Protective effects of phosphodiesterase-5 inhibitor (Tadalafil) in stress-induced anxiety, depression and gastric ulceration in rats

INTRODUCTION

Stress is a ubiquitous psychophysiological response that, when chronic, precipitates a spectrum of mental and physical disorders.^[1] Epidemiological data reveal that anxiety and depression affect approximately 27–28% of the global population, with even higher rates in socio-economically stressed cohorts.^[2,3] Concurrently, chronic psychological stress disrupts gastrointestinal homeostasis, predisposing to gastric ulceration through neuro-endocrine activation and mucosal oxidative injury.^[4] Current pharmacotherapies – selective serotonin reuptake inhibitors, benzodiazepines and proton-pump inhibitors – address individual facets of these comorbidities but seldom their shared pathophysiology.^[5]

The hypothalamic-pituitary-adrenal (HPA) axis orchestrates the mammalian stress response through corticotropin-releasing hormone and glucocorticoid release.^[6] Sustained activation engenders hippocampal neuronal loss, monoaminergic imbalance, microglial activation and systemic inflammation marked by elevated tumour necrosis factor-α (TNF-α) and interleukin-1β (IL-1β).^[7,8] These cytokines not only impair neuroplasticity but also compromise gastric mucosal defences by enhancing oxidative stress and attenuating mucus–bicarbonate secretion.^[1,9]

Phosphodiesterase-5 (PDE5) inhibitors – originally developed for erectile dysfunction – potently increase intracellular cyclic guanosine monophosphate (cGMP) by preventing its hydrolysis.^[10] Downstream activation of protein kinase G (PKG) enhances endothelial nitric-oxide production, vasodilation, anti-inflammatory signalling and neurotrophic gene expression.^[11] Pre-clinical studies demonstrate that PDE5 inhibition attenuates anxiety- and depression-like behaviours in rodents, an effect linked to hippocampal brain-derived neurotrophic factor (BDNF) up-regulation.^[12] In addition, cGMP-dependent vasodilation improves splanchnic perfusion, while suppression of TNF-α and oxidative stress confers gastroprotection against indomethacin- or ethanol-induced ulcers.^[13]

Tadalafil, distinguished by a 17.5-h half-life and high PDE5 selectivity, crosses the blood–brain barrier and exerts sustained tissue cGMP elevation.^[14,15] Yet, its integrated efficacy against stress-induced anxiety, depression-like states and gastric ulceration remains unexplored. We therefore investigated whether tadalafil could simultaneously mitigate behavioural dysfunction and mucosal injury in a validated rat model of chronic restraint stress (RS), benchmarking its effects against diazepam, imipramine and ranitidine.

TNF-α and IL-1β were chosen because these cytokines are central mediators of neuroinflammation and stress-induced behavioural and gastric changes, showing consistent correlation with HPA axis activation and neuroimmune signalling. C-reactive protein (CRP), while a systemic marker, is less sensitive in rodents and myeloperoxidase activity primarily reflects local neutrophil infiltration rather than central inflammatory processes relevant to stress physiology.

MATERIALS AND METHODS

RESULTS

Behavioural outcomes

EPM

RS markedly reduced open-arm exploration versus controls (OE: 2.67 ± 0.52 vs. 6.50 ± 0.84, P < 0.001; OT: 45.33 ± 6.41 s vs. 56.67 ± 3.72 s, P < 0.05), elevating anxiety index to 0.90 ± 0.02 (vs. 0.80 ± 0.01, P < 0.001). Diazepam restored OE and OT to control levels (anxiety index 0.72 ± 0.02, P < 0.001 vs. RS). Tadalafil produced a dose-dependent reversal: 2 mg/kg modestly increased OE (4.17 ± 0.75, P > 0.05), whereas 10 mg/kg achieved anxiolytic equivalence with diazepam (OE 6.67 ± 0.52; anxiety index 0.74 ± 0.02, P < 0.001 vs. RS). Linear regression confirmed a significant inverse dose–response for anxiety index (β = −0.0067, R2 = 0.576, P = 0.0003) [Table 1 and Figure 1].

Table 1: Effect of Tadalafil on Anxiety-Like Behaviour in the Elevated Plus Maze (EPM).

Group	Open Arm Entries	Closed Arm Entries	Time in Open Arms (s)	Time in Closed Arms (s)	Anxiety Index
Control	6.50±0.84	21.00±0.89	56.67±3.72	222.67±4.37	0.80±0.01
RS	2.67±0.52***	30.83±1.72***	45.33±6.41*	244.83±13.60*	0.90±0.02***
RS+Diazepam	7.17±0.41***	15.00±1.26***	63.67±3.14***	166.67±9.29***	0.72±0.02***
RS+Tadalafil (2 mg/kg)	4.17±0.75	21.83±1.47	55.00±5.37	206.00±3.90	0.79±0.02
RS+Tadalafil (5 mg/kg)	5.83±0.75*	19.50±1.64*	50.17±3.76	176.83±5.56*	0.78±0.01*
RS+Tadalafil (10 mg/kg)	6.67±0.52***	16.67±1.63***	59.00±5.48***	169.17±15.41***	0.74±0.02***

RS vs Control: significant increase in anxiety (***p<0.001). Diazepam vs RS: highly significant anxiety reduction (***p<0.001). Tadalafil 2 mg/kg: not significant vs RS. Tadalafil 5 mg/kg: mild reduction (*p<0.05 vs RS). Tadalafil 10 mg/kg: strong reduction (***p<0.001 vs RS). Values are expressed as mean±standard deviation (SD), n=6 per group. EPM: Elevated Plus Maze, RS: Restraint Stress, Time in arms is measured in seconds. Anxiety Index was calculated as 1 − ([Time in Open Arms/Total Time] + [Number of Entries in Open Arms/Total Entries])/2. Comparisons were made using one-way ANOVA followed by Tukey’s post hoc test

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Figure 1:

Effect of tadalafil on anxiety-like behaviour in the Elevated Plus Maze (Epm).

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OFT

RS suppressed locomotor activity (crossings 16.0 ± 5.9 vs. 43.5 ± 4.4, P < 0.001), rearings (6.17 ± 2.0 vs. 14.3 ± 1.4, P < 0.001) and increased latency to move (57.7 ± 6.7 s vs. 37.3 ± 2.7 s, P < 0.001). Tadalafil 10 mg/kg restored crossings (46.5 ± 3.9) and rearings (18.2 ± 3.2) to control levels while reducing latency to 13.8 ± 2.9 s (P < 0.001 vs. RS). Faecal pellet output, an autonomic stress indicator, fell from 5.17 ± 0.98 (RS) to 1.67 ± 1.03 (tadalafil 10 mg/kg, P < 0.05) [Table 2 and Figure 2].

Table 2: Effect of Tadalafil on Locomotor Behaviour in the Open-Field Test (OFT).

Group	Crossings	Rearing	Latency (s)	Fecal Pellets	Locomotor Activity
Control	43.5±4.37	14.33±1.37	37.33±2.73	2.0±1.55	57.83±3.87
RS	16.0±5.93***	6.17±2.04***	57.67±6.68***	5.17±0.98***	22.17±6.18***
RS+Imipramine	30.67±5.57**	14.33±2.66**	43.67±3.20**	2.17±1.72**	45.0±7.29**
RS+Tadalafil (2 mg)	36.0±4.98*	12.67±5.09	27.17±5.12**	3.17±1.33	48.67±5.28
RS+Tadalafil (5 mg)	40.83±4.71**	14.33±1.21**	23.67±4.84**	2.67±1.21*	55.17±4.62**
RS+Tadalafil (10 mg)	46.5±3.89***	18.17±3.19***	13.83±2.93***	1.67±1.03***	64.67±3.39***

RS vs Control: reduced activity and increased stress markers (***p<0.001). Imipramine vs RS: significant improvement (**p<0.01). Tadalafil 2 mg/kg: mild improvement (*p<0.05 crossings; **p<0.01 latency). Tadalafil 5 mg/kg: clear improvement (**p<0.01 vs RS). . Tadalafil 10 mg/kg: robust effect (***p<0.001 vs RS). All values are expressed as percentages of the maximum observed mean value for each respective parameter across all experimental groups. Specifically, for each group, the percentage was calculated using the formula:% = (Group Mean/Maximum Mean Observed Among All Groups) × 100. For example, the group’s mean latency to move was divided by the highest latency mean recorded among all groups, then multiplied by 100 to obtain “% Latency to Move.” This normalisation facilitates direct comparison of behavioural parameters such as latency to move, number of crossings, number of rearing, and faecal pellet count across groups. Lower latency and faecal count indicate reduced anxiety/depression, while higher crossings and rearings indicate improved locomotor and exploratory behaviour

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Figure 2:

Effect of tadalafil on locomotor behaviour in the Open-Field Test (OFT).

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Gastric ulceration

RS elevated UI to 7.24 ± 0.38 mm² from 2.09 ± 0.25 mm² in controls (P < 0.0001). Ranitidine decreased UI to 1.80 ± 0.21 mm² (P < 0.0001 vs. RS). Tadalafil produced a graded reduction: 2 mg/kg (2.90 ± 0.31 mm²), 5 mg/kg (2.48 ± 0.28 mm²) and 10 mg/kg (2.32 ± 0.19 mm²), the latter being statistically indistinguishable from ranitidine. Linear regression demonstrated a significant inverse relationship between tadalafil dose and UI (β = −0.0733, R² = 0.408, P = 0.0043) [Table 3 and Figure 4].

Table 3: Gastric Ulcer Index and Inflammatory Cytokine Levels.

Group	Ulcer Index (mm2)	TNF-α (pg/mg protein)	IL-1β (pg/mg protein)	Corticosterone (ng/mL)
Control	2.09±0.25	7.68±2.35	4.69±0.76	11.7±5.04
RS	7.24±0.38***	15.27±1.66***	16.09±2.71***	16.61±1.09***
RS+Ranitidine 150 mg/kg	1.80±0.21***	11.20±1.89	14.94±2.71	15.17±5.02
RS+Tadalafil (2 mg/kg)	2.90±0.31*	15.01±2.91	13.67±2.64	14.49±6.03
RS+Tadalafil (5 mg/kg)	2.48±0.28**	14.79±4.49*	11.88±2.70*	10.83±3.82**
RS+Tadalafil (10 mg/kg)	2.32±0.19***	13.40±4.20***	10.68±3.57***	11.88±1.76**

RS vs Control: significant increase in ulcer index, TNF-α, IL-1β, and corticosterone (***p<0.001).Ranitidine vs RS: strong protection against ulcers (***p<0.001). Tadalafil 2 mg/kg vs RS: reduced ulcer index (*p<0.05), no significant cytokinechange. Tadalafil 5 mg/kg vs RS: reduced ulcer index (**p<0.01), decreased TNF-α, IL-1β (*p<0.05), and corticosterone (**p<0.01). Tadalafil 10 mg/kg vs RS: marked reduction in ulcer index (***p<0.001), lowered TNF-α, IL-1βand corticosterone (***p<0.001). Values represent the mean Gastric Ulcer Index observed in each group following restraint stress. The index was calculated using the following formula:. Gastric Ulcer Index = (Number of ulcers×Severity score)/Total number of animals. The severity score was graded based on macroscopic observation of lesions on a scale of 0–3 (0=no lesion, 1=superficial mucosal erosion, 2=deep ulceration, 3=perforation). Ranitidine served as the standard gastroprotective agent. Tadalafil was administered at doses of 2 mg/kg, 5 mg/kg, and 10 mg/kg intraperitoneally, 30 minutes before stress exposure.Values are expressed as mean±SD, n=6 per group. TNF-αand IL-1βlevels were measured in brain tissue homogenates, and corticosterone in serum. RS: Restraint stress

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Figure 3:

Effect of tadalafil on inflammatory cytokine levels.

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Figure 4:

Effect of tadalafil on Gastric Ulcer Index.

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Inflammatory and endocrine markers

RS raised brain TNF-α to 15.27 ± 1.66 pg/mg protein from 7.68 ± 2.35 pg/mg (P < 0.001) and IL-1β to 16.09 ± 2.71 pg/mg from 4.69 ± 0.76 pg/mg (P < 0.001). Diazepam and imipramine each attenuated these cytokines (P < 0.01).

Tadalafil trended toward reductions (10 mg/kg: TNF-α 13.4 ± 4.2 pg/mg; IL-1β 10.7 ± 3.6 pg/mg), but regression slopes did not reach statistical significance (P = 0.449 and 0.109, respectively). Serum corticosterone levels were numerically elevated after RS (16.61 ± 1.09 ng/mL) but did not differ significantly among groups (F = 1.03, P = 0.427) [Table 3 and Figure 3].

DISCUSSION

The present study demonstrates, for the first time in an integrated model, that the long-acting PDE5 inhibitor tadalafil simultaneously mitigates RS-induced anxiety- and depression-like behaviours and attenuates gastric ulceration in a dose-dependent manner. Using validated behavioural paradigms, biochemical indices and macroscopic gastric assessment, we show that a 10 mg/kg dose of tadalafil achieves anxiolytic and gastroprotective efficacy comparable to standard reference drugs, while exerting modest anti-inflammatory effects.

Chronic RS reliably produced an anxiogenic phenotype— reduced open-arm exploration and increased anxiety index in the EPM, alongside diminished locomotion and exploratory drive in the OFT, mirroring findings in both rodent and human neuroimaging studies.^[16,20] Tadalafil dose-dependently reversed these deficits, with the 10 mg/kg dose restoring behavioural indices to levels indistinguishable from diazepam [Table 1]. These effects align with previous reports where PDE5 inhibition enhanced hippocampal cGMP and activated the PKG–CREB–BDNF cascade, thereby promoting synaptic plasticity and mood resilience.^[21,22] The absence of sedation (as evidenced by preserved or enhanced total crossings) supports a non-GABAergic mechanism, potentially circumventing the dependence liability associated with benzodiazepines.^[23]

RS increased gastric UI seven-fold, consistent with mucosal hypoperfusion, oxidative damage and inflammatory cytokine release.^[24] Tadalafil (10 mg/kg) reduced UI to values statistically equivalent to ranitidine [Table 3]. This protection is likely multifactorial: PDE5 inhibition elevates cGMP, enhancing nitric-oxide-mediated vasodilation in the gastric microvasculature,^[25] while concomitant suppression of TNF-α and IL-1β attenuates neutrophil infiltration and lipid peroxidation.^[19] Importantly, tadalafil conferred protection without acid suppression, distinguishing its mechanism from H2-receptor antagonists and potentially offering a safer long-term profile.^[9]

Although RS robustly elevated brain TNF-α and IL-1β, cytokine reductions with tadalafil did not achieve statistical significance across all doses, possibly reflecting the short treatment window or partial engagement of anti-inflammatory pathways. Serum corticosterone did not differ among groups, a finding consistent with adaptive HPA-axis desensitisation following repeated restraint^[16] and highlighting the need for time-course analyses in future studies.

The study strengths include (i) a validated chronic stress model with face, construct and predictive validity;^[16,26] (ii) parallel benchmarking against standard anxiolytic, antidepressant and anti-ulcer drugs; (iii) demonstration of clear dose–response relationships for both behavioural and gastric endpoints (R² = 0.576 and 0.408, respectively) and (iv) use of an FDA-approved drug with well-characterised pharmacokinetics. Collectively, the data support repurposing tadalafil as a multi-modal agent for stress-related neuro-gastrointestinal co-morbidities, addressing an unmet clinical need for integrated therapeutics.^[27,28]

Mechanistic pathways (cGMP–PKG–CREB, BDNF expression) were inferred rather than directly measured. Monoaminergic neurotransmitter quantification and additional depression-specific paradigms (e.g. forced swim or sucrose preference tests) would strengthen the antidepressant claim. Finally, chronic dosing studies and cross-species validation are warranted to confirm long-term safety and translational relevance.

Subsequent work should employ molecular assays (Western blotting, quantitative polymerase chain reaction) to confirm cGMP/PKG/BDNF signalling, extend the observation period to model chronic stress and progress to early-phase human trials in populations with comorbid anxiety and functional gastrointestinal disorders.

CONCLUSION

In a rat model of chronic RS, the PDE5 inhibitor tadalafil – most notably at 10 mg/kg – produced strong, dose-dependent anxiolytic and antidepressant-like effects, along with protection of the gastric mucosa from ulceration. These effects were comparable to those of standard drugs (diazepam, imipramine and ranitidine), respectively and occurred without signs of sedation. The findings suggest tadalafil as a potential therapeutic option for managing stress-related neuropsychiatric and gastrointestinal co-occurring disorders. Further mechanistic and translational studies are needed to validate these results and explore its clinical relevance in patients with multiple chronic conditions.