PTSD is a trauma-induced mental illness characterized by the intrusive reliving of past incidents, accompanied by negative thoughts, hyperarousal, avoidance, and terror (Enman et al., 2015; Lee et al., 2020). Concerning this, a distressing, potentially fatal incident can also result in the development of PTSD (Wilson et al., 2014). Symptoms associated with PTSD are dissociation, recurrent nightmares, recalls, intrusive thoughts, increased startle reaction, poor focus, interrupted sleep, and severely negative emotional states (Sherin and Nemeroff, 2022). Globally, the prevalence of PTSD among those who were exposed to trauma has been estimated to be present in 5.6% of the general population (White et al., 2023) while others have approximated it to be 9.2%–13.6% (Atwoli et al., 2015). During the COVID-19 pandemic, the overall pooled estimated incidence rate of PTSD from a total of 24 countries was as high as 17.52% (Yunitri et al., 2022).

PTSD involves structural and neurochemical abnormalities in different brain regions together with increased oxidative stress. The neurochemical abnormalities chiefly involve catecholamine imbalances in the neuroendocrine system (Cohen et al., 2009), and the amygdala, prefrontal cortex, and hippocampus are key brain areas associated with PTSD pathophysiology (Quinones et al., 2020). The frontal cortex is responsible for executive function and fear extinction, while the medial prefrontal cortex controls stress responses and the regulation of emotion via inhibitory control in association with its amygdaloidal connections. The prefrontal cortex also influences striatal brain activity to regulate habitual, orientated, and goal-directed behavior (Falconer et al., 2008; Piggott et al., 2019). Moreover, in models of PTSD, there is evidence of an adverse effect not only on hippocampal volume (Felmingham et al., 2009) but there are also reduced serotonin levels coupled with elevated noradrenaline concentrations reflecting an enhanced noradrenergic response (Wilson et al., 2014).

The striatum regulates goal-directed, motivated, and habitual behaviors that are disrupted in PTSD due to loss of inhibitory control of the pre-frontal cortex over the striatum (Piggott et al., 2019; Sambuco et al., 2021). Striatal monoamines and vitamin C were quantified because their levels are disrupted in the striatum during PTSD. Hence, serotonin modulates dopamine levels in the striatum (Krystal and Neumeister, 2009) which has a defined role in pre-frontal cortical-associated working memory (Landau et al., 2009; D’Ardenne et al., 2012).

In addition, aberrant serotonergic transmission disrupts the equilibrium between the amygdala and hippocampus, which increases angiogenesis (Lee et al., 2020). Others have shown that noradrenaline and dopamine levels are increased, and serotonin is reduced in the hippocampus, prefrontal cortex, and amygdala, which are key structures in the fear response and the regulation of emotions such as anxiety via hypothalamic stimulation (Krystal and Neumeister, 2009; Hoexter et al., 2012; Krishnamurthy et al., 2013). Furthermore, hypothalamic-pituitary-adrenocortical (HPA) dysregulation, oxidative stress, and monoamine (dopamine serotonin, and noradrenaline) imbalances are contributory elements in a PTSD model (Lee et al., 2020). Despite ongoing stress, patients with PTSD have elevated corticotrophin-releasing factor (CRF) and normal to decreased cortisol levels (Katrinli et al., 2022b). In this context, CRF and cortisol control noradrenaline release from the locus coeruleus resulting in an increased sympathetic tone and hallmark symptoms of PTSD, such as a heightened startle response and hyperarousal (DUNN et al., 2004; Seki et al., 2018).

Vitamin C is an aqueous-soluble vitamin with both antioxidant and neuroprotective properties. It has been shown to reduce memory impairment in animal models of PTSD, in addition to influencing catecholamines and corticosterone, these actions are thought to be derived from the prevention of oxidative stress in the brain (Alzoubi et al., 2020).

Adenosine is a neuromodulator and the adenosinergic system can be neuroprotective as well as neurodegenerative depending on how it is differentially manipulated, for example, by A₁ receptor activation, A_2A receptor antagonism or inhibition of adenosine kinase (Gomes et al., 2011). What is more, adenosine levels markedly increase during brain damage and this occurs most probably because of increased ATP consumption to maintain cell viability (Latini and Pedata, 2001; Gomes et al., 2011). In conjunction with this, adenosine and its metabolites have an established role in mood regulation and cognitive impairment (Elmenhorst et al., 2018; van Calker et al., 2019).

Both psychotherapy and pharmacotherapy are employed in the management and treatment of PTSD. Regarding pharmacotherapy, the FDA-approved drugs for PTSD as a first-line therapy, are the serotonin selective reuptake inhibitors (SSRIs). An α₁-adrenoceptor blocker, prazocin, has also been used off-label for trauma nightmares (Miller, 2008). However, a high relapse rate (>60 %) is associated with these drug therapies (Wahbeh et al., 2016; Akiki and Abdallah, 2019) and they tend to be used with caution, especially in older patients with lower tolerability (Draper and Berman, 2008). There is, therefore, a need for the development of novel agents with improved efficacy and limited toxicity for the management and treatment of PTSD.

Diosgenin is a hydrolysate of dioscin and can be extracted from Dioscorea plant tubers. Dioscin is a natural saponin, while diosgenin is a saponin derivative (Li et al., 2021). These two compounds are extensively present in Liliaceae, Dioscoreaceae, Leguminosae, Solanaceae, and Agavaceae plant families (Avula et al., 2014; Yuan et al., 2019; Semwal et al., 2022) with proven anti-atherosclerotic (Wang and Wang, 2022) and anti-asthmatic activities (Junchao et al., 2017). Both Diosgenin and Dioscin reduce oxidative stress by inhibiting ROS production and increasing superoxide dismutase expression (Qin et al., 2013). Dioscin also reduces RAGE and NOX4 expression yielding an anti-Alzheimer’s disease effect (Guan et al., 2022). Additionally, it has antidepressant-like activity and is neuroprotective, reducing inflammation by decreasing IL-6, IL-1β, and TNF-α levels (Yang et al., 2017; Qi et al., 2019).

The neuroprotective profile of diosgenin confers effectiveness against diabetes-induced neuropathy (Leng et al., 2020) and cerebral ischemic brain injury (Oyelaja-Akinsipo et al., 2020), whilst also possessing antioxidant and anti-inflammatory capacities (Yang et al., 2020) plus an ability to improve cognitive deficits in senescent mice (Chiu et al., 2011; Semwal et al., 2022). More recently, diosgenin has been shown to have a pharmacological propensity against chronic restraint stress-induced depression (Cui et al., 2023). Despite its clear neuroprotective and anti-inflammatory properties, the pharmacological potential of diosgenin has not been explored in a murine model of PTSD and this was the primary aim of this study using SPS in combination with short-term memory and anxiety/depression-like models.

We have evaluated the effect of treatment with diosgenin in the SPS rodent model of PTSD. Male mice were used in our experiments in congruence with other studies (Valdivieso et al., 2018; Lee et al., 2020), because they are more vulnerable than females, who have higher basal corticosterone levels, and male mice also react to stress less adaptably than females (Cohen and Yehuda, 2011). This particular model was employed because it produces stress-like responses arising from different source mechanisms, such as restraint for psychological stress, forced swimming for physiological stress, and ether to induce pharmacological stress (Lisieski et al., 2018). The protocol is intended to simulate the major corticosterone surge caused by the experience of a traumatic event (Armario et al., 2008). SPS is one of the most frequently used paradigms for evoking PTSD-related symptoms in rodents, including depression, anxiety, and impaired cognition (Souza et al., 2017).

In recent studies, mice exposed to SPS have been shown to display a higher anxiety index in the EPM paradigm (Figure 2), as well as decreased locomotor activity, incorrect transitions in grooming bouts, and increased rearing in the OFT (Figure 3), reflecting anxious behavior, increased immobility time in FST suggests that the animals are more fearful together with learned helplessness after experiencing trauma (Figure 4) and our findings are in accord with previous studies (Lee et al., 2018b; Lin et al., 2022; Sur and Lee, 2022), mirroring the anxiety and behavioral despair in PTSD patients. Diosgenin reduced the anxiety index (Figure 2E), the number of open arm entries (Figure 2A), diminished the percentage closed arm occupancy time at the highest dose (Figure 2D). These results are analogous to those obtained with the positive control, fluoxetine, where there was a correlation with lowered serotonin levels in the frontal cortex, hippocampus, and striatum, together with an increased anxiety index (Lee et al., 2020). Reduced serotonin levels in response to stress may be accompanied by aggression and mood changes, such as regret, depression, and anxiety since serotonin is centrally engaged in emotional and behavioral regulation, which influences aggressive behavior (Lee et al., 2020; Fluyau et al., 2022). In our study, diosgenin restored the otherwise SPS-reduced levels of serotonin in the hippocampus (Table 2), with very little effect on striatal serotonin (Table 3), conceivably signifying an anxiolytic-like effect also seen with fluoxetine. It is well documented that in rodent models fluoxetine is used as a positive control in the murine model of PTSD as it ameliorates the anxiety and depression-like symptoms associated with PTSD (Lee et al., 2018a; Oh et al., 2018; Lee et al., 2020). Moreover, fluoxetine after acute treatment has been reported to ameliorate the anxiety and depression-like behavior in PTSD (Oh et al., 2018) and chronic unpredictable stress (Ahmed et al., 2023). Diosgenin, like fluoxetine, not only increased locomotor activity (Figure 3A) but also reduced the incidence of rearing (Figure 3B) and incorrect transitions in grooming (Figure 3C). Excessive grooming or incorrect transitions in grooming patterns and rearing behaviors reflect anxiogenesis (Kruk et al., 1998; Kalueff et al., 2004; Nagarajan and Capecchi, 2023). These are typical rodent responses to stressful situations involving dysregulation of the HPA axis (Moody et al., 1988; Kinlein et al., 2019) resulting in high corticosterone levels, which ultimately give rise to abnormal stress processing (Patki et al., 2014). We observed that the serum corticosterone level was raised following SPS exposure, which was then reversed by diosgenin (Figure 7), and the degree of restoration even surpassed that of fluoxetine as the standard positive control. In humans, decreased peripheral cortisol levels are associated with PTSD, but these changes do not appear in the acute stage ensuing traumatic stress (Shalev et al., 2008; Videlock et al., 2008). In our study, plasma corticosterone levels were assessed during the initial stages after stress exposure, so there is no discord between our findings and those reporting that PTSD patients have lower cortisol levels (Mongeau et al., 1997; Morgese and Trabace, 2019). Diosgenin has reported antidepressant-like effect by decreasing serum pro-inflammatory cytokines and reducing the HPA axis activity (Cui et al., 2023). We have shown that diosgenin and fluoxetine both attenuated SPS elevated serum corticosterone to reverse HPA axis dysregulation (Figure 7). In addition, increased CRF or cortisol levels in response to stress or aversive stimuli results in noradrenaline release from locus coeruleus (LC) neurons (Yehuda et al., 1991; DUNN et al., 2004) leading to increased sympathetic tone and hallmark symptoms of PTSD, such as an increased startle response and hyperarousal (DUNN et al., 2004; Seki et al., 2018), and increased release of dopamine in the striatum, In addition to HPA axis dysregulation, a close relationship exists between monoamine dysfunction and PTSD-associated depression. Most antidepressants modulate the levels of both noradrenaline and serotonin in a time-dependent manner (Porsolt et al., 1977; Geracioti Jr et al., 2001) and these monoamines regulate each other through heteroreceptors. Negative feedback has been proposed because elevated serotonin levels cause noradrenaline production, which in turn blocks further serotonin release by activating α2A receptors (Mongeau et al., 1997). Hence, serotonergic receptors at noradrenergic terminals and α2A receptors (inhibitory in nature) on serotonergic terminals mediate this mechanism (Mongeau et al., 1997; Morgese and Trabace, 2019). Restoration of the HPA axis and monoamines in these brain regions resulted in the amelioration of SPS-induced anxiety and depression-like symptoms. Moreover, endogenous vitamin C levels were disrupted by SPS in the frontal cortex (Table 1), hippocampus (Table 2), and striatum (Table 3), and they were restored by diosgenin in these brain areas. Vitamin C affects signal transduction in the brain via modulation of neurotransmitter release and receptor binding (Hansen et al., 2014). During the synthesis of monoamines, Vitamin C serves as a co-factor (Rebec and Pierce, 1994; Kocot et al., 2017) and regulates dopamine and noradrenaline levels (May et al., 2012; Moritz et al., 2020). Vitamin C supplementation is associated with the protection of serotonin from oxidation, resulting in its increased uptake, receptor binding and turnover, with a likelihood of ameliorating anxiety/depression-like symptoms (Ballaz and Rebec, 2019). Reduced vitamin C levels in the brain are associated with a reduction in brain serotonin levels (Ward et al., 2013). SPS disrupts vitamin C levels in different brain regions, subsequently altering monoamine concentrations in the brain.

During neuropathological conditions, central adenosine levels are abruptly increased, exacerbating the ongoing pathology (van Calker et al., 2019). Adenosine is an additional neuromodulator involved in the pathophysiology of depression. It also plays a critical role in other biological processes, including the regulation of mood and behavior, by acting on various receptors such as the A₁, A_2A, A_2B, and A_3A subtypes (Pasquini et al., 2022). It is reported that adenosine and its analogs produced depressant-like behavioral effects in animal models of depression (Woodson et al., 1998; Hunter et al., 2003; Cunha et al., 2008). Thus, elevated adenosine levels extended the immobilization time in rats subjected to inescapable shocks as well as in the FST. Adenosine is converted to inosine in the presence of adenosine deaminase, which is further converted to hypoxanthine in the presence of purine nucleoside phosphorylase, then to xanthine, and finally to uric acid in the presence of xanthine oxidase. Inosine is formed inside the cell by adenosine deamination when the level of intracellular adenosine is high (Haskó et al., 2004). Activation of A₃ receptors by inosine has an additional anti-inflammatory effect (Haskó et al., 2004) and inosine also has neuroprotective activity (Saad et al., 2022). However, when inosine and hypoxanthine levels are abnormally high, they are ultimately metabolized to uric acid, which is responsible for the generation of hydrogen peroxide and ROS leading to neurodegeneration (Frenguelli and Dale, 2020). We observed a comparable outcome since the adenosine level was increased by SPS and subsequently reversed by diosgenin in the frontal cortex (Table 4) and striatum (Table 6). Additionally, inosine levels in the frontal cortex (Table 4) and hippocampus (Table 5) were boosted by SPS and then attenuated by diosgenin and fluoxetine concurring with the immobility time findings in the FST (Figure 3). However, no significant change was observed in hypoxanthine levels, which may be ascribed to the rapid conversion of hypoxanthine to xanthine in the presence of xanthine oxidase (Frenguelli and Dale, 2020).

Furthermore, Adenosine A_2A receptor activation modulates memory and learning through the presynaptic release of glutamate, and these adenosinergic receptor sub-types are also thought to play a role in memory and learning by facilitating synaptic plasticity (Chen, 2014). Increased adenosine and inosine levels in the frontal cortex following SPS exposure were then reduced by diosgenin, and this may well play a part in improving memory in the NOR task.

PTSD causes cognitive impairment, coupled with reduced recognition (Keller et al., 2015) and an impairment of spatial memory (Schoenfeld et al., 2019). Recognition memory is the capacity to identify something familiar when previously observed (Baxter, 2010) and it is used to evaluate rodent object recognition memory in NOR which if impaired, may reflect amnesia in humans (Baxter, 2010). NOR memory was reduced in the SPS-exposed animal group in our study, and this concurs with an earlier finding (Eagle et al., 2013). In this regard, it has been shown that damage to the frontal cortex and hippocampus, which are crucial to object recognition and spatial memory, can cause anterograde amnesia. Hence, diosgenin and donepezil both increased the DI (Figure 5A) and RI (Figure 5B) values degraded by SPS. Moreover, the reduction in memory impairment by diosgenin was comparable to that of donepezil, as the positive control. Donepezil is an FDA-approved drug clinically used to address cognitive problems. It is reported to augment fear extinction in PTSD and enhance memory, and has a neuroprotective role (Dong et al., 2009; Lim et al., 2016; Reid et al., 2018; Gomaa et al., 2021; Yanpallewar et al., 2022). In this respect, donepezil serves as a positive control in murine models of cognitive impairment (Arif et al., 2022).

It is well known that dopaminergic function is vital for the functioning of working memory. Thus, the dopamine concentration in the amygdala increases and activation of dopamine receptors is crucial for the storage of memories associated with fear. Dopamine concentrations in the striatum and prefrontal cortex mediate cognition and executive functioning, including attentiveness and the inhibitory response (Ding et al., 2023). In this context, overstimulation of the dopamine system in animal models is linked to both the strengthening of fear conditioning and delayed extinction of fear (Morrow et al., 1996). Moreover, high dopamine levels are associated with intensified fear conditioning responses to cues that resemble “trauma,” compromising the capacity to ignore unimportant stimuli and assign proper salience (Hoexter et al., 2012). Furthermore, neuronal serotonin transmission in the hippocampus plays a pivotal role in memory processing via long-term potentiation (LTP) and depression (LTD.) most likely involving 5-HT7 receptors (Sarkisyan and Hedlund, 2009). Diosgenin may exert its effect on NOR by modulating serotonin, particularly since it increased its concentration in the hippocampus and striatum in our study. Likewise, noradrenaline levels were modified in the frontal cortex, hippocampus, and striatum. In this regard, noradrenaline has an established role in memory formation, consolidation, and recovery (Hamidkhaniha et al., 2019). Additionally, dopamine levels were also increased in the frontal cortex, hippocampus, and striatum, which is in accord with previous studies; (Baxter, 2010; Wilson et al., 2014). In addition, donepezil decreased SPS disrupted dopamine levels in all three brain regions (Tables 1–3) suggesting that restoration of monoamines in distinct brain regions may be involved in the amelioration of SPS-induced cognitive impairment in the NOR test.

The Y-maze protocol is used for the assessment of spatial working memory by evaluating spontaneous alternations that reflect the functional interplay between the frontal cortex and hippocampus. We found a reduction in spontaneous alternations following the SPS protocol, which reflects negative cognition, and corroborates the findings of others (Fulco et al., 2022). Donepezil and diosgenin both increased the SPS reduction in spontaneous alternations (Figure 6A), improving the short-term working memory affected by SPS.

A convincing association between the pathophysiology of PTSD and oxidative stress has been reported in animal studies (Wilson et al., 2013; Miller et al., 2018; Li et al., 2020; De Munter et al., 2021) and human trials (Katrinli et al., 2022). The disrupted HPA axis in PTSD may also be involved in oxidative stress because high levels of glucocorticoids in response to excitotoxicity may result in an increased production of proinflammatory cytokines. Increased oxidative stress is responsible for neuronal damage in the frontal cortex and hippocampus (MacPherson et al., 2005), which is likely to impair learning. Vitamin C has an antioxidant potential that helps to oppose neurodegeneration in several ailments by conserving the antioxidant mechanism in the brain, imparting neuroprotection (Dwita et al., 2023), and ameliorating memory by restoring antioxidant mechanisms in the hippocampus. Through oxidation, vitamin C is hypothesized to shield cellular DNA, lipids, and proteins from oxidative damage caused by ROS and this can improve cognition (Alqudah et al., 2018). Additionally, conserving vitamin C levels have been associated with improved symptoms of PTSD ( Cuthrell, 2022 ). In light of this, an added issue concerning the study of vitamin C in our study is that it is a co-factor in the metabolism of monoamines (Hansen et al., 2014), so it has an interrelationship and impact on the range of neurotransmitters that have been measured.

Our observation that vitamin C levels were diminished in all three brain regions after SPS, probably contributed to memory impairment due to perturbed antioxidant mechanisms. This can be correlated with preclinical findings suggesting that deficient endogenous vitamin C levels result in oxidative stress and neurodegeneration (Ballaz and Rebec, 2019). Subsequently, both donepezil and diosgenin raised the otherwise SPS-reduced levels of vitamin C in the frontal cortex (Table 1) and striatum (Table 3) conceivably participating in improved memory in the NOR and Y maze tasks.

PTSD is known to be a comorbidity with depression, anxiety, and alcohol abuse, so its underlying mechanisms of action are complex and diverse (Cai et al., 2022). The disorder involves disruption of the HPA axis, inflammation, as well as modified neurotransmission, and neurotropic function (Aliev et al., 2020; Cai et al., 2022). This, diosgenin has neuroprotective and antioxidant activity, modulates neurotransmitters, is anti-apoptotic and anti-inflammatory, attenuates Ca²⁺ influx modulating neurotrophic factors, inhibits tau phosphorylation, and can regenerate neural networks (Sun et al., 2015). Some of these activities may well stem from the repression of TNF-α, IL-1β, and IL-6 levels and the NF-κB pathway (Zhang et al., 2017; Man et al., 2023) in addition to the aforementioned actions. Because of these diverse properties, diosgenin can generate mnemonic/antidepressant/anxiolytic outcomes in our behavioral paradigms following SPS exposure.

Accordingly, it may be postulated that it has conferred neuroprotection during PTSD induced by single prolonged stress through one or more of its recognized mechanisms. It might be proposed consequently, that diosgenin would be useful not only in the treatment of PTSD but also in the management of PTSD-related ailments.

In summary, our findings suggest that diosgenin has anxiolytic- and antidepressant-like effects in addition to enhancing memory in the NOR and Y-maze tasks after the SPS protocol. Furthermore, amelioration of these post-SPS behavioral parameters may be attributed to the normalization of monoamines and vitamin C in the frontal cortex, hippocampus, and striatum, as well as the reinstatement of plasma corticosterone levels and other actions. Such outcomes suggest that diosgenin may be a potential candidate for improving symptoms of PTSD.