Behavioral Outcomes in Rats Exposed to the CUMS ModelThe Open-field Test

Two-way ANOVA showed that the stress protocol and the different treatments (saline, perindopril and benazepril) had significant main effect on open field behavioral parameters: Latency time (stress: F(1,30) = 13.05, p = 0.0011; different treatments: F(2,30) = 18.60, p < 0.0001; stress by treatment: F(2,30) = 11.84, p = 0.0002), Ambulation Frequency (stress: F(1,30) = 4.33, p = 0.0461; different treatments: F(2,30) = 4.83, p = 0.0152; stress by treatment: F(2,30) = 11.28, p = 0.0002), Rearing frequency (stress: F(1,30) = 7.755, p = 0.0092; different treatments: F(2,30) = 7.092, p = 0.0030; stress by treatment: F(2,30) = 5.542, p = 0.0089). On the other hand, Grooming frequency statistical analysis showed that the stress protocol had significant main effect (stress: F(1,30) = 8.461, p = 0.0068; stress by treatment: F(2,30) = 13.03, p < 0.0001), without any significant main effect for different treatments (Fig. 2A-D).

Fig. 2

The effects of perindopril and benazepril) on the behavioral tests; Latency time (A), Ambulation frequency (B), Rearingfrequency (C), GroomingFrequency (D) in rats that were exposed to the mild, prolonged, unpredictable stress. The means ± standard deviation (n = 6) are used to display the data. Bonferroni’s multiple comparison test was used after a two-way ANOVA for statistical analysis. At a significance level of p < 0.05, Significant differences from non-stressed saline group (saline control group), Stressed saline group (CUMS group), and Stressed perindopril-treated group (perindopril-treated group)(10 mg/kg) are indicated by labels a, b, and c, respectively. The following is the acronyms: CUMS for chronic unpredictable mild stress

Post-hoc Bonferroni tests revealed that both benazepril and perindopril exhibited comparable efficacy in ameliorating CUMS-induced behavioral deficits, demonstrating similar antidepressant-like effects. Specifically, the CUMS group displayed significant behavioral alterations characteristic of a depressive-like state. Compared to the saline control group, the CUMS group showed a significant increase in latency time (322.38%) and a notable reduction in ambulation (63.80%), grooming frequencies (60.46%), and rearing (72.17%).

Both drug treatments effectively reversed these impairments induced by CUMS. The perindopril-treated group demonstrated a significant reduction in latency time (70.67%) and a significant increase in ambulation (159.50%), grooming frequencies (84.94%), and rearing (209.58%) compared to the CUMS group. Similarly, the benazepril-treated group also exhibited a significant reduction in latency time (88.34%) and a significant rise in ambulation (171.43%), grooming frequencies (100.11%), and rearing (279.04%) relative to the CUMS group.

Crucially, our statistical analysis confirmed no significant differences between the benazepril and perindopril-treated groups across any of these behavioral parameters, underscoring their comparable therapeutic impact.There is a significant difference between benazepril only group and saline control group in grooming frequency.

The Y-Maze Test

Short-term spatial memory function was evaluated using the Y-maze test (Fig. 3A&B). Two-way ANOVA showed that the stress protocol and the different treatments (saline, perindopril and benazepril) had significant main effect on Y-Maze parameters such as : SAP (stress: F(1,30) = 26.03, p < 0.0001; different treatments: F(2,30) = 23.96, p < 0.0001; stress by treatment: F(2,30) = 19.91, p < 0.0001), and TAE (stress: F(1,30) = 40.62, p < 0.0001; different treatments: F(2,30) = 11.71, p = 0.0002; stress by treatment: F(2,30) = 50.69, p < 0.0001).Our findings indicate that while both perindopril and benazepril effectively ameliorated CUMS-induced cognitive deficits, perindopril demonstrated a more robust impact on overall exploratory activity.

Fig. 3

The effects of perindopril and benazepril on the behavioral tests; spontaneous percentage of alternation (SAP) (A), Total Arm Entries count (TAE) (B), Immobility time (C), Struggling time (D) and Total time of grooming (E) in rats that were exposed to the mild, prolonged, unpredictable stress. The means ± standard deviation (n = 6)are used to display the data. Bonferroni’s multiple comparison test was used after a two-way ANOVA for statistical analysis. At a significance level of p < 0.05, Significant differences from non-stressed saline group (saline control group), Stressed saline group (CUMS group), and Stressed perindopril-treated group (perindopril-treated group)(10 mg/kg) are indicated by labels a, b, and c, respectively. The following are the acronyms: TAE stands for total arm entries, SAP for spontaneous percentage of alternation, and CUMS for chronic unpredictable mild stress

Specifically, the CUMS group demonstrated a significant decline in cognitive function compared to the saline control group, as evidenced by the decreased spontaneous percentage of alternation (SAP) and total arm entries (TAE) by 81.96% and 81.36%, respectively.

Conversely, both drug treatments significantly improved cognitive function relative to the CUMS group. Perindopril-treated rats showed a notable increase in SAP and TAE by 418.93% and 407.72%, respectively. Similarly, benazepril-treated rats also exhibited significant improvements, with SAP increasing by 476.27% and TAE by 243.35%. However, we did notice a curious distinction in their exploratory behavior: the benazepril group had a significantly lower TAE (32.38% reduction) compared to the perindopril group. This hints at a different effect on general activity, even though both drugs seemed equally effectiveat improving spatial alternation. On the other hand, there is a significant difference between perindoprilonly group and saline control group in TAE.

 The Forced Swimming Test

The forced swimming test, which assumes that immobility is a measure of behavioral despair, was used to track depressive-like behavior. Two-way ANOVA showed that the stress protocol and the different treatments (saline, perindopril and benazepril) had significant main effect on forced swimming test such as : Immobility time (stress: F(1,30) = 429.8, p < 0.0001; different treatments: F(2,30) = 5.162, p = 0.0118; stress by treatment: F(2,30) = 7.058, p = 0.0031), and Struggling time (stress: F(1,30) = 515.1, p < 0.0001; different treatments: F(2,30) = 7.203, p = 0.0028; stress by treatment: F(2,30) = 9.607, p = 0.0006).

Both perindopril and benazepril effectively reduced CUMS-induced behavioral despair, exhibiting comparable antidepressant-like effects. Specifically, the CUMS group demonstrated a considerably higher immobility time (249.43% increase) and a lower struggling time (78.91% decrease) when compared to the saline control group, confirming the induction of depressive-like behavior.

Conversely, both drug treatments significantly reversed these effects. The perindopril-treated group established a significant decrease in immobility time by 19.19% and an increase in struggling time by 106.91% in contrast to the CUMS group. Similarly, the benazepril-treated group also showed significant improvements, with a 21.21% decrease in immobility time and a 117.26% increase in struggling time.

Importantly, no significant differences were detectedbetween the benazepril- and perindopril-treated groups in terms of immobility or struggling times (Fig. 3C&D), indicating similar efficacy in alleviating behavioral despair.

The Splash Test

The splash test, as illustrated in Fig. 3E, was employed to track the self-care and motivational behavior index. Two-way ANOVA showed that the stress protocol and the different treatments (saline, perindopril and benazepril) had significant main effect on the total grooming time (stress: F(1,30) = 58.19, p < 0.0001; different treatments: F(2,30) = 11.38, p = 0.0002; stress by treatment: F(2,30) = 9.531, p = 0.0006).

Our findings reveal that both perindopril and benazepril significantly improved self-care behavior in CUMS-exposed rats, demonstrating comparable, robust effects.Specifically, the CUMS group exhibited a significant decrease in the time spent grooming by 91.80% compared to the saline control group, indicating impaired self-care and motivational deficits.

Conversely, both drug treatments notably reversed this deficit. The perindopril-treated group demonstrated a substantial increase in time spent grooming by 950.00% compared to the CUMS group. Similarly, the benazepril-treated group also showed a significant increase in grooming time by 586.67% relative to the CUMS group. Importantly, there was significant difference in the time spent grooming between the benazepril- and perindopril-treated groups.The benazepril-treated group exhibited a 38.25% decrease in grooming time compared to the perindopril-treated group.

 Histopathological ExaminationHematoxylin and Eosin Stain The Evaluation of the Cortical Tissues

Our histological examination of cortical tissues (Fig. 4A and Table 2) gave us some compelling evidence: both perindopril and benazepril effectively prevented CUMS-induced neuronal damage, maintaining normal neuronal histology comparable to healthy controls.

Fig. 4

The effects of Perindopril and benazepril on the different brain regions histological characteristics in rats that were exposed to the mild, prolonged, unpredictable stress using Hematoxylin and Eosin stain. Scale bar = 50 μm (x40 magnification) in the cortex, hippocampus/subiculum, and hippocampus/facia dentate and hilus, respectively; The following are the acronyms: N: for normal; ND: for neuron degeneration; and CUM: for Chronic Unpredictable Mild Stress

Table 2 The severity of histopathological changes in various brain regions of various experimental groups

Specifically, the saline control, perindopril-only, and benazepril-only groups all demonstrated normal histological features of neurons. In stark contrast, the CUMS group exhibited clear signs of neurodegeneration, characterized by degeneration and severe nuclear pyknosis in every neuron. On the other hand, both the benazepril- and perindopril-treated groups displayed normal neuronal histology. This powerfully demonstrates their protective effects against the changes CUMS caused. These findings together suggest that both treatments have a comparable ability to protect the neurons in the cortical region.

Evaluation of the Subiculum (hippocampus) Tissues

The subiculum region of the hippocampi of all experimental groups, even the CUMS-exposed animals, showed normal histological features. This finding suggests that the subiculum region was largely unaffected by the CUMS protocol or that the observed CUMS-induced damage was localized to other brain regions(Fig. 4B).

Evaluation of the Hilus and Fascia Dentate (hippocampus) Tissues

Histological evaluation of the hilus and fascia dentata regions within the hippocampus (Fig. 4C; Table 2) provided some critical insights into how perindopril and benazepril differed in protecting against CUMS-induced damage; it seemed perindopril offered more complete protection.

As expected, the saline control, perindopril-only, and benazepril-only groups did not exhibit any histological changes. Conversely, sections from the CUMS group displayed substantial nuclear pyknosis and degeneration in several neurons, highlighting significant neuronal harm.

On the other hand, neurons in sections from the perindopril-treated group showed no histological change, indicating complete neuroprotection. The benazepril-treated group, however, displayed only mild neurodegeneration and nuclear pyknosis, suggesting partial, but not complete, protection in these specific hippocampal regions.

Nissl Stain

Nissl stain expression in the hippocampus and cortex is depicted in Fig. 5. Two-way ANOVA showed that the stress protocol and different treatments (saline, perindopril and benazepril) had significant main effect on the percentage of degenerated neurons in the hippocampus (stress: F(1,12) = 452.1, p < 0.0001; different treatments: F(2,12) = 53.36, p < 0.0001; stress by treatment: F(2,12) = 56.84, p < 0.0001). Furthermore, two-way ANOVA showed that the stress protocol and different treatments (saline, perindopril and benazepril) had significant main effect on the percentage of degenerated neurons in the cortex (stress: F(1,12) = 708.0, p < 0.0001; different treatments: F(2,12) = 53.75, p < 0.0001; stress by treatment: F(2,12) = 115.1, p < 0.0001).

Fig. 5

The effects of Perindopril and benazepril on the different brain regions histological characteristics in rats that were exposed to the mild, prolonged, unpredictable stressusing Nissl’s Stain. A & B Photomicrographs showing the hippocampus and cortex nissl staining, respectively, Scale bar = 25 μm(x40 magnification). C & D The percent of the degenerated neurons to the total neurons by the quantitative image analysis for Nissl staining in the hippocampus and cortex regions, respectively.The means ± standard deviation (n = 3) are used to display the data. Bonferroni’s multiple comparison test was used after a two-way ANOVA for statistical analysis. At a significance level of p < 0.05, Significant differences from non-stressed saline group (saline control group), Stressed saline group (CUMS group), and Stressed perindopril-treated group (perindopril-treated group)(10 mg/kg) are indicated by labels a, b, and c, respectively. The acronym: CUMS is for for chronic unpredictable mild stress. Black and red arrows indicate intact and pyknotic neurons, respectively

Statistical analysis revealed that while perindopril offered complete protection against CUMS-induced neuronal damage in both the hippocampus and cerebral cortex, the neuroprotective effect of benazepril was more pronounced in the cortex than in the hippocampus.

Specifically, neurons in both the hippocampal and cerebral cortical regions of the saline control group, as well as the perindopril-only and benazepril-only groups, appeared normal. In stark contrast, the CUMS group displayed a large number of darkly stained, degenerating neurons in both the cerebral cortex and hippocampal regions.

The perindopril-treated group exhibited normal neurons in both the hippocampus and cerebral cortex, indicating a complete reversal of CUMS-induced damage in these areas. Conversely, while the cerebral cortex of the benazepril-treated group appeared normal, its hippocampus still displayed a few dark, degenerating neurons, suggesting partial neuroprotection in this specific region.

On the other hand, there is a significant difference between perindopril only group, benazepril only group and saline control group in cortical percent of degenerated neurons.

The Effects of Perindopril and Benazepril on Brain monoamine levels in the Rats Exposed To CUMS

As illustrated in Fig. 6, two-way ANOVA revealed that the stress protocol had significant main effect on hippocampal serotonin level (stress: F(1,30) = 86.89, p < 0.0001; stress by treatment: F(2,30) = 42.12, p < 0.0001). without any significant effect for treatments, Furthermore, two-way ANOVA showed that the stress protocol and the different treatments (saline, perindopril and benazepril) had significant main effect on cortical serotonin level (stress: F(1,30) = 34.76, p < 0.0001; different treatments: F(2,30) = 3.703, p = 0.0365; stress by treatment: F(2,30) = 20.12, p < 0.0001).On the other hand, it showed that the stress protocol and the different treatments (saline, perindopril and benazepril) had significant main effect on hippocampal dopamine level (stress: F(1,30) = 7.362, p = 0.0109; different treatments: F(2,30) = 7.344, p = 0.0025; stress by treatment: F(2,30) = 18.24, p < 0.0001). Furthermore, it showed the stress protocol and different treatments (saline, perindopril and benazepril) had significant main effect cortical dopamine level (stress: F(1,30) = 23.90, p < 0.0001; different treatments: F(2,30) = 13.67, p < 0.0001; stress by treatment: F(2,30) = 5.783, p = 0.0075.

Fig. 6

The effects of perindopril and benazepril on the monoamines, serotonin, and dopamine levels in rats’ brain that were exposed to the mild, prolonged, unpredictable stress. A & B Serotonin expression in the hippocampus and cortex regions, respectively, C & D Dopamine expression in the hippocampus and cortex regions, respectively. The means ± standard deviation (n = 6) are used to display the data. Bonferroni’s multiple comparison test was used after a two-way ANOVA for statistical analysis. At a significance level of p < 0.05, Significant differences from non-stressed saline group (saline control group), Stressed saline group (CUMS group), and Stressed perindopril-treated group (perindopril-treated group)(10 mg/kg) are indicated by labels a, b, and c, respectively. The acronyms: CUMS is for chronic unpredictable mild stress

These results indicate that while both perindopril and benazepril effectively reversed CUMS-induced reductions in serotonin and dopamine, there was a significant difference in the ability of benazepril and perindopril to restore serotonin and dopamine levels, in hippocampal levels.

Specifically, the CUMS group showed significantly lower levels of serotonin and dopamine compared to the saline control group. In the hippocampus, serotonin dropped by 58.87% and dopamine by 26.21%. In the cortex, serotonin was down by 62.75% and dopamine by 45.95%.

Both drug administrations markedly countered these deficits. Perindopril significantly raised serotonin and dopamine levels in the hippocampus by 46.92% and 38.16%, respectively, and in the cortex by 82.85% and 73.33%, respectively, when compared to the CUMS group. Benazepril also significantly increased these neurotransmitters in the hippocampus by 89.46% (serotonin) and 23.68% (dopamine), and in the cortex by 121.31% (serotonin) and 88.33% (dopamine).

A key distinction emerged in the hippocampus: benazepril markedly increased serotoninlevels by 28.96% and decreased dopamine levels by 10.48%, respectively, compared to perindopril-treated CUMS rats. While cortical dopamine levels, as well as cortical serotonin levels, did not significantly differ between the benazepril- and perindopril-treated groups, this specific finding highlights a unique benefit of benazepril in restoring hippocampal serotonin and perindopril in resorting hippocampal dopamine.

both benazepril only group and perindopril only group showed a significant difference in serotonin levels in the hippocampus compared to the saline control group. But in cortical serotonin level the benazepril only group showed a significant difference compared to saline control group.

The Effects of Perindopril and Benazepril on Brain Inflammatory Markers in the Rats Exposed to CUMS

Two-way ANOVA showed that the stress protocol and the different treatments (saline, perindopril and benazepril) had a significant main effect on hippocampal TNF-α levels(stress: F(1,30) = 91.05, p < 0.0001; different treatments: F(2,30) = 10.50, p = 0.0003; stress by treatment: F(2,30) = 19.38, p < 0.0001). Furthermore, on cortical TNF-α level(stress: F(1,30) = 64.80, p < 0.0001; different treatments: F(2,30) = 7.301, p = 0.0026; stress by treatment: F(2,30) = 15.77, p < 0.0001).

These statistical findings indicate both perindopril and benazepril effectively suppressed CUMS-induced increases in TNF-α expression in the hippocampus and cortex, demonstrating comparable anti-inflammatory effects.

As demonstrated in Fig. 7, the CUMS group exhibited a significant elevation in hippocampal and cortical TNF-α levels by 110.91% and 148.50%, respectively, compared to the saline control group. Conversely, both drug treatments significantly lowered TNF-α expression. The perindopril-treated group showed reductions in hippocampal and cortical TNF-α by 31.81% and 33.21%, respectively, when compared to the CUMS group. Similarly, the benazepril-treated group also significantly reduced TNF-α expression in the hippocampus by 33.60% and in the cortex by 41.14%.

Fig. 7

The effects of perindopril and benazepril on the TNF-α inflammatory indicators, in rats that were exposed to the mild, prolonged, unpredictable stress. A & B TNF-α expression in the hippocampus and cortex regions, respectively. The means ± standard deviation (n = 6) are used to display the data. Bonferroni’s multiple comparison test was used after a two-way ANOVA for statistical analysis. At a significance level of p < 0.05, Significant differences from non-stressed saline group (saline control group), Stressed saline group (CUMS group), and Stressed perindopril-treated group (perindopril-treated group)(10 mg/kg) are indicated by labels a, b, and c, respectively. The following are the acronyms: TNF-α stands for Tumor necrosis factor alpha, and CUMS for chronic unpredictable mild stress

Importantly, no statistically significant difference in cortical and hippocampal TNF-α levels was detected between the benazepril- and perindopril-treated CUMS groups, suggesting similar efficacy in modulating this pro-inflammatory cytokine.

Furthermore, the immunohistochemical evaluation of NF-κB expression in the hippocampus and cortex, as shown in Fig. 8A & B, provided additional insights. Two -way ANOVA showed that the stress protocol and the different treatments (saline, perindopril and benazepril) had significant main effect on hippocampal NF-κB expression (stress: F(1,12) = 627.2, p < 0.0001; different treatments: F(2,12) = 88.35, p < 0.0001; stress by treatment: F(2,12) = 80.15, p < 0.0001), and cortical NF-κB expression (stress: F(1,12) = 266.4, p < 0.0001; different treatments: F(2,12) = 88.45, p < 0.0001; stress by treatment: F(2,12) = 105.0, p < 0.0001).

Fig. 8

The effects of perindopril and benazepril on the expression of the Immunohisto-chemical inflammatory markers NF-қB in rats that were exposed to the mild, prolonged, unpredictable stress. A Photomicrograph, Scale bar = 25 μm(x40 magnification), illustrating NF-қB immunohisto-chemical staining in the hippocampus. B Photomicrograph, Scale bar = 25 μm, illustrating NF-қB immunohisto-chemical staining in the Cortex. C & D Quantitative image analysis for NF-қB expression as optical densities (OD) in the hippocampus and cortex regions, respectively.The means ± standard deviation (n = 3) are used to display the data. Bonferroni’s multiple comparison test was used after a two-way ANOVA for statistical analysis. At a significance level of p < 0.05, Significant differences from non-stressed saline group (saline control group), Stressed saline group (CUMS group), and Stressed perindopril-treated group (perindopril-treated group)(10 mg/kg) are indicated by labels a, b, and c, respectively. The following are the acronyms: NF-қB: stands for nuclear factor kappa- B, and CUMS for chronic unpredictable mild stress

These statistical findings indicate both perindopril and benazepril effectively suppressed CUMS-induced increases in NF-κB expression in the hippocampus and cortex, demonstrating comparable anti-inflammatory effects.

Notably, NF-κB expression in the CUMS group was notably higher than that of the saline control group, increasing by 116.36% in the hippocampus and 287.18% in the cortex. Both perindopril and benazepril administrations led to significant reductions in NF-κB expression compared to the CUMS group. Specifically, the perindopril-treated group demonstrated a 34.45% decrease in NF-κB expression in the hippocampal region and a 58.28% decrease in the cerebral cortex. Likewise, the benazepril-treated group also showed a substantial 34.45% decrease in NF-κB expression in the hippocampal region and a 60.26% decrease in the cerebral cortex.

Crucially, the benazepril-treated group showed no significantly lower level of NF-κB positive staining when compared to the perindopril-treated group, reinforcing their comparable ability to mitigate this key inflammatory mediator. No significant difference was observed between drugs-only groups (perindopril-only or benazepril-only) in all examined brain regions. Quantitative image analysis of NF-κB expression as optical densities (OD) in the hippocampus and cortex is further exhibited in Fig. 8C & D.

3.5 The Effects of Perindopril and Benazepril on Apoptosis indicator, caspase-3, in the Rats’ Brains Exposed To CUMS

The immunohistochemical evaluation of Caspase-3 expression in the hippocampus and cortex is depicted in Fig. 9A & B. Two -way ANOVA showed that the stress protocol and the different treatments (saline, perindopril and benazepril) had significant main effect on hippocampal caspase-3 expression (stress: F(1,12) = 459.1, p < 0.0001; different treatments: F(2,12) = 148.4, p < 0.0001; stress by treatment: F(2,12) = 183.6, p < 0.0001), and cortical caspase-3 expression (stress: F(1,12) = 263.9, p < 0.0001; different treatments: F(2,12) = 85.95, p < 0.0001; stress by treatment: F(2,12) = 125.1, p < 0.0001).

Fig. 9

The effects of perindopril and benazepril on the expression of the immunohisto-chemical apoptosis marker as caspase-3 expression in rats that were exposed to the mild, prolonged, unpredictable stress. A photomicrograph showing caspase-3 immunohistochemical expression in the hippocampus, with a scale bar of 25 μm and x40 magnification. B Photomicrograph showing caspase-3 immunohistochemical expression in the cortex, scale bar = 25 μm and x40 magnification. C & D Quantitative image analysis of the Hippocampaland Cortical optical densities (OD) for Caspase-3 expression, respectively.The means ± standard deviation (n = 3) are used to display the data. Bonferroni’s multiple comparison test was used after a two-way ANOVA for statistical analysis. At a significance level of p < 0.05 Significant differences from non-stressed saline group (saline control group), Stressed saline group (CUMS group), and Stressed perindopril-treated group (perindopril-treated group)(10 mg/kg) are indicated by labels a, b, and c, respectively. The following is the acronyms: CUMS stands for chronic unpredictable mild stress

These statistical findings indicate that both perindopril and benazepril effectively reduced CUMS-induced Caspase-3 expression, demonstrating comparable anti-apoptotic effects, though with notable differences in the extent of suppression.

Specifically, the saline control group, perindopril-only group, and benazepril-only group all displayed extremely low to negative Caspase-3 expression in both the hippocampus and cerebral cortex. In contrast, the CUMS group showed significantly higher levels of Caspase-3 expression in the cerebral cortex and hippocampal regions, by 444.83% and 247.83%, respectively, compared to the saline control group, indicating increased apoptosis.

Both treated groups significantly lowered Caspase-3 expression compared to the CUMS group. The perindopril-treated group exhibited limited Caspase-3 expression in the cerebral cortex and hippocampal regions, with reductions of 58.13% and 66.46%, respectively. The benazepril-treated group also showed a decrease in Caspase-3 expression, displaying mild positive staining in the cerebral cortex and hippocampal regions by 63.29% and 55.00%, respectively.

Importantly, there was no discernible change in Caspase-3 positive staining in the benazepril-treated group when compared to the perindopril-treated group, suggesting similar anti-apoptotic efficacy despite the numerical differences in reduction percentages. Furthermore, the quantitative image analysis of Caspase-3 expression as optical densities (OD) in hippocampus and cortex areas is shown in Fig. 9C & D.

The Effects of Perindopril and Benazepril on Brain RAS Levels in the Rats Exposed to CUMS

Two-way ANOVA showed that the stress protocol and the different treatments (saline, perindopril and benazepril) had significant main effect on hippocampal Ag II expression (stress: F(1,12) = 215.8, p < 0.0001; different treatments: F(2,12) = 26.65, p < 0.0001; stress by treatment: F(2,12) = 32.37, p < 0.0001), and cortical Ag II expression (stress: F(1,12) = 347.7, p < 0.0001; different treatments: F(2,12) = 25.45, p < 0.0001; stress by treatment: F(2,12) = 26.22, p < 0.0001). However, Two ANOVA showed that the stress protocol and the different treatments (saline, perindopril and benazepril) had significant main effect on Hippocampal ATR2 expression (stress: F(1,12) = 611.2, p < 0.0001; different treatments: F(2,12) = 34.30, p < 0.0001; stress by treatment: F(2,12) = 49.38, p < 0.0001), and cortical ATR2 expression (stress: F(1,12) = 190.5, p < 0.0001; different treatments: F(2,12) = 13.48, p = 0.0009; stress by treatment: F(2,12) = 19.37, p = 0.0002).

Our investigation into the brain’s Renin-Angiotensin System (RAS) components revealed that while both perindopril and benazepril effectively suppressed CUMS-induced increases in Ag II and ATR2 receptor levels, benazepril demonstrated a superior ability to reduce these markers in both the hippocampus and cortex.

As demonstrated in Fig. 10, the CUMS group exhibited a significant elevation in Ag II and ATR2 receptor levels compared to the saline control group. Specifically, hippocampal levels increased by 149.47% (Ag II) and 214.80% (ATR2), while cortical levels rose by 178.93% (Ag II) and 172.20% (ATR2).

Fig. 10

The effects of perindopril and benazepril on the AgII and ATR2 receptor brain contents in rats that were exposed to the mild, prolonged, unpredictable stress. A Ag II protein expression level and ATR2 protein expression level for hippocampal samples in different experimental groups compare to β- actin expression level for hippocampal samples, B Ag II protein expression level and ATR2 protein expression level for cortical samples in all groups as compare to expression level of β actin proein expression level for cortical samples. C & E The effect of perindopril and benazepril on the Hippocampal AgII and ATR2 respectively, in the CUMS-exposed rats. D & F The effect of perindopril and benazepril on the cortical Ag II and ATR2, respectively, in the CUMS-exposedrats. The means ± standard deviation (n = 3) are used to display the data. Bonferroni’s multiple comparison test was used after a two-way ANOVA for statistical analysis. At a significance level of p < 0.05, Significant differences from non-stressed saline group (saline control group), Stressed saline group (CUMS group), and Stressed perindopril-treated group (perindopril-treated group)(10 mg/kg) are indicated by labels a, b, and c, respectively. The following are the acronyms: CUMS stands for chronic unpredictable mild stress, Ag II: for Angiotensin II, and ATR2: for Angiotensin type 2 receptor

Conversely, both drug treatments significantly countered these increases. Perindopril administration considerably lowered brain Ag II and ATR2 receptor levels in the hippocampus by 20.25% and 20.30%, respectively, and in the cortex by 14.66% and 17.99%, respectively, when compared to the CUMS group. Benazepril administration also significantly decreased Ag II and ATR2 receptor levels, reducing them in the hippocampus by 46.08% and 40.77%, respectively, and in the cortex by 41.87% and 40.74%, respectively, relative to the CUMS group.

Notably, a key distinction emerged when comparing the two treatments: benazepril administration significantly decreased Ag II and ATR2 receptor levels in the hippocampus by 32.39% and 25.68%, respectively, and in the cortex by 31.88% and 27.74%, respectively, compared to the perindopril-treated groups. This really highlights benazepril’s superior ability to modulate these central RAS components when dealing with the effects of CUMS.