Dietary Phytoestrogen ameliorates Ovarian Toxicant–induced Neurotoxicity: Mechanistic and metabolic insights

Dietary Phytoestrogen ameliorates Ovarian Toxicant–induced Neurotoxicity: Mechanistic and metabolic insights | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Dietary Phytoestrogen ameliorates Ovarian Toxicant–induced Neurotoxicity: Mechanistic and metabolic insights Ambika Shandilya, Suhas Hajare, Dube Aakash Arwind, Chander Kant Giri, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9291892/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Most women undergoing accelerated ovarian failure experience cognitive-affective dysfunction, bioenergetic failure, amyloidogenic susceptibility, and persistent neuroinflammation, emerging chiefly from impaired estrogenic regulation. Despite the fact that ERT improves women's lives, it is not widely utilised due to the risk of thrombosis, cardiac issues, and endometrial cancer. The phytoestrogen diet (PED), renowned for its ERβ-centered neurorestorative potential, has shown considerable promise; however, the detailed mode of action remains largely underexplored. This study aims to elucidate the translational molecular mechanisms by which dietary phytoestrogen elicits neuroprotective benefits against ovatoxin-induced expedited cognitive ageing and neurodegenerative pathologies. We explored the therapeutic effects of PED using neurobehavioural paradigms, mitochondrial functional assessments, and estimates of neuronal atrophy and neuroinflammatory signalling via cell-specific dual immunofluorescence analyses. Additionally, we evaluated PED's efficacy in restoring the brain metabolite profile, identifying neurodegenerative signatures, and mitigating chronic neuroimmune transition. Results revealed that accelerated ovarian insufficiency exacerbated memory alterations and emotional instability, coinciding with decreased ER-β and BDNF expression, enhanced beta-amyloid deposition, and microgliosis-driven neuroimmune dysregulation. High-VIP metabolites reflected disruptions in steroid, sphingolipid, and fatty acid pathways, indicating that ovarian failure drives estrogen-dependent metabolic reprogramming. Our study demonstrated that PED rich in estrogen-mimicking isoflavones ameliorated mood and memory deficits by modulating beta-amyloid, reinstating mitochondrial integrity, rescuing the brain metabolome, and restoring neurotrophic signalling through ER-β activation in the mPFC. Therefore, PED is a promising candidate for treating neurocognitive decrements by potentiating ER-β activity, preserving mitochondrial integrity, and modulating the inflammopharmacological axis. Accelerated ovarian failure Brain Metabolomics Cognitive impairment Mitochondrial dynamics Neuroinflammation Phytoestrogen therapy Introduction The cessation of ovarian follicular function marks a critical inflexion point in female biological ageing, exerting profound effects on systemic physiology, neuroinflammation and overall brain health. A growing body of evidence suggests that premature or accelerated ovarian failure causes early cognitive decline, mood issues, memory impairment, and increased susceptibility to neurodegenerative diseases such as Alzheimer's disease, dementia, and cognitive rigidity (Voskuhl, Itoh et al. 2024, Mohammad, Finch et al. 2025, Yuan, Gong et al. 2025). The abrupt estrogen depletion accompanying premature ovarian failure disrupts neuroendocrine balance, amplifies neuronal cell senescence, neuroinflammatory signalling, and compromises mitochondrial integrity (Russell, Jones et al. 2019, Platholi, Marongiu et al. 2023, Wang, Mao et al. 2025), processes central to accelerated brain ageing and neurodegeneration. However, the precise molecular mechanisms linking ovarian insufficiency to accelerated neurodegeneration are still poorly understood. AOF creates an abrupt endocrine environment that may exacerbate age-related brain vulnerabilities. This differs from the gradual hormonal changes that occur during natural or physiological menopause. Chronic estrogen deprivation accelerates synaptic loss, reduces neuronal resilience, and triggers glial-mediated persistent neuroinflammation (Platholi, Marongiu et al. 2023, Voskuhl, Itoh et al. 2024, Marongiu, Platholi et al. 2025), all of which are hallmarks of neurodegenerative illness. Understanding these pathways is critical for determining female-specific patterns of premature brain ageing and identifying possibilities for therapeutic treatment to minimise neurodegenerative risk following early ovarian failure. AOF, which is often imitated using 4-vinylcyclohexene diepoxide (VCD), causes menopause to get underway immediately, resulting in expedited brain ageing, cognitive impairment, and increased susceptibility to neurodegenerative illnesses, including Alzheimer's disease (AD) (Mohammad, Finch et al. 2023, Mohammad, Finch et al. 2025). This situation is analogous to the rapid fall in estrogen and spike in FSH that occurs after ovarian failure. These changes may directly affect neurons (Blair, Bhatta et al. 2015), leading to neuroendocrine dysfunction, brain energy deficits, and increased indices of neuroinflammation. A study investigated structural brain abnormalities in people with idiopathic premature ovarian insufficiency (POI) and discovered that the ultrastructural changes found in POI participants' brains closely resemble those found in early dementia, particularly in areas linked to Alzheimer's disease (AD) (Marongiu 2019 , Yuan, Gong et al. 2025). AOF has been linked to increased amyloid-beta (Aβ) plaque deposition and tau phosphorylation, both of which are hallmarks of Alzheimer's disease pathology (Rosario, Carroll et al. 2006, Platholi, Marongiu et al. 2023, Marongiu, Platholi et al. 2025). A similar study utilising the VCD model found that POF reduces the discriminatory index in the NOR test, suggesting memory impairment. Ovarian failure also causes biochemical abnormalities linked with AD pathology, as shown by decreases in synaptic proteins such as SNAP25 (involved in neurotransmitter release) and NeuN (a marker of mature neurons), particularly during the perimenopausal transition (Mohammad, Finch et al. 2023, Mohammad, Finch et al. 2025). Furthermore, estrogen is a critical regulator of brain energy utilisation, and when AOF occurs, it leads to a loss of estrogen, resulting in severe impairment of mitochondrial functions and brain glucose metabolism. Recent evidence reveals that patients with accelerated ovarian ageing had lower ATP production and more mitochondrial DNA damage in their brains (Wang, Mao et al. 2025). Furthermore, estrogen deficiency increases the reactivity of microglia and astrocytes, resulting in a pro-inflammatory environment and immunogenic changes within the brain (Platholi, Marongiu et al. 2023; Voskuhl, Itoh et al. 2024). Finally, rapid depletion of ovarian hormones triggers a neuropathological cascade that disrupts brain metabolism, increases neuroinflammation, and accelerates the progression of AD, emphasising the importance of prompt intervention in cases of POF. These findings highlight the need for early interventions to reduce the long-term risks of cognitive decline and dementia in women with POI. Hormonal therapy (HT) may provide protection throughout the perimenopausal transition, but only if initiated early enough (within the window of opportunity) to prevent neuronal damage (Russell, Jones et al. 2019, Oppong-Gyebi, Metzger et al. 2022). Phytoestrogens have recently attracted increased scientific interest due to their well-established neurotrophic, neuroprotective, and neurorestorative potential (Gorzkiewicz, Bartosz et al. 2021, Oppong-Gyebi, Metzger et al. 2022). PED has a variety of pharmacological activities, including preventing neuronal cell death, combating free radicals, and decreasing inflammatory molecular signatures (Echeverria, Echeverria et al. 2021 ). It also promotes mitochondrial function and protects brain cells from various forms of oxidative and neuroimmune readouts (Zhao, Chen et al. 2002, Moreira, Silva et al. 2017, Ronchetti, Labombarda et al. 2025). PED is a promising option for treating neurodegenerative diseases due to its versatility. Nonetheless, PED, like many other natural compounds, is expected to act via multiple molecular pathways and targets, complicating understanding of its precise mechanism of action (Xu, Shi et al. 2008). While its neuroprotective effects have been demonstrated across a variety of experimental settings, the essential molecular pathways underlying these effects remain poorly understood and warrant further exploration. We used an integrative approach that included global brain metabolomics, neurobehavioral analyses, molecular biology methods, and cell-specific immunohistochemical analyses to predict and confirm the potential mitochondrial drug targets and neuroinflammatory signalling pathways that PED may engage in to improve cognitive outcomes. The purpose of this comprehensive investigation is to help us understand the therapeutic potential of PED and to identify the inflammopharmacological mechanisms underlying the relationship between endocrine failure and cerebral degeneration. This research provides a scientifically grounded alternative to ERT in AOF disease models by integrating evidence from maladaptive neuroinflammatory adaptations, mitochondrial drug targets, and global brain metabolic signalling pathways. Materials and Methods Experimental Animals: This experiment recruited female C57BL/6 mice aged 8 to 10 weeks and weighing 20 to 25 grams. The mice's living environment was meticulously controlled for light/dark cycles, temperature, and relative humidity. All animals were procured from the National Institute of Nutrition (NIN), Hyderabad and given a week to become accustomed to the amenities at the Central Animal House facility at NIPER, Hajipur. The animals were housed in groups of eight and given free access to a rodent chow diet and filtered water. To minimise the impact of daily fluctuations, behavioural evaluations were conducted during the light cycle, specifically from 9:00 to 14:00 hours . Ethical Statement: In accordance with Indian government regulations, the Institutional Animal Ethics Committee (IAEC) at NIPER, Hajipur, approved the study's experimental protocol ( IAEC Protocol no. NIPER-H/IAEC/66/23 ). All applicable guidelines of the institutional Committee for the Control and Supervision of Experiments on Animals (CCSEA) for the proper care, handling, and use of laboratory animals were strictly followed. Drugs and Chemicals: The ovarian toxin 4-vinylcyclohexene diepoxide (VCD) was obtained from Sigma-Aldrich, and cold-pressed pure corn oil (vehicle) was purchased from Deve Herbes on Amazon India. Ovotoxin VCD (Catalog #94956-100ML, Sigma Aldrich, purity >98% ) was dissolved in corn oil and administered at a dose of 160 mg/kg, at a volume of 10 ml/kg via the intraperitoneal ( i.p. ) route, once daily for 15 consecutive days (Niu, Miao et al. 2025). The detailed method of AOF induction is elaborated in Supplementary File 1 . Experimental Design: After confirming chemically-induced expedited ovarian failure and chronic estrogen depletion, the mice were arbitrarily separated into four groups (n=8 mice each group): (i) NCD group comprising of control mice, (ii) NCD+PED group, comprising of normal mice receiving phytoestrogen diet treatment (positive control), (iii) VCD group, 4-vinyl cyclohexene diepoxide challenged AOF mice, (iv) VCD+PED group, 4-vinyl cyclohexene diepoxide intoxicated mice, receiving phytoestrogen dietary treatment intervention. The PED was administered at ad libitum dosages for four weeks that were selected based on our previous findings. A detailed PED composition is available in Supplementary File 1 . After the behavioural tests, the mice were deeply anaesthetised using isoflurane (induction at 4–5%, maintenance at 1.5–2% in 100% oxygen) delivered via a precision vaporiser and euthanised via intracardiac perfusion. The saline-perfused brains were used for biochemical analysis (ELISA, immunoblotting, metabolomics analysis), whereas the PFA-perfused brains were used for immunohistochemical (IHC) and immunofluorescence (IF) assessments. A detailed experimental protocol schedule and treatment regimen are depicted in Figure 1. Fig. 1: Experimental timeline for inducing accelerated ovarian failure (AOF) in C57BL/6 mice. Fifteen concurrent intraperitoneal intoxication of ovatoxin VCD (at the dose of 160mg/kg) were used to induce POI in mice, followed by PED treatment intervention for four consecutive weeks. Twenty-four hours following the final behavioural test, the animals were deeply anaesthetised, euthanised, and their brains were transcardially perfused, carefully dissected, and stored at -80°C until they could be used for biochemical assessments like ELISA, western blotting, metabolomics analysis and immunohistochemical assessments. Behavioural Assessments: All groups of animals (with 8 mice per group) underwent a series of neurobehavioral assessments to evaluate mood, memory and cognitive functions, including the following tests in sequence: novel object recognition test (NORT) for assessing recognition memory, elevated plus maze test (EPMT) for assessing anxiety-like behavior, forced swim test (FST) for evaluating depressive-like behavior, and water maze test (WMT) for investigating spatial learning, navigation memory and cognitive flexibility. Following a week of acclimatisation period, the animals were familiarised with the arena in the absence of stimuli for 10 minutes each day over 2 days prior to the initiation of actual behavioural assessments (Kesharwani, Sree et al. 2025). A detailed protocol for behavioural tests conducted is provided in Supplementary File 1 . Animal Sacrifice, Method of Euthanasia and Tissue Harvesting for Biochemical Evaluation: Post-behavioural assessments, experimental animals were divided into two distinct cohorts for tissue harvesting. In the first cohort, mice were deeply anaesthetised with isoflurane (induction at 4–5%, maintenance at 1.5–2% in 100% oxygen) in an anaesthesia chamber. The depth of anaesthesia was strictly monitored by the loss of the pedal withdrawal reflex prior to the commencement of perfusion. Once the surgical plane of anaesthesia was confirmed by the absence of pedal reflex, animals were euthanised via decapitation while under the isoflurane, followed by intracardiac perfusion with 0.9% normal saline (NS). The saline-perfused brains were carefully harvested for immunoassays (ELISA), western blotting, and global metabolomic quantification. The second cohort was quickly flushed with NS, followed by transcardially perfusion with 4% paraformaldehyde (PFA). Decapitated, protein-fixed brain specimens were carefully removed from the skull, snap-frozen on ice, and stored at 4°C to be specifically used for further immunohistochemical (IHC) and immunofluorescence (IF) analyses (Kesharwani, Lahamge et al. 2025). A detailed description of the methodologies employed for tissue processing and biochemical measurements is provided in Supplementary File 1 . Biochemical Evaluation: Immunoassay (ELISA): A mouse-specific Uniovrsal E2 ELISA kit (ITLK01208) and a mouse aromatase quantitative sandwich ELISA kit (MBS456973) were used to measure 17β-estradiol and aromatase (CYP19A1/estrogen synthase) levels in hippocampal tissue homogenate. The sample absorbance was measured at 450nm using a Multimode Reader (Shandilya, Mehan et al. 2022). Immunoblotting: Hippocampal tissue was lysed on ice with RIPA lysis buffer and 1% protease inhibitor. Cell lysate protein concentrations were determined using a Merck BCA assay kit. Protein lysates (30-50μg) were separated on 8-12% Tris-glycine SDS-PAGE and transferred to 0.20μm PVDF membranes. Protein molecular weights were measured using a pre-stained protein ladder. After blocking membranes with 5% BSA (Sigma) for 2 hours at 37°C, primary antibodies against NDUFB8, MTCO1, ATP5A1, β-amyloid, ER-β, and BDNF were incubated overnight at 4°C on an orbital shaker. The next day, the membranes were washed three times with TBST and treated with secondary antibodies from the same species for 1 hour at 37°C. The immunoreactive bands were detected by enhanced chemiluminescence and photographed with the Molecular Imager ChemiDoc XRS System after three additional TBST washes. Normalisation loading control was beta-actin. Densitometric analysis was performed in ImageJ to quantify protein expression. Immunofluorescence Analysis: Three mice per group underwent intracardiac perfusion with a 0.9% normal saline flush followed by 4% PFA in 0.1M PBS (pH 7.4). After careful dissection, the brains were dehydrated in sucrose gradients until they sank, and then embedded in tissue-embedding media. Cutting 30μm-thick coronal slices of the prefrontal cortex and hippocampus using a Leica Cryostat, samples were collected sequentially on 24-well plates with 0.01 M PBS (including sodium azide). After three 5-minute washes with PBS (1X, pH=7.4), the mice's frozen brain tissue slices were blocked at room temperature for 30 minutes. Mouse monoclonal antibodies were used to assess the expression of markers, including AIF1/Iba1, HMGB1, TLR-4, NF-κB, CD68, and TREM2, in blocked sections incubated overnight at 4 °C. The next day, immunotagged sections were washed three times in 0.01 M PBS for 5 minutes each. After incubation with a secondary antibody from the same species for 30–60 minutes at 37°C in the dark, they were rewashed and stained with DAPI. A Nikon Eclipse AX/AXR confocal digital microscope was used to acquire Z-stacks (1µm intervals across brain tissue) at NIPER, Hajipur central imaging facility. Brain metabolome profiling through UPLC-MS/MS: A global LC-TQ-MS/MS-based mouse brain metabolome was searched for differentially regulated metabolites. MetaboAnalyst 6.0 (https://www.metaboanalyst.ca/) was used to stratify the raw data, deleting features with more than 50% missing values and imputing missing values to lower FDR. Analyses included fold change, PCA, PLS-DA, and univariate statistics (p 1 indicated metabolites that were differentiated. For DEM selection, FCs >±1.0 and p-values <0.05 were used to compare the control group. VIP ratings above 1.0 imply significance. The functional pathway enrichment is depicted using KEGG ortholog enrichment networks. Statistical Analysis: Data were presented as mean ± SEM. We examined (n=8) mice for neurobehavioural assessments and (n=3) for brain metabolomics, western blotting, and immunofluorescence. Experimental group metabolite differences were found using PCA or PLS-DA plots. To compare group behaviour, one-way, two-way, and three-way ANOVAs were used. All analyses used p-values < 0.05 and 95% CIs to assess statistical significance. Results PED restored local neuroestrogenic tone and modulated aromatase levels in VCD-mediated ovarian failure 17β-estradiol levels and aromatase concentrations were assessed in hippocampal tissue homogenate using an enzyme-linked immunoassay. A two-way ANOVA comparison revealed a significant decline in 17β-estradiol concentration, with significant interaction between VCD exposure and PED administration (F 1, 7 = 8.484, p= 0.0226), as well as significant primary effects of VCD induction (F 1, 7 = 194.9, p< 0.0001), and PED treatment (F 1, 7 = 8.778, p= 0.0210). Our post-hoc test is indicative of functional cerebral hypoestrogenism following VCD-triggered accelerated ovarian follicular depletion. Conversely, estradiol concentrations were modestly restored in the PED-treated groups compared with those in the VCD group ( Fig. 2A ). On the other hand, two-way ANOVA showed a compensatory upregulation of aromatase (CYP19A1) expression in the hippocampal homogenate of VCD-challenged AOF mice (p= 0.0019), reflecting an intrinsic attempt to counter chronic estrogen deprivation (a homeostatic neuroprotective response). We found significant interaction for aromatase expression between VCD-driven ovarian failure and PED administration (F 1, 7 = 18.27, p = 0.037), as well as significant main effects of VCD-induced ovarian intoxication (F 1, 7 = 23.33, p = 0.0019), and notable effect of PED treatment intervention (F 1, 7 = 56.70, p = 0.0001) on aromatase expression levels. Our post hoc multiple-comparison analysis showed that aromatase levels were substantially upregulated in the VCD-insulted mice group compared with the normal control group. However, PED supplementation partially normalised the dysregulated aromatase levels, consistent with a restoration of estrogenic tone rather than exaggerated compensatory neurosteroidogenesis, indicating compensatory drive ( Fig. 2B ). Fig. 2: Chronic VCD injections gradually reduced the 17β-estradiol levels (A) but increased aromatase expression (B) in the brains of AOF mice. Treatment with PED was effective in modulating this hormonal imbalance in VCD-challenged, follicular-depleted mice. Statistical analysis was conducted using a two-way ANOVA followed by post hoc Tukey’s multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001****p < 0.0001, (n = 3 assays per group). PED improved prefrontal cortex-dependent object recognition memory impairment in VCD-challenged AOF mice A two-way ANOVA did not find a significant interaction between the PED treatment and VCD-treated groups for total exploration time (F 1,7 = 0.6737, p = 0.4388), indicating that their combined effect did not significantly affect total exploration time for either object in the NOR test. Furthermore, neither PED treatment (F 1, 7 = 0.04763, p=0.8335) nor VCD challenge (F 1, 7 = 0.4602, p=0.5193; Fig. 3A ) yielded any significant effects on the total exploration time in the NOR task. This shows that neither VCD exposure nor PED therapeutic treatment impaired locomotor activity in mice. The three-way ANOVA analysis of percentage time spent examining new and familiar items indicated significant effects for novelty preference (F 1, 56 = 57.55, p<0.0001), novelty preference x VCD challenge (F 1, 56 = 4.881, p=0.0313), and novelty preference x PED treatment (p=0.0008). These findings demonstrate that both VCD exposure and PED therapy strongly influenced the percentage of time allocated to examining new items, as shown by the notable triple interaction among novelty preference, VCD, and PED treatment (F 1, 56 = 4.335, p=0.0419). Moreover, Tukey's multiple-comparison test indicated that both the NCD group and the NCD+PED groups showed a distinct preference for novelty, as evidenced by increased time spent examining new items (NCD: p<0.0001; NCD+PED: p<0.0005). The VCD group that did not receive PED treatment showed no preference for novel experiences (p = 0.9892). Nevertheless, mice in the VCD group that received PED treatment did not show these abnormalities and maintained their capacity to identify the unfamiliar item (p = 0.0008; Fig. 3B ). The two-way ANOVA analysis of DI revealed a significant interaction between PED therapy and VCD, indicating that their combined effect had a substantial impact on DI (F 1, 7 = 6.989, p = 0.0332). However, when considering the individual factors, PED treatment shows a significant modulatory effect on DI (F 1, 7 = 4.213, p=0.0792), while VCD also has a significant deleterious impact (F 1, 7 = 15.18, p=0.0059). Furthermore, Tukey’s multiple comparison test demonstrated that VCD-induced AOF mice treated with PED exhibited better intact memory and higher DI with respect to VCD-challenged animals (VCD vs VCD+PED, p<0.0001; Fig. 3C ). Fig. 3: PED treatment improves mPFC-dependent object recognition memory in AOF mice. NORT: The total exploration time did not vary across the groups (A). The reduction in novelty preference induced by the VCD challenge was significantly reversed by PED treatment, as evidenced by increased time spent exploring the novel object (B) and enhanced DI (C) in the NOR task. Total exploration time (A) and DI (C) were analysed by two-way ANOVA followed by Tukey’s multiple comparison test, while the percentage time spent exploring novel and familiar objects (B) was analysed by three-way ANOVA following Tukey’s multiple comparisons. * p < 0.05, **p < 0.01; ***p < 0.001; and ****p < 0.0001, (n = 8 mice per group). PED alleviated anxious behaviour and behavioural despair driven by chronic estrogen deficit in VCD-exposed AOF mice Subsequently, we used the EPM test, a well-known approach for measuring anxiety-like behaviour in rodents. An analysis of total time spent in the open arm revealed significant main effects of VCD induction (F 1, 7 = 31.76, p = 0.0008) and PED treatment intervention (F 1, 7 = 11.43, p = 0.0117), as well as a significant interaction between VCD and PED interventions (F 1, 7 = 14.91, p = 0.0062, Fig. 4A ). Furthermore, a two-way ANOVA analysis of the percentage of open-arm entries revealed a significant interaction between VCD insult and PED regimen (F 1, 7 = 8.366, p = 0.232), as well as significant main effects of VCD administration (F 1, 7 = 47.65, p = 0.0002) and PED treatment intervention (F 1, 7 = 14.57, p = 0.0066, Fig. 4B ). Our post hoc Tukey’s multiple-comparison test revealed that VCD mice treated with PED spent significantly more time (p=0.0074) in the open arms, and demonstrated a comparatively greater percentage of entries to the open arm (p=0.0278) than those treated with the vehicle. As a result, we can conclude that our PED treatment effectively mitigated accelerated ovarian failure-induced affective disorder ( Fig. 4A, 4B) . A two-way ANOVA was conducted to assess the effects of both the VCD exposure and PED therapeutic administration on the duration of immobility. The findings revealed a noteworthy two-way interaction between VCD induction and PED treatment groups (F1, 7 = 12.80, p = 0.009). Furthermore, significant main effects were observed for both VCD administration (F 1, 7 = 33.35, p = 0.0007) and PED treatment (F 1, 7 = 25.60, p = 0.0015). These outcomes suggest that exposure to VCD and/or PED significantly impacts the immobility duration in mice during the FST. Our post hoc analysis with Tukey’s multiple-comparison test also revealed that VCD-treated mice showed substantial depressive-like behaviour (p=0.0009), but PED administration significantly reduced the immobility duration (p = 0.0051; Fig. 4C ). The noteworthy reduction in the immobility time in the FST indicates a substantial decrease in depressive-like behaviour in VCD+PED-treated AOF mice. Fig. 4: PED intervention ameliorates anxiety and depressive-like behaviour in AOF mice. EPM: AOF mice exhibited increased affective dysfunction, as evidenced by decreased total time spent in the open arms (A) and a lower percentage of open-arm entries (B), which the PED treatment regimen effectively alleviated. FST: AOF mice displayed greater depressive-like behaviour, as evidenced by their enhanced time spent floating immobile (C). However, PED treatment significantly reversed the behavioural despair in VCD-triggered AOF mice. Data were analysed by two-way ANOVA followed by post-hoc Tukey’s multiple comparison test. * p < 0.05, **p < 0.01; ***p < 0.001; and ****p < 0.0001, (n = 8 mice per group). PED retrieved hippocampal-dependent spatial and navigation memory impairment and conferred cognitive flexibility in VCD-driven AOF mice The mice were subjected to a water-based navigation task in a circular pool filled with opaque water and a submerged platform to evaluate their hippocampal-dependent spatial memory. During the initial learning sessions (day 1-day 4), there were progressive, similar decreases in latencies to reach the platform (escape latency time) across all the groups, as indicated by a two-way ANOVA. In the final session (day 5), VCD-exposed mouse groups exhibited a significant increase in escape latency time (ELT) to locate the target quadrant compared with normal controls, indicating a significant impact of chronic estrogen deficiency induced by accelerated ovarian follicular depletion on memory retrieval. A two-way ANOVA revealed a significant interaction between training days and experimental groups (F 3.848, 26.94 = 6.538, p = 0.009), as well as significant main effects of learning days (F 2.515, 17.60 = 332.2, p < 0.0001) and groups (F 2.488, 17.42 = 56.51, p <0.0001, Fig. 5A ). This was further verified by comparing dwell intervals on the platform across the mouse groups. Unlike controls, mice subjected to a VCD insult spent significantly less time on the target platform than the normal mouse cohort (p=0.0004), indicating that exposure to chemically induced AOF progressively diminishes memory retention ability. A two-way ANOVA revealed a significant interaction between VCD challenge and PED intervention (F 1, 7 = 21.26, p = 0.0025), as well as significant main effects were observed for both VCD administration (F 1, 7 = 44.28, p = 0.0003) and PED treatments (F 1, 7 = 8.068, p = 0.0250, Fig. 5B ). The aforementioned findings were further supported by the number of crossings on the platform area. The mice that received the VCD intoxication had fewer platform area crossings than the control group (NCD v/s VCD: p = 0.0070; VCD v/s VCD+PED: p = 0.0188). A two-way ANOVA found a significant interaction between VCD challenge and PED intervention (F 1, 7 = 8.374, p = 0.0232), as well as significant primary effects for both VCD administration (F 1, 7 = 14.30, p = 0.0232) and PED treatments (F 1, 7 = 13.17, p = 0.0084, Fig. 5C ). On Day 6 th (24 hours post the last learning session), the target platform was removed for the probe test assessment to assess time spent by the animals in the target platform quadrant (TSTQ). Both control groups (NCD, NCD+PED) consistently preferred the platform quadrant over the other quadrants, but VCD-treated mice showed nearly equal preferences across all four quadrants (NCD v/s VCD: p=0.0024), with no distinct inclination toward the previous target zone, indicating a deficit in spatial learning and memory retrieval. A two-way ANOVA revealed a significant double-point interaction between VCD induction and the PED-treated groups (F 1, 7 = 12.90, p = 0.0088). Furthermore, significant main effects were observed for both VCD administration (F 1, 7 = 26.55, p = 0.0013) and PED treatments (F 1, 7 = 2.577, p = 0.1525, Fig. 5D). Interestingly, PED treatment generally showed a pattern of improvement in contextual memory retention ability, as reflected in increased time spent in the platform quadrant (VCD v/s VCD+PED: p=0.0066), substantially greater no.of platform area crossings (VCD v/s VCD+PED: p=0.0188) and comparatively enhanced dwell time on the platform (VCD v/s VCD+PED: p=0.0033), however, the extent to which this improvement was mechanistically translatable to a direct reversal of VCD-triggered cognitive deficits remained inconsistent, prompting further investigation into the underlying cellular and molecular mechanisms ( Fig. 5A-D) . Fig. 5: PED regimen mitigates hippocampal-dependent spatial learning impairment and cognitive rigidity in AOF mice. (A) escape latency time (ELT), (B) platform dwell time, (C) number of platform area crossings, and (D) time spent in the target quadrant (TSTQ) in the WMT. Data were analysed by two-way ANOVA followed by post-hoc Tukey’s multiple comparison test. * p < 0.05, **p < 0.01; ***p < 0.001; and ****p < 0.0001, (n = 8 mice per group). Cellular and Molecular Results: PED reversed the hippocampal mitochondrial bioenergetic failure in VCD-elicited POF mice Mitochondrial dysfunction is a key pathomechanism of neurodegeneration caused by VCD-provoked follicular depletion and ovarian failure, largely due to mitochondrial functional impairment, altered respiratory chain (OXPHOS) complexes and electron transport chain (ETC) enzyme dysfunction. To establish PED therapy's modulatory effects on mitochondrial functional signalling during chemically-induced ovarian senescence, comprehensive western blot analysis was performed on hippocampal tissue to determine the expression levels of key mitochondrial functional markers, including NDUFB8, MTCO1, and ATP5A1. The immunoblots were analysed using a two-way ANOVA with mean ± SEM and Tukey's multiple-comparison post-hoc test (n=3). The analysis revealed a significant double interaction between VCD induction and PED treatment (NDUFB8: F 1, 2 = 8939, p=0.0001; MTCO1: F 1, 2 = 1888, p=0.0005; ATP5A1: F 1, 2 = 211.4, p=0.0047), indicating a combined influence of these variables. Moreover, the VCD challenge in mice and the PED treatment intervention each exhibited significant individual effects. Notably, NDUFB8, MTCO1, and ATP5A1 expression decreased significantly (NDUFB8: F 1, 2 = 567.9, p=0.0018; MTCO1: F 1, 2 = 4517, p=0.0002; ATP5A1: F 1, 2 = 3028, p=0.0003) in the VCD-exposed AOF mice group compared with the normal control group. Conversely, treatment with PED effectively counteracted these mitochondrial functional anomalies, leading to significant preservation/restoration of the cerebral bioenergetic integrity (NDUFB8: F 1, 2 = 20.38, p=0.0457; MTCO1: F 1, 2 = 1081, p=0.0009; ATP5A1: F 1, 2 = 49.52, p=0.0196, Fig. 6A-C ). These ameliorative effects of PED demonstrate their potential to restore altered brain mitochondrial metabolism and enhance neuronal resilience in the context of AOF-induced follicular depletion, caused by the persistent challenge of ovarian intoxicants. PED suppresses amyloidogenic burden and reinstates ERβ–BDNF neuroprotective signalling in VCD-conditioned mice To interrogate the complex interplay among ovarian follicular depletion, neuroendocrine hormone loss, dysregulated estrogen receptor signalling, amyloid pathology, and neuronal atrophy, we sought to determine how premature ovarian insufficiency contributes to heightened AD susceptibility in women (Ansere, Ali-Mondal et al. 2021). Consistent with the reported findings, our immunoblotting results on hippocampal tissues demonstrated a significant interaction between VCD intoxication and PED treatment (β-amyloid: F 1, 2 = 68.39, p=0.0143; ER-β: F 1, 2 = 828.3, p=0.0012; BDNF: F 1, 2 = 54.38, p=0.0179), indicating a combined influence of these variables. Additionally, a marked upregulation of amyloid beta plaque deposition (F 1,2 = 451.5, p = 0.0022), concomitant with dynamically downregulated ER-β expression (F 1,2 = 228.5, p =0.0043) and significant inhibition of BDNF levels (F 1,2 = 292.7, p =0.0034) was found in the VCD-challenged mice group compared to the normal control mice group. Strikingly, administration of the PED therapeutic intervention partially normalised these molecular perturbations, attenuating beta-amyloid plaque deposition (F 1,2 = 66.00, p = 0.0148), while restoring BDNF abundance (F 1,2 = 89.37, p = 0.0110) and modulating ER-beta signalling (F 1,2 = 36.37, p = 0.0264) ( Fig. 6D-F ). These neuromodulatory outcomes collectively underscore PED’s capacity to suppress accelerated ovarian senescence-induced neurodegeneration and reinstate neuroprotective estrogen beta signalling, thereby substantiating its neurotrophic and neurorestorative efficacy in the context of AOF. Fig. 6: PED therapy elicits mitochondrial functional reconstitution, suppresses amyloidogenic burden, downregulates neuroinflammatory cascades, and reinstates estrogenic receptor β-mediated neurotrophic signalling in the hippocampus of AOF mice. (A) Representative immunoblots of mitochondrial respiratory chain complexes NDUFB8, MTCO1 and ATP5A1 in the hippocampal tissue homogenate of mice. Bar graph showing relative levels of (A) NDUFB8, (B) MTCO1, (C) ATP5A1, (D) β-amyloid, (E) ER-β, (F) BDNF, (G) Iba1, and (H) NF-ϏB. Data are represented as mean ± SEM. Statistical analysis was conducted by two-way ANOVA, followed by Tukey’s multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001, (n = 3 immunoblots per group). PED attenuates microgliosis-induced perpetuating neuroinflammatory loops and restores mPFC-mediated neuroimmune responses in AOF mice To further elucidate how microglial phenotypes are influenced by the gonadal hormone milieu and to better understand the neuromodulatory effects of PED treatment on cerebral inflammatory cascades following an ovatoxin VCD insult, double-labelling immunofluorescence staining was performed in the medial PFC. Interestingly, AOF-driven microglial inflammatory polarisation was examined by assessing the expression levels of key neuroinflammatory markers, including HMGB1, NF-κB, TLR-4, CD68, and TREM2, against Iba1-positive microglia. A two-way ANOVA indicated a significant interaction between VCD induction and PED treatment (HMGB1: F 1,2 = 187.8, p = 0.0053, NF-kB: F 1,2 = 44.25, p = 0.0219; TLR-4: F 1,2 = 585.2, p = 0.0017; CD68: F 1,2 = 51448, p < 0.0001; TREM2: F 1,2 = 72.89, p = 0.0134). The VCD-treated AOF mice group exhibited a marked augmentation in the fluorescence intensity of these microglial-mediated inflammatory markers, reflecting escalation of brain inflammatory phenotypic shift following accelerated ovarian senescence (Iba1+ HMGB1: F 1,2 = 964.5, p=0.0010; Iba1+NF-ϏB: F 1,2 = 1.702, p=0.0006; Iba1+TLR-4: F 1,2 =68.23, p = 0.0143, Iba1+CD68: F 1,2 = 96.19, p =0.0102; Iba1+TREM2: F 1,2 = 1003, p =0.0010). Conversely, treatment with PED resulted in a marked downregulation of neuroinflammatory indicators like HMGB1 (F 1,2 = 70.35, p =0.0139), NF-κB (F 1,2 =10.40, p =0.0842), TLR-4 (F 1,2 = 980.0, p =0.0010), CD68 (F 1,2 =74.87, p =0.0131), and TREM2 expression (F 1,2 = 574.1, p =0.0017), suggesting that PED treatment potentially mitigates microgliosis, forestalls innate immune signalling and attenuates self-propagating neuroimmune dysfunction in the mouse PFC in the context of POF ( Fig. 7A-E ). Fig. 7: The PFC of VCD-challenged AOF mice showed increased levels of inflammatory mediators, including HMGB1, NF-kB, TLR-4, CD68, and TREM2, whereas PED administration substantially inhibited the expression of these inflammaging markers. Representative double immunofluorescence staining of Iba1+ microglial cells expressing inflammatory markers (A) Iba1+HMGB1, (B) Iba1+NF-ϏB, (C) Iba1+TLR-4, (D) Iba1+CD68, (E) Iba1+TREM2, (Scale bar: 50μm). Analysis of fluorescence intensity of (A) HMGB1, (B) NF-ϏB, (C) TLR-4, (D) CD68, (E) TREM2. Data are represented as mean ± SEM. Statistical analysis was conducted by two-way ANOVA, followed by Tukey’s multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: non-significant, (n = 3 cerebral slices per group). PED confers metabolic neuroprotection in VCD ovatoxin-intoxicated AOF mice Statistical modelling of brain metabolome data using multivariate analysis identified differentially expressed metabolites and dysregulated metabolic pathways in VCD-challenged mice and evaluated the biochemical impact of PED treatment following VCD intoxication. In our metabolomic data, 45 DEMs were identified and quantified in NCD vs VCD samples, and 39 in VCD vs VCD+PED therapy samples. Comparing VCD-induced AOF mice to normal control mice, 31 metabolites were considerably elevated, and 14 were dramatically downregulated. Compared to (VCD+PED) group, VCD-intoxicated mice's brains had 24 upregulated and 15 downregulated metabolites. VIP-ranked metabolomic patterns show that VCD-induced premature ovarian failure severely alters estrogen-regulated brain metabolism. Dimethisterone, a high-impact metabolite, mostly maps to steroid and neurosteroid dysregulation, demonstrating reduced local estradiol synthesis and increased oxidative steroid turnover. In VCD-induced mice, the accumulation of fatty acyls (1-hexadecanoyl derivatives, 1-dodecanoyl-2-derivatives) and sphingolipid intermediates (apidiasphingosine, dehydrophytosphingosine, 1-hexadecanoyl species) indicates mitochondrial β-oxidation deficits, energy imbalance, pro-apoptotic signalling, and neuronal membrane instability. In VCD-insulted animals, elevated arachidonic acid–derived prostaglandins (PGD2 intermediates) indicate a lack of estrogenic control over inflammatory lipid signalling. In the VCD-challenged group, lower levels of ascorbate-related metabolites indicate a disturbed redox equilibrium and increased oxidative stress. These changes cause neuroenergetic stress, redox imbalance, and lipid-driven neuroinflammatory pathways, which underlie VCD-induced accelerated brain ageing ( Fig.8A ). However, estrogen-receptor-mediated metabolic repair of the AOF brain is shown by PED therapy's inhibition of VCD-induced anomalies in steroid metabolism, inflammatory lipid signalling, oxidative stress pathways, and membrane lipid homeostasis. PED restored 3-epi-5,6-trans steroidal derivative, attenuated lipid-driven neuroinflammation (O-arachidonoyl derivative; 9S,15S-dihydroxy lipid derivative), antioxidant buffering and estrogen-dependent redox control (2-amino-3-oxo derivative; 9-methoxy-hept derivative), neuronal membrane integrity and synaptic integrity (persediene), and metabolic efficiency and neuronal resilience ( Fig.8B). The most common DEMs were used for pathway mapping and enrichment analysis to identify metabolic pathways that differentiated control, VCD-challenged, and VCD+PED–treated mouse brains. Interestingly, VCD induction increased brain lipid-inflammation metabolic reprogramming, as indicated by the functional enrichment network. Glycerophospholipid metabolism had the largest node size and the highest connectivity in the pathway topology, indicating the most extensive alteration of neural membrane phospholipids, which may serve as precursors to persistent inflammatory mediators and cellular stress responses. A strong metabolic cluster for α-linolenic acid, linoleic acid, and arachidonic acid metabolism suggests coordinated abnormalities in polyunsaturated fatty acid metabolism, indicating lipid remodelling, immune activation, and the biosynthesis of inflammatory mediators. The arachidonic acid pathway is a key node in the network, producing eicosanoids (prostaglandins, leukotrienes) that activate the immune system and cause chronic neuronal inflammatory rewiring. The network's peripheral node, which illustrates sphingolipid metabolism, has been shown to affect ceramide-mediated downstream inflammatory processes, neuronal death, and immunological modulation, suggesting a role in the overall inflammatory metabolic landscape ( Fig. 8C) . Fig. 8: PED treatment intervention confers global metabolic neuroprotection in VCD-challenged AOF mice. Representative 2DPLS-DA plot, VIP score plot, biplot, and heat map visualisation of DEMs when comparing NCD against VCD-inducted group (A), and VCD against VCD+PED groups (B). The colour blue denotes downregulated/underexpressed metabolites, whereas red colour indicates upregulated/overexpressed metabolites in the VIP score plot. The heat clustering analysis highlights metabolites with significant increases or decreases in expression. Statistical cut-off was set at log2FC> 1 and p-values < 0.05. Sample size: (n = 3 in each group). The joint pathway enrichment network was derived from the KEGG database, and the circle size reflects the proportion of commonly enriched metabolites (C). Discussion The present study provides compelling behavioural, biochemical, and molecular evidence that persistent estrogen deficiency, modelled through VCD-induced accelerated ovarian failure (AOF), precipitates a broad neurobehavioural, neuroinflammatory and neurodegenerative phenotype encompassing cognitive impairment, affective dysregulation, mitochondrial dysfunction, amyloidogenic vulnerability, dysregulated estrogen signalling, neuronal atrophy, differential brain metabolome expression, microglial overactivation, and neuroinflammatory compromise. Importantly, our findings show that PED therapeutic intervention effectively combats these adverse impacts, implying that it has the potential to be a multi-targeted neurorestorative agent in estrogen-deficient states similar to premature ovarian insufficiency and perimenopausal brain ageing after chemically-induced, follicular-depleted, accelerated ovarian failure. While a rapid decline in systemic estrogen is a crucial indicator of accelerated ovarian failure, increasing research shows that the production of estrogen in the brain is essential for maintaining neuronal function and cognitive behaviour (Iqbal, Huang et al. 2024). In POI, brain 17β-estradiol levels decrease significantly, whereas neural aromatase levels increase to compensate. This leads to insufficient local estrogen synthesis, increased neuroinflammation, and quicker brain ageing. Although PED led to comparative increases in hippocampal estradiol levels, aromatase levels were partially reinstated. This observed trend suggests a partial restoration of intracerebral estrogenic tone, which is sufficient to activate downstream neuroprotective systems. It is noteworthy that aromatase activity is limited by the amounts of testosterone and androstenedione present; hence, E2 recovery is low. PED primarily reinstates prefrontal and hippocampal-dependent cognition, with little impact from locomotor factors. The novel object recognition (NOR) deficits observed in mice exposed to VCD indicate that the prefrontal cortex-hippocampal network is not functioning properly, a hallmark of cognitive ageing associated with low estrogen levels. Importantly, the absence of changes in overall exploration time demonstrates that locomotor or motivational limitations did not affect memory problems. The substantial three-way interaction between novelty preference, VCD induction, and PED treatment, as well as the restoration of the discrimination index (DI), demonstrates that PED recovers novelty preference and recognition memory processing, which were disrupted by ovarian hormone depletion. These findings are consistent with clinical data suggesting that estrogen insufficiency disproportionately affects executive and recognition memory domains, highlighting the paradigm's translational value (Mohammad, Finch et al. 2025, Abi-Ghanem, Opiela et al. 2026). Anxiety and depression-like symptoms are recognised as neuropsychiatric indications of menopausal transition and early ovarian failure. VCD-induced impairments in open-arm exploration in the EPMT and increased immobility in the FST are consistent with increased anxiety and behavioural despair in chronic estrogen-deficient conditions (Słopień 2018). PED treatment significantly improved these affective impairments, suggesting that its neuroprotective benefits go beyond cognitive functioning and encompass emotional regulation. These behavioural alterations are most likely the result of PED stabilising the limbic circuitry and the balance of monoaminergic and neurotrophic signals, both of which are highly influenced by estrogen receptor beta signalling. Spatial learning, navigation memory, and probe trial performance in the water maze test revealed that, whereas acquisition learning was mainly retained, chronic ovarian failure hampered memory retrieval and cognitive flexibility (Ramli, Yahaya et al. 2024). Mice treated with VCD did not spend as much time in the target quadrant (PQ) as in other quadrants, indicating that hippocampus-dependent processes for consolidating and retrieving memories were compromised. PED treatment improved target quadrant preference and platform dwell time, suggesting that hippocampal synaptic plasticity and adaptive learning had been restored. These findings are particularly significant, as cognitive dullness and difficulties with remembering information are clinically relevant early indicators of brain ageing and neurodegeneration following persistent ovarian senescence (Reuben, Karkaby et al. 2021). Surprisingly, PED markedly boosted the expression of critical mitochondrial electron transport chain proteins (NDUFB8, MTCO1, ATP5A1) that had been significantly reduced during VCD exposure, indicating that PED is an effective modulator of neuronal bioenergetics and brain energy metabolism (Torrens-Mas, Pons et al. 2020). The observed behavioural and cognitive improvements are most likely due to preservation of mitochondrial integrity, supporting the idea that phytoestrogenic medications have a major impact on mitochondrial functional reconstruction (Singh and Paramanik 2022). VCD-treated mice showed elevated amyloid beta deposition, downregulation of ER-β signalling, and reduction of BDNF, indicating a link between ovarian failure and increased susceptibility to Alzheimer's disease. These molecular alterations support what epidemiological and clinical research have found: early menopause and ovarian follicular depletion are associated with an increased risk of dementia (Karamitrou, Anagnostis et al. 2023, Liao, Cheng et al. 2023). PED reduces amyloid beta levels while increasing ER-β expression and BDNF synthesis, indicating that estrogen-dependent neurotrophic and anti-amyloidogenic mechanisms are reactivated. This conclusion is significant, as ER-β signalling has been linked to maintaining synapses (Srivastava, Woolfrey et al. 2010), safeguarding neuronal mitochondrial functional capacity (Wang, Mao et al. 2024), regulating neural differentiation (Varshney, Inzunza et al. 2017), neuronal inflammation, making PED a mitigative therapy that alters the course of a neurodegenerative pathology rather than merely treating symptoms. Chronic estrogen deprivation resulting from chemically induced accelerated ovarian failure induced significant microglial activation, as evidenced by increased HMGB1–TLR4–NFκB signalling and elevated expression of CD68 and TREM2, indicating a transition to a pro-inflammatory and phagocytic microglial phenotype (Acosta-Martínez 2020, Gao, Jiang et al. 2023). This inflammatory reprogramming was followed by considerable global brain metabolic reprogramming, emphasising the close link between increased neuroinflammaging and cerebral dysfunction in ovarian failure. Our PED therapy significantly inhibited these inflammatory cascades, mitigated microgliosis, and restored cerebral integrity, demonstrating complete preservation of the inflammaging unit. These results elucidate the mechanisms by which PED maintains neuronal homeostasis, mitigates mood disorders, and delays early cognitive decline in chronic estrogen-deprived environments, highlighting the pivotal role of the microglial–inflammatory interaction in the development of neurodegenerative pathophysiology (Gao, Jiang et al. 2023). Using metabolomic profiling, we also identified discrepancies in levels of pro-oxidative and inflammatory metabolites, as well as changes in numerous metabolic pathways, in the VCD-challenged mice group. Our metabolomics study, which mapped the metabolic pathways of VIP metabolites, found that VCD-induced AOF disrupts estrogen-regulated steroid, fatty acid, and inflammatory lipid metabolism, leading to mitochondrial dysfunction and lipid-mediated neuroinflammation in the brain. In the NCD mouse brain, coordinated steroidogenesis, lipid oxidation, membrane lipid signalling, and neuroenergetic function were observed while inhibiting inflammatory lipid cascades. VCD-induced ovarian failure results in a dysregulated metabolomic signature, including steroid depletion, mitochondrial fatty acid dysregulation, an excess of prostaglandins (PGF2α species, PGD2 derivatives, O-arachidonyl intermediates), sphingolipid imbalance, and oxidative stress, indicating estrogen-dependent metabolic collapse and rapid neuroinflammaging cascades. Our findings revealed that PED treatment stabilises steroid homeostasis, boosts neuroprotective ER signalling, combats lipid-driven neuroinflammation, and prevents oxidative-metabolic derailment by restoring antioxidant buffering, enhancing metabolism efficiency, and improving detox control. The joint pathway network analysis suggests that perturbations in membrane lipid metabolism and PUFA-derived inflammatory mediator pathways play a substantial role in the observed brain metabolomic changes. These results support the hypothesis that lipid-driven metabolic reprogramming may function as a critical upstream regulator of persistent neuronal inflammaging. These results align well with the currently evolving paradigm of immunometabolic regulation of neural inflammation in the context of brain ageing and neurodegeneration following chemically-induced ovarian senescence. Henceforth, this study identifies PED as a pleiotropic neuroprotective agent that targets mitochondrial dysfunction, neuroinflammation, amyloid pathology, neurotrophic collapse, and aberrant ER signalling—pathological pathways that intersect during perimenopause-induced accelerated brain ageing and neurodegeneration. We, for the first time, demonstrate compelling and outstanding mechanistic evidence on how PED effectively halts the neurobehavioral and molecular development of ovarian senescence-induced brain pathophysiology by restoring ERβ-mediated neuroprotective signalling and normalising microglial-mitochondrial-neuroimmune interactions (Ambika, Gajbhiye et al. 2025). Conclusion Profound hypoestrogenism caused by chemically-induced ovarian failure initiates a self-perpetuating neuroinflammatory and neurodegenerative cascade that includes microglial overactivation, mitochondrial malfunction, neuronal atrophy, and behavioural impairment (Zhou, Zhang et al. 2026). In a clinically relevant AOF model, PED effectively prevents HMGB1-TLR4-NFκB-induced microgliosis, restores mitochondrial integrity, and stabilises cognition (Li, Yang et al. 2023). PED preserves ERβ-dependent neuroimmune homeostasis and inflammopharmacological integrity, preventing self-perpetuating neuroinflammatory loops that accelerate brain ageing after chemically-induced early ovarian failure (Zhao, Mao et al. 2013). Henceforth, our results address a significant clinical gap in estrogen-associated neuroimmune dysregulation as a major contributor to perimenopausal brain vulnerability, and highlight PED as a potent immunomodulatory neuroprotective strategy during reproductive ageing. These findings advocate for integrating polyphenolic non-steroidal compounds into the clinical management of menopausal health to promote long-term cognitive resilience and overall metabolic homeostasis. Our evidences present groundbreaking translational implications, particularly for women experiencing early menopause or ovarian insufficiency, who are presently underrepresented in neuroprotective intervention studies. We propose that a bioactive phytoestrogen-enriched therapeutic strategy might be a safer, brain-targeted alternative to standard hormone replacement therapy, offering multidimensional neuroprotection without the systemic dangers associated with exogenous estrogen treatment. Abbreviations AOF: Accelerated ovarian failure, BDNF: Brain-derived neurotrophic factor, ER-β: Estrogen receptor beta, ERT: Estrogen replacement therapy, HMGB1: High mobility group box 1, NF-ϏB: Nuclear factor kappa B, PED: Phytoestrogen diet, PFC: Pre-frontal cortex, POF: Premature ovarian failure, TLR-4: Toll-like receptor-4, VCD: 4-vinylcyclohexene diepoxide Declarations Acknowledgements: The authors express their gratitude to the Department of Pharmaceuticals, Ministry of Chemicals and Fertilisers, for adequate funding and institutional support to carry out this research. Data availability statement: All data generated or analysed during this study are included in this article. There are no separate or additional files. Disclosure of potential conflicts of interest: The authors declare that they have no competing financial or personal interests. Ethical approval: All applicable institutional CCSEA and IAEC guidelines for the proper care, handling and use of laboratory animals were followed. Funding Declaration: This research was funded by the Department of Pharmaceuticals, Ministry of Chemicals and Fertilisers, Govt. of India. Clinical Trial Number: Not applicable. Contribution of authors: Ambika Shandilya (First Author): Pre-clinical survey, methodology, statistical data curation and analysis, writing original manuscript, review and editing, and formal analysis. Suhas Hajare: Investigation , validation. Dubey Aakash Arwind: Investigation, validation, visualisation . Chander Kant Giri: Validation and visualisation . Rahul Gajbhiye: Metabolomics . V. Ravichandiran: Supervision, resources, and funding acquisition . VK Parihar: Conceptualisation, project administration, supervision, editing, and funding acquisition . Note: All authors approved the final version of this study. References Abi-Ghanem C et al (2026) Loss of ovarian hormones is detrimental in early disease stages of mouse models of Alzheimer’s disease and multi-etiology dementia. Biology Sex Differences 17(1):4 Acosta-Martínez M (2020) Shaping microglial phenotypes through estrogen receptors: relevance to sex-specific neuroinflammatory responses to brain injury and disease. J Pharmacol Exp Ther 375(1):223–236 Ambika et al (2025) Unveiling the pioneering potential of phytoestrogens in promoting stem cell functions and neural regeneration in post-menopausal brain. SAGE PUBLICATIONS INC 2455 TELLER RD. CELL TRANSPLANTATION, THOUSAND OAKS. CA 91320 USA. Ansere VA et al (2021) Cellular hallmarks of aging emerge in the ovary prior to primordial follicle depletion. Mech Ageing Dev 194:111425 Blair JA et al (2015) Luteinizing hormone: Evidence for direct action in the CNS. Horm Behav 76:57–62 Echeverria V et al (2021) Estrogenic plants: to prevent neurodegeneration and memory loss and other symptoms in women after menopause. Front Pharmacol 12:644103 Gao C et al (2023) Microglia in neurodegenerative diseases: mechanism and potential therapeutic targets. Signal Transduct Target therapy 8(1):359 Gorzkiewicz J et al (2021) The potential effects of phytoestrogens: The role in neuroprotection. Molecules 26(10):2954 Iqbal J et al (2024) Role of estrogen in sex differences in memory, emotion and neuropsychiatric disorders. Mol Biol Rep 51(1):415 Karamitrou EK et al (2023) Early menopause and premature ovarian insufficiency are associated with increased risk of dementia: a systematic review and meta-analysis of observational studies. Maturitas 176:107792 Kesharwani A et al (2025) Potentiation of endocannabinoid signaling alleviates depressive-like behavior in diabetic mice. Pharmacol Research-Reports 3:100037 Kesharwani A et al (2025) Dehydrozingerone Improves Mood and Memory in Diabetic Mice via Modulating Core Neuroimmune Genes and Their Associated Proteins. ACS Pharmacol Translational Sci 8(6):1694–1710 Li X et al (2023) Calycosin attenuates the inflammatory damage of microglia induced by oxygen and glucose deprivation through the HMGB1/TLR4/NF-Κb signaling pathway: calycosin attenuates OGD/R damage in microglia. Acta Biochim Biophys Sin 55(9):1415 Liao H et al (2023) Association of earlier age at menopause with risk of incident dementia, brain structural indices and the potential mediators: a prospective community-based cohort study. EClinicalMedicine 60 Marongiu R (2019) Accelerated ovarian failure as a unique model to study peri-menopause influence on Alzheimer’s disease. Front Aging Neurosci 11:242 Marongiu R et al (2025) Perimenopause promotes neuroinflammation in select hippocampal regions in a mouse model of Alzheimer’s disease. bioRxiv: 2025.2003. 2014.643317. Mohammad A et al (2023) Accelerated Ovarian Failure (AOF) is an Effective Transitional Menopausal Model for the Study of Alzheimer’s Disease. Alzheimer's Dement 19:e071809 Mohammad A et al (2025) Accelerated ovarian failure results in brain alterations related to Alzheimer's disease that are not recovered by high-intensity interval training in mice. Alzheimer's Dement 21(2):e14463 Moreira AC et al (2017) Phytoestrogen coumestrol improves mitochondrial activity and decreases oxidative stress in the brain of ovariectomized Wistar-Han rats. J Funct Foods 34:329–339 Niu Y et al (2025) The 4-vinylcyclohexene dioxide induced mouse ovarian premature failure is caused by down regulation of IGF1R and triggering excessive autophagy. Stress Biology 5(1):21 Oppong-Gyebi A et al (2022) Long‐term hypogonadism diminishes the neuroprotective effects of dietary genistein in young adult ovariectomized rats after transient focal ischemia. J Neurosci Res 100(2):598–619 Platholi J et al (2023) Hippocampal glial inflammatory markers are differentially altered in a novel mouse model of perimenopausal cerebral amyloid angiopathy. Front Aging Neurosci 15:1280218 Ramli NZ et al (2024) Spatial learning and memory impairment at the post-follicular depletion state is associated with reduced hippocampal glucose uptake. Exp Gerontol 197:112607 Reuben R et al (2021) Menopause and cognitive complaints: are ovarian hormones linked with subjective cognitive. decline? Climacteric 24(4):321–332 Ronchetti S et al (2025) The phytoestrogen genistein improves hippocampal neurogenesis and cognitive impairment and decreases neuroinflammation in an animal model of metabolic syndrome. J Neuroendocrinol 37(2):e13480 Rosario ER et al (2006) Androgens regulate the development of neuropathology in a triple transgenic mouse model of Alzheimer's disease. J Neurosci 26(51):13384–13389 Russell JK et al (2019) role estrogen brain Cogn aging Neurother 16(3):649–665 Shandilya A et al (2022) Activation of IGF-1/GLP-1 signalling via 4-hydroxyisoleucine prevents motor neuron impairments in experimental ALS-rats exposed to methylmercury-induced neurotoxicity. Molecules 27(12):3878 Singh P, Paramanik V (2022) Neuromodulating roles of estrogen and phytoestrogens in cognitive therapeutics through epigenetic modifications during aging. Front Aging Neurosci 14:945076 Słopień R (2018) Mood disorders in women with premature ovarian insufficiency. Menopause Review/Przegląd Menopauzalny 17(3):124–126 Srivastava DP et al (2010) Estrogen receptor β activity modulates synaptic signaling and structure. J Neurosci 30(40):13454–13460 Torrens-Mas M et al (2020) Sexual hormones regulate the redox status and mitochondrial function in the brain. Pathological implications Redox biology 31:101505 Varshney MK et al (2017) Role of estrogen receptor beta in neural differentiation of mouse embryonic stem cells. Proceedings of the National Academy of Sciences 114(48): E10428-E10437 Voskuhl RR et al (2024) Mind the Gap: Estrogen Receptor-β in Astrocytes Is a Therapeutic Target to Prevent Cognitive Problems at Menopause [ID 2683385]. Obstet Gynecol 143(5S):2 Wang T et al (2024) Key role of ERβ in estrogenic regulation of neuronal mitochondrial function. Alzheimer's Dement 20:e086633 Wang T et al (2025) Accelerated midlife endocrine and bioenergetic brain aging in APOE4 females. Front Aging Neurosci 17:1632877 Xu X-W et al (2008) Effects of phytoestrogen on mitochondrial structure and function of hippocampal CA1 region of ovariectomized rats. Cell Mol Neurobiol 28(6):875–886 Yuan S et al (2025) Brain structural alterations in young women with premature ovarian insufficiency: Implications for dementia risk. Alzheimer's Dement 21(3):e70111 Zhao L et al (2002) Neuroprotective and neurotrophic efficacy of phytoestrogens in cultured hippocampal neurons. Experimental Biology Med 227(7):509–519 Zhao L et al (2013) Early intervention with an estrogen receptor β-selective phytoestrogenic formulation prolongs survival, improves spatial recognition memory, and slows progression of amyloid pathology in a female mouse model of Alzheimer's disease. J Alzheimer’s Disease 37(2):403–419 Zhou J et al (2026) Fertility under fire: how chemotherapy harms the ovaries and the science fighting back? Journal of Ovarian Research Figures Figures are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files GRAPHICALABSTRACT.docx SUPPLEMENTARYFILE1VCD.docx SUPPLEMENTARYFILE2BLOTS.docx Figures.pdf Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 24 Apr, 2026 Reviews received at journal 23 Apr, 2026 Reviews received at journal 16 Apr, 2026 Reviewers agreed at journal 15 Apr, 2026 Reviews received at journal 13 Apr, 2026 Reviewers agreed at journal 10 Apr, 2026 Reviewers agreed at journal 08 Apr, 2026 Reviewers invited by journal 08 Apr, 2026 Editor assigned by journal 07 Apr, 2026 Submission checks completed at journal 07 Apr, 2026 First submitted to journal 01 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9291892","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":622516582,"identity":"1d67cca9-d0f4-433b-a35c-f750f28fb760","order_by":0,"name":"Ambika Shandilya","email":"","orcid":"","institution":"National Institute of Pharmaceutical Education and Research","correspondingAuthor":false,"prefix":"","firstName":"Ambika","middleName":"","lastName":"Shandilya","suffix":""},{"id":622516583,"identity":"83631d8c-04ef-4a15-bbb4-7f6e2ce08d45","order_by":1,"name":"Suhas Hajare","email":"","orcid":"","institution":"National Institute of Pharmaceutical Education and Research","correspondingAuthor":false,"prefix":"","firstName":"Suhas","middleName":"","lastName":"Hajare","suffix":""},{"id":622516584,"identity":"28aeb162-2bef-4f65-8032-46c043bed758","order_by":2,"name":"Dube Aakash Arwind","email":"","orcid":"","institution":"National Institute of Pharmaceutical Education and Research","correspondingAuthor":false,"prefix":"","firstName":"Dube","middleName":"Aakash","lastName":"Arwind","suffix":""},{"id":622516585,"identity":"6e62e1fc-a2a6-4a99-85be-a76e016d7cf1","order_by":3,"name":"Chander Kant Giri","email":"","orcid":"","institution":"National Institute of Pharmaceutical Education and Research","correspondingAuthor":false,"prefix":"","firstName":"Chander","middleName":"Kant","lastName":"Giri","suffix":""},{"id":622516586,"identity":"ac51911d-2c58-4b3d-b630-b3df89c7cc5f","order_by":4,"name":"Rahul Gajbhiye","email":"","orcid":"","institution":"National Institute of Pharmaceutical Education and Research","correspondingAuthor":false,"prefix":"","firstName":"Rahul","middleName":"","lastName":"Gajbhiye","suffix":""},{"id":622516587,"identity":"2bff3646-a55e-4564-8680-b8a6177bf751","order_by":5,"name":"V. Ravichandiran","email":"","orcid":"","institution":"Delhi Pharmaceutical Sciences and Research University (DPSRU)","correspondingAuthor":false,"prefix":"","firstName":"V.","middleName":"","lastName":"Ravichandiran","suffix":""},{"id":622516588,"identity":"8d3c2b1e-dadf-4ec0-90e9-4d21feaff673","order_by":6,"name":"Vipan Kumar Parihar","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8ElEQVRIiWNgGAWjYDACZijNJgEkPoAY7KRoYZwBYjDjU44CgFqYeZANwQUMjvMe/Fy4wy6fT7r52WebX9vk+ZgZGD98zMGj5TBfsvTMM8mWbTLHjGfn9t02bGNmYJacuQ23FslmHgNp3jZmAzaJBGPm3J7bjEAtbMy8+LUY/+ZtqwdqSf/MbNlz256gFn5mHjOgLYeBWnKMmRl+3E4kQgtfmjXvmeMgLcWMvQ23k9uYGZvx+oWN/+zh27w7qg3kZ6RvZvjx57bt/Pbmgx8+4tHCwACMC8YGKJuxDUw24FSMqYXhDwHFo2AUjIJRMCIBAA2FRBGWdDG+AAAAAElFTkSuQmCC","orcid":"","institution":"National Institute of Pharmaceutical Education and Research","correspondingAuthor":true,"prefix":"","firstName":"Vipan","middleName":"Kumar","lastName":"Parihar","suffix":""}],"badges":[],"createdAt":"2026-04-01 12:27:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9291892/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9291892/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107705176,"identity":"d3ec4b90-1e23-41bc-9114-703947e5a6bc","added_by":"auto","created_at":"2026-04-24 09:09:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":320663,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9291892/v1/26ed7576-ed22-4c77-a982-c4190c838d09.pdf"},{"id":107012440,"identity":"0b8614ce-8edd-4b82-896d-db57447d4ef5","added_by":"auto","created_at":"2026-04-15 18:13:06","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":516145,"visible":true,"origin":"","legend":"","description":"","filename":"GRAPHICALABSTRACT.docx","url":"https://assets-eu.researchsquare.com/files/rs-9291892/v1/3517afbb6eea0e73422c1852.docx"},{"id":107482484,"identity":"c1560a11-7d90-4ddf-b68a-96916bceb26e","added_by":"auto","created_at":"2026-04-22 02:23:41","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":671399,"visible":true,"origin":"","legend":"","description":"","filename":"SUPPLEMENTARYFILE1VCD.docx","url":"https://assets-eu.researchsquare.com/files/rs-9291892/v1/53fe52fc0e7c54c3c051b300.docx"},{"id":107480813,"identity":"7d03edd8-e52a-4164-ab74-0ebdd25c9b41","added_by":"auto","created_at":"2026-04-22 02:13:42","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":872351,"visible":true,"origin":"","legend":"","description":"","filename":"SUPPLEMENTARYFILE2BLOTS.docx","url":"https://assets-eu.researchsquare.com/files/rs-9291892/v1/68fbb0f51bbb113373e06895.docx"},{"id":107480385,"identity":"954f6949-6801-4286-967d-5d98ac71db70","added_by":"auto","created_at":"2026-04-22 02:09:34","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":2642120,"visible":true,"origin":"","legend":"","description":"","filename":"Figures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9291892/v1/7060d312b85a8dec4237790f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Dietary Phytoestrogen ameliorates Ovarian Toxicant–induced Neurotoxicity: Mechanistic and metabolic insights","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe cessation of ovarian follicular function marks a critical inflexion point in female biological ageing, exerting profound effects on systemic physiology, neuroinflammation and overall brain health. A growing body of evidence suggests that premature or accelerated ovarian failure causes early cognitive decline, mood issues, memory impairment, and increased susceptibility to neurodegenerative diseases such as Alzheimer's disease, dementia, and cognitive rigidity (Voskuhl, Itoh et al. 2024, Mohammad, Finch et al. 2025, Yuan, Gong et al. 2025). The abrupt estrogen depletion accompanying premature ovarian failure disrupts neuroendocrine balance, amplifies neuronal cell senescence, neuroinflammatory signalling, and compromises mitochondrial integrity (Russell, Jones et al. 2019, Platholi, Marongiu et al. 2023, Wang, Mao et al. 2025), processes central to accelerated brain ageing and neurodegeneration. However, the precise molecular mechanisms linking ovarian insufficiency to accelerated neurodegeneration are still poorly understood. AOF creates an abrupt endocrine environment that may exacerbate age-related brain vulnerabilities. This differs from the gradual hormonal changes that occur during natural or physiological menopause. Chronic estrogen deprivation accelerates synaptic loss, reduces neuronal resilience, and triggers glial-mediated persistent neuroinflammation (Platholi, Marongiu et al. 2023, Voskuhl, Itoh et al. 2024, Marongiu, Platholi et al. 2025), all of which are hallmarks of neurodegenerative illness. Understanding these pathways is critical for determining female-specific patterns of premature brain ageing and identifying possibilities for therapeutic treatment to minimise neurodegenerative risk following early ovarian failure.\u003c/p\u003e \u003cp\u003eAOF, which is often imitated using 4-vinylcyclohexene diepoxide (VCD), causes menopause to get underway immediately, resulting in expedited brain ageing, cognitive impairment, and increased susceptibility to neurodegenerative illnesses, including Alzheimer's disease (AD) (Mohammad, Finch et al. 2023, Mohammad, Finch et al. 2025). This situation is analogous to the rapid fall in estrogen and spike in FSH that occurs after ovarian failure. These changes may directly affect neurons (Blair, Bhatta et al. 2015), leading to neuroendocrine dysfunction, brain energy deficits, and increased indices of neuroinflammation. A study investigated structural brain abnormalities in people with idiopathic premature ovarian insufficiency (POI) and discovered that the ultrastructural changes found in POI participants' brains closely resemble those found in early dementia, particularly in areas linked to Alzheimer's disease (AD) (Marongiu \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Yuan, Gong et al. 2025). AOF has been linked to increased amyloid-beta (Aβ) plaque deposition and tau phosphorylation, both of which are hallmarks of Alzheimer's disease pathology (Rosario, Carroll et al. 2006, Platholi, Marongiu et al. 2023, Marongiu, Platholi et al. 2025). A similar study utilising the VCD model found that POF reduces the discriminatory index in the NOR test, suggesting memory impairment. Ovarian failure also causes biochemical abnormalities linked with AD pathology, as shown by decreases in synaptic proteins such as SNAP25 (involved in neurotransmitter release) and NeuN (a marker of mature neurons), particularly during the perimenopausal transition (Mohammad, Finch et al. 2023, Mohammad, Finch et al. 2025). Furthermore, estrogen is a critical regulator of brain energy utilisation, and when AOF occurs, it leads to a loss of estrogen, resulting in severe impairment of mitochondrial functions and brain glucose metabolism. Recent evidence reveals that patients with accelerated ovarian ageing had lower ATP production and more mitochondrial DNA damage in their brains (Wang, Mao et al. 2025). Furthermore, estrogen deficiency increases the reactivity of microglia and astrocytes, resulting in a pro-inflammatory environment and immunogenic changes within the brain (Platholi, Marongiu et al. 2023; Voskuhl, Itoh et al. 2024). Finally, rapid depletion of ovarian hormones triggers a neuropathological cascade that disrupts brain metabolism, increases neuroinflammation, and accelerates the progression of AD, emphasising the importance of prompt intervention in cases of POF. These findings highlight the need for early interventions to reduce the long-term risks of cognitive decline and dementia in women with POI. Hormonal therapy (HT) may provide protection throughout the perimenopausal transition, but only if initiated early enough (within the window of opportunity) to prevent neuronal damage (Russell, Jones et al. 2019, Oppong-Gyebi, Metzger et al. 2022).\u003c/p\u003e \u003cp\u003ePhytoestrogens have recently attracted increased scientific interest due to their well-established neurotrophic, neuroprotective, and neurorestorative potential (Gorzkiewicz, Bartosz et al. 2021, Oppong-Gyebi, Metzger et al. 2022). PED has a variety of pharmacological activities, including preventing neuronal cell death, combating free radicals, and decreasing inflammatory molecular signatures (Echeverria, Echeverria et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). It also promotes mitochondrial function and protects brain cells from various forms of oxidative and neuroimmune readouts (Zhao, Chen et al. 2002, Moreira, Silva et al. 2017, Ronchetti, Labombarda et al. 2025). PED is a promising option for treating neurodegenerative diseases due to its versatility. Nonetheless, PED, like many other natural compounds, is expected to act via multiple molecular pathways and targets, complicating understanding of its precise mechanism of action (Xu, Shi et al. 2008). While its neuroprotective effects have been demonstrated across a variety of experimental settings, the essential molecular pathways underlying these effects remain poorly understood and warrant further exploration.\u003c/p\u003e \u003cp\u003eWe used an integrative approach that included global brain metabolomics, neurobehavioral analyses, molecular biology methods, and cell-specific immunohistochemical analyses to predict and confirm the potential mitochondrial drug targets and neuroinflammatory signalling pathways that PED may engage in to improve cognitive outcomes. The purpose of this comprehensive investigation is to help us understand the therapeutic potential of PED and to identify the inflammopharmacological mechanisms underlying the relationship between endocrine failure and cerebral degeneration. This research provides a scientifically grounded alternative to ERT in AOF disease models by integrating evidence from maladaptive neuroinflammatory adaptations, mitochondrial drug targets, and global brain metabolic signalling pathways.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eExperimental Animals:\u003c/strong\u003e This experiment recruited female C57BL/6 mice aged 8 to 10 weeks and weighing 20 to 25 grams. The mice\u0026apos;s living environment was meticulously controlled for light/dark cycles, temperature, and relative humidity. All animals were procured from the National Institute of Nutrition (NIN), Hyderabad and given a week to become accustomed to the amenities at the Central Animal House facility at NIPER, Hajipur. The animals were housed in groups of eight and given free access to a rodent chow diet and filtered water. To minimise the impact of daily fluctuations, behavioural evaluations were conducted during the light cycle, specifically from 9:00 to 14:00 hours\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Statement:\u0026nbsp;\u003c/strong\u003eIn accordance with Indian government regulations, the Institutional Animal Ethics Committee (IAEC) at NIPER, Hajipur, approved the study\u0026apos;s experimental protocol (\u003cstrong\u003eIAEC Protocol no. NIPER-H/IAEC/66/23\u003c/strong\u003e). All applicable guidelines of the institutional Committee for the Control and Supervision of Experiments on Animals (CCSEA) for the proper care, handling, and use of laboratory animals were strictly followed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDrugs and Chemicals:\u003c/strong\u003e The ovarian toxin 4-vinylcyclohexene diepoxide (VCD) was obtained from Sigma-Aldrich, and cold-pressed pure corn oil (vehicle) was purchased from Deve Herbes on Amazon India. Ovotoxin VCD (Catalog #94956-100ML, Sigma Aldrich, purity \u0026gt;98%\u003cstrong\u003e)\u0026nbsp;\u003c/strong\u003ewas dissolved in corn oil and administered at a dose of 160 mg/kg, at a volume of 10 ml/kg via the intraperitoneal (\u003cem\u003ei.p.\u003c/em\u003e) route, once daily for 15 consecutive days (Niu, Miao et al. 2025). \u003cem\u003eThe detailed method of AOF induction is elaborated in \u003cstrong\u003eSupplementary File 1\u003c/strong\u003e.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExperimental Design:\u003c/strong\u003e After confirming chemically-induced expedited ovarian failure and chronic estrogen depletion, the mice were arbitrarily separated into four groups (n=8 mice each group): (i) NCD group comprising of control mice, (ii) NCD+PED group, comprising of normal mice receiving phytoestrogen diet treatment (positive control), (iii) VCD group, 4-vinyl cyclohexene diepoxide challenged AOF mice, (iv) VCD+PED group, 4-vinyl cyclohexene diepoxide intoxicated mice, receiving phytoestrogen dietary treatment intervention. The PED was administered at \u003cem\u003ead libitum\u003c/em\u003e dosages for four weeks that were selected based on our previous findings. A detailed PED composition is available in \u003cstrong\u003e\u003cem\u003eSupplementary File 1\u003c/em\u003e\u003c/strong\u003e. After the behavioural tests, the mice were deeply anaesthetised using isoflurane (induction at 4\u0026ndash;5%, maintenance at 1.5\u0026ndash;2% in 100% oxygen) delivered via a precision vaporiser and euthanised via intracardiac perfusion. The saline-perfused brains were used for biochemical analysis (ELISA, immunoblotting, metabolomics analysis), whereas the PFA-perfused brains were used for immunohistochemical (IHC) and immunofluorescence (IF) assessments. \u003cstrong\u003e\u003cem\u003eA detailed experimental protocol schedule and treatment regimen are depicted in Figure 1.\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. 1:\u003c/strong\u003e Experimental timeline for inducing accelerated ovarian failure (AOF) in C57BL/6 mice. Fifteen concurrent intraperitoneal intoxication of ovatoxin VCD (at the dose of 160mg/kg) were used to induce POI in mice, followed by PED treatment intervention for four consecutive weeks. Twenty-four hours following the final behavioural test, the animals were deeply anaesthetised, euthanised, and their brains were transcardially perfused, carefully dissected, and stored at -80\u0026deg;C until they could be used for biochemical assessments like ELISA, western blotting, metabolomics analysis and immunohistochemical assessments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBehavioural Assessments:\u003c/strong\u003e All groups of animals (with 8 mice per group) underwent a series of neurobehavioral assessments to evaluate mood, memory and cognitive functions, including the following tests in sequence: novel object recognition test (NORT) for assessing recognition memory, elevated plus maze test (EPMT) for assessing anxiety-like behavior, forced swim test (FST) for evaluating depressive-like behavior, and water maze test (WMT) for investigating spatial learning, navigation memory and cognitive flexibility. Following a week of acclimatisation period, the animals were familiarised with the arena in the absence of stimuli for 10 minutes each day over 2 days prior to the initiation of actual behavioural assessments (Kesharwani, Sree et al. 2025). A detailed protocol for behavioural tests conducted is provided in \u003cstrong\u003e\u003cem\u003eSupplementary File 1\u003c/em\u003e\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimal Sacrifice, Method of Euthanasia and Tissue Harvesting for Biochemical Evaluation:\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePost-behavioural assessments, experimental animals were divided into two distinct cohorts for tissue harvesting. In the first cohort, mice were deeply anaesthetised with isoflurane (induction at 4\u0026ndash;5%, maintenance at 1.5\u0026ndash;2% in 100% oxygen) in an anaesthesia chamber. The depth of anaesthesia was strictly monitored by the loss of the pedal withdrawal reflex prior to the commencement of perfusion.\u003c/p\u003e\n\u003cp\u003eOnce the surgical plane of anaesthesia was confirmed by the absence of pedal reflex, animals were euthanised via decapitation while under the isoflurane, followed by intracardiac perfusion with 0.9% normal saline (NS). The saline-perfused brains were carefully harvested for immunoassays (ELISA), western blotting, and global metabolomic quantification. The second cohort was quickly flushed with NS, followed by transcardially perfusion with 4% paraformaldehyde (PFA). Decapitated, protein-fixed brain specimens were carefully removed from the skull, snap-frozen on ice, and stored at 4\u0026deg;C to be specifically used for further immunohistochemical (IHC) and immunofluorescence (IF) analyses (Kesharwani, Lahamge et al. 2025). \u003cem\u003eA detailed description of the methodologies employed for tissue processing and biochemical measurements is provided in \u003cstrong\u003eSupplementary File 1\u003c/strong\u003e.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiochemical Evaluation:\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunoassay (ELISA):\u003c/strong\u003e A mouse-specific Uniovrsal E2 ELISA kit (ITLK01208) and a mouse aromatase quantitative sandwich ELISA kit (MBS456973) were used to measure 17\u0026beta;-estradiol and aromatase (CYP19A1/estrogen synthase) levels in hippocampal tissue homogenate. The sample absorbance was measured at 450nm using a Multimode Reader (Shandilya, Mehan et al. 2022).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunoblotting:\u003c/strong\u003e Hippocampal tissue was lysed on ice with RIPA lysis buffer and 1% protease inhibitor. Cell lysate protein concentrations were determined using a Merck BCA assay kit. Protein lysates (30-50\u0026mu;g) were separated on 8-12% Tris-glycine SDS-PAGE and transferred to 0.20\u0026mu;m PVDF membranes. Protein molecular weights were measured using a pre-stained protein ladder. After blocking membranes with 5% BSA (Sigma) for 2 hours at 37\u0026deg;C, primary antibodies against NDUFB8, MTCO1, ATP5A1, \u0026beta;-amyloid, ER-\u0026beta;, and BDNF were incubated overnight at 4\u0026deg;C on an orbital shaker. The next day, the membranes were washed three times with TBST and treated with secondary antibodies from the same species for 1 hour at 37\u0026deg;C. The immunoreactive bands were detected by enhanced chemiluminescence and photographed with the Molecular Imager ChemiDoc XRS System after three additional TBST washes. Normalisation loading control was beta-actin. Densitometric analysis was performed in ImageJ to quantify protein expression.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence Analysis:\u003c/strong\u003e Three mice per group underwent intracardiac perfusion with a 0.9% normal saline flush followed by 4% PFA in 0.1M PBS (pH 7.4). After careful dissection, the brains were dehydrated in sucrose gradients until they sank, and then embedded in tissue-embedding media. Cutting 30\u0026mu;m-thick coronal slices of the prefrontal cortex and hippocampus using a Leica Cryostat, samples were collected sequentially on 24-well plates with 0.01 M PBS (including sodium azide). After three 5-minute washes with PBS (1X, pH=7.4), the mice\u0026apos;s frozen brain tissue slices were blocked at room temperature for 30 minutes. Mouse monoclonal antibodies were used to assess the expression of markers, including AIF1/Iba1, HMGB1, TLR-4, NF-\u0026kappa;B, CD68, and TREM2, in blocked sections incubated overnight at 4 \u0026deg;C. The next day, immunotagged sections were washed three times in 0.01 M PBS for 5 minutes each. After incubation with a secondary antibody from the same species for 30\u0026ndash;60 minutes at 37\u0026deg;C in the dark, they were rewashed and stained with DAPI. A Nikon Eclipse AX/AXR confocal digital microscope was used to acquire Z-stacks (1\u0026micro;m intervals across brain tissue) at NIPER, Hajipur central imaging facility.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBrain metabolome profiling through UPLC-MS/MS:\u003c/strong\u003e A global LC-TQ-MS/MS-based mouse brain metabolome was searched for differentially regulated metabolites. MetaboAnalyst 6.0 (https://www.metaboanalyst.ca/) was used to stratify the raw data, deleting features with more than 50% missing values and imputing missing values to lower FDR. Analyses included fold change, PCA, PLS-DA, and univariate statistics (p\u0026lt;0.05). VIP and log2FC \u0026gt; 1 indicated metabolites that were differentiated. For DEM selection, FCs \u0026gt;\u0026plusmn;1.0 and p-values \u0026lt;0.05 were used to compare the control group. VIP ratings above 1.0 imply significance. The functional pathway enrichment is depicted using KEGG ortholog enrichment networks.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis:\u0026nbsp;\u003c/strong\u003eData were presented as mean \u0026plusmn; SEM. We examined (n=8) mice for neurobehavioural assessments and (n=3) for brain metabolomics, western blotting, and immunofluorescence. Experimental group metabolite differences were found using PCA or PLS-DA plots. To compare group behaviour, one-way, two-way, and three-way ANOVAs were used. All analyses used p-values \u0026lt; 0.05 and 95% CIs to assess statistical significance.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePED restored local neuroestrogenic tone and modulated aromatase levels in VCD-mediated ovarian failure\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e17\u0026beta;-estradiol levels and aromatase concentrations were assessed in hippocampal tissue homogenate using an enzyme-linked immunoassay. A two-way ANOVA comparison revealed a significant decline in 17\u0026beta;-estradiol concentration, with significant interaction between VCD exposure and PED administration (F\u003csub\u003e1, 7\u003c/sub\u003e = 8.484, p= 0.0226), as well as significant primary effects of VCD induction (F\u003csub\u003e1, 7\u003c/sub\u003e = 194.9, p\u0026lt; 0.0001), and PED treatment (F\u003csub\u003e1, 7\u003c/sub\u003e = 8.778, p= 0.0210). Our post-hoc test is indicative of functional cerebral hypoestrogenism following VCD-triggered accelerated ovarian follicular depletion. Conversely, estradiol concentrations were modestly restored in the PED-treated groups compared with those in the VCD group (\u003cstrong\u003eFig. 2A\u003c/strong\u003e). On the other hand, two-way ANOVA showed a compensatory upregulation of aromatase (CYP19A1) expression in the hippocampal homogenate of VCD-challenged AOF mice (p= 0.0019), reflecting an intrinsic attempt to counter chronic estrogen deprivation (a homeostatic neuroprotective response). We found significant interaction for aromatase expression between VCD-driven ovarian failure and PED administration (F\u003csub\u003e1, 7\u003c/sub\u003e = 18.27, p = 0.037), as well as significant main effects of VCD-induced ovarian intoxication (F\u003csub\u003e1, 7\u003c/sub\u003e = 23.33, p = 0.0019), and notable effect of PED treatment intervention (F\u003csub\u003e1, 7\u003c/sub\u003e = 56.70, p = 0.0001) on aromatase expression levels. Our post hoc multiple-comparison analysis showed that aromatase levels were substantially upregulated in the VCD-insulted mice group compared with the normal control group. However, PED supplementation partially normalised the dysregulated aromatase levels, consistent with a restoration of estrogenic tone rather than exaggerated compensatory neurosteroidogenesis, indicating compensatory drive (\u003cstrong\u003eFig. 2B\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. 2:\u0026nbsp;\u003c/strong\u003eChronic VCD injections gradually reduced the 17\u0026beta;-estradiol levels (A) but increased aromatase expression (B) in the brains of AOF mice. Treatment with PED was effective in modulating this hormonal imbalance in VCD-challenged, follicular-depleted mice. Statistical analysis was conducted using a two-way ANOVA followed by post hoc Tukey\u0026rsquo;s multiple comparisons. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001****p \u0026lt; 0.0001, (n = 3 assays per group).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePED improved prefrontal cortex-dependent object recognition memory impairment in VCD-challenged AOF mice\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA two-way ANOVA did not find a significant interaction between the PED treatment and VCD-treated groups for total exploration time (F\u003csub\u003e1,7\u0026nbsp;\u003c/sub\u003e= 0.6737, p = 0.4388), indicating that their combined effect did not significantly affect total exploration time for either object in the NOR test. Furthermore, neither PED treatment (F\u003csub\u003e1, 7\u003c/sub\u003e = 0.04763, p=0.8335) nor VCD challenge (F\u003csub\u003e1, 7\u003c/sub\u003e = 0.4602, p=0.5193; \u003cstrong\u003eFig. 3A\u003c/strong\u003e) yielded any significant effects on the total exploration time in the NOR task. This shows that neither VCD exposure nor PED therapeutic treatment impaired locomotor activity in mice. The three-way ANOVA analysis of percentage time spent examining new and familiar items indicated significant effects for novelty preference (F\u003csub\u003e1, 56\u0026nbsp;\u003c/sub\u003e= 57.55, p\u0026lt;0.0001), novelty preference x VCD challenge (F\u003csub\u003e1, 56\u003c/sub\u003e = 4.881, p=0.0313), and novelty preference x PED treatment (p=0.0008). These findings demonstrate that both VCD exposure and PED therapy strongly influenced the percentage of time allocated to examining new items, as shown by the notable triple interaction among novelty preference, VCD, and PED treatment (F\u003csub\u003e1, 56\u0026nbsp;\u003c/sub\u003e= 4.335, p=0.0419). Moreover, Tukey\u0026apos;s multiple-comparison test indicated that both the NCD group and the NCD+PED groups showed a distinct preference for novelty, as evidenced by increased time spent examining new items (NCD: p\u0026lt;0.0001; NCD+PED: p\u0026lt;0.0005). The VCD group that did not receive PED treatment showed no preference for novel experiences (p = 0.9892). Nevertheless, mice in the VCD group that received PED treatment did not show these abnormalities and maintained their capacity to identify the unfamiliar item (p = 0.0008; \u003cstrong\u003eFig. 3B\u003c/strong\u003e). The two-way ANOVA analysis of DI revealed a significant interaction between PED therapy and VCD, indicating that their combined effect had a substantial impact on DI (F\u003csub\u003e1, 7\u0026nbsp;\u003c/sub\u003e= 6.989, p = 0.0332). However, when considering the individual factors, PED treatment shows a significant modulatory effect on DI (F\u003csub\u003e1, 7\u003c/sub\u003e = 4.213, p=0.0792), while VCD also has a significant deleterious impact (F\u003csub\u003e1, 7\u003c/sub\u003e = 15.18, p=0.0059). Furthermore, Tukey\u0026rsquo;s multiple comparison test demonstrated that VCD-induced AOF mice treated with PED exhibited better intact memory and higher DI with respect to VCD-challenged animals (VCD vs VCD+PED, p\u0026lt;0.0001; \u003cstrong\u003eFig.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e3C\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. 3:\u003c/strong\u003e PED treatment improves mPFC-dependent object recognition memory in AOF mice. NORT: The total exploration time did not vary across the groups (A). The reduction in novelty preference induced by the VCD challenge was significantly reversed by PED treatment, as evidenced by increased time spent exploring the novel object (B) and enhanced DI (C) in the NOR task. Total exploration time (A) and DI (C) were analysed by two-way ANOVA followed by Tukey\u0026rsquo;s multiple comparison test, while the percentage time spent exploring novel and familiar objects (B) was analysed by three-way ANOVA following Tukey\u0026rsquo;s multiple comparisons. * p \u0026lt; 0.05, **p \u0026lt; 0.01; ***p \u0026lt; 0.001; and ****p \u0026lt; 0.0001, (n = 8 mice per group).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePED alleviated anxious behaviour and behavioural despair driven by chronic estrogen deficit in VCD-exposed AOF mice\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSubsequently, we used the EPM test, a well-known approach for measuring anxiety-like behaviour in rodents. An analysis of total time spent in the open arm revealed significant main effects of VCD induction (F\u003csub\u003e1, 7\u003c/sub\u003e = 31.76, p = 0.0008) and PED treatment intervention (F\u003csub\u003e1, 7\u003c/sub\u003e = 11.43, p = 0.0117), as well as a significant interaction between VCD and PED interventions (F\u003csub\u003e1, 7\u003c/sub\u003e = 14.91, p = 0.0062, \u003cstrong\u003eFig. 4A\u003c/strong\u003e). Furthermore, a two-way ANOVA analysis of the percentage of open-arm entries revealed a significant interaction between VCD insult and PED regimen (F\u003csub\u003e1, 7\u003c/sub\u003e = 8.366, p = 0.232), as well as significant main effects of VCD administration (F\u003csub\u003e1, 7\u003c/sub\u003e = 47.65, p = 0.0002) and PED treatment intervention (F\u003csub\u003e1, 7\u0026nbsp;\u003c/sub\u003e= 14.57, p = 0.0066, \u003cstrong\u003eFig. 4B\u003c/strong\u003e). Our post hoc Tukey\u0026rsquo;s multiple-comparison test revealed that VCD mice treated with PED spent significantly more time (p=0.0074) in the open arms, and demonstrated a comparatively greater percentage of entries to the open arm (p=0.0278) than those treated with the vehicle. As a result, we can conclude that our PED treatment effectively mitigated accelerated ovarian failure-induced affective disorder (\u003cstrong\u003eFig. 4A, 4B)\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA two-way ANOVA was conducted to assess the effects of both the VCD exposure and PED therapeutic administration on the duration of immobility. The findings revealed a noteworthy two-way interaction between VCD induction and PED treatment groups (F1, 7 = 12.80, p = 0.009). Furthermore, significant main effects were observed for both VCD administration (F\u003csub\u003e1, 7\u003c/sub\u003e = 33.35, p = 0.0007) and PED treatment (F\u003csub\u003e1, 7\u003c/sub\u003e = 25.60, p = 0.0015). These outcomes suggest that exposure to VCD and/or PED significantly impacts the immobility duration in mice during the FST. Our post hoc analysis with Tukey\u0026rsquo;s multiple-comparison test also revealed that VCD-treated mice showed substantial depressive-like behaviour (p=0.0009), but PED administration significantly reduced the immobility duration (p = 0.0051; \u003cstrong\u003eFig. 4C\u003c/strong\u003e). The noteworthy reduction in the immobility time in the FST indicates a substantial decrease in depressive-like behaviour in VCD+PED-treated AOF mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. 4:\u003c/strong\u003e PED intervention ameliorates anxiety and depressive-like behaviour in AOF mice. EPM: AOF mice exhibited increased affective dysfunction, as evidenced by decreased total time spent in the open arms (A) and a lower percentage of open-arm entries (B), which the PED treatment regimen effectively alleviated.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFST: AOF mice displayed greater depressive-like behaviour, as evidenced by their enhanced time spent floating immobile (C). However, PED treatment significantly reversed the behavioural despair in VCD-triggered AOF mice. Data were analysed by two-way ANOVA followed by post-hoc Tukey\u0026rsquo;s multiple comparison test. * p \u0026lt; 0.05, **p \u0026lt; 0.01; ***p \u0026lt; 0.001; and ****p \u0026lt; 0.0001, (n = 8 mice per group).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePED retrieved hippocampal-dependent spatial and navigation memory impairment and conferred cognitive flexibility in VCD-driven AOF mice\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mice were subjected to a water-based navigation task in a circular pool filled with opaque water and a submerged platform to evaluate their hippocampal-dependent spatial memory. During the initial learning sessions (day 1-day 4), there were progressive, similar decreases in latencies to reach the platform (escape latency time) across all the groups, as indicated by a two-way ANOVA. In the final session (day 5), VCD-exposed mouse groups exhibited a significant increase in escape latency time (ELT) to locate the target quadrant compared with normal controls, indicating a significant impact of chronic estrogen deficiency induced by accelerated ovarian follicular depletion on memory retrieval. A two-way ANOVA revealed a significant interaction between training days and experimental groups (F\u003csub\u003e3.848, 26.94\u0026nbsp;\u003c/sub\u003e= 6.538, p = 0.009), as well as significant main effects of learning days (F\u003csub\u003e2.515, 17.60\u0026nbsp;\u003c/sub\u003e= 332.2, p \u0026lt; 0.0001) and groups (F\u003csub\u003e2.488, 17.42\u0026nbsp;\u003c/sub\u003e= 56.51, p \u0026lt;0.0001, \u003cstrong\u003eFig. 5A\u003c/strong\u003e).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThis was further verified by comparing dwell intervals on the platform across the mouse groups. Unlike controls, mice subjected to a VCD insult spent significantly less time on the target platform than the normal mouse cohort (p=0.0004), indicating that exposure to chemically induced\u0026nbsp;AOF progressively diminishes memory retention ability. A two-way ANOVA revealed a significant interaction between VCD challenge and PED intervention (F\u003csub\u003e1, 7\u0026nbsp;\u003c/sub\u003e= 21.26, p = 0.0025), as well as significant main effects were observed for both VCD administration (F\u003csub\u003e1, 7\u0026nbsp;\u003c/sub\u003e= 44.28, p = 0.0003) and PED treatments (F\u003csub\u003e1, 7\u0026nbsp;\u003c/sub\u003e= 8.068, p = 0.0250, \u003cstrong\u003eFig. 5B\u003c/strong\u003e). The aforementioned findings were further supported by the number of crossings on the platform area. The mice that received the VCD intoxication had fewer platform area crossings than the control group (NCD v/s VCD: p = 0.0070; VCD v/s VCD+PED: p = 0.0188). A two-way ANOVA found a significant interaction between VCD challenge and PED intervention (F\u003csub\u003e1, 7\u0026nbsp;\u003c/sub\u003e= 8.374, p = 0.0232), as well as significant primary effects for both VCD administration (F\u003csub\u003e1, 7\u0026nbsp;\u003c/sub\u003e= 14.30, p = 0.0232) and PED treatments (F\u003csub\u003e1, 7\u0026nbsp;\u003c/sub\u003e= 13.17, p = 0.0084, \u003cstrong\u003eFig. 5C\u003c/strong\u003e). \u0026nbsp;On Day 6\u003csup\u003eth\u003c/sup\u003e (24 hours post the last learning session), the target platform was removed for the probe test assessment to assess time spent by the animals in the target platform quadrant (TSTQ). Both control groups (NCD, NCD+PED) consistently preferred the platform quadrant over the other quadrants, but VCD-treated mice showed nearly equal preferences across all four quadrants (NCD v/s VCD: p=0.0024), with no distinct inclination toward the previous target zone, indicating a deficit in spatial learning and memory retrieval. A two-way ANOVA revealed a significant double-point interaction between VCD induction and the PED-treated groups (F\u003csub\u003e1, 7\u003c/sub\u003e = 12.90, p = 0.0088). Furthermore, significant main effects were observed for both VCD administration (F\u003csub\u003e1, 7\u0026nbsp;\u003c/sub\u003e= 26.55, p = 0.0013) and PED treatments (F\u003csub\u003e1, 7\u0026nbsp;\u003c/sub\u003e= 2.577, p = 0.1525, \u003cstrong\u003eFig. 5D).\u0026nbsp;\u003c/strong\u003eInterestingly, PED treatment generally showed a pattern of improvement in contextual memory retention ability, as reflected in increased time spent in the platform quadrant (VCD v/s VCD+PED: p=0.0066), substantially greater no.of platform area crossings (VCD v/s VCD+PED: p=0.0188) and comparatively enhanced dwell time on the platform (VCD v/s VCD+PED: p=0.0033), however, the extent to which this improvement was mechanistically translatable to a direct reversal of VCD-triggered cognitive deficits remained inconsistent, prompting further investigation into the underlying cellular and molecular mechanisms (\u003cstrong\u003eFig. 5A-D)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. 5:\u003c/strong\u003e PED regimen mitigates hippocampal-dependent spatial learning impairment and cognitive rigidity in AOF mice. (A) escape latency time (ELT), (B) platform dwell time, (C) number of platform area crossings, and (D) time spent in the target quadrant (TSTQ) in the WMT. Data were analysed by two-way ANOVA followed by post-hoc Tukey\u0026rsquo;s multiple comparison test. * p \u0026lt; 0.05, **p \u0026lt; 0.01; ***p \u0026lt; 0.001; and ****p \u0026lt; 0.0001, (n = 8 mice per group).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eCellular and Molecular Results:\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePED reversed the hippocampal mitochondrial bioenergetic failure in VCD-elicited POF mice\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMitochondrial dysfunction is a key pathomechanism of neurodegeneration caused by VCD-provoked follicular depletion and ovarian failure, largely due to mitochondrial functional impairment, altered respiratory chain (OXPHOS) complexes and electron transport chain (ETC) enzyme dysfunction. To establish PED therapy\u0026apos;s modulatory effects on mitochondrial functional signalling during chemically-induced ovarian senescence, comprehensive western blot analysis was performed on hippocampal tissue to determine the expression levels of key mitochondrial functional markers, including NDUFB8, MTCO1, and ATP5A1. The immunoblots were analysed using a two-way ANOVA with mean \u0026plusmn; SEM and Tukey\u0026apos;s multiple-comparison post-hoc test (n=3). The analysis revealed a significant double interaction between VCD induction and PED treatment (NDUFB8: F\u003csub\u003e1, 2\u003c/sub\u003e = 8939, p=0.0001; MTCO1: F\u003csub\u003e1, 2\u003c/sub\u003e = 1888, p=0.0005; ATP5A1: F\u003csub\u003e1, 2\u003c/sub\u003e = 211.4, p=0.0047), indicating a combined influence of these variables. Moreover, the VCD challenge in mice and the PED treatment intervention each exhibited significant individual effects. Notably, NDUFB8, MTCO1, and ATP5A1 expression decreased significantly (NDUFB8: F\u003csub\u003e1, 2\u003c/sub\u003e = 567.9, p=0.0018; MTCO1: F\u003csub\u003e1, 2\u003c/sub\u003e = 4517, p=0.0002; ATP5A1: F\u003csub\u003e1, 2\u003c/sub\u003e = 3028, p=0.0003) in the VCD-exposed AOF mice group compared with the normal control group. Conversely, treatment with PED effectively counteracted these mitochondrial functional anomalies, leading to significant preservation/restoration of the cerebral bioenergetic integrity (NDUFB8: F\u003csub\u003e1, 2\u003c/sub\u003e = 20.38, p=0.0457; MTCO1: F\u003csub\u003e1, 2\u003c/sub\u003e = 1081, p=0.0009; ATP5A1: F\u003csub\u003e1, 2\u003c/sub\u003e = 49.52, p=0.0196, \u003cstrong\u003eFig. 6A-C\u003c/strong\u003e). These ameliorative effects of PED demonstrate their potential to restore altered brain mitochondrial metabolism and enhance neuronal resilience in the context of AOF-induced follicular depletion, caused by the persistent challenge of ovarian intoxicants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePED suppresses amyloidogenic burden and reinstates ER\u0026beta;\u0026ndash;BDNF neuroprotective signalling in VCD-conditioned mice\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo interrogate the complex interplay among ovarian follicular depletion, neuroendocrine hormone loss, dysregulated estrogen receptor signalling, amyloid pathology, and neuronal atrophy, we sought to determine how premature ovarian insufficiency contributes to heightened AD susceptibility in women (Ansere, Ali-Mondal et al. 2021). Consistent with the reported findings, our immunoblotting results on hippocampal tissues demonstrated a significant interaction between VCD intoxication and PED treatment (\u0026beta;-amyloid: F\u003csub\u003e1, 2\u003c/sub\u003e = 68.39, p=0.0143; ER-\u0026beta;: F\u003csub\u003e1, 2\u003c/sub\u003e = 828.3, p=0.0012; BDNF: F\u003csub\u003e1, 2\u003c/sub\u003e = 54.38, p=0.0179), indicating a combined influence of these variables. Additionally, a marked upregulation of amyloid beta plaque deposition (F\u003csub\u003e1,2\u0026nbsp;\u003c/sub\u003e= 451.5, p = 0.0022), concomitant with dynamically downregulated ER-\u0026beta; expression (F\u003csub\u003e1,2\u0026nbsp;\u003c/sub\u003e= 228.5, p =0.0043) and significant inhibition of BDNF levels (F\u003csub\u003e1,2\u0026nbsp;\u003c/sub\u003e= 292.7, p =0.0034) was found in the VCD-challenged mice group compared to the normal control mice group. Strikingly, administration of the PED therapeutic intervention partially normalised these molecular perturbations, attenuating beta-amyloid plaque deposition (F\u003csub\u003e1,2\u0026nbsp;\u003c/sub\u003e= 66.00, p = 0.0148), while restoring BDNF abundance (F\u003csub\u003e1,2\u0026nbsp;\u003c/sub\u003e= 89.37, p = 0.0110) and modulating ER-beta signalling (F\u003csub\u003e1,2\u0026nbsp;\u003c/sub\u003e= 36.37, p = 0.0264) (\u003cstrong\u003eFig. 6D-F\u003c/strong\u003e). These neuromodulatory outcomes collectively underscore PED\u0026rsquo;s capacity to suppress accelerated ovarian senescence-induced neurodegeneration and reinstate neuroprotective estrogen beta signalling, thereby substantiating its neurotrophic and neurorestorative efficacy in the context of AOF.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. 6:\u003c/strong\u003e PED therapy elicits mitochondrial functional reconstitution, suppresses amyloidogenic burden, downregulates neuroinflammatory cascades, and reinstates estrogenic receptor \u0026beta;-mediated neurotrophic signalling in the hippocampus of AOF mice. (A) Representative immunoblots of mitochondrial respiratory chain complexes NDUFB8, MTCO1 and ATP5A1 in the hippocampal tissue homogenate of mice. Bar graph showing relative levels of (A) NDUFB8, (B) MTCO1, (C) ATP5A1, (D) \u0026beta;-amyloid, (E) ER-\u0026beta;, (F) BDNF, (G) Iba1, and (H) NF-ϏB. Data are represented as mean \u0026plusmn; SEM. Statistical analysis was conducted by two-way ANOVA, followed by Tukey\u0026rsquo;s multiple comparisons. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, (n = 3 immunoblots per group).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePED attenuates microgliosis-induced perpetuating neuroinflammatory loops and restores mPFC-mediated neuroimmune responses in AOF mice\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further elucidate how microglial phenotypes are influenced by the gonadal hormone milieu and to better understand the neuromodulatory effects of PED treatment on cerebral inflammatory cascades following an ovatoxin VCD insult, double-labelling immunofluorescence staining was performed in the medial PFC. Interestingly, AOF-driven microglial inflammatory polarisation was examined by assessing the expression levels of key neuroinflammatory markers, including HMGB1, NF-\u0026kappa;B, TLR-4, CD68, and TREM2, against Iba1-positive microglia. A two-way ANOVA indicated a significant interaction between VCD induction and PED treatment (HMGB1: F\u003csub\u003e1,2\u0026nbsp;\u003c/sub\u003e= 187.8, p = 0.0053, NF-kB: F\u003csub\u003e1,2\u0026nbsp;\u003c/sub\u003e= 44.25, p = 0.0219; TLR-4: F\u003csub\u003e1,2\u0026nbsp;\u003c/sub\u003e= 585.2, p = 0.0017; CD68: F\u003csub\u003e1,2\u0026nbsp;\u003c/sub\u003e= 51448, p \u0026lt; 0.0001; TREM2: F\u003csub\u003e1,2\u0026nbsp;\u003c/sub\u003e= 72.89, p = 0.0134). The VCD-treated AOF mice group exhibited a marked augmentation in the fluorescence intensity of these microglial-mediated inflammatory markers, reflecting escalation of brain inflammatory phenotypic shift following accelerated ovarian senescence (Iba1+ HMGB1: F\u003csub\u003e1,2\u0026nbsp;\u003c/sub\u003e= 964.5, p=0.0010; Iba1+NF-ϏB: F\u003csub\u003e1,2\u0026nbsp;\u003c/sub\u003e= 1.702, p=0.0006; Iba1+TLR-4: F\u003csub\u003e1,2\u003c/sub\u003e=68.23, p = 0.0143, Iba1+CD68: F\u003csub\u003e1,2\u0026nbsp;\u003c/sub\u003e= 96.19, p =0.0102; Iba1+TREM2: F\u003csub\u003e1,2\u0026nbsp;\u003c/sub\u003e= 1003, p =0.0010). Conversely, treatment with PED resulted in a marked downregulation of neuroinflammatory indicators like HMGB1 (F\u003csub\u003e1,2\u0026nbsp;\u003c/sub\u003e= 70.35, p =0.0139), NF-\u0026kappa;B (F\u003csub\u003e1,2\u0026nbsp;\u003c/sub\u003e=10.40, p =0.0842), TLR-4 (F\u003csub\u003e1,2\u0026nbsp;\u003c/sub\u003e= 980.0, p =0.0010), CD68 (F\u003csub\u003e1,2\u0026nbsp;\u003c/sub\u003e=74.87, p =0.0131), and TREM2 expression (F\u003csub\u003e1,2\u0026nbsp;\u003c/sub\u003e= 574.1, p =0.0017), suggesting that PED treatment potentially mitigates microgliosis, forestalls innate immune signalling and attenuates self-propagating neuroimmune dysfunction in the mouse PFC in the context of POF (\u003cstrong\u003eFig. 7A-E\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. 7:\u003c/strong\u003e The PFC of VCD-challenged AOF mice showed increased levels of inflammatory mediators, including HMGB1, NF-kB, TLR-4, CD68, and TREM2, whereas PED administration substantially inhibited the expression of these inflammaging markers. Representative double immunofluorescence staining of Iba1+ microglial cells expressing inflammatory markers (A) Iba1+HMGB1, (B) Iba1+NF-ϏB, (C) Iba1+TLR-4, (D) Iba1+CD68, (E) Iba1+TREM2, (Scale bar: 50\u0026mu;m). Analysis of fluorescence intensity of (A) HMGB1, (B) NF-ϏB, (C) TLR-4, (D) CD68, (E) TREM2. Data are represented as mean \u0026plusmn; SEM. \u0026nbsp; Statistical analysis was conducted by two-way ANOVA, followed by Tukey\u0026rsquo;s multiple comparisons. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001, ns: non-significant, (n = 3 cerebral slices per group).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePED confers metabolic neuroprotection in VCD ovatoxin-intoxicated AOF mice\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical modelling of brain metabolome data using multivariate analysis identified differentially expressed metabolites and dysregulated metabolic pathways in VCD-challenged mice and evaluated the biochemical impact of PED treatment following VCD intoxication. In our metabolomic data, 45 DEMs were identified and quantified in NCD vs VCD samples, and 39 in VCD vs VCD+PED therapy samples. Comparing VCD-induced AOF mice to normal control mice, 31 metabolites were considerably elevated, and 14 were dramatically downregulated. Compared to (VCD+PED) group, VCD-intoxicated mice\u0026apos;s brains had 24 upregulated and 15 downregulated metabolites.\u003c/p\u003e\n\u003cp\u003eVIP-ranked metabolomic patterns show that VCD-induced premature ovarian failure severely alters estrogen-regulated brain metabolism. Dimethisterone, a high-impact metabolite, mostly maps to steroid and neurosteroid dysregulation, demonstrating reduced local estradiol synthesis and increased oxidative steroid turnover. In VCD-induced mice, the accumulation of fatty acyls (1-hexadecanoyl derivatives, 1-dodecanoyl-2-derivatives) and sphingolipid intermediates (apidiasphingosine, dehydrophytosphingosine, 1-hexadecanoyl species) indicates mitochondrial \u0026beta;-oxidation deficits, energy imbalance, pro-apoptotic signalling, and neuronal membrane instability. In VCD-insulted animals, elevated arachidonic acid\u0026ndash;derived prostaglandins (PGD2 intermediates) indicate a lack of estrogenic control over inflammatory lipid signalling. In the VCD-challenged group, lower levels of ascorbate-related metabolites indicate a disturbed redox equilibrium and increased oxidative stress. These changes cause neuroenergetic stress, redox imbalance, and lipid-driven neuroinflammatory pathways, which underlie VCD-induced accelerated brain ageing (\u003cstrong\u003eFig.8A\u003c/strong\u003e). However, estrogen-receptor-mediated metabolic repair of the AOF brain is shown by PED therapy\u0026apos;s inhibition of VCD-induced anomalies in steroid metabolism, inflammatory lipid signalling, oxidative stress pathways, and membrane lipid homeostasis. PED restored 3-epi-5,6-trans steroidal derivative, attenuated lipid-driven neuroinflammation (O-arachidonoyl derivative; 9S,15S-dihydroxy lipid derivative), antioxidant buffering and estrogen-dependent redox control (2-amino-3-oxo derivative; 9-methoxy-hept derivative), neuronal membrane integrity and synaptic integrity (persediene), and metabolic efficiency and neuronal resilience\u0026nbsp;(\u003cstrong\u003eFig.8B).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe most common DEMs were used for pathway mapping and enrichment analysis to identify metabolic pathways that differentiated control, VCD-challenged, and VCD+PED\u0026ndash;treated mouse brains. Interestingly, VCD induction increased brain lipid-inflammation metabolic reprogramming, as indicated by the functional enrichment network. Glycerophospholipid metabolism had the largest node size and the highest connectivity in the pathway topology, indicating the most extensive alteration of neural membrane phospholipids, which may serve as precursors to persistent inflammatory mediators and cellular stress responses. A strong metabolic cluster for \u0026alpha;-linolenic acid, linoleic acid, and arachidonic acid metabolism suggests coordinated abnormalities in polyunsaturated fatty acid metabolism, indicating lipid remodelling, immune activation, and the biosynthesis of inflammatory mediators. The arachidonic acid pathway is a key node in the network, producing eicosanoids (prostaglandins, leukotrienes) that activate the immune system and cause chronic neuronal inflammatory rewiring. The network\u0026apos;s peripheral node, which illustrates sphingolipid metabolism, has been shown to affect ceramide-mediated downstream inflammatory processes, neuronal death, and immunological modulation, suggesting a role in the overall inflammatory metabolic landscape (\u003cstrong\u003eFig.\u003c/strong\u003e \u003cstrong\u003e8C)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. 8:\u0026nbsp;\u003c/strong\u003ePED treatment intervention confers global metabolic neuroprotection in VCD-challenged AOF mice. Representative 2DPLS-DA plot, VIP score plot, biplot, and heat map visualisation of DEMs when comparing NCD against VCD-inducted group (A), and VCD against VCD+PED groups (B). The colour blue denotes downregulated/underexpressed metabolites, whereas red colour indicates upregulated/overexpressed metabolites in the VIP score plot. The heat clustering analysis highlights metabolites with significant increases or decreases in expression. Statistical cut-off was set at log2FC\u0026gt; 1 and p-values \u0026lt; 0.05. Sample size: (n = 3 in each group). The joint pathway enrichment network was derived from the KEGG database, and the circle size reflects the proportion of commonly enriched metabolites (C).\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present study provides compelling behavioural, biochemical, and molecular evidence that persistent estrogen deficiency, modelled through VCD-induced accelerated ovarian failure (AOF), precipitates a broad neurobehavioural, neuroinflammatory and neurodegenerative phenotype encompassing cognitive impairment, affective dysregulation, mitochondrial dysfunction, amyloidogenic vulnerability, dysregulated estrogen signalling, neuronal atrophy, differential brain metabolome expression, microglial overactivation, and neuroinflammatory compromise. Importantly, our findings show that PED therapeutic intervention effectively combats these adverse impacts, implying that it has the potential to be a multi-targeted neurorestorative agent in estrogen-deficient states similar to premature ovarian insufficiency and perimenopausal brain ageing after chemically-induced, follicular-depleted, accelerated ovarian failure.\u003c/p\u003e\n\u003cp\u003eWhile a rapid decline in systemic estrogen is a crucial indicator of accelerated ovarian failure, increasing research shows that the production of estrogen in the brain is essential for maintaining neuronal function and cognitive behaviour (Iqbal, Huang et al. 2024). In POI, brain 17\u0026beta;-estradiol levels decrease significantly, whereas neural aromatase levels increase to compensate. This leads to insufficient local estrogen synthesis, increased neuroinflammation, and quicker brain ageing. Although PED led to comparative increases in hippocampal estradiol levels, aromatase levels were partially reinstated. This observed trend suggests a partial restoration of intracerebral estrogenic tone, which is sufficient to activate downstream neuroprotective systems. It is noteworthy that aromatase activity is limited by the amounts of testosterone and androstenedione present; hence, E2 recovery is low.\u003c/p\u003e\n\u003cp\u003ePED primarily reinstates prefrontal and hippocampal-dependent cognition, with little impact from locomotor factors. The novel object recognition (NOR) deficits observed in mice exposed to VCD indicate that the prefrontal cortex-hippocampal network is not functioning properly, a hallmark of cognitive ageing associated with low estrogen levels. Importantly, the absence of changes in overall exploration time demonstrates that locomotor or motivational limitations did not affect memory problems. The substantial three-way interaction between novelty preference, VCD induction, and PED treatment, as well as the restoration of the discrimination index (DI), demonstrates that PED recovers novelty preference and recognition memory processing, which were disrupted by ovarian hormone depletion. These findings are consistent with clinical data suggesting that estrogen insufficiency disproportionately affects executive and recognition memory domains, highlighting the paradigm\u0026apos;s translational value (Mohammad, Finch et al. 2025, Abi-Ghanem, Opiela et al. 2026). Anxiety and depression-like symptoms are recognised as neuropsychiatric indications of menopausal transition and early ovarian failure. VCD-induced impairments in open-arm exploration in the EPMT and increased immobility in the FST are consistent with increased anxiety and behavioural despair in chronic estrogen-deficient conditions (Słopień 2018). PED treatment significantly improved these affective impairments, suggesting that its neuroprotective benefits go beyond cognitive functioning and encompass emotional regulation. These behavioural alterations are most likely the result of PED stabilising the limbic circuitry and the balance of monoaminergic and neurotrophic signals, both of which are highly influenced by estrogen receptor beta signalling. Spatial learning, navigation memory, and probe trial performance in the water maze test revealed that, whereas acquisition learning was mainly retained, chronic ovarian failure hampered memory retrieval and cognitive flexibility (Ramli, Yahaya et al. 2024). Mice treated with VCD did not spend as much time in the target quadrant (PQ) as in other quadrants, indicating that hippocampus-dependent processes for consolidating and retrieving memories were compromised. PED treatment improved target quadrant preference and platform dwell time, suggesting that hippocampal synaptic plasticity and adaptive learning had been restored. These findings are particularly significant, as cognitive dullness and difficulties with remembering information are clinically relevant early indicators of brain ageing and neurodegeneration following persistent ovarian senescence (Reuben, Karkaby et al. 2021).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSurprisingly, PED markedly boosted the expression of critical mitochondrial electron transport chain proteins (NDUFB8, MTCO1, ATP5A1) that had been significantly reduced during VCD exposure, indicating that PED is an effective modulator of neuronal bioenergetics and brain energy metabolism (Torrens-Mas, Pons et al. 2020). The observed behavioural and cognitive improvements are most likely due to preservation of mitochondrial integrity, supporting the idea that phytoestrogenic medications have a major impact on mitochondrial functional reconstruction (Singh and Paramanik 2022). VCD-treated mice showed elevated amyloid beta deposition, downregulation of ER-\u0026beta; signalling, and reduction of BDNF, indicating a link between ovarian failure and increased susceptibility to Alzheimer\u0026apos;s disease. These molecular alterations support what epidemiological and clinical research have found: early menopause and ovarian follicular depletion are associated with an increased risk of dementia (Karamitrou, Anagnostis et al. 2023, Liao, Cheng et al. 2023). PED reduces amyloid beta levels while increasing ER-\u0026beta; expression and BDNF synthesis, indicating that estrogen-dependent neurotrophic and anti-amyloidogenic mechanisms are reactivated. This conclusion is significant, as ER-\u0026beta; signalling has been linked to maintaining synapses (Srivastava, Woolfrey et al. 2010), safeguarding neuronal mitochondrial functional capacity (Wang, Mao et al. 2024), regulating neural differentiation (Varshney, Inzunza et al. 2017), neuronal inflammation, making PED a mitigative therapy that alters the course of a neurodegenerative pathology rather than merely treating symptoms.\u003c/p\u003e\n\u003cp\u003eChronic estrogen deprivation resulting from chemically induced accelerated ovarian failure induced significant microglial activation, as evidenced by increased HMGB1\u0026ndash;TLR4\u0026ndash;NF\u0026kappa;B signalling and elevated expression of CD68 and TREM2, indicating a transition to a pro-inflammatory and phagocytic microglial phenotype (Acosta-Mart\u0026iacute;nez 2020, Gao, Jiang et al. 2023). This inflammatory reprogramming was followed by considerable global brain metabolic reprogramming, emphasising the close link between increased neuroinflammaging and cerebral dysfunction in ovarian failure. Our PED therapy significantly inhibited these inflammatory cascades, mitigated microgliosis, and restored cerebral integrity, demonstrating complete preservation of the inflammaging unit. These results elucidate the mechanisms by which PED maintains neuronal homeostasis, mitigates mood disorders, and delays early cognitive decline in chronic estrogen-deprived environments, highlighting the pivotal role of the microglial\u0026ndash;inflammatory interaction in the development of neurodegenerative pathophysiology (Gao, Jiang et al. 2023).\u003c/p\u003e\n\u003cp\u003eUsing metabolomic profiling, we also identified discrepancies in levels of pro-oxidative and inflammatory metabolites, as well as changes in numerous metabolic pathways, in the VCD-challenged mice group. Our metabolomics study, which mapped the metabolic pathways of VIP metabolites, found that VCD-induced AOF disrupts estrogen-regulated steroid, fatty acid, and inflammatory lipid metabolism, leading to mitochondrial dysfunction and lipid-mediated neuroinflammation in the brain. In the NCD mouse brain, coordinated steroidogenesis, lipid oxidation, membrane lipid signalling, and neuroenergetic function were observed while inhibiting inflammatory lipid cascades. VCD-induced ovarian failure results in a dysregulated metabolomic signature, including steroid depletion, mitochondrial fatty acid dysregulation, an excess of prostaglandins (PGF2\u0026alpha; species, PGD2 derivatives, O-arachidonyl intermediates), sphingolipid imbalance, and oxidative stress, indicating estrogen-dependent metabolic collapse and rapid neuroinflammaging cascades. Our findings revealed that PED treatment stabilises steroid homeostasis, boosts neuroprotective ER signalling, combats lipid-driven neuroinflammation, and prevents oxidative-metabolic derailment by restoring antioxidant buffering, enhancing metabolism efficiency, and improving detox control. The joint pathway network analysis suggests that perturbations in membrane lipid metabolism and PUFA-derived inflammatory mediator pathways play a substantial role in the observed brain metabolomic changes. These results support the hypothesis that lipid-driven metabolic reprogramming may function as a critical upstream regulator of persistent neuronal inflammaging. These results align well with the currently evolving paradigm of immunometabolic regulation of neural inflammation in the context of brain ageing and neurodegeneration following chemically-induced ovarian senescence.\u003c/p\u003e\n\u003cp\u003eHenceforth, this study identifies PED as a pleiotropic neuroprotective agent that targets mitochondrial dysfunction, neuroinflammation, amyloid pathology, neurotrophic collapse, and aberrant ER signalling\u0026mdash;pathological pathways that intersect during perimenopause-induced accelerated brain ageing and neurodegeneration. We, for the first time, demonstrate compelling and outstanding mechanistic evidence on how PED effectively halts the neurobehavioral and molecular development of ovarian senescence-induced brain pathophysiology by restoring ER\u0026beta;-mediated neuroprotective signalling and normalising microglial-mitochondrial-neuroimmune interactions (Ambika, Gajbhiye et al. 2025).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eProfound hypoestrogenism caused by chemically-induced ovarian failure initiates a self-perpetuating neuroinflammatory and neurodegenerative cascade that includes microglial overactivation, mitochondrial malfunction, neuronal atrophy, and behavioural impairment (Zhou, Zhang et al. 2026). In a clinically relevant AOF model, PED effectively prevents HMGB1-TLR4-NF\u0026kappa;B-induced microgliosis, restores mitochondrial integrity, and stabilises cognition (Li, Yang et al. 2023). PED preserves ER\u0026beta;-dependent neuroimmune homeostasis and inflammopharmacological integrity, preventing self-perpetuating neuroinflammatory loops that accelerate brain ageing after chemically-induced early ovarian failure (Zhao, Mao et al. 2013).\u003c/p\u003e\n\u003cp\u003eHenceforth, our results address a significant clinical gap in estrogen-associated neuroimmune dysregulation as a major contributor to perimenopausal brain vulnerability, and highlight PED as a potent immunomodulatory neuroprotective strategy during reproductive ageing. These findings advocate for integrating polyphenolic non-steroidal compounds into the clinical management of menopausal health to promote long-term cognitive resilience and overall metabolic homeostasis. Our evidences present groundbreaking translational implications, particularly for women experiencing early menopause or ovarian insufficiency, who are presently underrepresented in neuroprotective intervention studies. We propose that a bioactive phytoestrogen-enriched therapeutic strategy might be a safer, brain-targeted alternative to standard hormone replacement therapy, offering multidimensional neuroprotection without the systemic dangers associated with exogenous estrogen treatment.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAOF: Accelerated ovarian failure, BDNF: Brain-derived neurotrophic factor, ER-\u0026beta;: Estrogen receptor beta, ERT: Estrogen replacement therapy, HMGB1: High mobility group box 1, NF-ϏB: Nuclear factor kappa B, PED: Phytoestrogen diet, PFC: Pre-frontal cortex, POF: Premature ovarian failure, TLR-4: Toll-like receptor-4, VCD: 4-vinylcyclohexene diepoxide\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u0026nbsp;\u003c/strong\u003eThe authors express their gratitude to the Department of Pharmaceuticals, Ministry of Chemicals and Fertilisers, for adequate funding and institutional support to carry out this research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement:\u0026nbsp;\u003c/strong\u003eAll data generated or analysed during this study are included in this article. There are no separate or additional files.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclosure of potential conflicts of interest:\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no competing financial or personal interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval:\u0026nbsp;\u003c/strong\u003eAll applicable institutional CCSEA and IAEC guidelines for the proper care, handling and use of laboratory animals were followed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Declaration:\u003c/strong\u003e This research was funded by the Department of Pharmaceuticals, Ministry of Chemicals and Fertilisers, Govt. of India.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical Trial Number:\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContribution of authors: Ambika Shandilya (First Author):\u0026nbsp;\u003c/strong\u003ePre-clinical survey, methodology, statistical data curation and analysis, writing original manuscript, review and editing, and formal analysis. \u003cstrong\u003eSuhas Hajare:\u0026nbsp;\u003c/strong\u003eInvestigation\u003cstrong\u003e,\u0026nbsp;\u003c/strong\u003evalidation.\u003cstrong\u003e\u0026nbsp;Dubey Aakash Arwind:\u0026nbsp;\u003c/strong\u003eInvestigation,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003evalidation, visualisation\u003cstrong\u003e. Chander Kant Giri:\u0026nbsp;\u003c/strong\u003eValidation and visualisation\u003cstrong\u003e. Rahul Gajbhiye:\u0026nbsp;\u003c/strong\u003eMetabolomics\u003cstrong\u003e. V. Ravichandiran:\u0026nbsp;\u003c/strong\u003eSupervision, resources, and\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003efunding acquisition\u003cstrong\u003e. VK Parihar:\u0026nbsp;\u003c/strong\u003eConceptualisation, project administration, supervision, editing, and funding acquisition\u003cstrong\u003e. Note:\u003c/strong\u003e All authors approved the final version of this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbi-Ghanem C et al (2026) Loss of ovarian hormones is detrimental in early disease stages of mouse models of Alzheimer\u0026rsquo;s disease and multi-etiology dementia. Biology Sex Differences 17(1):4\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAcosta-Mart\u0026iacute;nez M (2020) Shaping microglial phenotypes through estrogen receptors: relevance to sex-specific neuroinflammatory responses to brain injury and disease. J Pharmacol Exp Ther 375(1):223\u0026ndash;236\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmbika et al (2025) Unveiling the pioneering potential of phytoestrogens in promoting stem cell functions and neural regeneration in post-menopausal brain. SAGE PUBLICATIONS INC 2455 TELLER RD. CELL TRANSPLANTATION, THOUSAND OAKS. CA 91320 USA.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAnsere VA et al (2021) Cellular hallmarks of aging emerge in the ovary prior to primordial follicle depletion. Mech Ageing Dev 194:111425\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBlair JA et al (2015) Luteinizing hormone: Evidence for direct action in the CNS. Horm Behav 76:57\u0026ndash;62\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEcheverria V et al (2021) Estrogenic plants: to prevent neurodegeneration and memory loss and other symptoms in women after menopause. Front Pharmacol 12:644103\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGao C et al (2023) Microglia in neurodegenerative diseases: mechanism and potential therapeutic targets. Signal Transduct Target therapy 8(1):359\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGorzkiewicz J et al (2021) The potential effects of phytoestrogens: The role in neuroprotection. Molecules 26(10):2954\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIqbal J et al (2024) Role of estrogen in sex differences in memory, emotion and neuropsychiatric disorders. Mol Biol Rep 51(1):415\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKaramitrou EK et al (2023) Early menopause and premature ovarian insufficiency are associated with increased risk of dementia: a systematic review and meta-analysis of observational studies. Maturitas 176:107792\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKesharwani A et al (2025) Potentiation of endocannabinoid signaling alleviates depressive-like behavior in diabetic mice. Pharmacol Research-Reports 3:100037\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKesharwani A et al (2025) Dehydrozingerone Improves Mood and Memory in Diabetic Mice via Modulating Core Neuroimmune Genes and Their Associated Proteins. ACS Pharmacol Translational Sci 8(6):1694\u0026ndash;1710\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi X et al (2023) Calycosin attenuates the inflammatory damage of microglia induced by oxygen and glucose deprivation through the HMGB1/TLR4/NF-Κb signaling pathway: calycosin attenuates OGD/R damage in microglia. Acta Biochim Biophys Sin 55(9):1415\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiao H et al (2023) Association of earlier age at menopause with risk of incident dementia, brain structural indices and the potential mediators: a prospective community-based cohort study. EClinicalMedicine 60\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarongiu R (2019) Accelerated ovarian failure as a unique model to study peri-menopause influence on Alzheimer\u0026rsquo;s disease. Front Aging Neurosci 11:242\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarongiu R et al (2025) Perimenopause promotes neuroinflammation in select hippocampal regions in a mouse model of Alzheimer\u0026rsquo;s disease. bioRxiv: 2025.2003. 2014.643317.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMohammad A et al (2023) Accelerated Ovarian Failure (AOF) is an Effective Transitional Menopausal Model for the Study of Alzheimer\u0026rsquo;s Disease. Alzheimer's Dement 19:e071809\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMohammad A et al (2025) Accelerated ovarian failure results in brain alterations related to Alzheimer's disease that are not recovered by high-intensity interval training in mice. Alzheimer's Dement 21(2):e14463\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoreira AC et al (2017) Phytoestrogen coumestrol improves mitochondrial activity and decreases oxidative stress in the brain of ovariectomized Wistar-Han rats. J Funct Foods 34:329\u0026ndash;339\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNiu Y et al (2025) The 4-vinylcyclohexene dioxide induced mouse ovarian premature failure is caused by down regulation of IGF1R and triggering excessive autophagy. Stress Biology 5(1):21\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOppong-Gyebi A et al (2022) Long‐term hypogonadism diminishes the neuroprotective effects of dietary genistein in young adult ovariectomized rats after transient focal ischemia. J Neurosci Res 100(2):598\u0026ndash;619\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePlatholi J et al (2023) Hippocampal glial inflammatory markers are differentially altered in a novel mouse model of perimenopausal cerebral amyloid angiopathy. Front Aging Neurosci 15:1280218\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRamli NZ et al (2024) Spatial learning and memory impairment at the post-follicular depletion state is associated with reduced hippocampal glucose uptake. Exp Gerontol 197:112607\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReuben R et al (2021) Menopause and cognitive complaints: are ovarian hormones linked with subjective cognitive. decline? Climacteric 24(4):321\u0026ndash;332\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRonchetti S et al (2025) The phytoestrogen genistein improves hippocampal neurogenesis and cognitive impairment and decreases neuroinflammation in an animal model of metabolic syndrome. J Neuroendocrinol 37(2):e13480\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRosario ER et al (2006) Androgens regulate the development of neuropathology in a triple transgenic mouse model of Alzheimer's disease. J Neurosci 26(51):13384\u0026ndash;13389\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRussell JK et al (2019) role estrogen brain Cogn aging Neurother 16(3):649\u0026ndash;665\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShandilya A et al (2022) Activation of IGF-1/GLP-1 signalling via 4-hydroxyisoleucine prevents motor neuron impairments in experimental ALS-rats exposed to methylmercury-induced neurotoxicity. Molecules 27(12):3878\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSingh P, Paramanik V (2022) Neuromodulating roles of estrogen and phytoestrogens in cognitive therapeutics through epigenetic modifications during aging. Front Aging Neurosci 14:945076\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSłopień R (2018) Mood disorders in women with premature ovarian insufficiency. Menopause Review/Przegląd Menopauzalny 17(3):124\u0026ndash;126\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSrivastava DP et al (2010) Estrogen receptor β activity modulates synaptic signaling and structure. J Neurosci 30(40):13454\u0026ndash;13460\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTorrens-Mas M et al (2020) Sexual hormones regulate the redox status and mitochondrial function in the brain. Pathological implications Redox biology 31:101505\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVarshney MK et al (2017) Role of estrogen receptor beta in neural differentiation of mouse embryonic stem cells. Proceedings of the National Academy of Sciences 114(48): E10428-E10437\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVoskuhl RR et al (2024) Mind the Gap: Estrogen Receptor-β in Astrocytes Is a Therapeutic Target to Prevent Cognitive Problems at Menopause [ID 2683385]. Obstet Gynecol 143(5S):2\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang T et al (2024) Key role of ERβ in estrogenic regulation of neuronal mitochondrial function. Alzheimer's Dement 20:e086633\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang T et al (2025) Accelerated midlife endocrine and bioenergetic brain aging in APOE4 females. Front Aging Neurosci 17:1632877\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu X-W et al (2008) Effects of phytoestrogen on mitochondrial structure and function of hippocampal CA1 region of ovariectomized rats. Cell Mol Neurobiol 28(6):875\u0026ndash;886\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYuan S et al (2025) Brain structural alterations in young women with premature ovarian insufficiency: Implications for dementia risk. Alzheimer's Dement 21(3):e70111\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao L et al (2002) Neuroprotective and neurotrophic efficacy of phytoestrogens in cultured hippocampal neurons. Experimental Biology Med 227(7):509\u0026ndash;519\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao L et al (2013) Early intervention with an estrogen receptor β-selective phytoestrogenic formulation prolongs survival, improves spatial recognition memory, and slows progression of amyloid pathology in a female mouse model of Alzheimer's disease. J Alzheimer\u0026rsquo;s Disease 37(2):403\u0026ndash;419\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou J et al (2026) Fertility under fire: how chemotherapy harms the ovaries and the science fighting back? Journal of Ovarian Research\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Figures","content":"\u003cp\u003eFigures are available in the Supplementary Files section. \u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"naunyn-schmiedebergs-archives-of-pharmacology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nsap","sideBox":"Learn more about [Naunyn-Schmiedeberg's Archives of Pharmacology](https://www.springer.com/journal/210)","snPcode":"210","submissionUrl":"https://submission.nature.com/new-submission/210/3","title":"Naunyn-Schmiedeberg's Archives of Pharmacology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Accelerated ovarian failure, Brain Metabolomics, Cognitive impairment, Mitochondrial dynamics, Neuroinflammation, Phytoestrogen therapy","lastPublishedDoi":"10.21203/rs.3.rs-9291892/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9291892/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMost women undergoing accelerated ovarian failure experience cognitive-affective dysfunction, bioenergetic failure, amyloidogenic susceptibility, and persistent neuroinflammation, emerging chiefly from impaired estrogenic regulation. Despite the fact that ERT improves women's lives, it is not widely utilised due to the risk of thrombosis, cardiac issues, and endometrial cancer. The phytoestrogen diet (PED), renowned for its ERβ-centered neurorestorative potential, has shown considerable promise; however, the detailed mode of action remains largely underexplored. This study aims to elucidate the translational molecular mechanisms by which dietary phytoestrogen elicits neuroprotective benefits against ovatoxin-induced expedited cognitive ageing and neurodegenerative pathologies. We explored the therapeutic effects of PED using neurobehavioural paradigms, mitochondrial functional assessments, and estimates of neuronal atrophy and neuroinflammatory signalling via cell-specific dual immunofluorescence analyses. Additionally, we evaluated PED's efficacy in restoring the brain metabolite profile, identifying neurodegenerative signatures, and mitigating chronic neuroimmune transition. Results revealed that accelerated ovarian insufficiency exacerbated memory alterations and emotional instability, coinciding with decreased ER-β and BDNF expression, enhanced beta-amyloid deposition, and microgliosis-driven neuroimmune dysregulation. High-VIP metabolites reflected disruptions in steroid, sphingolipid, and fatty acid pathways, indicating that ovarian failure drives estrogen-dependent metabolic reprogramming. Our study demonstrated that PED rich in estrogen-mimicking isoflavones ameliorated mood and memory deficits by modulating beta-amyloid, reinstating mitochondrial integrity, rescuing the brain metabolome, and restoring neurotrophic signalling through ER-β activation in the mPFC. Therefore, PED is a promising candidate for treating neurocognitive decrements by potentiating ER-β activity, preserving mitochondrial integrity, and modulating the inflammopharmacological axis.\u003c/p\u003e","manuscriptTitle":"Dietary Phytoestrogen ameliorates Ovarian Toxicant–induced Neurotoxicity: Mechanistic and metabolic insights","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-15 18:12:54","doi":"10.21203/rs.3.rs-9291892/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-24T06:17:17+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-23T17:24:28+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-16T09:50:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"149684410515809044107215839125318423746","date":"2026-04-15T22:40:52+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-13T18:47:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"104665142491746074063625691341580153297","date":"2026-04-10T10:37:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"196759858424495200089026791586105882980","date":"2026-04-08T12:25:32+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-08T09:55:34+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-08T02:54:21+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-08T02:54:12+00:00","index":"","fulltext":""},{"type":"submitted","content":"Naunyn-Schmiedeberg's Archives of Pharmacology","date":"2026-04-01T12:07:20+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"naunyn-schmiedebergs-archives-of-pharmacology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nsap","sideBox":"Learn more about [Naunyn-Schmiedeberg's Archives of Pharmacology](https://www.springer.com/journal/210)","snPcode":"210","submissionUrl":"https://submission.nature.com/new-submission/210/3","title":"Naunyn-Schmiedeberg's Archives of Pharmacology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"79984e81-3cb8-4035-8f42-fb621f1268b3","owner":[],"postedDate":"April 15th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-15T06:26:05+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-15 18:12:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9291892","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9291892","identity":"rs-9291892","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}