marked reduction in circulating melatonin levels. In the therapeutic group, treatment with the sacubitril/valsartan combination reduced these fluctuations, bringing values closer to those of the controls. Interestingly, in the early intervention group (Group IV), the day-to-evening variation across the three measured points was minimized, with blood pressure remaining baseline-like throughout. This stabilization suggests that early intervention may alleviate early-phase hemodynamic surges within this model. However, definitive conclusions regarding the preservation of true circadian rhythm or a reduced risk of future clinical cardiovascular events cannot be drawn without continuous 24-hour telemetry or structural tissue evaluations, which were outside the scope of this study.

​

Unlike studies performed in heart failure or high-renin conditions, sacubitril/valsartan treatment in our DOCA-salt model was associated with reduced serum angiotensin II concentrations. The DOCA-salt model is characterized by suppression of systemic renin-angiotensin system activity; therefore, sacubitril-mediated enhancement of natriuretic peptide signaling may have further inhibited renin release and angiotensin II generation. In addition, differences in analytical methodology, as Ang II was quantified by ELISA in the present study rather than equilibrium LC-MS/MS, may contribute to discrepancies between studies. 26,27

​

Our biochemical analyses revealed a substantial increase in serum endothelin-1 (ET-1) and angiotensin II (Ang II) in the untreated hypertensive group, paired with a significant decrease in serum melatonin. ET-1 is a potent endothelium-derived vasoconstrictor peptide. In the DOCA-salt rat model, elevated ET-1 levels increase vascular tone by binding to endothelin A (ETA) receptors on vascular smooth muscle cells. This effect is upregulated in the vascular wall under mineralocorticoid excess. 28 Previous in vitro research has shown that Ang II can stimulate the expression and secretion of ET-1, 29 and that these two peptides can act synergistically to exacerbate vasoconstriction in mineralocorticoid-induced hypertension. 30

​

However, the marked elevation of serum Ang II in our DOCA-salt group and its subsequent reduction by sacubitril/valsartan require careful mechanistic contextualization. The standard DOCA-salt model is traditionally characterized as a high-volume, salt-sensitive, low-renin model of hypertension, where systemic circulating components of the renin-angiotensin-aldosterone system (RAAS) are heavily suppressed by negative feedback from fluid retention. The observed increase in circulating Ang II in our protocol might stem from local tissue-level RAAS overactivation—particularly in the kidneys, vasculature, or central nervous system—that can spill over into the systemic circulation with prolonged exposure. Additionally, valsartan is an AT1 receptor blocker, a class of drug that typically causes a compensatory upward reflex in circulating Ang II due to the interruption of the negative feedback loop. The lower serum Ang II levels in our treated groups suggest that sacubitril/valsartan does not uniformly suppress the RAAS. Instead, simultaneous neprilysin inhibition by sacubitril increases natriuretic peptide levels, which may counterregulate systemic fluid balance, reduce sympathetic outflow, and ultimately modulate overall Ang II production in this specific high-salt state. To fully substantiate these pathways, future investigations should evaluate pre-analytical handling, local tissue RAAS expressions, and explicit ELISA cross-reactivities.

​

In our study, sacubitril/valsartan treatment significantly lowered serum ET-1 concentrations in the therapeutic group and limited its elevation in the preventive group. In the early-stage intervention group, serum ET-1 concentrations were kept low, suggesting that early limitation of these vasoconstrictors may help prevent the development of severe hypertension.

​

Regarding melatonin regulation, this pineal hormone plays a vital role in cardiovascular homeostasis via its antioxidant capacities. Melatonin production is highly time-dependent and sensitive to light and neurohumoral factors. In our protocol, blood collection for all experimental groups was strictly standardized and performed at exactly 15:00 under identical environmental light conditions. This eliminates confounding variations caused by sampling animals at shifting clock times or under varying light conditions. Elevated Ang II levels accompanied the reduced serum melatonin observed in the untreated hypertensive group. While renal Ang II does not cross an intact blood-brain barrier, hypertensive pathology can disrupt barrier integrity, potentially allowing peripheral peptides to interface with central structures and impact pineal synthesis. Following sacubitril/valsartan administration, serum Ang II decreased, while melatonin levels concurrently increased. In the early treatment group, where the onset of hypertension was attenuated, melatonin concentrations remained close to control values.

​

Several limitations must be acknowledged when interpreting these data. First, because tail-cuff plethysmography was limited to three time points per day, continuous diurnal profiling has not been verified. Second, while the term cardiovascular protection' is conceptually relevant, this study did not directly measure functional endothelial parameters, cardiac remodeling, oxidative stress markers, or renal structural injury. Therefore, the protective assertions made here are strictly limited to the observed modifications of serum biomarkers (ET-1, Ang II, melatonin) and the modulation of time-of-day blood pressure values.