Xiaoyao San intergenerational protects learning-memory ability and neurodevelopment in PPD offspring via the BDNF/mTOR signaling pathway

Xiaoyao San intergenerational protects learning-memory ability and neurodevelopment in PPD offspring via the BDNF/mTOR signaling pathway | 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 Xiaoyao San intergenerational protects learning-memory ability and neurodevelopment in PPD offspring via the BDNF/mTOR signaling pathway Hongxiao xie, Junping Ding, Yanning Jiang, Yixin Rui Rui, Zhiqiang Xie, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7515178/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract background As a principal TCM antidepressant intervention, Xiaoyao San (XYS) is extensively applied in postpartum depression (PPD) therapeutics. Nevertheless, fundamental research in this area remains limited, particularly insufficient investigations into the intergenerational effects of XYS on PPD treatment. Methods Female ICR mice received daily corticosterone (CORT) injections (PD1-PD21) to establish a PPD model, with concurrent XYS treatment (45, 30, 15 g/kg) for 21 days. Maternal caring behavior was assessed on PD4 and PD8. On PD22, behavioral tests were conducted, followed by ELISA quantification of serum CORT/ACTH/CRH and HE staining to evaluate hippocampal neuronal damage. Offspring mice underwent morris water maze and Y-maze tests on PD28 to assess cognition, with histological/molecular analyses (HE, immunofluorescence, Golgi staining, western blot) for neurodevelopment. Western blot quantified hippocampal BDNF/mTOR pathway proteins, validated via ANA-12 inhibition. Results XYS enhanced maternal care, reduced anxiety/depression-like behaviors in PPD mice. XYS conferred transgenerational protection of learning and memory functions in offspring mice, as evidenced by: reduced escape latency, increased platform crossings/spontaneous alternation. Besides, XYS treatment attenuated hippocampal neuronal damage in PPD offspring, promoted neurogenesis (increased DCX + /NeuN + cells in DG region), dendritic complexity (higher spine density/length), and synaptic protein expression. Maternal XYS administration upregulated BDNF/mTOR pathway. ANA-12 confirmed the pivotal role of BDNF/mTOR pathway in mediating XYS's transgenerational neuroprotective and learning and memory ability. Conclusion XYS alleviated postpartum depression in mice and conferred transgenerational neuroprotection via BDNF/mTOR activation, preserving offspring cognition. This supports XYS's clinical potential for interrupting intergenerational psychiatric disorders. Xiaoyao San Postpartum depression Offspring Learning and memory Neurogenesis Synapse BDNF Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 1 Introduction Postpartum depression (PPD), as defined in the Diagnostic and Statistical Manual of Mental Disorders (DSM-V), is a subtype of major depressive disorder that emerges within 4 to 6 weeks after pregnancy or childbirth[ 1 ]. It is the most prevalent psychiatric condition during the puerperium, with global incidence rates ranging from 15–30%[ 2 ]. PPD significantly compromises maternal-infant bonding and caregiving, as evidenced by decreased breastfeeding frequency, diminished emotional responsiveness, and impaired mother-infant attachment. These disruptions have been associated with adverse outcomes in the offspring, including emotional dysregulation, cognitive deficits, and developmental delays[ 3 – 5 ]. Neurodevelopment encompasses the acquisition and refinement of motor, sensory, cognitive, linguistic, psychosocial, and self-regulatory capacities throughout the lifespan. A critical neurodevelopmental window occurs during early postnatal stages, particularly from conception through the first two years[ 6 ]. PPD impairs maternal care behaviors in lactating animals, thereby creating early-life adversity during offspring development[ 7 ]. Animal studies indicate that maternal PPD compromises caregiving behaviors, inducing postnatal adversity that disrupts offspring hippocampal neurogenesis and synaptic plasticity. This is evidenced by reduced doublecortin (DCX) expression-a biomarker of immature hippocampal neurons - correlating with depression-like phenotypes and cognitive impairment[ 8 – 11 ]. As a key neurotrophin, brain-derived neurotrophic factor (BDNF) critically regulates hippocampal neuronal proliferation, synaptic formation, and apoptosis[ 12 ]. BDNF further modulates higher-order cognitive functions including learning and memory consolidation[ 13 , 14 ]. Emerging evidence indicates BDNF regulates mTOR-dependent autophagy via the TrkB/PI3K/Akt pathway, subsequently modulating hippocampal synaptic integrity and cognitive function [ 15 ]. Moreover, elevated BDNF levels and activation of the PI3K/AKT/mTOR signaling cascade have been linked to reductions in neuroinflammation and oxidative stress, contributing to cognitive resilience Timely pharmacological intervention following PPD onset is essential to mitigate its adverse consequences, particularly those affecting offspring. Although selective serotonin reuptake inhibitors (SSRIs) remain the first-line pharmacotherapy, they are associated with safety concerns in lactating mothers. For instance, fluoxetine can be secreted into breast milk, resulting in reduced maternal body weight, altered milk composition, and long-lasting growth deficits in the offspring[ 16 ]. Brexanolone (Zulresso), a neuroactive steroid recently approved by the U.S. FDA, has shown efficacy in treating PPD but also carries risks such as excessive sedation and loss of consciousness[ 17 ]. Animal data further indicate that exposure to high doses of brexanolone during gestation may lead to teratogenic and neurobehavioral abnormalities in the offspring [ 18 ]. Given the limitations of current treatments, traditional Chinese medicine (TCM) and integrative therapies have gained attention for their clinical efficacy and favorable safety profile in managing PPD[ 19 , 20 ]. Rooted in centuries of clinical practice, TCM emphasizes the interplay between maternal emotional states and fetal health, captured in classical doctrines such as “external influences shape internal states, and these states form the basis for the offspring” and “injury to the mother’s qi harms the child’s qi.” Accordingly, TCM treatment philosophies like “treating the mother to protect the child” and “preventive treatment of disease” reflect a longstanding focus on intergenerational health. Recent experimental studies have validated these concepts, showing that pharmacological interventions in PPD mothers—such as ketamine or salidroside—can indirectly promote neurodevelopment and reduce depressive behaviors in the offspring[ 10 , 21 ].Traditional Chinese medical (TCM) theory has long emphasized the link between maternal emotional health and offspring development, as reflected in classical tenets such as “external influences shape internal states, and these states form the basis for the offspring,” and “if the mother’s qi is injured, the child’s qi will also be harmed.” These principles underscore the potential intergenerational consequences of maternal psychological disturbances. TCM practitioners have proposed treatment concepts such as "treating the mother to protect the child," "treating the mother for the child's illness," and "preventive treatment of disease". Recent experimental studies have validated these concepts, showing that pharmacological interventions in PPD mothers—such as ketamine or salidroside—can indirectly promote neurodevelopment and reduce depressive behaviors in the offspring[ 10 , 21 ]. Salidroside can prevent learning disabilities in offspring caused by pregnancy-induced hypertension by intervening in the Wnt/SKp2 pathway[ 21 ]. Xiaoyao San (XYS), a classical TCM formula originating from the Formulary of the Bureau of Peaceful Benevolent Dispensaries , has demonstrated antidepressant properties via multiple molecular pathways. Previous studies have shown that XYS attenuates HPA axis hyperactivity[ 22 , 23 ], modulates monoaminergic neurotransmission[ 24 ], and suppresses oxidative stress and inflammation in key brain regions[ 25 ]. Clinically, XYS is widely prescribed for PPD due to its ability to harmonize liver and spleen functions and restore qi-blood balance. Numerous clinical trials have demonstrated its effectiveness in alleviating depressive symptoms, especially when used in combination with conventional antidepressants or other supportive therapies, with minimal side effects[ 26 – 28 ]. Animal studies further support its role in restoring neurotransmitter balance and ameliorating depression-like behaviors in PPD models[ 29 ]. However, despite its established antidepressant effects, the potential transgenerational neuroprotective actions of XYS remain underexplored. In this study, we employed a corticosterone-induced PPD mouse model to investigate the maternal and intergenerational effects of XYS. We focused on hippocampal neuroplasticity and the BDNF/mTOR signaling pathway to evaluate the efficacy of XYS in alleviating depressive-like behaviors in mothers and promoting neurodevelopment and cognitive function in offspring. This research not only advances our understanding of XYS as a multi-target intervention for PPD but also provides a scientific foundation for its clinical application in protecting maternal and offspring mental health. Ultimately, our findings aim to support early-stage, transgenerational interventions and expand the global relevance of traditional Chinese medicine in the field of neuropsychiatry. 2 Materials and methods 2.1 Reagents The eight herbal components of XYS are Bupleuri Radix (Lingnan Traditional Chinese Medicine Co., Ltd, 20220201), Angelicae Sinensis Radix , Paeoniae Alba Radix , Atractylodes Macrocephala Rhizoma , Glycyrrhizae Radix et Rhizoma , Poria (Sichuan Xin lotus traditional Chinese medicine co., Ltd, D2112001, 2206152, 2205057, 2204046, D2206022), Mentha Haplocalycis Herba (Jiangsu Donglian Drug Co., Ltd, 20210227), and Zingiber (purchased from the local market). They were identified by Professor Wang Guangzhi from the School of Pharmacy, Chengdu University of Traditional Chinese Medicine. The quality control substance of Paeoniflorin in the Chinese Pharmacopoeia (2020 edition) for Xiaoyaosan was purchased from Chengdu Kroma Biotechnology Co., Ltd. Corticosterone was purchased from MedChemExpress (MCE, NJ, USA). Sertraline hydrochloride (SERT) purchased from Pfizer (Wuxi, Jiangsu, China). The BDNF inhibitor (ANA-12) was purchased from Selleck (Texas, USA). Enzyme-linked immunosorbent assay (ELISA) kits were purchased from Elabscience (Wuhan,Hubei,China). 2.2 Animals Thirty-six female and eighteen male adult ICR mice (8-week-old) were sourced from SPF Biotechnology Co., Ltd. (Beijing, China; Certification: SCXK-[Jing]-2019-0010). Animals were acclimatized for 7 days under controlled conditions: 22 ± 1°C, 40–60% humidity, 12-hour light/dark cycle, with ad libitum access to food and water. The experimental procedures followed the guidelines of the Committee for Animal Care and Use of Laboratory Animals of the College of Pharmacy, Chengdu University of Traditional Chinese Medicine (approval No. TCM-2017-312). 2.3 Preparations of XYS The XYS extracts preparation was carried out as previously described[ 30 ]. We mixed Bupleuri , Angelicae , Paeoniae , Atractylodes , Poria , Zingiber , Glycyrrhizae , and Mentha in the ratio of 6:6:6:6:6:6:3:1 (w/w, with a total weight of 1320 g). Add the mixture of raw herbs with 8 times the amount of water and soak for 30 minutes,followed by 1 h boiling at 100°C. Two additional extractions were performed with 6 volumes of water for 40 and 30 minutes, respectively. Combine the three filtrates and concentrate under reduced pressure to 1.5 g/m L. According to the Chinese Pharmacopoeia (2020 Edition), the content of paeoniflorin in the water decoction of Xiaoyaosan was quantified using high-performance liquid chromatography (HPLC). Specifically, 8 mg of the paeoniflorin reference standard was dissolved in 2.5 mL of 50% methanol to prepare a stock solution with a concentration of 3.2 mg/mL. A standard curve was then established by the method of serial dilution.Chromatographic separation was achieved on a Hypersil GOLD C18 column (250 mm × 4.6 mm, 5 µm, Thermo Scientific) with a mobile phase of 0.1% phosphoric acid and acetonitrile (86:14, v/v), flow rate of 1.0 mL/min, column temperature of 30°C, and detection wavelength of 230 nm. 2.4 Establishment of the PPD model and drug treatment Female and male mice were co-housed in Plexiglas cages with a sex ratio of 1:2 (one female to two males). Each morning, we checked the female mice for the presence of a vaginal plug and monitored their body weight to confirm pregnancy. Once confirmed, pregnant dams were immediately transferred to individual cages. The day of delivery was designated as postnatal day 0 (PD0). To ensure standardized litters, we culled the offspring to retain exactly 8 pups per dams (4 males and 4 females). Dams were randomly allocated to six experimental groups. (n = 6/group): CON, PPD, SERT (25 mg/kg, i.g.), XYS_H (45 g/kg, i.g.), XYS_M (30 g/kg, i.g.), and XYS_L (15 g/kg, i.g.). From PD1-PD21 (for 21 days), except the CON group, all groups received daily intraperitoneal injections of CORT (40 mg/kg) between 9:00–10:00. Six hours post-CORT, mice received corresponding drug treatments; CON and PPD groups received vehicles. The experimental workflow is illustrated in Fig. 2 A. 2.5 Offspring group and drug treatment Experiment 1: The iterative effect of XYS on the learning and memory ability of PPD offspring mice and a preliminary investigation of its mechanisms. Throughout development, offspring were not exposed to any drug treatments. On PD21, one male and one female pup per litter were randomly selected (n = 12/group), grouped according to their maternal treatment (CON-F1, PPD-F1, SERT-F1, XYS-H-F1, XYS-M-F1, XYS-L-F1). Males and females were housed separately. Experimental workflow is shown in Fig. 4 A. Data from offspring within the same group were pooled for joint analysis. The specific detail experimental workflow of offspring mice is depicted schematically in Figure.4A. Experient 2: Reverse validation of the effect of BDNF on neuroplasticity in XYS iteratively protected PPD offspring mice using the ANA-12, a BDNF receptor inhibitor. On PD21, we selected offspring (n = 10/group, 5 males and 5 females) were divided into five groups: CON-F1, PPD-F1, XYS-F1, CON + A-F1, and XYS + A-F1. ANA-12 (0.5 mg/kg/day, i.p.) was administered to “+A” groups for 14 days from PD21. Other groups received saline[ 31 , 32 ]. Workflow is shown in Fig. 8 A. Preparation of ANA-12 inhibitor injection: 0.32 mg of the drug was dissolved in 320 µ L of DMSO solution to make a mother liquor of 1 mg/m L. Then take 320 µ L of DMSO mother liquor, add 2560 µ L of PEG300 and 320 µ L of Tween 80, and mix until clarified. Then adding 3200 µ L of ddH2O, mixing until clarified and set aside[ 31 , 32 ] 2.6 Behavioral testing of maternal mice 2.6.1 Maternal care behavior Maternal care assessment followed established protocols[ 33 ]. Behavioral observations were conducted twice daily in the home cage (13:00–14:00 and 18:00–19:00) on postnatal days 4 and 8. Each 10-minute observation session commenced ≥ 2 hours post-injection. Maternal care behaviors recorded included three primary categories: (a) nursing postures, comprising arched-back nursing (active nursing with dorsally curved posture), blanket nursing (resting atop pups in nursing position), and passive nursing (side-lying position while nursing); (b) pup-directed licking, defined as licking behavior occurring independently of nursing; and (c) nest building, characterized by active rearrangement of nesting material. Video recordings were subsequently analyzed to quantify the duration of each behavior for each dam. Total maternal care time (sum of nursing, pup licking, and nest-construction durations) was calculated from two daily sessions using the formula: Maternal care time = (a) + (b) + (c). 2.6.2 Sucrose preference test (SPT) The sucrose preference test (SPT) evaluated murine anhedonia—a core depressive symptom. Prior to testing, a 48h habituation phase exposed mice to 1% sucrose and water bottles in individual cages, with bottle positions swapped at 12-h intervals. The test phase used pre-weighed bottles rotated identically every 12h.After 24 h, fluid consumption was measured. Sucrose preference was calculated as:[sucrose intake/(sucrose intake + water intake)]×100% [ 34 ]. 2.6.3 Forced swimming test (FST) FST/TST sustained immobility signifies behavioral despair with compromised escape drive. Mice underwent 6 min trials in cylindrical water tanks (25 cm depth, 24 ± 1°C). After 2 min habituation, immobility (defined as floating with only essential movements for flotation) during t = 2-6min was scored by SMART 3.0 tracking system [ 35 ]. 2.6.4 Tail suspension test (TST) Tail suspension test (TST) was conducted in black-walled enclosures (18 cm width × 18.5 cm height). Mice were suspended vertically 8 cm above the base using adhesive tape placed 2.5 cm proximal to the tail tip. Automated quantification of immobility duration focused on the terminal 4-min interval of 6 min trials, excluding the initial 120-s acclimatization period. 2.6.5 Open field test (OFT) The open field apparatus was a 47 cm × 47 cm square chamber. Mice were initially placed in a corner. After a 2-min acclimation period, total distance moved and central area duration (23.5 × 23.5 cm inner zone) during the final 4 min of the 6 min test were recorded by tracking software. The arena was cleaned with 75% ethanol between trials to remove residual odors. 2.6.6 Elevated plus maze test (EPMT) The elevated plus maze (EPM) apparatus featured two open and two closed arms (50 × 10 cm each) intersecting at a central platform (10 × 10 cm). Closed arms had 40-cm high opaque walls. The maze was elevated 40 cm, and mice started from the central platform facing a closed arm. After a 2 min acclimation period, open arm duration during the subsequent 5 min test phase was recorded by tracking software. The apparatus was cleaned with 75% ethanol between trials to eliminate residual odors. 2.7 Behavioral testing of offspring mice 2.7.1 Morris water maze test The Morris water maze consisted of a circular tank (120 cm diameter × 50 cm height) filled with 23–25°C ink-mixed water, divided into four quadrants marked by distinct geometric cues (triangle, circle, trapezoid, quadrilateral). A 5-cm diameter escape platform was submerged ~ 2 cm below the water surface at a target quadrant center. During spatial acquisition training (PD28-PD32), mice underwent daily two-trial sessions (30-min inter-trial interval) where they were gently released from the platform-opposite quadrant and allowed 60 s to locate the hidden platform, remaining on it for 15 s; unsuccessful trials recorded 60 s escape latency. On day 6 (probe trial), after platform removal, mice explored for 120 s with recording of: target quadrant latency,platform crossing and swim speed. All behaviors were video-tracked via SMART 3.0(Zhang et al., 2021). 2.7.2 Y-maze test Spontaneous alternation in the Y-maze was quantified to evaluate spatial learning and short-term memory. The apparatus comprised three identical arms (35 × 10 × 15 cm; L×W×H) designated A, B, and C, radially extending at 120° intervals. Mice were introduced at the distal end of arm A for an 8-min unreinforced exploration period. Arm entry sequences and frequencies were recorded visually. Alternation events were defined as consecutive entries into all three distinct arms (e.g., ABC, BCA). Valid entries required complete hind-paw insertion into an arm. Inter-trial cleaning with 75% ethanol eliminated odor cues. The calculation formula of spontaneous alternation percentage is: alternations (%) = alternate times/(total arm entries-2)×100, high alternate percentage was regarded as good spatial memory[ 36 ]. 2.8 Tissue collection and tissue processing Twenty-four hours post-behavioral assessment, all mice underwent pentobarbital via intraperitoneal injection. Orbital blood sampling was followed by centrifugation (4°C, 3500 rpm, 15 min) for serum isolation. Serum aliquots underwent cryostorage at -80°C. Hippocampal tissues were ice-dissected, vitrified in liquid nitrogen, and cryobanked at -80°C. Selected whole brains were fixed in 4% paraformaldehyde or Golgi-Cox solution. 2.9 Adrenal gland index of maternal mice Maternal mice were weighed at PD24, and blood was collected from the orbits after pentobarbital anesthesia, followed by decapitation and execution. The adrenal glands were bilaterally excised via abdominal dissection, cleared of connective tissue and adipose debris with forceps/scissors, dried on filter paper, and weighed. Adrenal index (%) = [gland weight (mg) / body weight (g)] × 100%. 2.10 Enzyme-linked immunosorbent assay (Elisa) Serum corticosterone (CORT), adrenocorticotropic hormone (ACTH), and corticotropin-releasing hormone (CRH) levels in dams, alongside brain-derived neurotrophic factor (BDNF) levels in adolescent offspring, were measured using commercial ELISA kits per manufacturer protocols. 2.11 HE staining Fixed tissues were paraffin-embedded, sectioned, stained with hematoxylin-eosin, and imaged under 20× and 100× magnification. Neuronal morphology in hippocampal regions was analyzed using CaseViewer software. 2.12 Immunofluorescent staining Following antigen retrieval, brain sections were washed in phosphate-buffered saline (PBS, pH 7.4) and blocked using 3% bovine serum albumin (BSA) for 30 min. Sections were then incubated overnight at 4°C with primary antibodies:anti- Neuronal nuclei (NeuN, 1:100, Cat No.GB13138-1 Servicebio) and anti-doublecortin (DCX, 1:200, Cat No.ab207175 Abcam). After PBS washes, sections were exposed to secondary antibodies - goat anti-mouse IgG (1:400, Cat No.GB25301, Servicebio) and goat anti-rabbit IgG (1:300, Cat No. GB21303, Servicebio) - for 50 min at room temperature under dark conditions. Following additional washes, nuclei were counterstained with DAPI (10 min, RT, dark). Sections then underwent autofluorescence quenching (5 min, RT, dark), thorough deionized water rinsing (10 min), and final imaging via panoramic slide scanner. The hippocampal dentate gyrus (DG) was delineated in 40x images using CaseViewer software, with DCX + and NeuN + immunofluorescence intensity within DG quantified through ImageJ analysis. 2.13 Golgi-cox Staining Mice brain tissue was dissected into 2–3 mm³ pieces and rinsed repeatedly with saline. Tissue pieces were then immersed in Golgi-Cox staining solution, protected from light, and maintained for 14 days. Following three washes in distilled water, tissues were transferred to 80% ammonia solution overnight. Sections (100 µm thick) were prepared using an oscillating slicer and subsequently stained. Dendritic spine density and length in CA1, CA3, and DG neurons were quantified using Image-Pro Plus 6.0 (400×). Analyses focused on intact central neurons within each image, measuring spines along 30–90 µm segments of secondary/tertiary dendrites. Spine counts and length per 10 µm were calculated. 2.14 Western blotting Total protein from Hippocampal tissue were quantified with a BCA assay kit (Beyotime Biotechnology). Following SDS-PAGE separation, proteins were transferred to PVDF membranes. Following blocking with 5% nonfat milk (1.5 h), membranes were incubated overnight at 4°C with primary antibodies targeting: BDNF (1:1000, Cat. No. 47808S, Cell Signaling Technology), TrkB (rabbit pAb), phospho-TrkB (Y817), PI3K p85α (1:2000, Cat. No. T40115F, Abmart), phospho-PI3K p85α/γ (Tyr467/199) (1:2000, Cat. No. T40065F, Abmart), AKT1/2/3 (1:2000, Cat. No. T55561F, Abmart), phospho-AKT (Ser473) (1:2000, Cat. No. T40067F, Abmart), mTOR (1:1000, Cat. No. 2983S, Cell Signaling Technology), phospho-mTOR (Ser2448) (1:1000, Cat. No. 5536S, Cell Signaling Technology), P70 S6 Kinase (1:2000, Cat. No. TA6226, Abmart), synaptophysin (1:2000, Cat. No. T55273S, Abmart), PSD95/DLG4 (1:6000, Cat. No. 20665-1-AP, Proteintech), GAPDH (1:2000, Cat. No. GB11002-100, Servicebio), β-tubulin (1:2000, Cat. No. GB11017-100, Servicebio), and β-actin (1:2000, Cat. No. GB15003-100, Servicebio). After washing, blots were incubated with HRP-conjugated goat anti-rabbit IgG secondary antibody (1:5000, Cat. No. BA1054,Boster Bio) for 1.5 h at room temperature. Protein bands were visualized using ECL substrate (Merck Millipore), with chemiluminescent signals acquired via ChemiDoc XRS + system (Bio-Rad). Quantitative analysis was performed in Image Lab 6.0 by measuring grayscale values of target and reference protein bands, with target protein expression normalized to internal controls (β-actin/GAPDH/β-tubulin). 2.15 Statistical analysis Data analysis utilized SPSS 26.0, with results expressed as mean ± SD. Following outlier removal via normality assessment, group differences were analyzed by one-way ANOVA (normally distributed, homoscedastic data) or Kruskal-Wallis test (non-parametric distributions). We established P < 0.05 as the statistical significance criterion for all analyses. 3 Results 3.1 XYS quality control According to the content determination standard of Xiaoyao Pill (water pill) in the first prescription preparation and single flavor preparation of Chinese Pharmacopoeia (2020 edition), paeoniflorin was selected as the quality control object of XYS decoction. The result of HPLC was that the extract powder of 1 g XYS contained 10.20 mg paeoniflorin (C23H28O11), indicating that the content of paeoniflorin in XYS was in line with the content determination standard in Xiaoyao Pill (water pill) of the 2020 edition of Chinese Pharmacopoeia (no less than 2.5 mg per 1 g of paeoniflorin) (Fig. 1 A, 1 B). 3.2 XYS increased maternal nursing and improved anxiety- and depression-like behavior of PPD maternal mice Maternal care behaviors in dams were assessed on postnatal days 4 and 8 to evaluate offspring nursing performance. Maternal care assessment was discontinued after PD8 due to emerging pup mobility compromising observational reliability. Compared with the CON group, the maternal caring time of maternal mice showed a decreasing trend at PD4 (Fig. 2 B, P > 0.05), and significantly decreased at PD8 in PPD group (Fig. 2 B, P < 0.01). The maternal mice spent more time to care their offspring than PPD group at PD8 in XYS_H and XYS_M group (Fig. 2 B, P < 0.05), suggesting postpartum administration XYS could ameliorate CORT-induced deficits in the maternal caring behavior of PPD mice. Depressive-like behaviors in dams were evaluated using SPT, TST and FST. Sucrose preference rate is a key indicator for detecting symptoms of pleasure deficit in depression, and TST and FST were used to evaluate the state of behavioral despair in mice. The PPD maternal mice presented notable decreased sucrose preference rate and increased immobility time compared to the CON group (Fig. 2 C, P < 0.01; Fig. 2 D, P < 0.05; Fig. 2 E, P < 0.01). Compared to PPD group, sucrose preference rate significantly elevated in SPT and immobility time declined in FST of XYS_H, and XYS_M group’s mice (Fig. 2 C, P < 0.01; Fig. 2 E, P < 0.01 or P < 0.05). In the TST, the immobility time decreased in the XYS_H and XYS_L group (Fig. 2 D, P < 0.05). OFT and EPMT was subjected to assess anxiety-like behavior of maternal mice. PPD dams exhibited significantly reduced center zone duration and locomotion in the OFT relative to controls. (Fig. 2 F, 2 G, 2 H, P < 0.001). In the EPMT, the PPD mice spent less time in open arm (Fig. 2 I, 2 J, P < 0.01). Compared PPD group, the time spent in the centre zone and total distance were significantly increased in XYS_H and XYS_M group (Fig. 2 F, 2 G, 2 H, P < 0.05 or P < 0.001). The time spent in open arm was elevated in the XYS_H, XYS_M and XYS_L group (Fig. 2 I, 2 J, P < 0.05 or P < 0.01 or P < 0.001). These findings demonstrate XYS ameliorates CORT-induced anxiety- and depression-like phenotypes in PPD dams. 3.3 Administration of XYS suppressed HPA axis dysfunction and reduced neuronal damage in the hippocampus of PPD maternal mice In this study, the adrenal index was used to roughly reflect the HPA axis activity. When HPA axis activity is increased, hypersecretion of the adrenal cortex occurs with tissue hypertrophy and weight gain, whereas when HPA axis activity is decreased, adrenal cortical secretory activity is diminished and tissue atrophy and weight loss occur. Adrenal index was higher in the PPD group of maternal mice compared to the CON group (Fig. 3 A, P < 0.05); and it was significantly lower in the XYS_H group than in the PPD group (Fig. 3 A, P < 0.05). In addition, we measured the levels of serum HPA axis hormones of maternal mice. The levels of CORT, ACTH, and CRH were significantly higher in the PPD maternal mice compared with the CON group (Fig. 3 B, 3 C, 3 D, P < 0.01 or P < 0.001). Compared with the PPD group, CORT, ACTH, and CRH levels were significantly reduced in the XYS_H group (Fig. 3 B, 3 C, 3 D, P < 0.05 or P < 0.01), and CORT and CRH levels were significantly reduced in the XYS_M group (Fig. 3 B, 3 C, 3 D, P < 0.05 or P < 0.01). Moreover, XYS treatment dramatically ameliorated hippocampal neuronal damage in PPD mother mice. In the CON group, maternal mice had abundant neurons in the hippocampus, tightly arranged, normal neuronal morphology and structure, clear cytoplasmic demarcation of cytosolic nuclei, obvious nucleoli, and no obvious pathological changes were seen. However, in PPD group, there were different degrees of degeneration and necrosis of vertebral cells in the hippocampus, nuclear atrophy and deep staining, and the cytosol was obviously smaller (as shown by the green arrows in the figure), which could be seen as obvious pathological damage. After the treatment with XYS, there were a small number of pyramidal cells with degeneration and necrosis in the hippocampal area, nuclear atrophy and deep staining, and some pathological damage was visible of the maternal mice in XYS_H, XYS_M and XYS_L groups (Fig. 3 E). 3.4 XYS intergenerationally protected the learning and memory ability of PPD offspring mice Escape latency progressively declined across training days during spatial acquisition in all offspring group. On day 5, the escape latency of male and female offspring mice in the PPD-F1 group was higher than CON-F1 group, while the escape latency of both male and female offspring mice in the XYS_H-F1, XYS_M-F1, and XYS_L-F1 groups showed a tendency to decrease compared with PPD-F1 group (Fig. 4 B, 4 C and 4 D, P > 0.05). Compared to day 1, the escape latency on day 5 decreased significantly in male offspring mice in the CON-F1 and XYS_H-F1 groups, as well as in female offspring mice in the CON-F1 and SERT-F1 groups (Fig. 4 C, 4 D, P < 0.05). In the spatial exploration experiment, compared to the CON-F1 group, both sexes of PPD-F1 offspring showed decreased platform crossings and reduced target quadrant occupancy time (%) (Fig. 4 B, 4 E and 4 F, P < 0.05 or P < 0.01). Compared with the PPD-F1 group, the numbers of crossing the original platform area and the percentage of time spent in the target quadrant were significantly increased of male and female offspring in XYS_H-F1 group (Fig. 4 B, 4 E and 4 F, P < 0.05). The percentage of time spent in the target quadrant was significantly elevated of male offspring in XYS_M-F1 group (Fig. 4 B, 4 F, P 0.05). In the Y-maze test, compared with the CON-F1 group, both male and female offspring mice had significantly lower spontaneous alternations in the PPD-F1 group (Fig. 4 H, P < 0.001). While compared with the PPD-F1 group, the spontaneous alternation rate was significantly higher in male and female offspring mice in the XYS_H-F1 group (Fig. 4 H, P < 0.05), and in male offspring mice in the XYS_M-F1 group (Fig. 4 H, P < 0.05). It is suggested that postpartum administration of XYS to PPD maternal mice intergenerationally improved the ability of spatial learning and memory in offspring mice. Behavior analyses of offspring demonstrated congruent trends in learning and memory-related behaviors between male and female offspring. Thus, subsequent analyses were performed on pooled male and female offspring data. 3.5 XYS intergenerationally impacted neurodevelopment of PPD offspring mice Postpartum treatment with XYS to PPD maternal mice was able to exert an iterative protective effect on offspring neurons and avoided pathological damage to hippocampal neurons in offspring mice. Hippocampal neurons in CON-F1 mice exhibited dense packing, intact morphology, and distinct nuclear boundaries without observable pathology. PPD-F1 hippocampal neurons demonstrated degenerative changes: nuclear pyknosis with hyperchromasia, cytoplasmic shrinkage (Fig. 5 , green arrows), and necrotic foci, confirming significant neuropathological injury In the hippocampus of the offspring of mice in the XYS_H-F1 group, the XYS_M-F1 group, the XYS_L-F1 group, only a small number of neuronal cells had poorly defined cytoplasmic nuclei, and nucleolus was not obvious (as shown by the green arrows in the figure), and no obvious pathological damage was seen (Fig. 5 A). Postpartum treatment with XYS to PPD maternal mice iteratively protects neurogenesis in the hippocampal DG region and increases the positive expression of neonatal neuronal markers in offspring mice. Compared with the CON-F1 group, DCX + immunoreactivity was significantly diminished in the hippocampal dentate gyrus of PPD-F1 offspring. (Fig. 5 B, 5 C, P 0.05). Compared with the PPD-F1 group, the expression of DCX + and NeuN + in the hippocampal DG region was enhanced in the offspring mice of the XYS_H-F1 group (Fig. 5 B, 5 C, 5 D, P < 0.05). Postpartum treatment of XYS to PPD maternal mice iteratively improved dendritic spine growth and synaptic protein expression in PPD offspring. Dendritic spine density and dendritic spine length were significantly lower in the hippocampal region in the PPD-F1 group, compared to the CON-F1 group (Fig. 6 A, 6 B, 6 C, P < 0.01). In contrast to the PPD-F1 group, dendritic spine density and dendritic spine length were higher in the hippocampal region in the XYS_H-F1 group (Fig. 6 A, 6 B, 6 C, P < 0.01 or P < 0.001). Additionally, the levels of hippocampal synapse-associated proteins SYP and PSD95 proteins were reduced in the PPD-F1 group compared with the CON-F1 group (Fig. 6 D, 6 E, 6 F, P < 0.05 or P < 0.01). Compared with PPD-F1 group, hippocampal SYP and PSD95 protein levels were significantly increased in XYS_H-F1 and XYS_M -F1 groups (Fig. 6 D, 6 E, 6 F, P < 0.05 or P < 0.01), and hippocampal SYP protein levels were significantly increased in XYS_L -F1 group (Fig. 6 D, 6 E, P < 0.05). 3.6 XYS intergenerationally impacted hippocampal BDNF/mTOR signaling pathway of PPD offspring mice Postpartum treatment with XYS to PPD maternal mice iteratively increased serum BDNF levels in offspring mice. Compared with the CON-F1 group, the serum BDNF level in the offspring mice of the PPD-F1 group were significantly decreased (Fig. 7 B, P < 0.01). The serum BDNF levels in the offspring mice of XYS_H-F1 and XYS_M-F1 groups were significantly increased when compared to the PPD-F1 group (Fig. 7 B, P < 0.05 or P < 0.01). Additionally, the protein analysis results revealed that, hippocampal BDNF, p-TrkB/TrkB, p-PI3K/PI3K, p-AKT/AKT, p-mTOR/mTOR, and P70 ribosomal protein S6 kinase (P70S6K) protein levels were significantly decreased in the PPD-F1 group compared with the CON-F1 group (Fig. 7 A, 7 C, 7 D, 7 E, 7 F, 7 G, 7 H, P < 0.05 or P < 0.01 or P < 0.001). Compared with PPD-F1 group, hippocampal BDNF, p-TrkB/TrkB, p-PI3K /PI3K, p-AKT/AKT, p-mTOR/mTOR, and P70S6K protein levels were significantly higher in XYS_H-F1, (Fig. 7 A, 7 C, 7 D, 7 E, 7 F, 7 G, 7 H, P < 0.05 or P < 0.01), hippocampal BDNF, p-TrkB/TrkB, p-AKT/AKT protein levels were higher in XYS_M-F1(Fig. 7 A, 7 C, 7 D, 7 F, P < 0.01 or P < 0.001), hippocampal BDNF, p-TrkB/TrkB, p-AKT/AKT, and p-mTOR/mTOR protein levels were significantly higher in and XYS_L-F1 groups(Fig. 7 A, 7 C, 7 D, 7 F, 7 G, P < 0.05 or P < 0.01). It is suggested that postpartum XYS treatment given to PPD maternal mice could intergenerationally activate the hippocampal BDNF/mTOR signaling pathway in offspring mice. 3.7 BDNF receptor inhibitor ANA-12 suppressed the intergenerationally protective effect of XYS in neurodevelopment of PPD offspring mice During spatial acquisition training, escape latencies progressively declined across all offspring groups with successive training days. By day 5, CON + A-F1 offspring showed moderately prolonged latencies versus CON-F1 counterparts. (Fig. 8 B, 8 C, P > 0.05). There was a trend for the avoidance latency of the offspring mice in the XYS + A-F1 group to be elevated compared to the XYS-F1 group (Fig. 8 B, 8 C, P > 0.05). Moreover, escape latencies significantly decreased from day 1 to day 5 in CON-F1, XYS-F1, and CON + A-F1 progeny (Fig. 8 C, P < 0.05, P 0.05). During the spatial exploration experimental phase, CON + A-F1 offspring showed significantly fewer platform crossings and lower target quadrant dwell times than CON-F1 controls. (Fig. 8 B, 8 D, 8 E P < 0.01). Compared to XYS-F1 offspring, XYS + A-F1 offspring exhibited significantly fewer entries into and spent a lower percentage of time in the target quadrant (original platform location) (Fig. 8 B, 8 D, 8 E P 0.05). These results showed that intraperitoneal injection of ANA-12 reversed the improvement in the learning and memory ability of the offspring of PPD maternal mice given postpartum treatment with XYS. Compared with CON-F1 group, BDNF, p-TrkB/TrkB, p-PI3K /PI3K, p-AKT/AKT, and p-mTOR/mTOR protein levels were significantly decreased in the hippocampus of the offspring mice in the CON + A-F1 group (Fig. 9 A, 9 B, 9 C, 9 D, 9 E, 9 F, P < 0.05, P < 0.01 or P < 0.001). Compared with XYS-F1 group, BDNF, p-TrkB/TrkB, p-PI3K /PI3K, p-AKT/AKT, and p-mTOR/mTOR protein levels were significantly decreased in XYS + A-F1 group (Fig. 9 A, 9 B, 9 C, 9 D, 9 E, 9 F, P < 0.05 or P < 0.01 or P < 0.001). It is indicated that intraperitoneal injection of the BDNF receptor inhibitor into offspring mice reversed the activating effect of postpartum administration of XYS treatment to PPD maternal mice on the BDNF/mTOR signaling pathway in offspring. DCX⁺ labeling in the hippocampal DG of CON + A-F1 group was lower (Fig. 9 G, 9 I, P 0.05). Compared with the XYS-F1 group, DCX + expression in the hippocampal DG area was decreased in the XYS + A-F1 group (Fig. 9 G, 9 I, P 0.05). These findings demonstrate that ANA-12-mediated BDNF receptor inhibition impairs hippocampal DG neurogenesis in offspring, primarily suppressing DCX + expression. Critically, this intervention abolishes the transgenerational neuroprotective effects of maternal XYS treatment on offspring hippocampal neurogenesis in the PPD model. 4 Discussion Postpartum depression (PPD) is a multifactorial disorder influenced by social, psychological, and biological factors, including genetic predisposition, inflammation, neurotransmitter dysregulation, and hormonal imbalances[ 17 ]. Based on its etiology, various animal models have been developed to simulate PPD, such as hormone-simulated pregnancy models, chronic corticosterone (CORT) induction, perinatal stress exposure, chronic social stress, maternal-infant separation, and prenatal stress paradigms[ 1 ]. Current research on maternal PPD's offspring effects predominantly employs models established through: chronic corticosterone exposure, gestational/postpartum stress paradigms, or maternal separation protocols. The adverse effects of maternal PPD on offspring have been confirmed in multiple PPD models[ 37 – 39 ]. Notably, PPD pathogenesis is closely linked to HPA axis dysfunction. CORT, a pivotal hormone within this axis, critically mediates negative feedback regulation. Elevated CORT levels impair this feedback mechanism, leading to increased secretion of HPA-related hormones and depression-like behaviors in animals[ 40 ]. Additionally, multiple studies have demonstrated that administering high levels of CORT to postpartum female mice impairs maternal care behaviors and induces postpartum depression-like behaviors[ 7 , 33 , 41 ]. Our prior findings revealed tha CORT-induced PPD in mice not only impairs maternal behaviors but also leads to significant cognitive deficits in their offspring[ 11 ] Chronic CORT treatment in postpartum dams induced HPA axis hyperfunction and elevated serum CORT, ACTH, and CRH, consistent with prior studies. Treated mice showed: reduced maternal care time, decreased sucrose preference, prolonged immobility in TST/FST, and diminished center time (OFT) plus open arm time (EPMT). These changes confirm successful modeling of PPD-related anxiety- and depression-like behaviors. Beyond maternal pathology, this study reinforces the intergenerational impact of PPD on offspring neurodevelopment. Clinical and preclinical evidence has demonstrated that maternal PPD increases the risk of emotional, cognitive, and behavioral abnormalities in offspring, including impaired attention regulation, heightened stress reactivity, and reduced IQ scores[ 42 , 43 ]. For example, Amani et al. (2021) reported that offspring of prenatally stressed mothers exhibited decreased sucrose preference, impaired recognition memory, elevated CORT levels, and reduced hippocampal BDNF expression. Existing evidence indicates maternal PPD compromises spatial cognition and emotional regulation in adolescent male offspring[ 38 ]. As a highly plastic structure, the hippocampus exhibits significant stress sensitivity and critically regulates learning and memory processes[ 44 ]. Hippocampal neurogenesis and synaptic plasticity serve as critical indicators of neural plasticity[ 45 ]. DCX and NeuN are markers of neuronal differentiation: DCX labels neuronal progenitor cells and immature newborn neurons, while NeuN is expressed in mature neurons that subsequently integrate into neural circuits to modulate cognitive functions[ 14 ]. Studies have shown that chronic social defeat stress impairs social memory and spatial object recognition memory in mice, accompanied by reduced numbers of Ki67 + and DCX + cells (markers of neuronal proliferation and differentiation) in DG region of the hippocampus[ 46 ]. The synaptic protein PSD95 functions as a core scaffold within the postsynaptic membrane and is vital for storing neuronal information. SYP, a phosphoprotein located on the presynaptic membrane, regulates presynaptic vesicle quantity and neurotransmitter release[ 47 , 48 ]. Maternal malnutrition-induced cognitive dysfunction in adult offspring has been linked to decreased expression of PSD95 and SYP in the hippocampal CA1, CA2, CA3, and DG regions[ 49 ]. Dendritic spines form the morphological basis of synaptic plasticity. Various chronic stressors could reduce hippocampal dendritic spine density and length, decrease synaptic protein expression, and induce learning and memory impairments in rats[ 50 ]. Maternal PPD is increasingly associated with offspring cognitive deficits. Experimental CORT models show diminished DCX + immunoreactivity in the dorsal hippocampus of both adolescent progeny and adult female offspring (Gobinath et al., 2016). Our previous work demonstrated that CORT-induced PPD in mice not only impairs maternal behaviors but also leads to significant cognitive deficits in their offspring[ 11 ]. Our research found that administering high levels of CORT to postpartum maternal ICR mice to induce PPD prolonged the escape latency of male and female offspring mice in finding the platform during the Morris water maze test. It also reduced the number of crossing the original platform area and the percentage of movement time spent in the target quadrant, while decreasing the spontaneous alternation rate in the Y-maze test. Notably, the behavioral changes showed consistent trends in both male and female offspring, without significant differences between the sexes. Additionally, the results revealed that offspring of PPD-affected maternal mice not only exhibited reduced expression of DCX + and NeuN+, markers for newborn neurons in the DG region of the hippocampus, but also had decreased dendritic spine density, spine length, and synaptic protein expression in the hippocampus. These findings confirm that maternal PPD impaired learning and memory abilities as well as neurodevelopment in offspring mice, identifying it as a risk factor in the growth and developmental processes of subsequent generations. XYS has been confirmed to exhibit definite curative effects in the treatment of PPD and can produce intergenerational protective effects on offspring. In ancient Chinese medical literature, there is no specific terminology for PPD. Later physicians classified PPD into the categories of "depression syndrome", "visceral agitation (Zangzao,)", and " Baihe disease" based on its clinical manifestations. The pathogenesis of PPD involves postpartum deficiency of qi and blood. When blood fails to nourish the heart, it leads to mental restlessness. Excessive sorrow and anxiety damaging the heart and spleen, or emotional injuries causing liver qi stagnation that transforms into disease[ 51 ]. Therefore, the primary therapeutic principles for PPD are to soothe the liver and relieve depression, nourish blood, and strengthen the spleen. XYS, composed of eight medicinal herbs, is a classic formula for soothing the liver, relieving depression, nourishing blood, and strengthening the spleen. Clinical trials have shown that XYS effectively alleviates depressive symptoms during pregnancy, postpartum, and menopause, with minimal side effec[ 19 , 52 ]. Furthermore, combined therapy with fluoxetine and XYS demonstrated superior outcomes in PPD patients compared to fluoxetine alone[ 53 ]. In animal models, XYS improves depression-like behaviors, enhances learning and memory performance, increases dendritic complexity, and activates signaling pathways such as ERα-PI3K in the hippocampus[ 54 , 55 ]. Despite growing evidence of its maternal antidepressant efficacy, few studies have addressed its intergenerational effects on offspring. In accordance with the TCM principle of "treating the mother to benefit the child," In this study, PPD mother mice were treated with XYS during the postpartum lactation period. These results suggest that XYS can not only directly exert antidepressant effects on maternal PPD mice themselves, but also indirectly protect offspring mice through maternal administration, preventing the adverse effects of maternal PPD on the neurodevelopment, as well as learning and memory abilities of offspring. The study demonstrated that XYS significantly alleviated anxiety- and depression-like behaviors in maternal PPD mice, while also ameliorating HPA axis hyperactivity and neuronal damage. In addition, maternal treatment with XYS can improve the learning and memory abilities of offspring mice across generations. In the Morris water maze, PPD offspring mice of both sexes exhibited significantly reduced escape latencies during spatial acquisition training. Furthermore, during the probe test, these mice demonstrated increased platform crossings and spent a greater percentage of time in the target quadrant. Moreover, in the Y-maze test, it enhances the spontaneous alternation rate in both male and female offspring mice. In addition, maternal treatment with XYS can intergenerationally improve the learning and memory abilities of offspring mice, reduce the escape latency of male and female PPD offspring mice during the spatial acquisition training phase of the Morris water maze test, increase the number of crossings into the original platform area and the percentage of movement time spent in the target quadrant during the spatial exploration test phase, and increase the spontaneous alternation rate of male and female offspring mice in the Y-maze test. Moreover, maternal treatment with XYS can also improve the indicators related to hippocampal neurogenesis and synaptic function in offspring mice. The intergenerational protection conferred by XYS on offspring mice might involve BDNF/mTOR pathway modulation (Fig. 10 ). BDNF has emerged as a key regulator of neuronal development and synaptic plasticity, which plays a crucial role in the regulation of learning and memory abilities[ 56 , 57 ]. BDNF binds to its receptor TrkB, which can activate the downstream PI3K/ AKT/ mTOR signaling pathway[ 13 ]. This process increased the expression of neuronal differentiation markers DCX and NeuN in the DG region of the hippocampus, thereby regulating neuronal growth and differentiation, promoting neurogenesis, and affecting cognitive functions[ 14 ]. The binding of BDNF to TrkB can also activate downstream targets such as AKT, extracellular signal-regulated kinase (ERK), and cAMP response element-binding protein (CREB) to protect neuronal survival and regulate neural plasticity[ 58 ]. Stress can reduce BDNF, downregulate its downstream protein mTOR, decrease the expression of the synaptic protein PSD95, and damage synaptic morphology[ 13 ]. Additionally, BDNF can regulate the downstream P70S6K protein level by activating the mTOR signaling pathway, thereby upregulating synaptic protein levels and improving synaptic function and cognitive dysfunction[ 59 ]. Studies show maternal PPD lowers offspring hippocampal BDNF, suppressing downstream signaling and causing cognitive impairment. The PPD model established by prenatal stress downregulates BDNF levels in the whole brain of offspring, leading to cognitive function deficits in offspring[ 9 ]. Furthermore, studies have found that the BDNF/AKT/mTOR signaling pathway in the hippocampus of adult offspring from a PPD model induced by pre-pregnancy chronic unpredictable mild stress (CUMS) is impaired[ 10 ]. In the PPD model constructed by pre-pregnancy restraint stress, the AKT/mTOR signaling pathway in the hippocampus of offspring is inhibited during adolescence and adulthood, with downregulated expression of phosphorylated proteins of AKT, mTOR, and P70S6K[ 60 ]. Therefore, this study hypothesizes that regulating the BDNF/mTOR signaling pathway may be one of the pathways for preventing and treating cognitive impairment in offspring. This study demonstrated inhibition of the hippocampal BDNF/mTOR signaling pathway in offspring of the PPD model, with significantly downregulated protein levels of BDNF, p-TrkB/TrkB, p-PI3K/PI3K, p-AKT/AKT, p-mTOR/mTOR, and P70S6K. Treatment of PPD maternal mice with XYS increased BDNF levels in the serum and hippocampus of their offspring mice, activated the hippocampal BDNF/mTOR signaling pathway, and upregulated the levels of related proteins. To further validate the critical role of BDNF and its downstream signaling pathway in the intergenerational protective effect of XYS on learning and memory ability in PPD offspring, ANA-12 (a BDNF receptor antagonist) was used to inhibit BDNF receptors in this study. Experimental results showed that after inhibition of BDNF receptors in offspring mice, the BDNF/mTOR signaling pathway was suppressed, which reversed the intergenerational protective effect of XYS on offspring mice. Concurrently, offspring mice exhibited significant cognitive deficits in spatial learning and memory consolidation. This was paralleled by diminished DCX⁺ immunoreactivity, suggesting BDNF mediates the intergenerational preservation of neural plasticity and cognitive functions through XYS intervention. 5 Conclusion This study confirms that PPD can adversely affect offspring neurodevelopment, consistent with previous findings. XYS, a classical traditional Chinese medicine formula with multi-component and multi-target characteristics, not only ameliorates depressive-like behaviors in PPD mouse model but also confers significant intergenerational protective effects on their offspring. In our experiments, high-dose XYS exerted the most pronounced protective effects on hippocampal neurogenesis and cognitive performance in the offspring. These benefits were associated with the intergenerational protective effects of XYS were mediated through BDNF/mTOR signaling activation, as evidenced by upregulated hippocampal expression of BDNF, p-TrkB/TrkB, and downstream PI3K/AKT/mTOR/P70S6K pathway components in offspring. We hypothesize that the intergenerational protective effects of XYS may be mediated through two complementary mechanisms: (1) indirect modulation of maternal endocrine levels that improves maternal-infant interactions;(2) direct transmission of active components through maternal milk that influence neurodevelopment in the offspring. Future investigations will delineate the compositional dynamics of maternal milk and elucidate the bioactive mediators underlying these functional effects. This work also contributes to improve population quality and promoting theoretical innovation and clinical expansion of TCM in the transgenerational intervention of mental disorders. Abbreviations ACTH, adrenocorticotropic hormone; AKT, protein kinase B; BDNF, brain-derived neurotrophic factor; CORT, corticosterone; CREB, cAMP response element-binding protein; CRH, corticotropin-releasing hormone; CUMS, chronic unpredictable mild stress; X, doublecortin; DG, dentate gyrus; Elisa, enzyme-linked immunosorbent assay; EPMT, elevated plus maze test; ERK, extracellular signal-regulated kinase; FST, forced swimming test; HE, hematoxylineosin staining; HPA, hypothalamic-pituitary-adrenal; mTOR, mammalian target of rapamycin; NeuN, neuronal nuclear antigen; OFT, open field test; P70S6K, P70 ribosomal protein S6 kinase.; PD, postpartum day; PI3K, phosphoinositide 3-kinase; PPD, postpartum depression; PSD95, postsynaptic density protein 95; SPT, sucrose preference test; SYP, synaptophysin; TrkB, tropomyosin-related kinase B; TST, tail suspension test; XYS, Xiaoyao San; Declarations Ethics declarations Ethics approval and consent to participate The animal experiment followed the guidelines of the Committee for Animal Care and Use of Laboratory Animals of the College of Pharmacy, Chengdu University of Traditional Chinese Medicine (approval No. TCM-2017-312). Consent for publication Not applicable. Availability of data and materials The data used to support this research are available from the corresponding author upon request. Competing interests The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Funding This work was supported by the University–Hospital Joint Innovation Fund Project of ChengDu TCM (LH 202402016). Authors' contributions H X designed and conceived this study. H X completed data analysis, experimentation and manuscript writing. J D, Y J, R Y, Z X, X Z, Q Z carried out experiments. J Z, L C provided suggestions for the research. Q Y reviewed the manuscript. N Z, R L conceived and designed the experiment and reviewed the manuscript. Acknowledgements Not applicable. Authors' information Hongxiao Xie 1 # and Junping Ding 1 # These authors contributed equally to this work and share first authorship. * Corresponding authors: Correspondence to:Nan Zeng, Qiong Yi, Rong Liu State Key Laboratory of Southwestern Chinese Medicine Resources, School of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan, 611137, China; Hongxiao Xie, Junping Ding, Yanning Jiang, Yixin Rui, Zhiqiang Xie, Xiumeng Zhang, Jiuseng Zeng, Quanrun Zhu, Li Chen, Nan Zeng, Rong Liu Department of Pharmacy, The First People's Hospital of Shuangliu District, West China (Airport) Hospital of Sichuan University, Chengdu,Sichuan, 610200, China Zhiqiang Xie, Xiumeng Zhang Department of Pharmacy, Clinical Medical College and the First Affiliated Hospital of Chengdu Medical College, Chengdu, Sichuan,610500, China Li Chen Meishan Hospital of Chengdu University of TCM, Meishan,Sichuan, 620010, China Qiong Yi References Mir FR, Pollano A, Rivarola MA. 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09:07:21","extension":"xml","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":193445,"visible":true,"origin":"","legend":"","description":"","filename":"c28b4af6f1b64eefbe28e19d7303b4a11structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7515178/v1/6f912583ddc13e6a2543651f.xml"},{"id":94091820,"identity":"9d0b770c-9369-4d8a-8baa-3765f817e275","added_by":"auto","created_at":"2025-10-22 09:10:04","extension":"html","order_by":25,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":211830,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7515178/v1/979ce3b77f89d9dba269d8c2.html"},{"id":94091860,"identity":"f3abb749-6ed9-446e-9e25-a23fd922d900","added_by":"auto","created_at":"2025-10-22 09:10:34","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":60748,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe quality control map of XYS. (A) \u003c/strong\u003eHigh performance liquid chromatography (HPLC) map of Paeoniflorin standard solution.\u003cstrong\u003e (B)\u003c/strong\u003eHPLC map of XYS test solution.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7515178/v1/31dd4b490d8a3f4444022a95.png"},{"id":94091763,"identity":"6f4c7baa-45ac-4a30-a565-2c7a56bf3722","added_by":"auto","created_at":"2025-10-22 09:09:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":6055619,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eXYS increased maternal nursing and improved anxiety- and depression-like behavior of PPD maternal mice. (A) \u003c/strong\u003eTimeline of the experimental procedure of maternal mice. \u003cstrong\u003e(B) \u003c/strong\u003eMaternal caring time in the maternal behavior test, n=6.\u003cstrong\u003e (C)\u003c/strong\u003e Sucrose preference in the SPT, n=6. \u003cstrong\u003e(D)\u003c/strong\u003e Immobility time in the TST, n=6. \u003cstrong\u003e(E)\u003c/strong\u003e Immobility time in the FST, n=6. \u003cstrong\u003e(F)\u003c/strong\u003e Representative tracking plots of the OFT.\u003cstrong\u003e (G) \u003c/strong\u003eTime spent in centre zone in the OFT, n=6.\u003cstrong\u003e (H)\u003c/strong\u003e Total distance traveled in the OFT, n=6. \u003cstrong\u003e(I) \u003c/strong\u003eTime spent in open arm in the EPMT, n=6.\u003cstrong\u003e (J) \u003c/strong\u003eRepresentative tracking plots of the EPMT. n: number in per group. Results are presented as means ± SD. PPD vs CON,\u003csup\u003e #\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05; \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01; \u003csup\u003e###\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001; XYS vs PPD, SERT vs PPD, *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05; **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01; ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001; ns, non-significant difference.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7515178/v1/c79a7855aca7a92ad305a30a.png"},{"id":94091744,"identity":"994ba632-7f74-4eab-a246-3354fc1e02e5","added_by":"auto","created_at":"2025-10-22 09:09:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":5700965,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eXYS inhibited HPA axis hyperfunction and ameliorated hippocampal neuronal damage of PPD maternal mice. (A)\u003c/strong\u003eThe changes in adrenal index of maternal mice, n=6. \u003cstrong\u003e(B) \u003c/strong\u003eThe level of CORT in the serum, n=6.\u003cstrong\u003e (C) \u003c/strong\u003eThe level of ACTH in the serum, n=6.\u003cstrong\u003e (D) \u003c/strong\u003eThe level of CRH in the serum, n=6.\u003cstrong\u003e (E) \u003c/strong\u003eRepresentative images of neuronal cell changes in the hippocampus with HE staining. Scale bar: 100 μm and 20μm. n: number in per group. Results are presented as means ± SD. PPD vs CON,\u003csup\u003e #\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05; \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01; \u003csup\u003e###\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001; XYS vs PPD, SERT vs PPD, *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05; **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01; ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001; ns, non-significant difference.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7515178/v1/2a967c8cd92f45f007ccd12a.png"},{"id":94091797,"identity":"d377aaaf-111e-4ebf-9be1-e359b798afd7","added_by":"auto","created_at":"2025-10-22 09:09:55","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":5867746,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eXYS intergenerationally protected the learning and memory ability of PPD offspring mice. (A)\u003c/strong\u003e Timeline of the experimental procedure of offspring mice in Experient 1. \u003cstrong\u003e(B) \u003c/strong\u003eRepresentative tracking plots of the morris water maze test.\u003cstrong\u003e (C) \u003c/strong\u003eEscape latency in the morris water maze test during training period of male offspring, n=6.\u003cstrong\u003e (D) \u003c/strong\u003eEscape latency in the morris water maze test during training period of female offspring, n=6.\u003cstrong\u003e (E) \u003c/strong\u003eNumbers of crossing platform in the morris water maze test of male and female offspring, n=6. \u003cstrong\u003e(F) \u003c/strong\u003ePercentage of time spent in the target quadrant in the morris water maze test of male and female offspring, n=6. \u003cstrong\u003e(G) \u003c/strong\u003eSwimming speed in the morris water maze test of male and female offspring, n=6. \u003cstrong\u003e(H) \u003c/strong\u003eSpontaneous alternation in the Y-maze test of male and female offspring, n=6. n: number in per group. Results are presented as means ± SD. PPD-F1 vs CON-F1,\u003csup\u003e #\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05; \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01; \u003csup\u003e###\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001; XYS-F1 vs PPD-F1, SERT-F1 vs PPD-F1, *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05; **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01; ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001; ns, non-significant difference. Compared with the 1st day training in this group, \u003csup\u003e▲\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05. As the trends of female and male offspring showed similar trends, the figure only presents the representative trajectory of male offspring.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7515178/v1/201a3c08fa551d54d3135da3.png"},{"id":94091817,"identity":"62dc9503-ca03-4eef-9db6-c9185c714d7f","added_by":"auto","created_at":"2025-10-22 09:10:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":8894438,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eXYS intergenerationally impacted neurodevelopment of PPD offspring mice. (A)\u003c/strong\u003e Representative images of neuronal cell changes in the hippocampus with HE staining. Scale bar: 100 μm and 20μm. \u003cstrong\u003e(B) \u003c/strong\u003eRepresentative images of the CON and CORT with double immunostaining of DCX\u003csup\u003e+ \u003c/sup\u003e(red) and NeuN\u003csup\u003e+\u003c/sup\u003e (green) cells, scale bar: 50 μm.\u003cstrong\u003e (C) \u003c/strong\u003eQuantification of DCX\u003csup\u003e+\u003c/sup\u003e cells, n=4. \u003cstrong\u003e(D) \u003c/strong\u003eQuantification of NeuN\u003csup\u003e+\u003c/sup\u003e cells, n=4. n: number in per group. Results are presented as means ± SD. PPD-F1 vs CON-F1,\u003csup\u003e #\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05; \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01; \u003csup\u003e###\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001; XYS-F1 vs PPD-F1, SERT-F1 vs PPD-F1, *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05; **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01; ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001; ns, non-significant difference.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7515178/v1/a0131e8671f5ca4aa4e21144.png"},{"id":94091848,"identity":"0500f3b3-b71f-40fa-8798-706726a1be65","added_by":"auto","created_at":"2025-10-22 09:10:21","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":5838968,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eXYS intergenerationally impacted neurodevelopment of PPD offspring mice. (A)\u003c/strong\u003e Representative images of neuronal spine changes in the hippocampus with Golgi staining. Scale bar: 10 μm. \u003cstrong\u003e(B) \u003c/strong\u003eQuantification of neuronal spine density in the hippocampus, n=12 (4×3).\u003cstrong\u003e (C) \u003c/strong\u003eQuantification of neuronal spine length in the hippocampus, n=12 (4×3).\u003cstrong\u003e (D) \u003c/strong\u003eRepresentative western blots analyzing PSD95 and SYP protein expression of offspring hippocampus; A: CON-F; B: PPD-F1; C: SERT-F1; D: XYS_H-F1; E: XYS_M-F1; F: XYS_L-F1.\u003cstrong\u003e (E) \u003c/strong\u003eQuantification of PSD95 protein expression of offspring hippocampus, n=3. \u003cstrong\u003e(F) \u003c/strong\u003eQuantification of SYP protein expression of offspring hippocampus, n=3. n: number in per group. Results are presented as means ± SD. PPD-F1 vs CON-F1,\u003csup\u003e #\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05; \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01; \u003csup\u003e###\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001; XYS-F1 vs PPD-F1, SERT-F1 vs PPD-F1, *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05; **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01; ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001; ns, non-significant difference.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7515178/v1/ce812d593e070753a4ff4116.png"},{"id":94091795,"identity":"09e16b0f-b9d8-474b-a193-3efe562875d8","added_by":"auto","created_at":"2025-10-22 09:09:53","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":4007376,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eXYS intergenerationally impacted hippocampal BDNF/mTOR signaling pathway of PPD offspring mice. (A)\u003c/strong\u003e Representative western blots analyzing protein expression of BDNF/mTOR signaling pathway of offspring hippocampus; A: CON-F; B: PPD-F1; C: SERT-F1; D: XYS_H-F1; E: XYS_M-F1; F: XYS_L-F1.\u0026nbsp; \u003cstrong\u003e(B) \u003c/strong\u003eThe level of serum BDNF of offspring, n=6.\u003cstrong\u003e (C) \u003c/strong\u003eQuantification of BDNF protein expression of offspring hippocampus, n=3.\u003cstrong\u003e (D) \u003c/strong\u003eQuantification of p-TrkB/TrkB protein expression of offspring hippocampus, n=3.\u003cstrong\u003e (E) \u003c/strong\u003eQuantification of p-PI3K/PI3K protein expression of offspring hippocampus, n=3. \u003cstrong\u003e(F) \u003c/strong\u003eQuantification of p-AKT/AKT protein expression of offspring hippocampus, n=3. \u003cstrong\u003e(G) \u003c/strong\u003eQuantification of p-mTOR/mTOR protein expression of offspring hippocampus, n=3. \u003cstrong\u003e(H) \u003c/strong\u003eQuantification of P70S6K protein expression of offspring hippocampus, n=3. n: number in per group. Results are presented as means ± SD. PPD-F1 vs CON-F1, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05; \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01; \u003csup\u003e###\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001; XYS-F1 vs PPD-F1, SERT-F1 vs PPD-F1, *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05; **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01; ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001; ns, non-significant difference\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7515178/v1/905db73df685272b2353a425.png"},{"id":94091831,"identity":"b5d2e4bf-e0f5-4002-b759-4dfc6a85861a","added_by":"auto","created_at":"2025-10-22 09:10:10","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":5283529,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBDNF receptor inhibitor ANA-12 suppressed the intergenerationally protective effect of XYS in neurodevelopment of PPD offspring mice. (A)\u003c/strong\u003e Timeline of the experimental procedure of offspring mice Experient 2. \u003cstrong\u003e(B) \u003c/strong\u003eRepresentative tracking plots of the morris water maze test.\u003cstrong\u003e (C) \u003c/strong\u003eEscape latency in the morris water maze test during training period of offspring, n=10.\u003cstrong\u003e (D) \u003c/strong\u003eNumbers of crossing platform in the morris water maze test of offspring, n=10.\u003cstrong\u003e (E) \u003c/strong\u003ePercentage of time spent in the target quadrant in the morris water maze test of offspring, n=10. \u003cstrong\u003e(F) \u003c/strong\u003eSwimming speed in the morris water maze test of male and female offspring, n=10. n: number in per group. Results are presented as means ± SD. PPD-F1, CON+A-F1 vs CON-F1,\u003csup\u003e #\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05; \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01; \u003csup\u003e###\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001; XYS-F1 vs PPD-F1, *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05; **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01; ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001; XYS+A-F1 vs XYS-F1, \u003csup\u003e\u0026amp;\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05; \u003csup\u003e\u0026amp;\u0026amp;\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01; \u003csup\u003e\u0026amp;\u0026amp;\u0026amp;\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001; ns, non-significant difference.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7515178/v1/8e3f0c9dc8e6bed17d5ceab8.png"},{"id":94091758,"identity":"8244d757-4ad6-44dc-93de-c92d8c5a8474","added_by":"auto","created_at":"2025-10-22 09:09:42","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":6455739,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBDNF receptor inhibitor ANA-12 suppressed the intergenerationally protective effect of XYS in neurodevelopment of PPD offspring mice. (A)\u003c/strong\u003e Representative western blots analyzing protein expression of BDNF/mTOR signaling pathway of offspring hippocampus. \u003cstrong\u003e(B)\u003c/strong\u003e Quantification of BDNF protein expression of offspring hippocampus, n=3; A: CON-F1; B: PPD-F1; C: XYS-F1; D: CON+A-F1; E: XYS+A-F1.\u003cstrong\u003e (C)\u003c/strong\u003e Quantification of p-TrkB/TrkB protein expression of offspring hippocampus, n=3.\u003cstrong\u003e (D) \u003c/strong\u003eQuantification of p-PI3K/PI3K protein expression of offspring hippocampus, n=3.\u003cstrong\u003e (E) \u003c/strong\u003eQuantification of p-AKT/AKT protein expression of offspring hippocampus, n=3.\u003cstrong\u003e (F) \u003c/strong\u003eQuantification of p-mTOR/mTOR protein expression of offspring hippocampus, n=3.\u003cstrong\u003e (G) \u003c/strong\u003eQuantification of DCX\u003csup\u003e+\u003c/sup\u003e cells, n=3.\u003cstrong\u003e (H)\u003c/strong\u003e Quantification of NeuN\u003csup\u003e+\u003c/sup\u003e cells, n=3.\u003cstrong\u003e (I) \u003c/strong\u003eRepresentative images of the CON and CORT with double immunostaining of DCX\u003csup\u003e+\u003c/sup\u003e (red) and NeuN\u003csup\u003e+\u003c/sup\u003e (green) cells, scale bar: 50 μm.\u003cstrong\u003e \u003c/strong\u003en: number in per group. Results are presented as means ± SD. PPD-F1, CON+A-F1 vs CON-F1,\u003csup\u003e #\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05; \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01; \u003csup\u003e###\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001; XYS-F1 vs PPD-F1, *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05; **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01; ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001; XYS+A-F1 vs XYS-F1, \u003csup\u003e\u0026amp;\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05; \u003csup\u003e\u0026amp;\u0026amp;\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01; \u003csup\u003e\u0026amp;\u0026amp;\u0026amp;\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001; ns, non-significant difference.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-7515178/v1/c2b2f338ce660c503476a4c4.png"},{"id":94091811,"identity":"790d2af5-5bf7-4aa9-a184-d1e602915a55","added_by":"auto","created_at":"2025-10-22 09:09:57","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":1482456,"visible":true,"origin":"","legend":"\u003cp\u003eXYS ameliorated maternal caring behavior and depressive-like behaviors in PPD maternal mouse. XYS could also exert intergenerational protection to learning-memory ability and neurodevelopment of offspring via mediating BDNF/mTOR signaling activation, as evidenced by upregulated hippocampal expression of BDNF, p-TrkB/TrkB, and downstream PI3K/AKT/mTOR/P70S6K pathway components in offspring. (Created in https://BioRender.com).\u003c/p\u003e","description":"","filename":"floatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-7515178/v1/5b023e5219ef25697ef274e0.png"},{"id":99789980,"identity":"acf9bf69-411a-49d4-92f7-89152413346d","added_by":"auto","created_at":"2026-01-08 12:51:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":51568658,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7515178/v1/18c25f28-7761-4293-9b58-943fdaf06089.pdf"},{"id":94091879,"identity":"93b70fb2-1833-46e5-b6fc-d9b4745b7993","added_by":"auto","created_at":"2025-10-22 09:10:42","extension":"pptx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":11490293,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalfigureWB20250831.pptx","url":"https://assets-eu.researchsquare.com/files/rs-7515178/v1/90eb9547c30bd12487492129.pptx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Xiaoyao San intergenerational protects learning-memory ability and neurodevelopment in PPD offspring via the BDNF/mTOR signaling pathway","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003ePostpartum depression (PPD), as defined in the \u003cem\u003eDiagnostic and Statistical Manual of Mental Disorders\u003c/em\u003e (DSM-V), is a subtype of major depressive disorder that emerges within 4 to 6 weeks after pregnancy or childbirth[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. It is the most prevalent psychiatric condition during the puerperium, with global incidence rates ranging from 15\u0026ndash;30%[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. PPD significantly compromises maternal-infant bonding and caregiving, as evidenced by decreased breastfeeding frequency, diminished emotional responsiveness, and impaired mother-infant attachment. These disruptions have been associated with adverse outcomes in the offspring, including emotional dysregulation, cognitive deficits, and developmental delays[\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eNeurodevelopment encompasses the acquisition and refinement of motor, sensory, cognitive, linguistic, psychosocial, and self-regulatory capacities throughout the lifespan. A critical neurodevelopmental window occurs during early postnatal stages, particularly from conception through the first two years[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. PPD impairs maternal care behaviors in lactating animals, thereby creating early-life adversity during offspring development[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Animal studies indicate that maternal PPD compromises caregiving behaviors, inducing postnatal adversity that disrupts offspring hippocampal neurogenesis and synaptic plasticity. This is evidenced by reduced doublecortin (DCX) expression-a biomarker of immature hippocampal neurons - correlating with depression-like phenotypes and cognitive impairment[\u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAs a key neurotrophin, brain-derived neurotrophic factor (BDNF) critically regulates hippocampal neuronal proliferation, synaptic formation, and apoptosis[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. BDNF further modulates higher-order cognitive functions including learning and memory consolidation[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Emerging evidence indicates BDNF regulates mTOR-dependent autophagy via the TrkB/PI3K/Akt pathway, subsequently modulating hippocampal synaptic integrity and cognitive function [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Moreover, elevated BDNF levels and activation of the PI3K/AKT/mTOR signaling cascade have been linked to reductions in neuroinflammation and oxidative stress, contributing to cognitive resilience\u003c/p\u003e\u003cp\u003eTimely pharmacological intervention following PPD onset is essential to mitigate its adverse consequences, particularly those affecting offspring. Although selective serotonin reuptake inhibitors (SSRIs) remain the first-line pharmacotherapy, they are associated with safety concerns in lactating mothers. For instance, fluoxetine can be secreted into breast milk, resulting in reduced maternal body weight, altered milk composition, and long-lasting growth deficits in the offspring[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Brexanolone (Zulresso), a neuroactive steroid recently approved by the U.S. FDA, has shown efficacy in treating PPD but also carries risks such as excessive sedation and loss of consciousness[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Animal data further indicate that exposure to high doses of brexanolone during gestation may lead to teratogenic and neurobehavioral abnormalities in the offspring [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eGiven the limitations of current treatments, traditional Chinese medicine (TCM) and integrative therapies have gained attention for their clinical efficacy and favorable safety profile in managing PPD[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Rooted in centuries of clinical practice, TCM emphasizes the interplay between maternal emotional states and fetal health, captured in classical doctrines such as \u0026ldquo;external influences shape internal states, and these states form the basis for the offspring\u0026rdquo; and \u0026ldquo;injury to the mother\u0026rsquo;s qi harms the child\u0026rsquo;s qi.\u0026rdquo; Accordingly, TCM treatment philosophies like \u0026ldquo;treating the mother to protect the child\u0026rdquo; and \u0026ldquo;preventive treatment of disease\u0026rdquo; reflect a longstanding focus on intergenerational health. Recent experimental studies have validated these concepts, showing that pharmacological interventions in PPD mothers\u0026mdash;such as ketamine or salidroside\u0026mdash;can indirectly promote neurodevelopment and reduce depressive behaviors in the offspring[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].Traditional Chinese medical (TCM) theory has long emphasized the link between maternal emotional health and offspring development, as reflected in classical tenets such as \u0026ldquo;external influences shape internal states, and these states form the basis for the offspring,\u0026rdquo; and \u0026ldquo;if the mother\u0026rsquo;s qi is injured, the child\u0026rsquo;s qi will also be harmed.\u0026rdquo; These principles underscore the potential intergenerational consequences of maternal psychological disturbances. TCM practitioners have proposed treatment concepts such as \"treating the mother to protect the child,\" \"treating the mother for the child's illness,\" and \"preventive treatment of disease\". Recent experimental studies have validated these concepts, showing that pharmacological interventions in PPD mothers\u0026mdash;such as ketamine or salidroside\u0026mdash;can indirectly promote neurodevelopment and reduce depressive behaviors in the offspring[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Salidroside can prevent learning disabilities in offspring caused by pregnancy-induced hypertension by intervening in the Wnt/SKp2 pathway[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eXiaoyao San (XYS), a classical TCM formula originating from the \u003cem\u003eFormulary of the Bureau of Peaceful Benevolent Dispensaries\u003c/em\u003e, has demonstrated antidepressant properties via multiple molecular pathways. Previous studies have shown that XYS attenuates HPA axis hyperactivity[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], modulates monoaminergic neurotransmission[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], and suppresses oxidative stress and inflammation in key brain regions[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Clinically, XYS is widely prescribed for PPD due to its ability to harmonize liver and spleen functions and restore qi-blood balance. Numerous clinical trials have demonstrated its effectiveness in alleviating depressive symptoms, especially when used in combination with conventional antidepressants or other supportive therapies, with minimal side effects[\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Animal studies further support its role in restoring neurotransmitter balance and ameliorating depression-like behaviors in PPD models[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. However, despite its established antidepressant effects, the potential transgenerational neuroprotective actions of XYS remain underexplored.\u003c/p\u003e\u003cp\u003eIn this study, we employed a corticosterone-induced PPD mouse model to investigate the maternal and intergenerational effects of XYS. We focused on hippocampal neuroplasticity and the BDNF/mTOR signaling pathway to evaluate the efficacy of XYS in alleviating depressive-like behaviors in mothers and promoting neurodevelopment and cognitive function in offspring. This research not only advances our understanding of XYS as a multi-target intervention for PPD but also provides a scientific foundation for its clinical application in protecting maternal and offspring mental health. Ultimately, our findings aim to support early-stage, transgenerational interventions and expand the global relevance of traditional Chinese medicine in the field of neuropsychiatry.\u003c/p\u003e"},{"header":"2 Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Reagents\u003c/h2\u003e\u003cp\u003eThe eight herbal components of XYS are \u003cem\u003eBupleuri Radix\u003c/em\u003e (Lingnan Traditional Chinese Medicine Co., Ltd, 20220201), \u003cem\u003eAngelicae Sinensis Radix\u003c/em\u003e, \u003cem\u003ePaeoniae Alba Radix\u003c/em\u003e, \u003cem\u003eAtractylodes Macrocephala Rhizoma\u003c/em\u003e, \u003cem\u003eGlycyrrhizae Radix et Rhizoma\u003c/em\u003e, \u003cem\u003ePoria\u003c/em\u003e (Sichuan Xin lotus traditional Chinese medicine co., Ltd, D2112001, 2206152, 2205057, 2204046, D2206022), \u003cem\u003eMentha Haplocalycis Herba\u003c/em\u003e (Jiangsu Donglian Drug Co., Ltd, 20210227), and \u003cem\u003eZingiber\u003c/em\u003e (purchased from the local market). They were identified by Professor Wang Guangzhi from the School of Pharmacy, Chengdu University of Traditional Chinese Medicine. The quality control substance of Paeoniflorin in the Chinese Pharmacopoeia (2020 edition) for Xiaoyaosan was purchased from Chengdu Kroma Biotechnology Co., Ltd. Corticosterone was purchased from MedChemExpress (MCE, NJ, USA). Sertraline hydrochloride (SERT) purchased from Pfizer (Wuxi, Jiangsu, China). The BDNF inhibitor (ANA-12) was purchased from Selleck (Texas, USA). Enzyme-linked immunosorbent assay (ELISA) kits were purchased from Elabscience (Wuhan,Hubei,China).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Animals\u003c/h2\u003e\u003cp\u003eThirty-six female and eighteen male adult ICR mice (8-week-old) were sourced from SPF Biotechnology Co., Ltd. (Beijing, China; Certification: SCXK-[Jing]-2019-0010). Animals were acclimatized for 7 days under controlled conditions: 22\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, 40\u0026ndash;60% humidity, 12-hour light/dark cycle, with ad libitum access to food and water. The experimental procedures followed the guidelines of the Committee for Animal Care and Use of Laboratory Animals of the College of Pharmacy, Chengdu University of Traditional Chinese Medicine (approval No. TCM-2017-312).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Preparations of XYS\u003c/h2\u003e\u003cp\u003eThe XYS extracts preparation was carried out as previously described[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. We mixed \u003cem\u003eBupleuri\u003c/em\u003e, \u003cem\u003eAngelicae\u003c/em\u003e, \u003cem\u003ePaeoniae\u003c/em\u003e, \u003cem\u003eAtractylodes\u003c/em\u003e, \u003cem\u003ePoria\u003c/em\u003e, \u003cem\u003eZingiber\u003c/em\u003e, \u003cem\u003eGlycyrrhizae\u003c/em\u003e, and \u003cem\u003eMentha\u003c/em\u003e in the ratio of 6:6:6:6:6:6:3:1 (w/w, with a total weight of 1320 g). Add the mixture of raw herbs with 8 times the amount of water and soak for 30 minutes,followed by 1 h boiling at 100\u0026deg;C. Two additional extractions were performed with 6 volumes of water for 40 and 30 minutes, respectively. Combine the three filtrates and concentrate under reduced pressure to 1.5 g/m L. According to the Chinese Pharmacopoeia (2020 Edition), the content of paeoniflorin in the water decoction of Xiaoyaosan was quantified using high-performance liquid chromatography (HPLC). Specifically, 8 mg of the paeoniflorin reference standard was dissolved in 2.5 mL of 50% methanol to prepare a stock solution with a concentration of 3.2 mg/mL. A standard curve was then established by the method of serial dilution.Chromatographic separation was achieved on a Hypersil GOLD C18 column (250 mm \u0026times; 4.6 mm, 5 \u0026micro;m, Thermo Scientific) with a mobile phase of 0.1% phosphoric acid and acetonitrile (86:14, v/v), flow rate of 1.0 mL/min, column temperature of 30\u0026deg;C, and detection wavelength of 230 nm.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Establishment of the PPD model and drug treatment\u003c/h2\u003e\u003cp\u003eFemale and male mice were co-housed in Plexiglas cages with a sex ratio of 1:2 (one female to two males). Each morning, we checked the female mice for the presence of a vaginal plug and monitored their body weight to confirm pregnancy. Once confirmed, pregnant dams were immediately transferred to individual cages. The day of delivery was designated as postnatal day 0 (PD0). To ensure standardized litters, we culled the offspring to retain exactly 8 pups per dams (4 males and 4 females). Dams were randomly allocated to six experimental groups. (n\u0026thinsp;=\u0026thinsp;6/group): CON, PPD, SERT (25 mg/kg, i.g.), XYS_H (45 g/kg, i.g.), XYS_M (30 g/kg, i.g.), and XYS_L (15 g/kg, i.g.). From PD1-PD21 (for 21 days), except the CON group, all groups received daily intraperitoneal injections of CORT (40 mg/kg) between 9:00\u0026ndash;10:00. Six hours post-CORT, mice received corresponding drug treatments; CON and PPD groups received vehicles. The experimental workflow is illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Offspring group and drug treatment\u003c/h2\u003e\u003cp\u003e\u003cb\u003eExperiment 1: The iterative effect of XYS on the learning and memory ability of PPD offspring mice and a preliminary investigation of its mechanisms.\u003c/b\u003e Throughout development, offspring were not exposed to any drug treatments. On PD21, one male and one female pup per litter were randomly selected (n\u0026thinsp;=\u0026thinsp;12/group), grouped according to their maternal treatment (CON-F1, PPD-F1, SERT-F1, XYS-H-F1, XYS-M-F1, XYS-L-F1). Males and females were housed separately. Experimental workflow is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA. Data from offspring within the same group were pooled for joint analysis. The specific detail experimental workflow of offspring mice is depicted schematically in Figure.4A.\u003c/p\u003e\u003cp\u003e\u003cb\u003eExperient 2: Reverse validation of the effect of BDNF on neuroplasticity in XYS iteratively protected PPD offspring mice using the ANA-12, a BDNF receptor inhibitor.\u003c/b\u003e On PD21, we selected offspring (n\u0026thinsp;=\u0026thinsp;10/group, 5 males and 5 females) were divided into five groups: CON-F1, PPD-F1, XYS-F1, CON\u0026thinsp;+\u0026thinsp;A-F1, and XYS\u0026thinsp;+\u0026thinsp;A-F1. ANA-12 (0.5 mg/kg/day, i.p.) was administered to \u0026ldquo;+A\u0026rdquo; groups for 14 days from PD21. Other groups received saline[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Workflow is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA.\u003c/p\u003e\u003cp\u003ePreparation of ANA-12 inhibitor injection: 0.32 mg of the drug was dissolved in 320 \u0026micro; L of DMSO solution to make a mother liquor of 1 mg/m L. Then take 320 \u0026micro; L of DMSO mother liquor, add 2560 \u0026micro; L of PEG300 and 320 \u0026micro; L of Tween 80, and mix until clarified. Then adding 3200 \u0026micro; L of ddH2O, mixing until clarified and set aside[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Behavioral testing of maternal mice\u003c/h2\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003e2.6.1 Maternal care behavior\u003c/h2\u003e\u003cp\u003eMaternal care assessment followed established protocols[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Behavioral observations were conducted twice daily in the home cage (13:00\u0026ndash;14:00 and 18:00\u0026ndash;19:00) on postnatal days 4 and 8. Each 10-minute observation session commenced\u0026thinsp;\u0026ge;\u0026thinsp;2 hours post-injection. Maternal care behaviors recorded included three primary categories: (a) nursing postures, comprising arched-back nursing (active nursing with dorsally curved posture), blanket nursing (resting atop pups in nursing position), and passive nursing (side-lying position while nursing); (b) pup-directed licking, defined as licking behavior occurring independently of nursing; and (c) nest building, characterized by active rearrangement of nesting material. Video recordings were subsequently analyzed to quantify the duration of each behavior for each dam. Total maternal care time (sum of nursing, pup licking, and nest-construction durations) was calculated from two daily sessions using the formula: Maternal care time = (a) + (b) + (c).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section3\"\u003e\u003ch2\u003e2.6.2 Sucrose preference test (SPT)\u003c/h2\u003e\u003cp\u003eThe sucrose preference test (SPT) evaluated murine anhedonia\u0026mdash;a core depressive symptom. Prior to testing, a 48h habituation phase exposed mice to 1% sucrose and water bottles in individual cages, with bottle positions swapped at 12-h intervals. The test phase used pre-weighed bottles rotated identically every 12h.After 24 h, fluid consumption was measured. Sucrose preference was calculated as:[sucrose intake/(sucrose intake\u0026thinsp;+\u0026thinsp;water intake)]\u0026times;100% [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section3\"\u003e\u003ch2\u003e2.6.3 Forced swimming test (FST)\u003c/h2\u003e\u003cp\u003eFST/TST sustained immobility signifies behavioral despair with compromised escape drive. Mice underwent 6 min trials in cylindrical water tanks (25 cm depth, 24\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C). After 2 min habituation, immobility (defined as floating with only essential movements for flotation) during t\u0026thinsp;=\u0026thinsp;2-6min was scored by SMART 3.0 tracking system [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\u003ch2\u003e2.6.4 Tail suspension test (TST)\u003c/h2\u003e\u003cp\u003eTail suspension test (TST) was conducted in black-walled enclosures (18 cm width \u0026times; 18.5 cm height). Mice were suspended vertically 8 cm above the base using adhesive tape placed 2.5 cm proximal to the tail tip. Automated quantification of immobility duration focused on the terminal 4-min interval of 6 min trials, excluding the initial 120-s acclimatization period.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\u003ch2\u003e2.6.5 Open field test (OFT)\u003c/h2\u003e\u003cp\u003eThe open field apparatus was a 47 cm \u0026times; 47 cm square chamber. Mice were initially placed in a corner. After a 2-min acclimation period, total distance moved and central area duration (23.5 \u0026times; 23.5 cm inner zone) during the final 4 min of the 6 min test were recorded by tracking software. The arena was cleaned with 75% ethanol between trials to remove residual odors.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section3\"\u003e\u003ch2\u003e2.6.6 Elevated plus maze test (EPMT)\u003c/h2\u003e\u003cp\u003eThe elevated plus maze (EPM) apparatus featured two open and two closed arms (50 \u0026times; 10 cm each) intersecting at a central platform (10 \u0026times; 10 cm). Closed arms had 40-cm high opaque walls. The maze was elevated 40 cm, and mice started from the central platform facing a closed arm. After a 2 min acclimation period, open arm duration during the subsequent 5 min test phase was recorded by tracking software. The apparatus was cleaned with 75% ethanol between trials to eliminate residual odors.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Behavioral testing of offspring mice\u003c/h2\u003e\u003cdiv id=\"Sec16\" class=\"Section3\"\u003e\u003ch2\u003e2.7.1 Morris water maze test\u003c/h2\u003e\u003cp\u003eThe Morris water maze consisted of a circular tank (120 cm diameter \u0026times; 50 cm height) filled with 23\u0026ndash;25\u0026deg;C ink-mixed water, divided into four quadrants marked by distinct geometric cues (triangle, circle, trapezoid, quadrilateral). A 5-cm diameter escape platform was submerged\u0026thinsp;~\u0026thinsp;2 cm below the water surface at a target quadrant center. During spatial acquisition training (PD28-PD32), mice underwent daily two-trial sessions (30-min inter-trial interval) where they were gently released from the platform-opposite quadrant and allowed 60 s to locate the hidden platform, remaining on it for 15 s; unsuccessful trials recorded 60 s escape latency. On day 6 (probe trial), after platform removal, mice explored for 120 s with recording of: target quadrant latency,platform crossing and swim speed. All behaviors were video-tracked via SMART 3.0(Zhang et al., 2021).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section3\"\u003e\u003ch2\u003e2.7.2 Y-maze test\u003c/h2\u003e\u003cp\u003eSpontaneous alternation in the Y-maze was quantified to evaluate spatial learning and short-term memory. The apparatus comprised three identical arms (35 \u0026times; 10 \u0026times; 15 cm; L\u0026times;W\u0026times;H) designated A, B, and C, radially extending at 120\u0026deg; intervals. Mice were introduced at the distal end of arm A for an 8-min unreinforced exploration period. Arm entry sequences and frequencies were recorded visually. Alternation events were defined as consecutive entries into all three distinct arms (e.g., ABC, BCA). Valid entries required complete hind-paw insertion into an arm. Inter-trial cleaning with 75% ethanol eliminated odor cues. The calculation formula of spontaneous alternation percentage is: alternations (%)\u0026thinsp;=\u0026thinsp;alternate times/(total arm entries-2)\u0026times;100, high alternate percentage was regarded as good spatial memory[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Tissue collection and tissue processing\u003c/h2\u003e\u003cp\u003eTwenty-four hours post-behavioral assessment, all mice underwent pentobarbital via intraperitoneal injection. Orbital blood sampling was followed by centrifugation (4\u0026deg;C, 3500 rpm, 15 min) for serum isolation. Serum aliquots underwent cryostorage at -80\u0026deg;C. Hippocampal tissues were ice-dissected, vitrified in liquid nitrogen, and cryobanked at -80\u0026deg;C. Selected whole brains were fixed in 4% paraformaldehyde or Golgi-Cox solution.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e2.9 Adrenal gland index of maternal mice\u003c/h2\u003e\u003cp\u003eMaternal mice were weighed at PD24, and blood was collected from the orbits after pentobarbital anesthesia, followed by decapitation and execution. The adrenal glands were bilaterally excised via abdominal dissection, cleared of connective tissue and adipose debris with forceps/scissors, dried on filter paper, and weighed. Adrenal index (%) = [gland weight (mg) / body weight (g)] \u0026times; 100%.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e2.10 Enzyme-linked immunosorbent assay (Elisa)\u003c/h2\u003e\u003cp\u003eSerum corticosterone (CORT), adrenocorticotropic hormone (ACTH), and corticotropin-releasing hormone (CRH) levels in dams, alongside brain-derived neurotrophic factor (BDNF) levels in adolescent offspring, were measured using commercial ELISA kits per manufacturer protocols.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e2.11 HE staining\u003c/h2\u003e\u003cp\u003eFixed tissues were paraffin-embedded, sectioned, stained with hematoxylin-eosin, and imaged under 20\u0026times; and 100\u0026times; magnification. Neuronal morphology in hippocampal regions was analyzed using CaseViewer software.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003e2.12 Immunofluorescent staining\u003c/h2\u003e\u003cp\u003eFollowing antigen retrieval, brain sections were washed in phosphate-buffered saline (PBS, pH 7.4) and blocked using 3% bovine serum albumin (BSA) for 30 min. Sections were then incubated overnight at 4\u0026deg;C with primary antibodies:anti- Neuronal nuclei (NeuN, 1:100, Cat No.GB13138-1 Servicebio) and anti-doublecortin (DCX, 1:200, Cat No.ab207175 Abcam). After PBS washes, sections were exposed to secondary antibodies - goat anti-mouse IgG (1:400, Cat No.GB25301, Servicebio) and goat anti-rabbit IgG (1:300, Cat No. GB21303, Servicebio) - for 50 min at room temperature under dark conditions. Following additional washes, nuclei were counterstained with DAPI (10 min, RT, dark). Sections then underwent autofluorescence quenching (5 min, RT, dark), thorough deionized water rinsing (10 min), and final imaging via panoramic slide scanner. The hippocampal dentate gyrus (DG) was delineated in 40x images using CaseViewer software, with DCX\u0026thinsp;+\u0026thinsp;and NeuN\u0026thinsp;+\u0026thinsp;immunofluorescence intensity within DG quantified through ImageJ analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\u003ch2\u003e2.13 Golgi-cox Staining\u003c/h2\u003e\u003cp\u003eMice brain tissue was dissected into 2\u0026ndash;3 mm\u0026sup3; pieces and rinsed repeatedly with saline. Tissue pieces were then immersed in Golgi-Cox staining solution, protected from light, and maintained for 14 days. Following three washes in distilled water, tissues were transferred to 80% ammonia solution overnight. Sections (100 \u0026micro;m thick) were prepared using an oscillating slicer and subsequently stained.\u003c/p\u003e\u003cp\u003eDendritic spine density and length in CA1, CA3, and DG neurons were quantified using Image-Pro Plus 6.0 (400\u0026times;). Analyses focused on intact central neurons within each image, measuring spines along 30\u0026ndash;90 \u0026micro;m segments of secondary/tertiary dendrites. Spine counts and length per 10 \u0026micro;m were calculated.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003e2.14 Western blotting\u003c/h2\u003e\u003cp\u003eTotal protein from Hippocampal tissue were quantified with a BCA assay kit (Beyotime Biotechnology). Following SDS-PAGE separation, proteins were transferred to PVDF membranes. Following blocking with 5% nonfat milk (1.5 h), membranes were incubated overnight at 4\u0026deg;C with primary antibodies targeting: BDNF (1:1000, Cat. No. 47808S, Cell Signaling Technology), TrkB (rabbit pAb), phospho-TrkB (Y817), PI3K p85α (1:2000, Cat. No. T40115F, Abmart), phospho-PI3K p85α/γ (Tyr467/199) (1:2000, Cat. No. T40065F, Abmart), AKT1/2/3 (1:2000, Cat. No. T55561F, Abmart), phospho-AKT (Ser473) (1:2000, Cat. No. T40067F, Abmart), mTOR (1:1000, Cat. No. 2983S, Cell Signaling Technology), phospho-mTOR (Ser2448) (1:1000, Cat. No. 5536S, Cell Signaling Technology), P70 S6 Kinase (1:2000, Cat. No. TA6226, Abmart), synaptophysin (1:2000, Cat. No. T55273S, Abmart), PSD95/DLG4 (1:6000, Cat. No. 20665-1-AP, Proteintech), GAPDH (1:2000, Cat. No. GB11002-100, Servicebio), β-tubulin (1:2000, Cat. No. GB11017-100, Servicebio), and β-actin (1:2000, Cat. No. GB15003-100, Servicebio). After washing, blots were incubated with HRP-conjugated goat anti-rabbit IgG secondary antibody (1:5000, Cat. No. BA1054,Boster Bio) for 1.5 h at room temperature. Protein bands were visualized using ECL substrate (Merck Millipore), with chemiluminescent signals acquired via ChemiDoc XRS\u0026thinsp;+\u0026thinsp;system (Bio-Rad). Quantitative analysis was performed in Image Lab 6.0 by measuring grayscale values of target and reference protein bands, with target protein expression normalized to internal controls (β-actin/GAPDH/β-tubulin).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\u003ch2\u003e2.15 Statistical analysis\u003c/h2\u003e\u003cp\u003eData analysis utilized SPSS 26.0, with results expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Following outlier removal via normality assessment, group differences were analyzed by one-way ANOVA (normally distributed, homoscedastic data) or Kruskal-Wallis test (non-parametric distributions). We established \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 as the statistical significance criterion for all analyses.\u003c/p\u003e\u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec27\" class=\"Section2\"\u003e\u003ch2\u003e3.1 XYS quality control\u003c/h2\u003e\u003cp\u003eAccording to the content determination standard of Xiaoyao Pill (water pill) in the first prescription preparation and single flavor preparation of Chinese Pharmacopoeia (2020 edition), paeoniflorin was selected as the quality control object of XYS decoction. The result of HPLC was that the extract powder of 1 g XYS contained 10.20 mg paeoniflorin (C23H28O11), indicating that the content of paeoniflorin in XYS was in line with the content determination standard in Xiaoyao Pill (water pill) of the 2020 edition of Chinese Pharmacopoeia (no less than 2.5 mg per 1 g of paeoniflorin) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA,\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\u003ch2\u003e3.2 XYS increased maternal nursing and improved anxiety- and depression-like behavior of PPD maternal mice\u003c/h2\u003e\u003cp\u003eMaternal care behaviors in dams were assessed on postnatal days 4 and 8 to evaluate offspring nursing performance. Maternal care assessment was discontinued after PD8 due to emerging pup mobility compromising observational reliability. Compared with the CON group, the maternal caring time of maternal mice showed a decreasing trend at PD4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, P\u0026thinsp;\u0026gt;\u0026thinsp;0.05), and significantly decreased at PD8 in PPD group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). The maternal mice spent more time to care their offspring than PPD group at PD8 in XYS_H and XYS_M group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), suggesting postpartum administration XYS could ameliorate CORT-induced deficits in the maternal caring behavior of PPD mice.\u003c/p\u003e\u003cp\u003eDepressive-like behaviors in dams were evaluated using SPT, TST and FST. Sucrose preference rate is a key indicator for detecting symptoms of pleasure deficit in depression, and TST and FST were used to evaluate the state of behavioral despair in mice. The PPD maternal mice presented notable decreased sucrose preference rate and increased immobility time compared to the CON group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Compared to PPD group, sucrose preference rate significantly elevated in SPT and immobility time declined in FST of XYS_H, and XYS_M group\u0026rsquo;s mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In the TST, the immobility time decreased in the XYS_H and XYS_L group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). OFT and EPMT was subjected to assess anxiety-like behavior of maternal mice. PPD dams exhibited significantly reduced center zone duration and locomotion in the OFT relative to controls. (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). In the EPMT, the PPD mice spent less time in open arm (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Compared PPD group, the time spent in the centre zone and total distance were significantly increased in XYS_H and XYS_M group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The time spent in open arm was elevated in the XYS_H, XYS_M and XYS_L group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.01 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). These findings demonstrate XYS ameliorates CORT-induced anxiety- and depression-like phenotypes in PPD dams.\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.3 Administration of XYS suppressed HPA axis dysfunction and reduced neuronal damage in the hippocampus of PPD maternal mice\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn this study, the adrenal index was used to roughly reflect the HPA axis activity. When HPA axis activity is increased, hypersecretion of the adrenal cortex occurs with tissue hypertrophy and weight gain, whereas when HPA axis activity is decreased, adrenal cortical secretory activity is diminished and tissue atrophy and weight loss occur. Adrenal index was higher in the PPD group of maternal mice compared to the CON group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05); and it was significantly lower in the XYS_H group than in the PPD group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In addition, we measured the levels of serum HPA axis hormones of maternal mice. The levels of CORT, ACTH, and CRH were significantly higher in the PPD maternal mice compared with the CON group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Compared with the PPD group, CORT, ACTH, and CRH levels were significantly reduced in the XYS_H group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and CORT and CRH levels were significantly reduced in the XYS_M group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e\u003cp\u003eMoreover, XYS treatment dramatically ameliorated hippocampal neuronal damage in PPD mother mice. In the CON group, maternal mice had abundant neurons in the hippocampus, tightly arranged, normal neuronal morphology and structure, clear cytoplasmic demarcation of cytosolic nuclei, obvious nucleoli, and no obvious pathological changes were seen. However, in PPD group, there were different degrees of degeneration and necrosis of vertebral cells in the hippocampus, nuclear atrophy and deep staining, and the cytosol was obviously smaller (as shown by the green arrows in the figure), which could be seen as obvious pathological damage. After the treatment with XYS, there were a small number of pyramidal cells with degeneration and necrosis in the hippocampal area, nuclear atrophy and deep staining, and some pathological damage was visible of the maternal mice in XYS_H, XYS_M and XYS_L groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e\u003ch2\u003e3.4 XYS intergenerationally protected the learning and memory ability of PPD offspring mice\u003c/h2\u003e\u003cp\u003eEscape latency progressively declined across training days during spatial acquisition in all offspring group. On day 5, the escape latency of male and female offspring mice in the PPD-F1 group was higher than CON-F1 group, while the escape latency of both male and female offspring mice in the XYS_H-F1, XYS_M-F1, and XYS_L-F1 groups showed a tendency to decrease compared with PPD-F1 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Compared to day 1, the escape latency on day 5 decreased significantly in male offspring mice in the CON-F1 and XYS_H-F1 groups, as well as in female offspring mice in the CON-F1 and SERT-F1 groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In the spatial exploration experiment, compared to the CON-F1 group, both sexes of PPD-F1 offspring showed decreased platform crossings and reduced target quadrant occupancy time (%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Compared with the PPD-F1 group, the numbers of crossing the original platform area and the percentage of time spent in the target quadrant were significantly increased of male and female offspring in XYS_H-F1 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The percentage of time spent in the target quadrant was significantly elevated of male offspring in XYS_M-F1 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In addition, the swimming speed of male and female offspring mice in each group did not show remarkable difference (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG, P\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e\u003cp\u003eIn the Y-maze test, compared with the CON-F1 group, both male and female offspring mice had significantly lower spontaneous alternations in the PPD-F1 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). While compared with the PPD-F1 group, the spontaneous alternation rate was significantly higher in male and female offspring mice in the XYS_H-F1 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and in male offspring mice in the XYS_M-F1 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). It is suggested that postpartum administration of XYS to PPD maternal mice intergenerationally improved the ability of spatial learning and memory in offspring mice. Behavior analyses of offspring demonstrated congruent trends in learning and memory-related behaviors between male and female offspring. Thus, subsequent analyses were performed on pooled male and female offspring data.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec30\" class=\"Section2\"\u003e\u003ch2\u003e3.5 XYS intergenerationally impacted neurodevelopment of PPD offspring mice\u003c/h2\u003e\u003cp\u003ePostpartum treatment with XYS to PPD maternal mice was able to exert an iterative protective effect on offspring neurons and avoided pathological damage to hippocampal neurons in offspring mice. Hippocampal neurons in CON-F1 mice exhibited dense packing, intact morphology, and distinct nuclear boundaries without observable pathology. PPD-F1 hippocampal neurons demonstrated degenerative changes: nuclear pyknosis with hyperchromasia, cytoplasmic shrinkage (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, green arrows), and necrotic foci, confirming significant neuropathological injury In the hippocampus of the offspring of mice in the XYS_H-F1 group, the XYS_M-F1 group, the XYS_L-F1 group, only a small number of neuronal cells had poorly defined cytoplasmic nuclei, and nucleolus was not obvious (as shown by the green arrows in the figure), and no obvious pathological damage was seen (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003ePostpartum treatment with XYS to PPD maternal mice iteratively protects neurogenesis in the hippocampal DG region and increases the positive expression of neonatal neuronal markers in offspring mice. Compared with the CON-F1 group, DCX\u0026thinsp;+\u0026thinsp;immunoreactivity was significantly diminished in the hippocampal dentate gyrus of PPD-F1 offspring. (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and no significant change were observed in the expression of NeuN+ (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Compared with the PPD-F1 group, the expression of DCX\u0026thinsp;+\u0026thinsp;and NeuN\u0026thinsp;+\u0026thinsp;in the hippocampal DG region was enhanced in the offspring mice of the XYS_H-F1 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003cp\u003ePostpartum treatment of XYS to PPD maternal mice iteratively improved dendritic spine growth and synaptic protein expression in PPD offspring. Dendritic spine density and dendritic spine length were significantly lower in the hippocampal region in the PPD-F1 group, compared to the CON-F1 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA,\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). In contrast to the PPD-F1 group, dendritic spine density and dendritic spine length were higher in the hippocampal region in the XYS_H-F1 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA,\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Additionally, the levels of hippocampal synapse-associated proteins SYP and PSD95 proteins were reduced in the PPD-F1 group compared with the CON-F1 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Compared with PPD-F1 group, hippocampal SYP and PSD95 protein levels were significantly increased in XYS_H-F1 and XYS_M -F1 groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and hippocampal SYP protein levels were significantly increased in XYS_L -F1 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec31\" class=\"Section2\"\u003e\u003ch2\u003e3.6 XYS intergenerationally impacted hippocampal BDNF/mTOR signaling pathway of PPD offspring mice\u003c/h2\u003e\u003cp\u003ePostpartum treatment with XYS to PPD maternal mice iteratively increased serum BDNF levels in offspring mice. Compared with the CON-F1 group, the serum BDNF level in the offspring mice of the PPD-F1 group were significantly decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). The serum BDNF levels in the offspring mice of XYS_H-F1 and XYS_M-F1 groups were significantly increased when compared to the PPD-F1 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e\u003cp\u003eAdditionally, the protein analysis results revealed that, hippocampal BDNF, p-TrkB/TrkB, p-PI3K/PI3K, p-AKT/AKT, p-mTOR/mTOR, and P70 ribosomal protein S6 kinase (P70S6K) protein levels were significantly decreased in the PPD-F1 group compared with the CON-F1 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.01 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Compared with PPD-F1 group, hippocampal BDNF, p-TrkB/TrkB, p-PI3K /PI3K, p-AKT/AKT, p-mTOR/mTOR, and P70S6K protein levels were significantly higher in XYS_H-F1, (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), hippocampal BDNF, p-TrkB/TrkB, p-AKT/AKT protein levels were higher in XYS_M-F1(Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), hippocampal BDNF, p-TrkB/TrkB, p-AKT/AKT, and p-mTOR/mTOR protein levels were significantly higher in and XYS_L-F1 groups(Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). It is suggested that postpartum XYS treatment given to PPD maternal mice could intergenerationally activate the hippocampal BDNF/mTOR signaling pathway in offspring mice.\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.7 BDNF receptor inhibitor ANA-12 suppressed the intergenerationally protective effect of XYS in neurodevelopment of PPD offspring mice\u003c/b\u003e\u003c/p\u003e\u003cp\u003eDuring spatial acquisition training, escape latencies progressively declined across all offspring groups with successive training days. By day 5, CON\u0026thinsp;+\u0026thinsp;A-F1 offspring showed moderately prolonged latencies versus CON-F1 counterparts. (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB, \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC, P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). There was a trend for the avoidance latency of the offspring mice in the XYS\u0026thinsp;+\u0026thinsp;A-F1 group to be elevated compared to the XYS-F1 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB, \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC, P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Moreover, escape latencies significantly decreased from day 1 to day 5 in CON-F1, XYS-F1, and CON\u0026thinsp;+\u0026thinsp;A-F1 progeny (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), whereas the escape latency of the offspring mice in the PPD-F1 and XYS\u0026thinsp;+\u0026thinsp;A-F1 groups only tended to decrease on the 5th day of training (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC, P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). During the spatial exploration experimental phase, CON\u0026thinsp;+\u0026thinsp;A-F1 offspring showed significantly fewer platform crossings and lower target quadrant dwell times than CON-F1 controls. (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB, \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD, \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Compared to XYS-F1 offspring, XYS\u0026thinsp;+\u0026thinsp;A-F1 offspring exhibited significantly fewer entries into and spent a lower percentage of time in the target quadrant (original platform location) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB, \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD,\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). There was no significant difference in swimming speed of offspring mice in each group (8F, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). These results showed that intraperitoneal injection of ANA-12 reversed the improvement in the learning and memory ability of the offspring of PPD maternal mice given postpartum treatment with XYS.\u003c/p\u003e\u003cp\u003eCompared with CON-F1 group, BDNF, p-TrkB/TrkB, p-PI3K /PI3K, p-AKT/AKT, and p-mTOR/mTOR protein levels were significantly decreased in the hippocampus of the offspring mice in the CON\u0026thinsp;+\u0026thinsp;A-F1 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA, \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB, \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eC, \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eD, \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eE, \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eF, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Compared with XYS-F1 group, BDNF, p-TrkB/TrkB, p-PI3K /PI3K, p-AKT/AKT, and p-mTOR/mTOR protein levels were significantly decreased in XYS\u0026thinsp;+\u0026thinsp;A-F1 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA, \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB, \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eC, \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eD, \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eE, \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eF, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.01 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). It is indicated that intraperitoneal injection of the BDNF receptor inhibitor into offspring mice reversed the activating effect of postpartum administration of XYS treatment to PPD maternal mice on the BDNF/mTOR signaling pathway in offspring.\u003c/p\u003e\u003cp\u003eDCX⁺ labeling in the hippocampal DG of CON\u0026thinsp;+\u0026thinsp;A-F1 group was lower (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eG, \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eI, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and there was a trend of decreasing expression of NeuN\u003csup\u003e+\u003c/sup\u003e than the CON-F1 group group (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eH, \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eI, P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Compared with the XYS-F1 group, DCX\u003csup\u003e+\u003c/sup\u003e expression in the hippocampal DG area was decreased in the XYS\u0026thinsp;+\u0026thinsp;A-F1 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eG, \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eI, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and there was a trend of decreased level of NeuN\u003csup\u003e+\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eH, \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eI, P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). These findings demonstrate that ANA-12-mediated BDNF receptor inhibition impairs hippocampal DG neurogenesis in offspring, primarily suppressing DCX\u0026thinsp;+\u0026thinsp;expression. Critically, this intervention abolishes the transgenerational neuroprotective effects of maternal XYS treatment on offspring hippocampal neurogenesis in the PPD model.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003ePostpartum depression (PPD) is a multifactorial disorder influenced by social, psychological, and biological factors, including genetic predisposition, inflammation, neurotransmitter dysregulation, and hormonal imbalances[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Based on its etiology, various animal models have been developed to simulate PPD, such as hormone-simulated pregnancy models, chronic corticosterone (CORT) induction, perinatal stress exposure, chronic social stress, maternal-infant separation, and prenatal stress paradigms[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Current research on maternal PPD's offspring effects predominantly employs models established through: chronic corticosterone exposure, gestational/postpartum stress paradigms, or maternal separation protocols. The adverse effects of maternal PPD on offspring have been confirmed in multiple PPD models[\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Notably, PPD pathogenesis is closely linked to HPA axis dysfunction. CORT, a pivotal hormone within this axis, critically mediates negative feedback regulation. Elevated CORT levels impair this feedback mechanism, leading to increased secretion of HPA-related hormones and depression-like behaviors in animals[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Additionally, multiple studies have demonstrated that administering high levels of CORT to postpartum female mice impairs maternal care behaviors and induces postpartum depression-like behaviors[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Our prior findings revealed tha CORT-induced PPD in mice not only impairs maternal behaviors but also leads to significant cognitive deficits in their offspring[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eChronic CORT treatment in postpartum dams induced HPA axis hyperfunction and elevated serum CORT, ACTH, and CRH, consistent with prior studies. Treated mice showed: reduced maternal care time, decreased sucrose preference, prolonged immobility in TST/FST, and diminished center time (OFT) plus open arm time (EPMT). These changes confirm successful modeling of PPD-related anxiety- and depression-like behaviors.\u003c/p\u003e\u003cp\u003eBeyond maternal pathology, this study reinforces the intergenerational impact of PPD on offspring neurodevelopment. Clinical and preclinical evidence has demonstrated that maternal PPD increases the risk of emotional, cognitive, and behavioral abnormalities in offspring, including impaired attention regulation, heightened stress reactivity, and reduced IQ scores[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. For example, Amani et al. (2021) reported that offspring of prenatally stressed mothers exhibited decreased sucrose preference, impaired recognition memory, elevated CORT levels, and reduced hippocampal BDNF expression. Existing evidence indicates maternal PPD compromises spatial cognition and emotional regulation in adolescent male offspring[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAs a highly plastic structure, the hippocampus exhibits significant stress sensitivity and critically regulates learning and memory processes[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Hippocampal neurogenesis and synaptic plasticity serve as critical indicators of neural plasticity[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. DCX and NeuN are markers of neuronal differentiation: DCX labels neuronal progenitor cells and immature newborn neurons, while NeuN is expressed in mature neurons that subsequently integrate into neural circuits to modulate cognitive functions[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Studies have shown that chronic social defeat stress impairs social memory and spatial object recognition memory in mice, accompanied by reduced numbers of Ki67\u0026thinsp;+\u0026thinsp;and DCX\u0026thinsp;+\u0026thinsp;cells (markers of neuronal proliferation and differentiation) in DG region of the hippocampus[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. The synaptic protein PSD95 functions as a core scaffold within the postsynaptic membrane and is vital for storing neuronal information. SYP, a phosphoprotein located on the presynaptic membrane, regulates presynaptic vesicle quantity and neurotransmitter release[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Maternal malnutrition-induced cognitive dysfunction in adult offspring has been linked to decreased expression of PSD95 and SYP in the hippocampal CA1, CA2, CA3, and DG regions[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Dendritic spines form the morphological basis of synaptic plasticity. Various chronic stressors could reduce hippocampal dendritic spine density and length, decrease synaptic protein expression, and induce learning and memory impairments in rats[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Maternal PPD is increasingly associated with offspring cognitive deficits. Experimental CORT models show diminished DCX\u0026thinsp;+\u0026thinsp;immunoreactivity in the dorsal hippocampus of both adolescent progeny and adult female offspring (Gobinath et al., 2016). Our previous work demonstrated that CORT-induced PPD in mice not only impairs maternal behaviors but also leads to significant cognitive deficits in their offspring[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eOur research found that administering high levels of CORT to postpartum maternal ICR mice to induce PPD prolonged the escape latency of male and female offspring mice in finding the platform during the Morris water maze test. It also reduced the number of crossing the original platform area and the percentage of movement time spent in the target quadrant, while decreasing the spontaneous alternation rate in the Y-maze test. Notably, the behavioral changes showed consistent trends in both male and female offspring, without significant differences between the sexes. Additionally, the results revealed that offspring of PPD-affected maternal mice not only exhibited reduced expression of DCX\u0026thinsp;+\u0026thinsp;and NeuN+, markers for newborn neurons in the DG region of the hippocampus, but also had decreased dendritic spine density, spine length, and synaptic protein expression in the hippocampus. These findings confirm that maternal PPD impaired learning and memory abilities as well as neurodevelopment in offspring mice, identifying it as a risk factor in the growth and developmental processes of subsequent generations.\u003c/p\u003e\u003cp\u003eXYS has been confirmed to exhibit definite curative effects in the treatment of PPD and can produce intergenerational protective effects on offspring. In ancient Chinese medical literature, there is no specific terminology for PPD. Later physicians classified PPD into the categories of \"depression syndrome\", \"visceral agitation (Zangzao,)\", and \" Baihe disease\" based on its clinical manifestations. The pathogenesis of PPD involves postpartum deficiency of qi and blood. When blood fails to nourish the heart, it leads to mental restlessness. Excessive sorrow and anxiety damaging the heart and spleen, or emotional injuries causing liver qi stagnation that transforms into disease[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Therefore, the primary therapeutic principles for PPD are to soothe the liver and relieve depression, nourish blood, and strengthen the spleen. XYS, composed of eight medicinal herbs, is a classic formula for soothing the liver, relieving depression, nourishing blood, and strengthening the spleen.\u003c/p\u003e\u003cp\u003eClinical trials have shown that XYS effectively alleviates depressive symptoms during pregnancy, postpartum, and menopause, with minimal side effec[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Furthermore, combined therapy with fluoxetine and XYS demonstrated superior outcomes in PPD patients compared to fluoxetine alone[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. In animal models, XYS improves depression-like behaviors, enhances learning and memory performance, increases dendritic complexity, and activates signaling pathways such as ERα-PI3K in the hippocampus[\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Despite growing evidence of its maternal antidepressant efficacy, few studies have addressed its intergenerational effects on offspring.\u003c/p\u003e\u003cp\u003eIn accordance with the TCM principle of \"treating the mother to benefit the child,\" In this study, PPD mother mice were treated with XYS during the postpartum lactation period. These results suggest that XYS can not only directly exert antidepressant effects on maternal PPD mice themselves, but also indirectly protect offspring mice through maternal administration, preventing the adverse effects of maternal PPD on the neurodevelopment, as well as learning and memory abilities of offspring. The study demonstrated that XYS significantly alleviated anxiety- and depression-like behaviors in maternal PPD mice, while also ameliorating HPA axis hyperactivity and neuronal damage. In addition, maternal treatment with XYS can improve the learning and memory abilities of offspring mice across generations. In the Morris water maze, PPD offspring mice of both sexes exhibited significantly reduced escape latencies during spatial acquisition training. Furthermore, during the probe test, these mice demonstrated increased platform crossings and spent a greater percentage of time in the target quadrant. Moreover, in the Y-maze test, it enhances the spontaneous alternation rate in both male and female offspring mice. In addition, maternal treatment with XYS can intergenerationally improve the learning and memory abilities of offspring mice, reduce the escape latency of male and female PPD offspring mice during the spatial acquisition training phase of the Morris water maze test, increase the number of crossings into the original platform area and the percentage of movement time spent in the target quadrant during the spatial exploration test phase, and increase the spontaneous alternation rate of male and female offspring mice in the Y-maze test. Moreover, maternal treatment with XYS can also improve the indicators related to hippocampal neurogenesis and synaptic function in offspring mice.\u003c/p\u003e\u003cp\u003eThe intergenerational protection conferred by XYS on offspring mice might involve BDNF/mTOR pathway modulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e). BDNF has emerged as a key regulator of neuronal development and synaptic plasticity, which plays a crucial role in the regulation of learning and memory abilities[\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. BDNF binds to its receptor TrkB, which can activate the downstream PI3K/ AKT/ mTOR signaling pathway[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. This process increased the expression of neuronal differentiation markers DCX and NeuN in the DG region of the hippocampus, thereby regulating neuronal growth and differentiation, promoting neurogenesis, and affecting cognitive functions[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The binding of BDNF to TrkB can also activate downstream targets such as AKT, extracellular signal-regulated kinase (ERK), and cAMP response element-binding protein (CREB) to protect neuronal survival and regulate neural plasticity[\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Stress can reduce BDNF, downregulate its downstream protein mTOR, decrease the expression of the synaptic protein PSD95, and damage synaptic morphology[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Additionally, BDNF can regulate the downstream P70S6K protein level by activating the mTOR signaling pathway, thereby upregulating synaptic protein levels and improving synaptic function and cognitive dysfunction[\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Studies show maternal PPD lowers offspring hippocampal BDNF, suppressing downstream signaling and causing cognitive impairment. The PPD model established by prenatal stress downregulates BDNF levels in the whole brain of offspring, leading to cognitive function deficits in offspring[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Furthermore, studies have found that the BDNF/AKT/mTOR signaling pathway in the hippocampus of adult offspring from a PPD model induced by pre-pregnancy chronic unpredictable mild stress (CUMS) is impaired[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In the PPD model constructed by pre-pregnancy restraint stress, the AKT/mTOR signaling pathway in the hippocampus of offspring is inhibited during adolescence and adulthood, with downregulated expression of phosphorylated proteins of AKT, mTOR, and P70S6K[\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Therefore, this study hypothesizes that regulating the BDNF/mTOR signaling pathway may be one of the pathways for preventing and treating cognitive impairment in offspring.\u003c/p\u003e\u003cp\u003eThis study demonstrated inhibition of the hippocampal BDNF/mTOR signaling pathway in offspring of the PPD model, with significantly downregulated protein levels of BDNF, p-TrkB/TrkB, p-PI3K/PI3K, p-AKT/AKT, p-mTOR/mTOR, and P70S6K. Treatment of PPD maternal mice with XYS increased BDNF levels in the serum and hippocampus of their offspring mice, activated the hippocampal BDNF/mTOR signaling pathway, and upregulated the levels of related proteins. To further validate the critical role of BDNF and its downstream signaling pathway in the intergenerational protective effect of XYS on learning and memory ability in PPD offspring, ANA-12 (a BDNF receptor antagonist) was used to inhibit BDNF receptors in this study. Experimental results showed that after inhibition of BDNF receptors in offspring mice, the BDNF/mTOR signaling pathway was suppressed, which reversed the intergenerational protective effect of XYS on offspring mice. Concurrently, offspring mice exhibited significant cognitive deficits in spatial learning and memory consolidation. This was paralleled by diminished DCX⁺ immunoreactivity, suggesting BDNF mediates the intergenerational preservation of neural plasticity and cognitive functions through XYS intervention.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"5 Conclusion","content":"\u003cp\u003eThis study confirms that PPD can adversely affect offspring neurodevelopment, consistent with previous findings. XYS, a classical traditional Chinese medicine formula with multi-component and multi-target characteristics, not only ameliorates depressive-like behaviors in PPD mouse model but also confers significant intergenerational protective effects on their offspring. In our experiments, high-dose XYS exerted the most pronounced protective effects on hippocampal neurogenesis and cognitive performance in the offspring. These benefits were associated with the intergenerational protective effects of XYS were mediated through BDNF/mTOR signaling activation, as evidenced by upregulated hippocampal expression of BDNF, p-TrkB/TrkB, and downstream PI3K/AKT/mTOR/P70S6K pathway components in offspring.\u003c/p\u003e\u003cp\u003eWe hypothesize that the intergenerational protective effects of XYS may be mediated through two complementary mechanisms: (1) indirect modulation of maternal endocrine levels that improves maternal-infant interactions;(2) direct transmission of active components through maternal milk that influence neurodevelopment in the offspring. Future investigations will delineate the compositional dynamics of maternal milk and elucidate the bioactive mediators underlying these functional effects. This work also contributes to improve population quality and promoting theoretical innovation and clinical expansion of TCM in the transgenerational intervention of mental disorders.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eACTH, adrenocorticotropic hormone; AKT, protein kinase B; BDNF, brain-derived neurotrophic factor; CORT, corticosterone; CREB, cAMP response element-binding protein; CRH, corticotropin-releasing hormone; CUMS, chronic unpredictable mild stress; X, doublecortin; DG, dentate gyrus; Elisa, enzyme-linked immunosorbent assay; EPMT, elevated plus maze test; ERK, extracellular signal-regulated kinase; FST, forced swimming test; HE, hematoxylineosin staining; HPA, hypothalamic-pituitary-adrenal; mTOR, mammalian target of rapamycin; NeuN, neuronal nuclear antigen; OFT, open field test; P70S6K, P70 ribosomal protein S6 kinase.; PD, postpartum day; PI3K, phosphoinositide 3-kinase; PPD, postpartum depression; PSD95, postsynaptic density protein 95; SPT, sucrose preference test; SYP, synaptophysin; TrkB, tropomyosin-related kinase B; TST, tail suspension test; XYS, Xiaoyao San;\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe animal experiment followed the guidelines of the Committee for Animal Care and Use of Laboratory Animals of the College of Pharmacy, Chengdu University of Traditional Chinese Medicine (approval No. TCM-2017-312).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data used to support this research are available from the corresponding author upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the University\u0026ndash;Hospital Joint Innovation Fund Project of ChengDu TCM (LH 202402016).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eH X designed and conceived this study. H X completed data analysis, experimentation and manuscript writing. J D, Y J, R Y, Z X, X Z, Q Z carried out experiments. J Z, L C provided suggestions for the research. Q Y reviewed the manuscript. N Z, R L conceived and designed the experiment and reviewed the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHongxiao Xie\u003csup\u003e1\u003cstrong\u003e#\u0026nbsp;\u003c/strong\u003e\u003c/sup\u003eand\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eJunping Ding\u003csup\u003e1\u003cstrong\u003e#\u003c/strong\u003e\u003c/sup\u003eThese authors contributed equally to this work and share first authorship.\u003c/p\u003e\n\u003cp\u003e* Corresponding authors: Correspondence to:Nan Zeng,\u0026nbsp;Qiong Yi, Rong Liu\u003c/p\u003e\n\u003cp\u003eState Key Laboratory of Southwestern Chinese Medicine Resources, School of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan, 611137, China;\u003c/p\u003e\n\u003cp\u003eHongxiao Xie, Junping Ding,\u0026nbsp;Yanning Jiang,\u0026nbsp;Yixin Rui,\u0026nbsp;Zhiqiang Xie,\u0026nbsp;Xiumeng Zhang,\u0026nbsp;Jiuseng Zeng,\u0026nbsp;Quanrun Zhu, Li Chen,\u0026nbsp;Nan Zeng,\u0026nbsp;Rong Liu\u003c/p\u003e\n\u003cp\u003eDepartment of Pharmacy, The First People\u0026apos;s Hospital of Shuangliu District, West China (Airport) Hospital of Sichuan University, Chengdu,Sichuan, 610200, China\u003c/p\u003e\n\u003cp\u003eZhiqiang Xie,\u0026nbsp;Xiumeng Zhang\u003c/p\u003e\n\u003cp\u003eDepartment of Pharmacy, Clinical Medical College and the First Affiliated Hospital\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eof Chengdu Medical College, Chengdu, Sichuan,610500, China\u003c/p\u003e\n\u003cp\u003eLi Chen\u003c/p\u003e\n\u003cp\u003eMeishan Hospital of Chengdu University of TCM, Meishan,Sichuan, 620010, China\u003c/p\u003e\n\u003cp\u003eQiong Yi\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMir FR, Pollano A, Rivarola MA. 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Cisplatin induces BDNF downregulation in middle-aged female rat model while BDNF enhancement attenuates cisplatin neurotoxicity. Experimental Neurology. 2024;375:114717. \u003c/li\u003e\n\u003cli\u003eWang B, Zhao Y, Hu S, Wang J. Discussion on the Treatment of Female Postpartum Depression with Traditional Chinese Medicin. Journal of Huanghe S\u0026amp;T College. 2023;25:37\u0026ndash;9. \u003c/li\u003e\n\u003cli\u003ePan J, Wang Y, Gao Z, Xue X, Wang N, Lv Y, et al. Research Progress on Classical Chinese Medicine Formulas for the Treatment of Depression. Modernization of Traditional Chinese Medicine and Materia Medica-World Science and Technology. 2022;24:2809\u0026ndash;16. \u003c/li\u003e\n\u003cli\u003eZhang J. Analysis of the therapeutic effect of fluoxetine combined with Xiaoyao San on postpartum depression. Electronic Journal of Practical Gynecological Endocrinology. 2021;8:55\u0026ndash;7. \u003c/li\u003e\n\u003cli\u003eLiu, Ge F, Yang H, Shi H, Lu W, Sun Z, et al. 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Proceedings of the National Academy of Sciences. 2021;118:e2017841117. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Xiaoyao San, Postpartum depression, Offspring, Learning and memory, Neurogenesis, Synapse, BDNF","lastPublishedDoi":"10.21203/rs.3.rs-7515178/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7515178/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ebackground\u003c/h2\u003e\u003cp\u003eAs a principal TCM antidepressant intervention, Xiaoyao San (XYS) is extensively applied in postpartum depression (PPD) therapeutics. Nevertheless, fundamental research in this area remains limited, particularly insufficient investigations into the intergenerational effects of XYS on PPD treatment.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eFemale ICR mice received daily corticosterone (CORT) injections (PD1-PD21) to establish a PPD model, with concurrent XYS treatment (45, 30, 15 g/kg) for 21 days. Maternal caring behavior was assessed on PD4 and PD8. On PD22, behavioral tests were conducted, followed by ELISA quantification of serum CORT/ACTH/CRH and HE staining to evaluate hippocampal neuronal damage. Offspring mice underwent morris water maze and Y-maze tests on PD28 to assess cognition, with histological/molecular analyses (HE, immunofluorescence, Golgi staining, western blot) for neurodevelopment. Western blot quantified hippocampal BDNF/mTOR pathway proteins, validated via ANA-12 inhibition.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eXYS enhanced maternal care, reduced anxiety/depression-like behaviors in PPD mice. XYS conferred transgenerational protection of learning and memory functions in offspring mice, as evidenced by: reduced escape latency, increased platform crossings/spontaneous alternation. Besides, XYS treatment attenuated hippocampal neuronal damage in PPD offspring, promoted neurogenesis (increased DCX\u003csup\u003e+\u003c/sup\u003e/NeuN\u003csup\u003e+\u003c/sup\u003e cells in DG region), dendritic complexity (higher spine density/length), and synaptic protein expression. Maternal XYS administration upregulated BDNF/mTOR pathway. ANA-12 confirmed the pivotal role of BDNF/mTOR pathway in mediating XYS's transgenerational neuroprotective and learning and memory ability.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eXYS alleviated postpartum depression in mice and conferred transgenerational neuroprotection via BDNF/mTOR activation, preserving offspring cognition. This supports XYS's clinical potential for interrupting intergenerational psychiatric disorders.\u003c/p\u003e","manuscriptTitle":"Xiaoyao San intergenerational protects learning-memory ability and neurodevelopment in PPD offspring via the BDNF/mTOR signaling pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-22 08:59:00","doi":"10.21203/rs.3.rs-7515178/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9ccd0509-f3b8-454b-b763-dd390c3472ea","owner":[],"postedDate":"October 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-05T01:39:14+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-22 08:59:00","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7515178","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7515178","identity":"rs-7515178","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}