ABSTRACT

   The apple was found to be involved in antidepressant properties. Thus,
   this study aimed to explore the effects of apple extract (AE) on
   depression and to elucidate the underlying mechanisms. Our network
   pharmacology analysis indicated that AE may exert its antidepressant
   effects through the regulation of inflammatory responses and the cAMP
   signaling pathway. Behavioral tests showed that AE significantly
   improved spatial learning, exploratory behavior, anhedonia, and despair
   in CUMS mice. Biochemical analyses revealed that AE increased synaptic
   density in the hippocampus, enhanced brain levels of
   5‐hydroxytryptamine (5‐HT) and gamma‐aminobutyric acid (GABA), and
   reduced the expression of pro‐inflammatory cytokines TNF‐α, IL‐1β, and
   IL‐6 in both serum and brain tissue. In vitro, AE demonstrated
   significant neuroprotective effects against CORT‐induced cytotoxicity.
   Furthermore, AE treatment upregulated the levels of cAMP,
   phosphorylated PKA (pPKA), and phosphorylated CREB (pCREB) while
   downregulating PTGS2 expression, suggesting that the
   cAMP/PKA/CREB/PTGS2 signaling pathway is a key mechanism underlying the
   antidepressant effects of AE. Therefore, our study indicated that AE
   can improve cognitive dysfunction and alleviate depressive‐like
   behavior by targeting the cAMP/PKA/CREB/PTGS2 signaling pathway in CUMS
   mice. This study paved the way for the clinical application of AE as a
   novel treatment for depression and provided new targeted therapeutic
   strategies.

   Keywords: apple extract, cAMP, CUMS, inflammatory response, network
   pharmacology
     __________________________________________________________________

   This study aimed to investigate the effects of apple extract (AE) on
   depression and its underlying mechanisms, revealing that AE may exert
   antidepressant effects by regulating inflammatory responses and the
   cAMP signaling pathway. Behavioral tests and biochemical analyses
   demonstrated that AE significantly improved depressive‐like behaviors
   and cognitive dysfunction in CUMS mice by increasing synaptic density
   in the hippocampus, enhancing brain levels of 5‐hydroxytryptamine and
   gamma‐aminobutyric acid, and reducing pro‐inflammatory cytokines. The
   findings indicate that the cAMP/PKA/CREB/PTGS2 signaling pathway plays
   a crucial role in the antidepressant effects of AE, paving the way for
   its potential clinical application as a novel treatment for depression.

   graphic file with name FSN3-13-e70408-g002.jpg

1. Introduction

   Depression is a mental disorder characterized by high prevalence, high
   relapse rates, and elevated suicide risk. Clinically, it manifests as
   marked and sustained emotional disturbances, diminished appetite,
   anhedonia (loss of interest or pleasure in most activities), cognitive
   slowing, and social withdrawal. In severe cases, patients may exhibit
   hallucinations and are at risk of self‐injurious behaviors or suicide,
   posing a substantial threat to public health (Kim et al. [32]2024; Yuan
   et al. [33]^2020). Currently, widely accepted theories regarding the
   pathophysiology of depression encompass the neurotransmitter
   hypothesis, the hypothalamic–pituitary–adrenal (HPA) axis hypothesis,
   the inflammatory hypothesis, the neuroplasticity hypothesis, and the
   epigenetic hypothesis, among others. However, these hypotheses do not
   fully elucidate the complex pathophysiological mechanisms underlying
   depressive disorders (Cui et al. [34]2024). It is estimated that by
   2030, depression will become the leading cause of the global disease
   burden, thereby imposing significant psychological stress and economic
   burdens on families and society (Malhi and Mann [35]2018). Depression
   exhibits a high degree of heterogeneity, with its etiology and
   pathophysiological mechanisms being exceedingly complex. This
   complexity makes it challenging to elucidate the pharmacological
   mechanisms of antidepressant treatments, which severely limits the
   development and application of effective medications. Therefore,
   researching the mechanisms of antidepressant action, developing new
   antidepressant drugs, and reducing the incidence of depression hold
   significant importance for both societal well‐being and economic
   development.

   Currently, the treatment of depression primarily relies on
   pharmacotherapy, and a variety of antidepressant medications have been
   developed. However, pharmacological treatments often come with
   significant limitations, including strong dependency, notable side
   effects, and in some cases, treatment‐resistant depression (Carvalho
   et al. [36]2016; Duman and Aghajanian [37]^2012). Therefore, the search
   for and development of complementary and alternative medicines that can
   prevent or treat depression is of great significance for improving the
   health of patients with depression. Traditional Chinese Medicine (TCM)
   herbs, due to their multi‐component, multi‐target, multi‐signal
   pathway, and multi‐effect characteristics, along with relatively low
   toxicity and side effects, align well with the multifactorial,
   multi‐mechanistic, and multi‐theoretical nature of depression. This
   makes TCM, especially the food products with both medicinal and edible
   properties as complementary and alternative therapies, a focal point in
   the research of antidepressant therapies. Among these, the apple, which
   is the fruit of a deciduous tree belonging to the Rosaceae family,
   originally native to Europe and central Asia, is commonly cultivated in
   Yunnan, Sichuan, Tibet, Liaoning, and Gansu provinces in China, and is
   widely enjoyed by people. The city of Zhaotong in Yunnan Province
   boasts unique natural resources and climatic conditions, producing
   apple with distinctive qualities characterized as being “early, sweet,
   fragrant, crisp, and colorful.” After nearly a century of introduction,
   cultivation, and development, Zhaotong has become the largest
   high‐quality apple production base in southern China. In ancient
   Chinese medical literature, such as “Ming Yi Bie Lu” (Famous
   Physicians' Additional Records), the apple was recorded as having
   medicinal value. As a fruit with both medicinal and dietary uses, the
   apple possesses high nutritional and economic value. It contains a
   large amount of bioactive substances, including flavonoids, phenolic
   acids, and triterpenes. The main flavonoids found in the apple include
   flavones, flavonols, flavanols, anthocyanins, and chalcones. To date,
   35 types of flavonoids have been identified in the apple, such as
   catechin, epicatechin, and quercetin. Additionally, 21 types of
   phenolic acids, including p‐coumaroylquinic acid, quinic acid,
   chlorogenic acid, and caffeic acid, and 24 types of triterpenes, such
   as ursolic acid and β‐sitosterol, have been discovered (Lee
   et al. [38]2003; Schieber et al. [39]^2001; Wang, Wang, Liu,
   et al. [40]^2024). Due to its rich content of various bioactive
   components, the apple exhibits multiple pharmacological activities.
   Research has shown that the apple extract possesses a range of
   biological functional properties, including the ability to scavenge
   free radicals, anti‐inflammatory effects, antioxidant activity,
   inhibition of tumor cell proliferation, and anti‐aging properties (He
   and Liu [41]2008; Li et al. [42]^2019; Luo et al. [43]^2016; Xiang
   et al. [44]^2011; Zielinska et al. [45]^2019). Indeed, the apple
   contains a variety of bioactive components that play important roles in
   the prevention of chronic diseases and in promoting overall health.
   However, there is limited reporting specifically on the antidepressant
   effects of apple. In TCM, depression falls under the category of
   Yuzheng, with the primary pathological location being the liver.
   According to the Suwen, one of the foundational texts of TCM, “anger
   injures the liver.” The liver is responsible for storing blood and
   regulating the smooth flow of Qi. When the liver functions properly, Qi
   and blood circulate harmoniously, leading to a balanced and cheerful
   mood. Conversely, dysfunction of the liver can lead to abnormal
   emotional states. Clinical observations and anecdotal evidence
   suggested that the apple may have liver‐nourishing and detoxifying
   properties. Furthermore, it has been noted that when patients with
   depression inhale the apple aroma, they experience a reduction in
   feelings of oppression, an improvement in mood, and a relief from
   insomnia symptoms. These observations indicated that the apple may
   possess potential antidepressant properties. To summarize, while the
   apple is well‐known for its general health benefits and its role in
   preventing chronic diseases, emerging evidence suggests that it may
   also have specific benefits for mental health, particularly in the
   context of depression. The liver‐nourishing and mood‐enhancing effects
   of apple, as observed in TCM, provide a promising area for further
   research into their potential as a complementary and alternative
   therapy for depression (“Health Benefits of Apples,” [46]^2013). An
   increasing body of evidence supports the antidepressant effects of
   individual components found in apple, such as chlorogenic acid and
   epicatechin, in mouse models of depression (Martínez‐Damas
   et al. [47]^2021; Song et al. [48]^2019). Moreover, proanthocyanidin B2
   has been demonstrated to decrease blood–brain barrier (BBB) leakage and
   enhance the diminished expression of the tight junction protein zonula
   occludens‐1 (ZO‐1) in the cortex and striatum, thus reinforcing the
   BBB. This suggests that proanthocyanidin B2 may also have
   antidepressant effects (Wu et al. [49]2015). Recent studies have
   demonstrated that apple polyphenol extracts improve depressive‐like
   behaviors in mice fed a high‐sugar diet by inhibiting inflammation in
   the gut‐brain axis (Xie, Wu, et al. [50]^2024). Another study indicated
   that apple polyphenol extracts alleviate depressive‐like behaviors in
   high‐sugar diet‐fed mice through the farnesoid X receptor
   (FXR)‐mediated hepatoenteric‐brain axis bile acid circulation (Xie,
   Shang, et al. [51]^2024). However, these studies have focused on the
   antidepressant effects of specific components in the apple, which may
   not fully reflect the interactions and synergistic effects of the
   multiple bioactive compounds present in the apple. Therefore,
   investigating the impact of apple extract mixtures on depression could
   provide new insights that might not be achievable with individual
   components alone. This holistic approach could help in understanding
   the comprehensive therapeutic potential of apple in the context of
   depression and may lead to the development of more effective,
   natural‐based treatments.

   Network pharmacology integrates systems biology, polypharmacology, and
   computational analysis techniques to construct a
   “drug‐component‐target‐disease” interaction network. This approach
   reduces the blindness of research and enhances research efficiency. By
   building multi‐level networks, it systematically elucidates the
   mechanisms of drug actions at the gene, molecular, and cellular levels,
   laying the foundation for the development of new drugs (Zhang
   et al. [52]2023). PharmMapper is a tool that integrates databases such
   as DrugBank, BindingDB, Target Bank, and PPTD to perform reverse
   pharmacophore modeling for target prediction. It can rapidly predict
   the targets of drug molecules (Wang et al. [53]2017). Computer‐Aided
   Drug Design (CADD) is a method that utilizes computer technology and
   simulation software to design, screen, and optimize drug molecules
   based on the structure and function of their targets. CADD is a crucial
   technique in modern drug discovery and development. This study
   initially aimed to establish a predictive model for the antidepressant
   activity of apple using network pharmacology, to predict the potential
   targets and mechanisms of action of the antidepressant components in
   the apple, thereby providing a theoretical basis for further research.

   Therefore, building upon previous research, this study will integrate
   network pharmacology with traditional pharmacodynamic techniques and
   high‐throughput sequencing technologies to systematically elucidate the
   antidepressant mechanisms of apple from Zhaotong, Yunnan. This
   comprehensive approach aims to provide a scientific basis for the safe
   and extensive clinical application of apple and to offer a novel
   perspective for the development and utilization of apple in the
   Zhaotong region of Yunnan.

2. Materials and Methods

2.1. Screening of Active Ingredients of Apple and Related Targets

   We conducted a literature review using the CNKI and PubMed databases to
   gather information on the active components of apple. The canonical
   SMILES of the relevant compounds were input into the Traditional
   Chinese Medicine Integrated Database (TCMID), ECTM, and Swiss ADME
   ([54]http://www.swissadme.ch/) to select compounds with high
   gastrointestinal absorption and favorable drug‐like properties as the
   target compounds. Subsequently, the canonical SMILES of these target
   compounds were input into the Swiss Target Prediction database
   ([55]http://www.swisstargetprediction.ch/), with the species set to “
   Homo sapiens .” Targets with a probability greater than 0.7 were
   selected as the potential targets of the active components in apple.

2.2. Screening of Depression‐Related Targets

   Depression targets were retrieved from GeneCards, DrugBank, Therapeutic
   Target Database, OMIM and DisGeNET databases using the keyword
   “Depression”. The species was restricted to “ Homo sapiens ”. The
   duplicate targets were removed, and the names were standardized using
   the UniProt database.

2.3. Acquisition of Apple‐Depression Overlapping Targets

   Targets related to apples and those associated with depression were
   input into Venny2.1.0 to create an overlapping target set for further
   analysis.

2.4. Establishment of Protein–Protein Interaction (PPI) and Screening of Core
Active Targets

   The overlapping targets obtained were uploaded to the STRING database,
   specifying “ Homo sapiens ” as the protein species and setting the
   minimum required interaction score to the highest confidence level
   (0.900). Isolated nodes were removed, and the online tools were used to
   predict the interactions among the genes. The results were visualized
   as a Protein–Protein Interaction (PPI) network for the antidepressant
   activity of apple using Cytoscape 3.9.1 software. Topological analysis
   of the PPI network was carried out using the CentiScape 2.2 plugin
   within Cytoscape 3.9.1. The size and color intensity of the nodes
   represent the Degree values, and the thickness of the edges reflects
   the Combined Score. The top 6 nodes ranked by Degree value were
   selected as the core target network, and the core active targets were
   identified.

2.5. GO and KEGG Pathway Enrichment Analyses

   The targets overlapping between apple‐related and depression‐related
   genes were uploaded to the Metascape database. GO enrichment analysis
   and KEGG pathway enrichment analysis were then conducted on these
   intersecting targets.

2.6. Preparation of Apple Extract

   The apples ( Malus pumila Mill. cv. Ruixue) were harvested in Zhaotong
   City, Yunnan Province, China. The apples underwent thorough washing
   with water, were shade‐dried to remove surface moisture, and
   subsequently ground into a fine powder. A quantity of this dried powder
   (50 g) was subjected to extraction using 70% ethanol (5 L) for a
   duration of 12 h. After the extraction process, the resulting liquid
   was concentrated using a rotary evaporator over a period of 2 h. To
   ensure complete removal of ethanol, the concentrated extract was
   diluted with water and subjected to another round of concentration.
   Following this, the processed liquid was carefully filtered through
   filter paper to remove any residual particulates. The filtrate was then
   placed on a freezing tray where it remained frozen for 24 h.
   Subsequently, the frozen material underwent freeze‐drying for an
   extended period of 36 h, ultimately yielding a completely dried apple
   extract weighing 10 g. This final product was then dissolved in a
   solvent system (10% DMSO + 40% PEG300 + 5% Tween‐80 + 45% Saline) and
   then diluted 2‐fold with saline for experimental use.

2.7. Chemical Composition Analysis

   The composition of 70% ethanol extracts from the “Ruixue” variety of
   Malus pumila Mill. apples was quantitatively evaluated via
   High‐Performance Liquid Chromatography (HPLC). An HPLC setup from
   Shimadzu (Kyoto, Japan) was employed. The data collection process
   relied on the Shimadzu Lab Solution software. This separation occurred
   at room temperature, with detection occurring at a wavelength of
   273 nm. The mobile phase was composed of water containing 0.1%
   trifluoroacetic acid (designated as A) and acetonitrile (designated as
   B), and gradient elution was applied in the following manner: within
   0–15 min, the proportion of B was 5%–10%; from 15–30 min, it was
   10%–30%; during 30–50 min, 30%–50%; in 50–70 min, 50%–80%; from
   70–75 min, 80%–100%; between 75–85 min, it remained at 100%; from
   85–95 min, it decreased from 100% to 5%; and from 95–100 min, it stayed
   at 5%. The flow rate was set at 1 mL per minute, and the temperature
   was maintained at 37°C. The main compounds identified in the 70%
   ethanol extract of “Ruixue” apples were determined. These major
   compounds were epicatechin (14.50% ± 1.27%), caffeic acid
   (3.40% ± 0.35%), gallic acid (2.60% ± 0.28%), phloretin
   (2.95% ± 0.25%), and quercetin (0.90% ± 0.08%).

2.8. Animals

   Thirty 8‐week‐old male C57BL/6J mice, each weighing around 23 ± 2 g,
   were sourced from the Yunnan University Experimental Animal Centre
   (approval number SYXK (Dian) 2021–0002). These mice were housed in
   groups of 5 to 6 per cage under controlled environmental conditions: a
   temperature of 22°C ± 2°C, relative humidity of 55%, and a 12‐h
   light/dark cycle. Food and water were provided ad libitum throughout
   the study, except during specific experimental procedures. All
   procedures were reviewed and approved by the Zhaotong University Animal
   Ethics Committee (approval number 20220003A8), ensuring compliance with
   stringent ethical standards. These procedures were carried out
   following the guidelines set forth by the National Institutes of Health
   (NIH) Guide for the Care and Use of Laboratory Animals, which are
   internationally recognized for promoting the humane treatment of
   animals in research, and all personnel involved held valid
   certifications for working with laboratory animals.

2.9. Animal Grouping and Drug Administration

   Before the experiment, the animals were subjected to a one‐week
   acclimation period.

   Following this, the male C57BL/6J mice were randomly allocated into
   three distinct groups: normal control group, CUMS group, and CUMS + AE
   (administered at a dose of 500 mg/kg) group. All mice received
   physiological saline daily for three days before the experiment to
   familiarize them with oral gavage (i.g.). The apple extract was
   administered via intragastric gavage at a constant volume and given
   30 min before the initiation of the CUMS protocol. Throughout the
   5‐week experimental period, these treatments were administered daily.

2.10. CUMS Procedure

   The CUMS paradigm was conducted as previously described with
   modifications (Nollet et al. [56]2019). Briefly, all groups, excluding
   the control group, were exposed to a total of 13 chronic stressors.
   These included tail pinch (2 min), shaking the mouse cage (15 min),
   exposure to a novel object (24 h), swimming in 4°C ice water (5 min),
   physical restraint (3 h), tilting the cage at a 45° angle (24 h),
   olfactory irritation (24 h), food deprivation (24 h), exposure to
   strobe lighting (5 h), reversal of day and night cycles (24 h), moist
   bedding (24 h), water deprivation (24 h), and mixed housing conditions
   (24 h). Stressors were randomly assigned over a period of 7 weeks, with
   one or two unpredictable stimuli given to the animals each day. Care
   was taken to ensure that no stressor was repeated on consecutive days,
   preventing the animals from anticipating the stimuli.

2.11. Behavioral Tests

   Behavioral assessments were performed following 5 weeks of CUMS
   exposure by an evaluator who was unaware of the treatment groups.

2.11.1. Morris Water Maze (MWM)

   The Morris Water Maze (MWM) was used to assess the spatial cognitive
   function of the mice as described in our previous study (Li
   et al. [57]2020). Briefly, the experimental setup included a circular
   stainless steel pool with a diameter of 120 cm and a height of 40 cm.
   The pool's interior was painted black and divided into four equal
   quadrants. It was filled with water to a depth of 30 cm, which was kept
   at a temperature of 22°C ± 2°C. A circular escape platform, measuring
   10 cm in diameter, was positioned in one of the quadrants designated as
   the target quadrant, submerged approximately 1.5 cm beneath the water
   surface. The behavioral test was structured into two main phases: an
   acquisition training phase followed by a spatial probe test. During the
   acquisition training, subjects were required to locate the hidden
   platform using visual cues surrounding the pool. This phase helped
   assess their ability to learn and remember the platform's location over
   multiple trials. In the subsequent spatial probe test, the platform was
   removed, and the animals' search patterns were analyzed to evaluate
   their retention and recall of the platform's previous position.
   Throughout both phases, detailed data such as the swim path, swim
   distance, and swim time were meticulously recorded using a
   high‐resolution camera system coupled with automated tracking and
   analysis software.

2.11.2. Open Field Test (OFT)

   The Open Field Test (OFT) was often used to reflect the curiosity,
   exploration, and locomotion of mice in a new environment, and the
   experimental procedure was as described previously (Wang
   et al. [58]2019). Briefly, the test system comprised an open‐field
   apparatus with dimensions of 50 × 50 × 50 cm, monitored by a digital
   camera positioned directly above its center. Each mouse was placed
   individually at the center of the apparatus and given the opportunity
   to explore freely for a total duration of 6 min. Following an initial
   2‐min acclimation period, the number of crossings was recorded during
   the subsequent 4 min. To ensure cleanliness and prevent any olfactory
   cues from previous trials, the apparatus was thoroughly cleaned with
   75% alcohol between each trial, removing excrement and odors.

2.11.3. Sucrose Preference Test (SPT)

   The Sucrose Preference Test (SPT) was utilized to evaluate anhedonia,
   or the loss of pleasure in mice. The SPT was conducted as previously
   described, with minor modifications (Liu et al. [59]2018). Briefly,
   prior to the test, the mice underwent a 24‐h acclimation period to a 1%
   sucrose solution (w/v). Following acclimation, the mice were subjected
   to a 12‐h period of water and food deprivation. Subsequently, each
   mouse was individually housed and given access to two bottles: one
   containing tap water and the other 1% sucrose solution, for a duration
   of 12 h, with the positions of the bottles switched after 6 h to
   prevent positional bias. After the 12‐h period, the water bottle was
   removed, and the consumption volumes of both the sucrose solution and
   plain water were recorded. Sucrose preference, which represents
   anhedonia, was then calculated using the formula: sucrose preference
   index (%) = sucrose solution consumption/total fluid intake × 100%.

2.11.4. Forced Swim Test (FST)

   The Forced Swim Test (FST) was conducted as previously described (Duman
   et al. [60]2007; Porsolt et al. [61]^1977). Briefly, each mouse was
   individually introduced into a cylindrical water tank measuring 30 cm
   in height and 40 cm in diameter. The water temperature was maintained
   at 22°C ± 2°C to ensure consistent experimental conditions. Mice were
   allowed to remain in the tank for a total duration of 6 min. During
   this period, immobility time was recorded specifically during the final
   4 min. Immobility was defined as the mouse positioning its body
   perpendicular to the water surface, with only its nose above water, or
   when the mouse floated almost motionless on the water's surface. This
   behavioral assessment aimed to evaluate the level of despair or
   depressive‐like behavior in the mice. By focusing on the last 4 min,
   the test minimized the impact of initial stress reactions and provided
   a more accurate measure of sustained immobility, which is indicative of
   reduced escape‐related behaviors.

2.11.5. Tail Suspension Test (TST)

   The Tail Suspension Test (TST) was performed to evaluate behavioral
   despair of mice as previously described, with minor modifications
   (Steru et al. [62]1985). Briefly, each mouse was suspended
   approximately 20 cm above the ground, with the rope positioned about
   1 cm from the tip of the tail. The test duration was 6 min. During this
   period, any mouse that remained completely motionless was considered
   immobile. The total immobility time was specifically recorded during
   the last 4 min of the observation to assess behavioral despair. To
   elaborate further, this method aimed to evaluate the level of despair
   in mice by focusing on their immobility behavior. By excluding the
   initial 2 min and recording only the final 4 min, the test minimized
   the influence of initial stress reactions and provided a more accurate
   measure of sustained immobility.

2.12. Tissue Sample Collection

   After behavioral tests, the mice were deeply anesthetized by CO[2]. On
   day 60, different sample materials were collected from each group of
   mice. Blood was first drawn from the mice's eyes using a capillary tube
   and transferred to EP tubes, where it was allowed to clot at room
   temperature for 1 h. The samples were then centrifuged at 4000 rpm for
   15 min at 4°C. The resulting serum was carefully aspirated and stored
   at −20°C for further analysis. Simultaneously, brain tissues were
   rapidly harvested and snap‐frozen in liquid nitrogen before being
   stored at −80°C. For histological analysis, mice were deeply
   anesthetized using CO[2] and then transcardially perfused with 20 mL of
   pre‐cooled phosphate‐buffered saline (1× PBS, pH 7.4). Brain tissues
   intended for immunofluorescence assays were extracted and post‐fixed
   overnight in the same PBS solution. These tissues were subsequently
   dehydrated through a graded series of sucrose solutions at 4°C to
   prepare them for sectioning and staining. The isolated tissues were
   then embedded in optimal cutting temperature (OCT) compound.

2.13. Immunofluorescence Assay

   The immunofluorescence assay was conducted according to the protocol
   previously described by us, with minor modifications (Li
   et al. [63]2024). Briefly, brain tissues were slowly frozen and then
   sectioned into serial coronal slices, each 20 μm thick. The sections
   were rinsed three times to remove any debris and permeabilized with
   0.1% Triton X‐100 for 20 min to allow better penetration of antibodies.
   Following this, the sections were incubated in a blocking solution
   containing 1.5% fetal bovine serum for 1 h at room temperature to
   minimize non‐specific binding. Subsequently, the sections were
   incubated overnight at 4°C with primary antibodies: PSD‐95 (1:500,
   ab18258, Abcam) and Synaptophysin (1:400, ab8049, Abcam). After another
   round of PBS rinsing, the sections were incubated with
   fluorescent‐conjugated secondary antibodies. The immunofluorescence
   slides were imaged using a confocal laser scanning microscope (LSM980,
   Carl Zeiss).

2.14. Enzyme‐Linked Immunosorbent Assay (ELISA)

   Following the completion of the behavioral tests, the enzyme‐linked
   immunosorbent assay (ELISA) was performed in accordance with the
   manufacturer's guidelines. ELISA was used to measure levels of
   inflammatory mediators, including TNF‐α (Solarbio, SEKM‐0034), IL‐6
   (Solarbio, SEKM‐0007) and IL‐1β (Solarbio, SEKM‐0002), PTGS2 (Solarbio,
   SEKM‐0156) and cAMP (Solarbio, SEKSM‐0017) in brain tissue and serum of
   mice.

2.15. Western Blot

   The protein sample lysates or precipitates were denatured in sodium
   dodecyl sulfate (1 × SDS) sample buffer and subsequently separated by
   10% SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE). Following
   separation, the proteins were transferred onto a polyvinylidene
   difluoride (PVDF) membrane. The membrane was then incubated with
   appropriate primary antibodies to detect specific proteins. Anti‐PKA
   (1:1000, ab32514, Abcam) and anti‐phospho‐PKA (1:1000, ab238951,
   Abcam), anti‐CREB (1:1000, ab32515, Abcam) and anti‐phospho‐CREB
   (1:1000, ab32096, Abcam) were primary antibodies. Each antibody was
   diluted according to the manufacturer's recommendations and applied to
   the membrane to identify the respective proteins of interest. After
   incubation with the primary antibodies and subsequent washing steps to
   remove unbound antibodies, the membrane was incubated with the
   HRP‐labeled secondary antibody (1:4000, A0545, Sigma). Protein
   visualization was achieved using enhanced chemiluminescence, and their
   densities were quantified using ImageJ software (NIH).

2.16. Cell Culture and Treatments

   Neuro‐2a (N2a) cells were cultured in high‐glucose Dulbecco's Modified
   Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS),
   100 U/mL penicillin, and 100 mg/mL streptomycin. The cells were seeded
   at a density of 1 × 10^5 cells/mL and maintained at 37°C in a
   humidified atmosphere containing 5% CO[2] for 24 h. All cell lines used
   in our study were confirmed to be mycoplasma‐free through PCR testing.
   Plated N2a cells were then exposed to corticosterone (CORT) at various
   concentrations (0, 100, 200, 400, 600, and 800 μmol/L), with a purity
   of 95%, for 24 h. This treatment was designed to assess the effects of
   different CORT concentrations on the cells.

2.17. Cell Viability Assay

   Cell viability was measured using a Cell Counting Kit‐8 (CCK‐8)
   (Beyotime, C0039). Briefly, inoculate an appropriate amount of cells
   into 96‐well plates and culture them to allow the cells to adhere to
   the wall and grow to a suitable state. Perform corresponding drug
   treatments on cells in different groups. After the cells in each group
   are intervened with drugs for the corresponding time, discard the
   original medium in the wells. Add 100 μL of fresh medium to each well,
   and then add 10 μL of CCK‐8 solution to each well. Place the 96‐well
   plate back into the cell incubator and continue culturing for 2 h to
   ensure sufficient reaction between the CCK‐8 solution and the cells.
   After the culture is completed, use a microplate reader to measure the
   absorbance values of each group at a wavelength of 450 nm. The
   experimental results are expressed as the ratios of the absorbance
   values of other groups to that of the normal group.

2.18. Statistical Analysis

   Comparisons among the three groups were conducted using one‐way
   analysis of variance (ANOVA), followed by the Newman–Keuls test for
   post hoc comparisons. All statistical analyses were performed using
   SPSS 20.0 and Prism 9.0 (GraphPad Software). For the quantification of
   immunostaining, assessments were carried out in a blinded manner to
   minimize bias. Data were expressed as mean ± standard deviation (SD),
   ensuring clear representation of the central tendency and variability
   of the measurements. Statistical significance was set at p < 0.05,
   meaning that any p values less than 0.05 were considered indicative of
   statistically significant differences.

3. Results

3.1. Based on Network Pharmacology, the Potential Targets and Mechanisms of
Action for the Antidepressant Effects of AE Have Been Predicted

   Through the integration of five databases—TCMID, HERB, ETCM, HIT, and
   TCMIP—along with supplementary literature from CNKI and PubMed, 17
   active components of apples were identified, with detailed information
   listed in Table [64]1. Using the PharmMapper database and after
   removing duplicates, standardized gene names were obtained via the
   UniProt database, resulting in 389 potential targets for the effective
   components of apple. A total of 1341 depression‐related genes were
   retrieved from the Genecards database (with a Relevance score ≥ 3). By
   intersecting the potential targets of apple's active components with
   depression‐related targets, 118 potential anti‐depressive targets of
   apple's active components were identified (Figure [65]1A).
   Protein–Protein Interaction (PPI) analysis was conducted on these 118
   intersecting targets, followed by the selection of the top 6 nodes
   (AKT1, TNF, ESR1, CASP3, PTGS2, and EGFR) based on Degree value using
   the CentiScape 2.2 plugin in Cytoscape software, forming the core
   target network (Figure [66]1B). To better understand the biological
   functions and pathways associated with the 118 potential targets, we
   conducted both Gene Ontology (GO) enrichment analysis and Kyoto
   Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Together,
   these complementary analyses provide a comprehensive view of the
   functional roles and pathway associations of the 118 potential targets.
   GO analysis revealed that the anti‐depressive effects of apple are
   mainly enriched in biological processes such as response to hypoxia,
   response to xenobiotic stimulus, inflammatory response, and positive
   regulation of gene expression (Figure [67]1C). KEGG pathway analysis
   indicated that the key pathways through which apple exerts its
   anti‐depressive effects include pathways in cancer, cAMP signaling
   pathway, calcium signaling pathway, and neuroactive ligand‐receptor
   interaction (Figure [68]1D). The aforementioned predictive results
   suggested that the apple may play a crucial role in antidepressant
   mechanisms, which is associated with inflammatory responses, and the
   cAMP signaling pathway is likely a key pathway through which apple
   exerts its antidepressant effects.

TABLE 1.

   Major active components of apples.
   Component CAS number Canonical SMILES
   Quercetin 117‐39‐5 C1=CC(=C(C=C1C2=C(C(=O)C3=C(C=C(C=C3O2)O)O)O)O)O
   Phloretin 60‐82‐2 C1=CC(=CC=C1CCC(=O)C2=C(C=C(C=C2O)O)O)O
   Taxifolin 480‐18‐2 C1=CC(=C(C=C1C2C(C(=O)C3=C(C=C(C=C3O2)O)O)O)O)O
   Anthocyanidin 13306‐05‐3 C1=CC(=C(C=C1C2=[O+]C3=CC(=CC(=C3C=C2O)O)O)O)O
   Methyl protocatechuate 2150‐43‐8 COC(=O)C1=CC(=C(C=C1)O)O
   Catechin 154‐23‐4 C1C(C(OC2=CC(=CC(=C21)O)O)C3=CC(=C(C=C3)O)O)O
   Epicatechin 490‐46‐0 C1C(C(OC2=CC(=CC(=C21)O)O)C3=CC(=C(C=C3)O)O)O
   Gallic acid 149‐91‐7 C1=C(C=C(C(=C1O)O)O)C(=O)O
   Caffeic acid 331‐39‐5 C1=CC(=C(C=C1C=CC(=O)O)O)O
   Ferulic acid 1135‐24‐6 COC1=C(C=CC(=C1)C=CC(=O)O)O
   Maleic acid 110‐16‐7 C(=CC(=O)O)C(=O)O
   p‐Coumaric acid 4501‐31‐9 C1=CC(=CC=C1C=CC(=O)O)O
   Cinnamic acid 140‐10‐3 C1=CC=C(C=C1)C=CC(=O)O
   2α‐Hydroxyursolic acid 4547‐24‐4
   CC1CCC2(CCC3(C(=CCC4C3(CCC5C4(CC(C(C5(C)C)O)O)C)C)C2C1C)C)C(=O)O
   β‐Sitosterol 83‐46‐5
   CCC(CCC(C)C1CCC2C1(CCC3C2CC=C4C3(CCC(C4)O)C)C)C(C)C
   Ethyl 3,4‐Dihydroxybenzoate 3943‐89‐3 CCOC(=O)C1=CC(=C(C=C1)O)O
   2(R)‐Hydroxybutaned‐ioic acid 143435‐96‐5 C(C(C(=O)O)O)C(=O)O
   [69]Open in a new tab

FIGURE 1.

   FIGURE 1
   [70]Open in a new tab

   Prediction of potential targets and mechanisms of action for the
   antidepressant effects of apples. (A) Venn diagram showing the
   intersection between the targets of apple's active components and
   depression‐associated disease targets. (B) Protein–Protein Interaction
   (PPI) network of the intersected targets between the targets of apple's
   active components and depression‐associated disease targets. (C) Gene
   Ontology (GO) enrichment analysis. (D) Kyoto Encyclopedia of Genes and
   Genomes (KEGG) pathway enrichment analysis.

3.2. Effect of AE Treatment on Cognitive Functions and Depression‐Like
Behaviors in CUMS Mice

   To explore the impact of AE on cognitive function and depression‐like
   behaviors induced by chronic unpredictable mild stress (CUMS) in mice,
   we conducted a series of behavioral tests such as MWM, OFT, SPT, TST,
   and FST. The detailed experimental flow chart is illustrated in
   Figure [71]2A. During the acquisition training phase of the MWM,
   different groups displayed varying levels of spatial learning ability
   over the 5‐day training period. Treatment with AE significantly
   decreased the escape latency (Figure [72]2B). Specifically, the CUMS
   group showed significantly longer escape latencies compared to the
   control group on days 3–5, whereas the AE‐treated group exhibited
   notably shorter escape latencies relative to the CUMS group
   (Figure [73]2B). In the probe test, CUMS‐exposed mice crossed the
   target platform less frequently and spent a significantly lower
   percentage of time in the target quadrant compared to the control mice.
   Conversely, the AE group demonstrated a significantly higher frequency
   of crossing the target platform and a greater percentage of time spent
   in the target quadrant compared to the CUMS group (Figure [74]2C,D). In
   the OFT, CUMS‐exposed mice had a significantly reduced number of grid
   crossings compared to the control group, while AE administration
   significantly increased the number of grid crossings compared to the
   CUMS group (Figure [75]2E). For the SPT, CUMS‐exposed mice consumed
   significantly less sucrose than control mice, indicating anhedonia.
   However, treatment with AE significantly reversed this reduction in
   sucrose preference and alleviated anhedonia symptoms in CUMS mice
   (Figure [76]2F). The desperation state in mice was assessed using the
   immobility time in both the TST and FST (Figure [77]2G,H). CUMS
   stimulation led to a significant increase in immobility time in both
   tests compared to the control group (Figure [78]2G,H). Consistent with
   expectations, AE treatment notably reduced the immobility duration and
   improved signs of hopelessness in the AE group compared to the CUMS
   group (Figure [79]2G,H). Overall, these behavioral tests demonstrated
   that AE treatment not only enhanced cognitive performance but also
   produced antidepressant‐like effects in the CUMS‐induced depression
   model. This comprehensive evaluation underscores the potential
   therapeutic benefits of AE in mitigating both cognitive deficits and
   depressive symptoms.

FIGURE 2.

   FIGURE 2
   [80]Open in a new tab

   AE treatment ameliorates cognitive dysfunction and depression‐like
   behaviors in CUMS mice. (A) Diagram of experimental schedule. (B) The
   latency to the platform of 5‐day acquisition training in MWM. (C) The
   frequency across the target quadrant in the probe test. (D) The
   percentage of time spent in the target quadrant in the probe test. (E)
   Number of crossings of central area in OFT. (F) Percentage of sucrose
   consumption in SPT. (G‐H) The immobility in the TST (G) and FST (H).
   All data are presented as the mean ± SD. n = 6 mice per group, ^##
   p < 0.01, ^### p < 0.001, and ^#### p < 0.0001 vs. Control group,
   **p < 0.01, ***p < 0.001, and ****p < 0.0001 vs. CUMS group.

3.3. Effect of AE Treatment on Synaptic Plasticity and Neurotransmitter in
CUMS Mice

   To determine whether AE influenced synaptic growth in vivo, we
   performed immunofluorescence assays using hippocampal tissues from
   mice, focusing on the proteins PSD‐95 and synaptophysin. These markers
   are crucial for assessing synaptic integrity and density. In the
   hippocampal tissues of CUMS‐exposed mice, both the relative
   fluorescence intensities and protein levels of PSD‐95 and synaptophysin
   were notably lower compared to those in the control group. In AE
   treatment, the relative fluorescence intensities of synaptophysin
   significantly increased, but the increase of PSD‐95 was limited,
   pointing to elevated neuronal synaptic density in vivo (Figure [81]3A).
   As the most extensive cohesive neurotransmitter systems in the brain,
   5‐hydroxytryptamine (5‐HT, also known as serotonin) and
   gamma‐aminobutyric acid (GABA) play crucial roles in regulating a wide
   array of biological functions. These include mood regulation, sleep
   patterns, appetite control, and energy balance. Therefore, we examined
   the levels of neurotransmitters 5‐HT and GABA in mouse brain tissue.
   Compared to the control, the levels of neurotransmitters 5‐HT and GABA
   were significantly decreased in CUMS mice. The AE intervention led to a
   significant increase in the levels of 5‐HT (Figure [82]3B) and GABA
   (Figure [83]3C) in brain tissue compared to the CUMS group. Together,
   these results strongly suggested that AE plays essential roles in
   promoting synaptic plasticity and balancing neurotransmitter systems.

FIGURE 3.

   FIGURE 3
   [84]Open in a new tab

   AE plays essential roles in regulating synaptic plasticity and
   neurotransmitter levels in CUMS mice. (A) Immunofluorescence assay of
   PSD‐95 and synaptophysin in mice hippocampal tissues. Image analyses
   and quantification were performed using ImageJ software. (B and C) The
   levels of neurotransmitters 5‐HT (B) and GABA (C) in mouse brain
   tissue. All data were presented as the mean ± SD. n = 6 mice per group,
   n.s. > 0.05, **p < 0.01 and ****p < 0.0001 vs. CUMS group.

3.4. Effect of AE Treatment on Inflammatory Cytokine in CUMS Mice

   The pro‐inflammatory factors such as TNF‐α, IL‐1β, and IL‐6 are
   classical markers of inflammation frequently studied in the context of
   depression. These cytokines play significant roles in mediating
   inflammatory responses, which have been linked to the development and
   progression of depressive symptoms. To investigate the impact of AE on
   inflammation, we measured the levels of TNF‐α, IL‐1β, and IL‐6 in both
   serum and brain tissue of mice. Our analysis revealed that AE treatment
   effectively reduced the concentrations of these pro‐inflammatory
   cytokines in serum compared to the CUMS group. In serum, compared to
   the control, levels increased in CUMS mice; compared to the CUMS group,
   they could be downregulated in the AE group (Figure [85]4A–C). The
   analysis of inflammatory factor content in the brain tissues of mice
   demonstrated that AE significantly lowered the levels of TNF‐α, IL‐1β,
   and IL‐6. Specifically, compared to the control group, the brain tissue
   of CUMS‐exposed mice showed a marked increase in TNF‐α, IL‐1β, and IL‐6
   levels. However, when compared to the CUMS group, the AE‐treated group
   exhibited a significant reduction in these pro‐inflammatory markers
   (Figure [86]4D–F). These findings indicated that AE possesses
   antidepressant properties by effectively reducing the expression of
   inflammatory factors.

FIGURE 4.

   FIGURE 4
   [87]Open in a new tab

   AE inhibited CUMS‐induced inflammatory responses. (A–C) Levels of TNF‐α
   (A), IL‐1β (B), and IL‐6 (C) in serum. (D–F) Levels of TNF‐α (D), IL‐1β
   (E), and IL‐6 (F) in brain tissue. All data are presented as the
   mean ± SD. n = 6 mice per group, ****p < 0.0001 vs. CUMS group.

3.5. Effects of AE on CORT‐Induced Neurotoxicity

   To determine the neuroprotective effects of AE against cell
   cytotoxicity induced by high‐concentration CORT, we established an
   in vitro model mimicking stress‐induced depression. This study found
   that CORT at concentrations of 200, 400, 600, and 800 μmol/L led to a
   significant reduction in N2a cell viability. Specifically, exposure to
   600 μmol/L CORT decreased cell viability to about 50%, making it the
   concentration chosen for further experiments (Figure [88]5A).
   Subsequently, we examined the cytotoxicity and protective effects of
   different AE concentrations. Intriguingly, AE treatments at 12.5, 25,
   50, and 100 μmol/L notably reduced CORT‐induced cytotoxicity in a
   dose‐dependent manner (Figure [89]5B). Based on these results, we opted
   for 50 μmol/L AE in subsequent experiments involving the 600 μmol/L
   CORT‐induced depression cell model. In this model, compared to the
   control cells, the levels of 5‐HT and GABA were significantly lower,
   while TNF‐α, IL‐1β, and IL‐6 levels were markedly higher in the
   CORT‐induced depression cell model. These alterations mirrored those
   observed in CUMS‐induced depressed mice. Importantly, AE treatment
   reversed these changes, restoring the levels of 5‐HT and GABA and
   decreasing the levels of TNF‐α, IL‐1β, and IL‐6 (Figure [90]5C–G). The
   above results indicated that AE provides substantial neuroprotection
   against CORT‐induced cytotoxicity in vitro. By elevating key
   neurotransmitter levels such as 5‐HT and GABA and lowering
   pro‐inflammatory cytokines like TNF‐α, IL‐1β, and IL‐6, AE shows
   promise as a therapeutic agent for mitigating neuronal damage caused by
   chronic stress.

FIGURE 5.

   FIGURE 5
   [91]Open in a new tab

   AE exerted significant neuroprotective effects against CORT‐induced
   cytotoxicity. To determine the protective effect of AE on CORT‐induced
   cytotoxicity in N2a cells, we measured the cell viability at various AE
   concentrations. (A) N2a cells were exposed to CORT at different
   concentrations (0, 100, 200, 400, 600 and 800 μmol/L) for 24 h and then
   were determined by CCK‐8 assay. (B) N2a cells were exposed to different
   concentrations of AE (12.5, 25, 50, 100 μmol/L) and 600 μmol/L CORT for
   24 h. Quantification of 5‐HT (C), GABA (D), TNF‐α (E), IL‐1β (F), and
   IL‐6 (G) in cell culture supernatant harvested from the 600 μmol/L
   CORT‐induced depression‐like cell model intervened with AE (50 μmol/L).
   All data were presented as the mean ± SD. n = 6 cell culture
   supernatants per group, ^## p < 0.01, ^### p < 0.001, ^#### p < 0.0001
   vs. control group, ***p < 0.001, ****p < 0.0001 vs. CORT‐induced
   depression‐like cell model group.

3.6. Effects of AE on cAMP/PKA/CREB/PTGS2 Signaling Pathway

   To better understand the molecular mechanisms responsible for the
   protective effects of AE in CUMS mice, we utilized enzyme‐linked
   immunosorbent assay (ELISA) to quantify the levels of cyclic AMP (cAMP)
   in both serum and brain tissue. At the same time, Western blot was used
   to detect the levels of phosphorylated PKA (pPKA) and phosphorylated
   CREB (pCREB) in brain tissue. Moreover, ELISA was used to detect the
   levels of cAMP and PTGS2 in serum and brain tissue. Our results showed
   that cAMP, pPKA, and pCREB levels were suppressed in CUMS mice, but the
   expression level of PTGS2 was significantly higher compared to the
   control group. In the CUMS+AE group, coupled with the elevation of
   cAMP, pPKA, and pCREB levels, AE treatment significantly reduced PTGS2
   level compared to the CUMS group (Figure [92]6A–F). Interestingly,
   compared to the control cells, the level of PTGS2 was elevated in the
   CORT‐induced depression cell model. These results were consistent with
   those observed in CUMS‐induced depressed mice and were reversed by AE
   treatment (Figure [93]6G). Our research indicated that the
   cAMP/PKA/CREB/PTGS2 signaling pathway may play a crucial role in the
   antidepressant‐like effects of AE.

FIGURE 6.

   FIGURE 6
   [94]Open in a new tab

   AE acts on the cAMP/PKA/CREB signaling pathway to inhibit PTGS2. (A and
   B) The expression levels of cAMP and PTGS2 were detected by ELISA in
   serum (A) and brain tissue (B). (C) Western blot bands: Band 1, control
   group; Band 2, CUMS group; Band 3, AE (50 μmol/L) + CUMS group.
   Phosphorylated PKA (pPKA) levels were normalized to total PKA levels.
   (D) Western blot bands: Band 1, control group; Band 2, CUMS group; Band
   3, AE (50 μmol/L) + CUMS group. Phosphorylated CREB (pCREB) levels were
   normalized to total CREB levels. (E–G) Levels of PTGS2 in serum (E),
   brain tissue (F) and cell culture supernatant (G). All data were
   presented as the mean ± SD. n = 6 mice per group, ****p < 0.0001 vs.
   CUMS group.

4. Discussion

   Depression, as one of the major diseases threatening human health, has
   garnered increasing attention and concern. Western medical treatments
   for depression are based on theories such as the monoamine
   neurotransmitter hypothesis and receptor hypotheses, but their
   mechanisms of action remain unclear, and they are often associated with
   a slow onset of efficacy and numerous adverse reactions. Therefore,
   screening effective alternative strategies is urgent. Studies have
   proved the obvious antidepressant effects of dietary polyphenols such
   as resveratrol, quercetin, and chlorogenic acid, particularly the
   effects on depression‐related inflammation (Tayab et al. [95]2022; Wang
   et al. [96]^2022). Apple polyphenol extracts (APE), an active mixture
   rich in active monomers like chlorogenic acid, quercetin, and
   proanthocyanin B2, exhibit strong anti‐inflammatory, antioxidant, and
   antiaging biological activities (Li et al. [97]2019). Recent studies
   have shown that apple polyphenol extracts improve depressive‐like
   behaviors in high‐sugar diet‐fed mice by inhibiting inflammation in the
   gut‐brain axis (Xie, Wu, et al. [98]^2024). Another study also
   demonstrated that apple polyphenol extracts alleviate depressive‐like
   behaviors in high‐sugar diet‐fed mice by modulating bile acid
   circulation through the liver‐gut‐brain axis mediated by the Farnesoid
   X Receptor (FXR) (Xie, Shang, et al. [99]^2024). However, these studies
   have focused on the antidepressant effects of specific components in
   the apple, which may not fully reflect the interactions among multiple
   active components present in the apple. Therefore, this study seeks to
   uncover the antidepressant mechanisms of AE using a combination of
   network pharmacology analysis and traditional pharmacological
   techniques.

   In this study, we identified 17 active components of AE through
   database screening. Subsequently, we retrieved 389 targets linked to AE
   and 1341 targets connected to depression. Among these, 118 common
   targets shared by both AE and depression were selected for subsequent
   analysis. PPI network analysis identified AKT1, TNF, ESR1, CASP3,
   PTGS2, and EGFR as the core targets of AE for antidepressant treatment.
   GO analysis of the 118 candidate targets indicated a notable enrichment
   in biological processes such as the response to xenobiotic stimulus and
   the inflammatory response. Increasing evidence has supported the
   antidepressant effects of the individual components of AE (e.g.,
   chlorogenic acid and epicatechin) in a mouse model of depression
   (Martínez‐Damas et al. [100]^2021; Song et al. [101]^2019).
   Additionally, procyanidin B2 has been proven to decrease blood–brain
   barrier (BBB) leakage and restore the decreased expression of a tight
   junction protein, zonula occludens‐1 (ZO‐1), in both the cortex and
   striatum to attenuate BBB disruption in ischemic rats, suggesting that
   procyanidin B2 might also have an antidepressant effect (Wu
   et al. [102]2015). APE has been reported to effectively ameliorate
   sugary‐diet‐induced depression‐like behaviors in CUMS mice by reducing
   inflammation within the gut‐brain axis (Xie, Wu, et al. [103]^2024).
   KEGG pathway analysis indicated that the candidate targets are
   significantly enriched in pathways like the cAMP signaling pathway.
   Growing evidence suggested that these pathways play a crucial role in
   the development and progression of depression (Gao et al. [104]2022; Li
   et al. [105]^2009). These insights suggested that AE may effectively
   alleviate depression primarily by modulating the cAMP signaling
   pathway. This hypothesis highlights the necessity for further research
   to clarify the precise roles of these pathways in the antidepressant
   mechanisms of AE. AE contains a diverse array of compounds, each with
   multiple targets and pharmacological effects relevant to depression.
   This multifaceted composition may lead to synergistic effects,
   potentially reducing the side effects commonly associated with
   single‐action antidepressants. Our findings indicated that AE improves
   depressive‐like behavior in CUMS mice through several mechanisms,
   including the regulation of synaptic plasticity, enhancement of
   synaptic levels of monoamines, and inhibition of inflammatory factor
   expression. The CORT‐induced nerve injury cell model, which can
   simulate the neurotransmitter deficits, oxidative stress, inflammatory
   response, and neuronal loss associated with depression (Pan
   et al. [106]2023; Wang, Wang, Wang, et al. [107]^2024; Wang, Hu, Liu,
   et al. [108]^2024), is suitable for research on the effective
   mechanisms of drug relating to neurotransmitter imbalance or
   inflammatory cytokine changes in vitro. Our results demonstrated that
   50 μmol/L AE was the optimal concentration for treating N2a cell injury
   induced by 600 μmol/L CORT in vitro. Furthermore, the protective
   effects of AE were associated with the promotion of neurotransmitter
   synthesis and inhibition of inflammatory reactions. In our study, AE
   has demonstrated therapeutic efficacy in both in vivo and in vitro
   depression models by correcting imbalances in neurotransmitters and
   inflammation. This dual‐action approach highlights AE's potential as a
   comprehensive treatment for depression, addressing multiple underlying
   mechanisms simultaneously.

   Intracellular signal transduction pathways are currently a focus and
   challenge in the research of antidepressant mechanisms. Cyclic
   adenosine monophosphate (cAMP) is a crucial second messenger within
   cells, mediating the actions of various intracellular hormones,
   neurotransmitters, and other signaling molecules. Adenylyl cyclase (AC)
   is a key enzyme in G protein‐coupled signaling pathways, converting
   adenosine triphosphate (ATP) into the second messenger cAMP. cAMP then
   activates protein kinase A (PKA), which in turn phosphorylates various
   proteins, including transcription factors, ion channels, cytoskeletal
   elements, and enzymes. One of these phosphorylated transcription
   factors is the cAMP response element‐binding protein (CREB). CREB is a
   nuclear protein composed of 341 amino acids. It plays a critical role
   in neuronal development, regeneration, synapse formation, and cellular
   repair, making it one of the key factors in regulating central nervous
   system function (Dinevska et al. [109]2024). It is an important
   molecular target in the treatment of depression. Recent studies have
   suggested that alterations in hippocampal CREB are closely related to
   the pathogenesis of depression and the effectiveness of antidepressant
   therapy. The expression and activation of hippocampal CREB are
   influenced by various signaling molecules and pathways. Multiple
   signaling pathways activated by antidepressants ultimately lead to the
   phosphorylation of the nuclear transcription factor CREB, thereby
   regulating the expression of many genes that promote cell growth,
   proliferation, survival, and neural plasticity, such as brain‐derived
   neurotrophic factor (BDNF), nerve growth factor (NGF), vascular
   endothelial growth factor (VEGF), insulin‐like growth factor I (IGF‐I),
   and activin, thus exerting antidepressant effects (McCauslin
   et al. [110]^2006). This indicates that CREB is a key protein for the
   efficacy of many antidepressant drugs and can be activated by various
   signaling pathways. As a transcription factor, CREB participates in the
   transition from short‐term to long‐term memory, and its phosphorylation
   is regulated by multiple signaling pathways, including the classic
   AC/cAMP/PKA pathway (Dowlatshahi et al. [111]1998). The AC/cAMP/PKA
   pathway plays a significant role in mood regulation and is one of the
   earliest and most extensively studied signal transduction pathways in
   antidepressant research. The general process of the cAMP signaling
   cascade is as follows: exogenous stimuli cause the release of
   neurotransmitters (such as serotonin), which bind to corresponding
   receptors on the cell membrane (such as G protein‐coupled receptors). G
   proteins can regulate the activity of AC, catalyzing the conversion of
   ATP to cAMP, which in turn activates PKA, leading to the
   phosphorylation of CREB. The phosphorylation of CREB enables the
   induction and regulation of related genes (Shaywitz and
   Greenberg [112]1999). Studies have found that antidepressants can
   activate G protein coupling in blood cells of depressed patients,
   specifically increasing the coupling of Gs protein with adenylyl
   cyclase (Gs‐AC) (Grønli et al. [113]2004). cAMP is currently recognized
   as one of the key signaling pathways associated with antidepressant
   effects. Under normal physiological conditions, cAMP is synthesized
   from ATP through the enzymatic action of AC and is mainly degraded by
   type IV phosphodiesterase (PDE4). The functional states of AC and PDE4
   together maintain the stability of cAMP levels in neurons. Branski
   et al. discovered that the PKA agonist 8‐Br‐cAMP could dose‐dependently
   decrease the immobility time of rats in the forced swim test,
   indicating its antidepressant properties. This study provided the first
   animal evidence of the antidepressant action of a PKA agonist (Brański
   et al. [114]2008). Li et al. used the specific PDE4 inhibitor rolipram
   to inhibit the hydrolysis of cAMP in mice. After continuous
   administration for 17–23 days, the levels of cAMP and pCREB in the
   hippocampus were upregulated, reducing the immobility time in the
   forced swim and tail suspension tests, similar to the behavioral
   changes observed after fluoxetine administration. This suggests that
   the activation of the cAMP pathway in the hippocampus plays a positive
   role in improving depressive symptoms (Li et al. [115]2009), consistent
   with Branski's findings. Clinical studies have confirmed that the
   levels of cAMP and PKA activity in the prefrontal cortex of suicide
   victims with depression are reduced, along with the expression of
   neuroprotective and synaptic plasticity molecules activated by cAMP or
   PKA, such as Rap‐1 (Dwivedi et al. [116]2006). Another clinical study
   found that PKA activity is decreased in peripheral blood and
   post‐mortem brain tissue samples from depressed patients, particularly
   in those with severe depression and suicidal tendencies (Shelton
   et al. [117]2009). Many studies have indicated that the cAMP signaling
   pathway, through intracellular PKA, plays a crucial role in
   antidepressant therapy (Gao et al. [118]2022). Chang Shen Hua volatile
   oil (CSHVO) improves CORT‐induced injury in the PC12 cell model and
   alleviates depression‐like behaviors in a CUMS combined with orphan
   rearing model in rats. The antidepressant mechanism of CSHVO is
   associated with the modulation of the cAMP‐PKA‐CREB signaling pathway
   (Zhang et al. [119]2024). These research findings suggested that the
   cAMP signaling pathway is downregulated in depressed patients, and
   antidepressant treatment can upregulate the function of the cAMP
   pathway. In our study, compared to the control group, the expression of
   cAMP, PKA, and CREB in the brain tissue of the depression model mice
   was significantly decreased, consistent with previous literature
   reports. After 5 weeks of intraperitoneal administration of AE, the
   protein expression of cAMP, PKA, and CREB was significantly increased,
   indicating that the mechanism of AE's antidepressant effect is related
   to the cAMP/PKA/CREB signaling pathway.

   PTGS2 (Prostaglandin‐Endoperoxide Synthase 2, also known as COX‐2) is
   an inducible enzyme that plays a crucial role in the inflammatory
   response. There is a close association between PTGS2 and depression,
   involving multiple aspects, including neuroinflammation,
   neurotransmitter metabolism, neuroplasticity, and
   hypothalamic–pituitary–adrenal (HPA) axis function (He
   et al. [120]2022). Previous studies have demonstrated that COX‐2 and
   PGE2 are upregulated in a rat model of depression, and inhibition of
   the COX‐2/PGE2 pathway can mitigate the inflammatory response in the
   hippocampus and effectively alleviate depressive behaviors (Song
   et al. [121]2018). Studies have shown that kaempferol exerts
   antidepressant effects, at least partially through the inhibition of
   the COX‐2/PGE2 pathway, which modulates neuroinflammation,
   neurotransmitter imbalance, and impaired neurogenesis (Zhu
   et al. [122]2023). Another study found that bergapten alleviates
   depression‐like behavior by inhibiting COX‐2 activity and the
   NF‐κB/MAPK signaling pathway in microglia (Yan et al. [123]2023).
   Additionally, a recent study also found that quercetin inhibits
   neuronal ferroptosis and enhances immune responses in breast
   cancer‐related depression (BCRD) mice by targeting the lipid
   metabolism‐related gene PTGS2 (Zhu et al. [124]2024). In our study, it
   was found that the relative protein expression of PTGS2 in the model
   group was markedly elevated compared to the normal control group,
   aligning with earlier research findings. This increase in PTGS2 levels
   underscores its potential role in the pathophysiology of depression.
   After 5 weeks of AE treatment, the relative protein expression of PTGS2
   in the treated group was significantly reduced compared to the model
   group, indicating a substantial impact of AE on this inflammatory
   marker. Thus, our study suggested that alongside the improvement of
   cognitive functions and depression‐like behaviors, AE treatment showed
   a regulatory effect on the cAMP/PKA/CREB/PTGS2 signaling pathway in
   CUMS mice.

5. Conclusions

   In summary, our study demonstrated that cAMP/PKA/CREB/PTGS2 is one of
   the potential signaling pathways for depression treatment. Through
   targeting the cAMP/PKA/CREB/PTGS2 signaling pathway, AE can regulate
   synaptic plasticity and neurotransmitters, inhibit inflammatory
   responses, and reduce neuronal toxicity, thereby improving cognitive
   dysfunction and alleviating depressive‐like behavior in CUMS mice.
   These findings highlighted the potential of AE as a promising treatment
   option for depression. The regulatory effect of AE on the
   cAMP/PKA/CREB/PTGS2 signaling pathway suggested its ability to address
   multiple aspects of depression. However, further research is essential
   to fully elucidate the specific mechanisms involved and to optimize
   dosage and administration strategies for more effective utilization of
   AE in depression therapy.

Author Contributions

   Zhu Li: conceptualization (lead), data curation (equal), formal
   analysis (equal), funding acquisition (lead), investigation (equal),
   methodology (equal), project administration (lead), visualization
   (equal), writing – original draft (equal), writing – review and editing
   (lead). Yue Chen: conceptualization (equal), data curation (equal),
   formal analysis (equal), investigation (equal), methodology (equal),
   writing – original draft (equal). Shunqiang Yang: conceptualization
   (equal), data curation (equal), investigation (equal), writing – review
   and editing (equal). Huihuang Shi: investigation (equal), visualization
   (equal), writing – original draft (equal). Jizhe Cui: visualization
   (equal), writing – original draft (supporting).

Conflicts of Interest

   The authors declare no conflicts of interest.

Acknowledgments