Abstract

   Depression is a complex mental disorder, affecting approximately 280
   million individuals globally. The pathobiology of depression is not
   fully understood, and the development of new treatments is urgently
   needed. Dihydromyricetin (DHM) is a natural flavanone, mainly
   distributed in Ampelopsis grossedentata. DHM has demonstrated a
   protective role against cardiovascular disease, diabetes, liver
   disease, cancer, kidney injury and neurodegenerative disorders. In the
   present study, we examined the protective effect of DHM against
   depression in a chronic depression mouse model induced by
   corticosterone (CORT). Animals exposed to CORT displayed
   depressive-like behaviors; DHM treatment reversed these behaviors.
   Network pharmacology analyses showed that DHM’s function against
   depression involved a wide range of targets and signaling pathways,
   among which the inflammation-linked targets and signaling pathways were
   critical. Western blotting showed that CORT-treated animals had
   significantly increased levels of the advanced glycation end product
   (AGE) and receptor of AGE (RAGE) in the hippocampus, implicating
   activation of the AGE-RAGE signaling pathway. Furthermore,
   enzyme-linked immunosorbent assay (ELISA) detected a marked increase in
   the production of proinflammatory cytokines, interleukin-1 beta
   (IL-1β), IL-6 and tumor necrosis factor-alpha (TNFα) in the hippocampus
   of CORT-treated mice. DHM administration significantly counteracted
   these CORT-induced changes. These findings suggest that protection
   against depression by DHM is mediated by suppression of
   neuroinflammation, predominantly via the AGE-RAGE signaling pathway.

   Keywords: depression, dihydromyricetin, inflammation, network
   pharmacology, AGE-RAGE signaling pathway

1. Introduction

   Depression is a common and severe psychiatric disease with a prevalence
   of 5% in adults, affecting more than 350 million individuals across the
   world [[40]1]. It is a complex mental disorder, associated with
   psychological, environmental and genetic factors [[41]2]. Although
   there have been a great many studies, the underlying molecular
   mechanisms of depression formation and progression are poorly
   understood. Currently, a variety of antidepressants are available,
   e.g., tricyclics and monoamines, which have slow onset, low efficacy
   and undesirable side effects. Furthermore, less than two-thirds of
   patients respond to pharmacotherapy, while 70% of treated patients did
   not achieve full remission [[42]3]. Therefore, there is an urgent need
   to develop more natural antidepressants with reliable efficacy and
   fewer side effects.

   Dihydromyricetin (DHM) is a flavonoid produced from Ampelopsis
   grossedentata (A. grossedentata), which is also present in other
   medicinal plants, e.g., Cedrus deodara and Hovenia dulcis, and in some
   plant-based foods, such as grapes [[43]4]. DHM has demonstrated
   anti-inflammatory, anti-oxidative damage, anti-alcoholic and
   anti-cancer effects, and alleviates pathology in diabetes, skin damage,
   liver, kidney and cardiac injury [[44]4,[45]5]. Additionally, DHM has
   also shown neuroprotection in neurodegenerative disorders, e.g.,
   Alzheimer’s disease and Parkinson’s disease [[46]5,[47]6]. Recently,
   DHM has been shown to have anti-depressive properties in rodents with
   diabetic and depressive symptoms [[48]7]. However, the
   antidepressant-like effects of DHM have not been fully elucidated,
   especially in the chronic corticosterone-induced depression model.

   Corticosterone is a rodent stress hormone, which is homologous to human
   cortisol. Corticosterone mediates the hypothalamic–pituitary–adrenal
   (HPA) axis and regulates brain functions in rodents [[49]8]. Chronic
   corticosterone injection causes dysfunction of the HPA axis and induces
   depressive-like behaviors, including social withdrawal, anhedonia and
   despair, as well as increased pro-inflammatory responses and
   upregulated pro-inflammatory cytokines in rodents [[50]3,[51]8,[52]9].
   Chronic administration of corticosterone in rodents causes impaired
   circadian rhythms, increased oxidative damage and inflammation,
   decreased production of neurotrophic factors (e.g., brain-derived
   neurotrophic factor, BDNF) and reduced hippocampal neurogenesis, which
   are supposed to contribute to depression [[53]3]. This chronic
   corticosterone exposure-induced depressive model has the advantages of
   simple replication and strong stability, and avoids the potential
   habituation effect of rodents to stress. This model has been widely
   used to examine the antidepressant-like role of drugs in rodents
   [[54]3]. In the current study, we used the corticosterone-induced
   depressive mouse model to examine the effects of DHM on depression-like
   behavior and to investigate the underlying pathological mechanisms.

2. Material and Methods

2.1. Animals

   Seven-week-old male Institute of Cancer Research (ICR) mice (30 ± 2 g
   weight) were purchased from Hunan SJA Laboratory Animal Co. Ltd.
   (Hunan, China) and pair-housed in a standard environment with
   sufficient water and food provision. Prior to the commencement of the
   experiments, the mice were petted for 5 min per day for a week by the
   experimenter. All animal treatment procedures were performed following
   the Guidance for the Care and Use of Laboratory Animals, University of
   South China.

2.2. Chemicals

   Dihydromyricetin (Shanghai Yuanye Bio-Technology Co., Ltd., Shanghai,
   China) was dissolved in 0.9% saline containing 0.2% dimethylsulfoxide
   (DMSO). Corticosterone (Shanghai Aladdin Biochemical Technology Co.,
   Ltd., Shanghai, China) was dissolved in 0.9% saline containing 0.1%
   DMSO and 1% Tween 80. Both dihydromyricetin (intraperitoneally, i.p.)
   and corticosterone (subcutaneously, s.c.) were selected at a dose of 20
   mg/kg body weight in current experiments.

2.3. Animal Treatment

   The timeline of drug treatment is shown in [55]Figure 1. Animals were
   randomly divided into three groups: control group (Control, n = 10),
   corticosterone group (CORT, n = 10) and corticosterone +
   dihydromyricetin group (CORT+DHM, n = 10). Animals in the control group
   received an injection (s.c.) of 0.9% saline containing 0.1% DMSO and 1%
   Tween 80 for 23 consecutive days (day 1–day 23) and an additional
   injection (i.p.) of 0.9% saline containing 0.2% DMSO for 7 consecutive
   days (day 18–day 24); The animals in the CORT group received 23
   consecutive s.c. injections of CORT (20 mg/kg body weight, day1–day
   23); animals in the CORT + DHM group received 23 consecutive s.c.
   injections of CORT (day 1–day 23) and 7 consecutive i.p. injections of
   DHM (20 mg/kg body weight, day 18–day 24). For behavior experiments, an
   open field test (OFT) and forced swimming test (FST) were conducted
   from 10:00 to 14:00 on day 22, while a sucrose preference test (SPT)
   was carried out at all times on days 23 and 24.

Figure 1.

   [56]Figure 1
   [57]Open in a new tab

   Schematic of the experimental design showing the timeline of drug
   injection and behavior tasks. CORT, corticosterone; DHM,
   dihydromyricetin; OFT, open field test; FST, forced swimming test; SPT,
   sucrose preference test.

2.4. Behavior Tasks

2.4.1. Open Field Test (OFT)

   For the OFT, mice were randomly placed into open field apparatus (50 ×
   50 × 70 cm) for 5 min. The total distance traveled was measured using
   behavioral analysis software (Anymaze 6.16).

2.4.2. Forced Swimming Test (FST)

   For the FST, mice were individually placed into a plexiglass cylinder
   (25 cm high, 15 cm in diameter) containing 17 cm deep water (27 ± 1 °C)
   for 6 min. The behavior of these animals was video-recorded, and the
   immobility time was analyzed.

2.4.3. Sucrose Preference Test (SPT)

   The SPT was performed according to a previous description [[58]9].
   Briefly, the SPT was divided into two phases (habituation and test
   phases). During the habituation phase, the mice were exposed to two
   identical bottles containing 1% sucrose solution for 24 h. The next 24
   h was the test phase, during which mice were presented with two
   identical bottles, one containing 1% sucrose solution and the other
   containing only water. Sucrose preference was designated as the volume
   of sucrose intake/(sucrose intake + water intake) × 100% during the
   test phase.

2.5. Western Blotting

   To avoid the possible influence of the behavioral tests on expression
   of target proteins, another cohort of animals was set up and received
   same treatment as described in [59]Section 2.3 without the behavior
   task. These animals were killed 1 h after the final DHM treatment on
   day 24. The entire hippocampus tissues were carefully dissected and
   lysed in the RIPA buffer with protease inhibitors and phosphatase
   inhibitors. The concentration of lysed samples was measured using a
   commercial kit following the manufacturer’s protocol. An amount of 50
   µg protein of individual samples was separated using sodium
   dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and
   transferred onto PVDF (polyvinylidene difluoride) membranes. After
   blocking with 3% BSA at room temperature for 1 h, the membranes were
   exposed to anti-AGE, anti-RAGE (Abcam, Cambridge, UK, 1:1000) or
   anti-β-actin (Proteintech, Rosemont, IL, USA, 1:5000) antibodies at
   room temperature for 2 h, followed by exposure to secondary antibodies
   for 2 h. The signals were detected using the ChemiDoc XRS imaging
   system (Bio-Rad, Hercules, CA, USA) and the intensity of targeted bands
   was quantified and normalized to β-actin.

2.6. Enzyme-Linked Immunosorbent Assay (ELISA)

   IL-1β (Cat. KE10003), IL-6 (Cat. KE10007) and TNFα (Cat. KE10002) ELISA
   kits were purchased from Proteintech ([60]https://www.ptglab.com/,
   accessed on 5 July 2022). The procedure was performed according to the
   manufacturer’s guidance. Briefly, 100mg mouse hippocampus tissue from
   individual animals was homogenized in 1 mL extraction reagent
   containing 1 mM phenylmethylsulfonyl fluoride and centrifuged at
   10,000× g for 10 min at 4 °C. The supernatants were collected, and the
   concentration was determined using a commercial kit. An amount of 100
   µL of standards or samples was added to the wells of capture
   antibody-coated microplate and incubated for 1 h at 37 °C. The liquid
   was removed, and the plate was washed four times with 1× washing
   buffer. An amount of 100 µL 1× detection antibody was added to each
   well and incubated for 1hr at 37 °C. The excess antibodies were washed
   away, and the plate was washed four times with 1× washing buffer. An
   amount of 100 µL of 1× horseradish peroxidase (HRP)-conjugated antibody
   was added to each well and was incubated for 40 min at 37°C. Excess
   antibody was washed away with 1× washing buffer. An amount of 100 µL of
   3,3’,5,5’-Tetramethylbenzidine (substrate for HRP) was added to each
   well and incubated in darkness for 15 min at 37 °C. Additionally, 100
   µL of stop solution was added to each well and the signal was
   determined using a microplate reader.

2.7. Network Pharmacology Analyses

2.7.1. Prediction of DHM and Depression Targets

   We used the PubChem database ([61]https://pubchem.ncbi.nlm.nih.gov/,
   accessed on 15 August 2022) to search the structures of canonical
   SMILES strings of DHM, which were imported into the Swiss Target
   Prediction platform ([62]http://www.swisstargetpredict.ch/index.php,
   accessed on 15 August 2022) and TargetNet database
   ([63]http://targetnet.scbdd.com/calcnet/index/, accessed on 15 August
   2022) to foreshadow their molecular targets.

   The keyword “depression” was used for entering into the Genecards
   database ([64]https://www.genecards.org/, accessed on 18 August 2022)
   (relevance score >1 was selected), TTD database
   ([65]https://db.idrblab.org/ttd/, accessed on 18 August 2022) and OMIM
   database ([66]https://omim.org/, accessed on 18 August 2022) and
   depression-related genes (species “Homo sapiens”) were screened.

2.7.2. Construction of Venn Diagram and DHM Targets Depression Network

   The common targets between DHM and depression were determined and
   visualized using the online tool Venny 2.1
   ([67]https://bioinfogp.cnb.csic.es/tools/venny/, accessed on 18 August
   2022). Subsequently, the common targets were imported into Cytoscape
   software (version 3.7.2, [68]https://cytoscape.org/, accessed on 18
   August 2022) to construct a DHM targets depression network.

2.7.3. Construction of Protein–Protein Interaction (PPI) Network

   The 75 intersection targets between DHM and depression were input to
   the STRING database ([69]https://string-db.org/ver.11.0, accessed on 18
   August 2022) to identify the PPI relationships. In the current study,
   the data analysis mode was set as “multiple proteins”, the species were
   selected as “Homo Sapiens”, the combined scores ≥ 0.4 and the
   disconnected nodes were hidden. The network graphics were downloaded,
   and the analyzed results were saved as TSV format files and imported
   into Cytoscape 3.7.2 software for the network construction.

2.7.4. Gene Ontology (GO) Function and Kyoto Encyclopedia of Genes and
Genomes (KEGG) Pathway Enrichment

   The intersecting targets were also imported into the R Studio software
   ([70]https://www.rstudio.com/, accessed on 22 August 2022) and the
   “clusterProfiler” R package
   ([71]https://bioconductor.org/packages/release/bioc/html/clusterProfile
   r.html, accessed on 22 August 2022) to annotate the gene ontology (GO)
   enrichment and KEGG pathways. The related biological processes and
   signaling pathways were identified via GO enrichment and KEGG pathway
   enrichment analyses. The p-value < 0.05 was set as the screening
   threshold of enrichment molecular analysis. The top ten GO terms in
   biological process, function, and cellular component and top twenty
   KEGG pathways were imported into the R Studio software
   ([72]https://www.rstudio.com/, accessed on 22 August 2022) and
   visualized.

2.8. Statistical Analysis

   All the data in the present experiment are expressed as mean ±SEM and
   analyzed by Sigma Plot 12.5 software using a one-way ANOVA (analysis of
   variance) test, followed by a post hoc Fisher LSD test. Data were
   regarded as significant if the p-value < 0.05.

3. Results

3.1. Dihydromyricetin Alleviated CORT-Induced Depressive-like Behaviors in
Mice

   We first investigated the effect of DHM on depressive-like behavior in
   animals receiving chronic injections. As shown in [73]Figure 2, animals
   in the control group, the CORT group, or the CORT+DHM group had no
   significant differences in distance traveled in the open field test
   (OFT) (p > 0.05, [74]Figure 2A). In the FST, the CORT group showed
   significantly increased immobility time compared with the control
   group, while DHM-treated animals had significantly less immobility time
   (p < 0.001, [75]Figure 2B) compared to the CORT group; in the SPT, the
   animals in the CORT group had a significant decrease in sucrose
   preference, compared to the control group (p < 0.001, [76]Figure 2C).
   Animals in the CORT + DHM group showed significantly increased sucrose
   preference (p < 0.001, [77]Figure 2C) compared to the CORT group.

Figure 2.

   [78]Figure 2
   [79]Open in a new tab

   Effect of DHM administration on depression-like behaviors induced by
   CORT in mice. (A) Total distance travelled by the three groups in the
   open field test (OFT) (n = 10). (B) Immobility time of the three groups
   in the forced swimming test (FST) (n = 10). (C) Sucrose preference of
   the three groups in the sucrose preference test (SPT) (n = 5). Data are
   expressed as mean ± SEM; *** p < 0.001, vs. the control group; ^# p <
   0.05, ^### p < 0.001, vs. the CORT group. CORT, corticosterone; DHM,
   dihydromyricetin.

3.2. Target Prediction of DHM in Depression

   To elucidate the underlying mechanisms of DHM against depression, a
   network pharmacology approach was applied. A total of 72 genes from the
   Swiss Target Prediction platform and 114 genes from the TargetNet
   database were identified as DHM targets. After removing duplicate
   genes, the final number of targeted genes was 148 ([80]Table S1). A
   total of 3231 genes from the GenecCards database, 560 genes from the
   OMIM database and 53 genes from the TTD database were identified. All
   collected genes were combined and deduplicated, and 3659 target genes
   were predicted ([81]Table S2). Subsequently, 75 potential DHM targets
   for depression were predicted in the Venny database
   ([82]http://bioinfogp.cnb.csis.es/tools/venny/index.html, accessed on
   18 August 2022) ([83]Figure 3, [84]Table S3).

Figure 3.

   [85]Figure 3
   [86]Open in a new tab

   The intersection of Dihydromyricetin targets and depression targets.

3.3. PPI Network of DHM Targets in Depression

   We constructed a PPI network by importing 75 potential targets into the
   STRING database; the network was visualized using Cytoscape software
   Version 3.9.1. [87]Figure 4 shows the 73 nodes and their interactions.
   Two targets, CMP-N-acetylneuraminate-beta-1,4-galactoside
   alpha-2,3-sialyltransferase (ST3GAL3) and galanin receptor type 3
   (GALR3) are not presented in [88]Figure 4, due to the fact that they
   were not connected to this network. In the network, the node represents
   the relevant gene, with the size and color of the node representing the
   value of the free degree. Proteins with a “degree” value higher than 31
   were selected as the significant targets, which included proto-oncogene
   tyrosine-protein kinase (SRC), heat shock protein 90 alpha family class
   A member 1 (HSP90AA1), estrogen receptor 1 (ESR1), hypoxia inducible
   factor 1 subunit alpha (HIF1A), vascular endothelial growth factor A
   (VEGFA), silent information regulator 1 (Sirt1) and prostaglandin
   endoperoxide synthase 2 (PTGS2), all of which may play an important
   role in DHM’s action against depression.

Figure 4.

   [89]Figure 4
   [90]Open in a new tab

   Seventy-three intersection targets predicted by Protein–protein
   interaction (PPI) network analysis. The size and color of the nodes
   indicate the size of the node degree value. The larger the node and the
   deeper the color red, the higher the degree value of the node.

3.4. GO and KEGG Pathway Enrichment Analyses for DHM-Targeted Gene Function
and Signaling Pathways

   Related biological processes and signaling pathways of DHM targets were
   identified using GO enrichment and KEGG pathway enrichment analyses. A
   total of 1503 GO terms, including 1316 of biological process (BP), 65
   of cellular component (CC), and 122 of molecular function (MF), were
   acquired. The top 10 terms of BP, CC, and MF are shown in [91]Figure 5.
   The data demonstrate that these potential targets are predominantly
   associated with the activities of neurotransmitter receptors,
   transmitter-gated ion channel and nuclear (steroid) receptors, which
   mediate depression [[92]9,[93]10,[94]11,[95]12,[96]13,[97]14]; these
   target genes are also possibly involved in the binding of transcription
   factors and transcription coactivators, which may mediate the
   development of depression [[98]15]. These potential targets function
   mainly in the (post)synaptic membrane, GABA-ergic membrane and
   (organelle) outer membrane, where receptor-mediated signal
   transductions may regulate the development of depression
   [[99]16,[100]17]. The targets possibly play a critical role in response
   to steroid hormone levels, regulation of inflammation and response to
   oxygen levels, which are known to be involved in the development of
   depression [[101]18,[102]19,[103]20]. Additionally, the KEGG pathway
   enrichment analysis was performed to uncover DHM-mediated functional
   mechanisms. Among the mapped top 20 signaling pathways ([104]Figure 6),
   the retrograde endocannabinoid signaling, Rap1 signaling, AGE-RAGE
   signaling, Estrogen signaling, oxygen reactive species signaling and
   VEGF signaling pathways are particularly associated with the
   development and progression of depression
   [[105]21,[106]22,[107]23,[108]24,[109]25,[110]26].

Figure 5.

   [111]Figure 5
   [112]Open in a new tab

   The top 10 enriched terms as shown in the GO enrichment. The x-axis
   represents the number of enriched genes in each term; the y-axis
   represents enriched terms in each category. BP: biological process; CC:
   cellular component; and MF: Molecular function.

Figure 6.

   [113]Figure 6
   [114]Open in a new tab

   The top 20 dihydromyricetin (DHM)-mediated signaling pathways predicted
   by the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment
   analysis.

3.5. Inhibition of AGE-RAGE Signaling Mediated Inflammation in CORT-Exposed
Mouse Hippocampus

   We next examined one of the mapped top signaling pathways, AGE-RAGE, in
   the hippocampus of mice chronically treated with CORT. We found that
   the levels of AGE and RAGE proteins were significantly increased in the
   CORT-treated animals, compared to the control animals; DHM-treated
   animals had significantly lower levels of AGE and RAGE proteins
   compared to animals treated with CORT alone ([115]Figure 7A,B and
   [116]Figure S1). Activation of the AGE-RAGE signaling pathway can lead
   to upregulation of proinflammatory cytokines [[117]27]. Consequently,
   we examined the production of IL-1β, IL-6 and TNFα in the hippocampus
   of these animals by ELISA. The levels of these proinflammatory
   cytokines were markedly increased in CORT-treated mice compared to the
   control animals; mice co-treated with DHM had a marked decrease in the
   expression of these cytokines ([118]Figure 7C) compared to those
   treated with CORT alone.

Figure 7.

   [119]Figure 7
   [120]Open in a new tab

   Inhibition of AGE-RAGE-mediated inflammation by DHM in the hippocampus
   of CORT-treated mice. (A) Representative immunoblots of hippocampal
   AGE, RAGE. (B) Quantification of AGE, RAGE by normalizing to β-actin.
   Data are expressed as mean ± SEM (n = 4 mice/group). *** p < 0.001, vs.
   the control group; ^# p < 0.05, ^## p < 0.01, vs. the CORT group. (C)
   Levels of proinflammatory cytokines, IL-1β, IL-6 and TNFα detected by
   ELISA. Data are expressed as mean ± SEM (n = 5 mice/group). **** p <
   0.0001. CORT, corticosterone; DHM, dihydromyricetin.

4. Discussion

   In the current study, we demonstrated that DHM relieved chronic
   CORT-induced depression-like behaviors in mice, supporting the notion
   that DHM has therapeutic potential for patients with depressive
   disorders. Additionally, network pharmacology analyses predicted that
   DHM’s efficacy against depression is possibly mediated by different
   targets and signaling pathways, of which the AGE-RAGE was further
   examined. We confirmed that the AGE-RAGE signaling pathway was
   activated and expression of proinflammatory cytokines was upregulated
   in the hippocampus of mice exposed to CORT, and that co-treatment with
   DHM counteracted these changes.

   Earlier studies have demonstrated that DHM alleviates depression-like
   behaviors in the lipopolysaccharides (LPS)-induced acute depression
   mouse model and in the chronic unpredictable mild stress-induced
   depression model [[121]28,[122]29]. DHM has also demonstrated
   antidepression effects in a diabetic neuropathic pain and depression
   comorbidity model [[123]7,[124]30]. Consistently, our data showed that
   chronic CORT injection resulted in increased immobility time in the
   FST, while DHM administration exerted an anti-immobility effect.
   Considering the effect of the level of locomotor activity on immobility
   time in the FST, the distance traveled by the mice was measured to
   evaluate locomotor activity in the OFT. Our data show that all the
   groups traveled an equivalent distance, indicating that neither CORT
   nor DHM administration influenced the level of locomotor activity.
   These data further strengthen the anti-despair role of DHM.
   Additionally, our data also show that chronic CORT injection resulted
   in decreased sucrose preference behavior in SPT, while DHM treatment
   boosted sucrose preference in chronic CORT-exposed mice, suggesting an
   anti-anhedonia role of DHM.

   Network pharmacology is a system biology approach that is used to
   identify potential drug candidates and drug targets and to predict
   their functional mechanisms [[125]31]. Recently, it has been widely
   applied in the discovery of antidepressants from natural products
   including medicinal plants and traditional Chinese medicine
   [[126]32,[127]33]. Network pharmacology analyses showed that DHM had
   multiple targets associated with its anti-depression function. Among
   these core targets, SIRT1, HIF1A, ESR1, VEGFA, HSP90AA1 and PTGS2 are
   well documented to be associated with depression
   [[128]26,[129]28,[130]34,[131]35,[132]36,[133]37]. Furthermore, the
   KEGG enrichment approach mapped DHM-mediated signaling pathways which
   are possibly involved in the pathogenesis of depression. One of these
   pathways, AGE-RAGE, has also been suggested by the network pharmacology
   approach to be involved in the efficacy of herb medicines against
   chronic pain-related depression [[134]32,[135]33]. Previous studies
   have also demonstrated that the alleviation of depressive-like
   behaviors in rodent models by natural products (p-coumaric acid and
   melatonin) is mediated by the AGE-RAGE signaling pathway
   [[136]9,[137]38].

   Inflammation plays a critical role in the development and progression
   of depression [[138]19]. DHM has demonstrated an anti-inflammation
   capacity in various types of diseases by inactivating the NLRP-3 and
   NF-κB pathways and decreasing the production of proinflammatory
   cytokines such as IL-1β, IL-6 and TNFα [[139]39]. Wei et al. reported
   that DHM reversed depressive-like behavior in LPS-treated rodents by
   blocking the TLR4/Akt/HIF1a/NLRP3 pathway, inactivating the NF-κB
   pathway and decreasing the expression of proinflammatory factors
   [[140]28]. In the chronic unpredicted mild stress-induced depression
   mouse model, DHM’s action against depression is associated with the
   upregulation of brain-derived neurotrophic factor (BDNF) and inhibition
   of inflammation in the hippocampus [[141]29]. Similarly, DHM treatment
   also upregulates BDNF expression and decreases the expression of
   proinflammatory cytokines in the hippocampus of rats with depression
   and diabetic neuropathic pain [[142]30]. The AGE-RAGE signaling pathway
   also regulates inflammation via downstream activation of the NF-κB
   pathway and the promotion of proinflammatory cytokine generation
   [[143]27]. In the current study, we found that DHM treatment decreased
   the levels of AGE, RAGE and proinflammatory factors in CORT-treated
   mouse hippocampus, suggesting that the AGE-RAGE signaling pathway was
   inactivated. Previous studies have reported that BDNF inhibits
   hippocampus inflammation in type 1 diabetic mice via the inactivation
   of the RAGE–NF-κB pathway [[144]40]. Since DHM has been shown to
   enhance BDNF expression in two other depression rodent models
   [[145]29,[146]30], it is reasonable to conclude that DHM also
   stimulates BDNF expression, controls the AGE-RAGE–NF-κB axis, and
   inhibits inflammation in the hippocampus of CORT-exposed mice, which is
   worthy of further investigation.

   Depression is also a common clinical feature presented in
   neurodegenerative disorders, including Alzheimer’s disease (AD) and
   Parkinson’s disease (PD) [[147]41,[148]42]. The depressive phenotypes
   in AD or PD patients are similar to that of depressive patients without
   AD or PD. Both AD and major depression have overlapping disease
   mechanisms [[149]43,[150]44]. DHM may therefore offer benefits to
   patients with neurodegenerative diseases.

   In conclusion, our present study showed that DHM eased depression-like
   behaviors in chronic CORT-treated animals. The protection of DHM
   against depression may be associated with multiple targets and
   signaling pathways, among which the AGE-RAGE pathway possibly plays a
   central role. The data suggest that DHM has therapeutic potential for
   treating patients with depression.

Supplementary Materials

   The following supporting information can be downloaded at:
   [151]https://www.mdpi.com/article/10.3390/cells11233730/s1; Figure S1:
   the image of the original Western blot for [152]Figure 7A; Table S1:
   Targets of dihydromyricetin; Table S2: Targets of depression; Table S3:
   Intersecting targets.
   [153]Click here for additional data file.^ (509.2KB, zip)

Author Contributions

   J.H., B.C., H.W., S.H., X.Y. and Y.Y. carried out the experiments and
   network pharmacology analyses. X.Y. and X.S. analyzed the data. X.Y.,
   J.R., Z.H. and X.S. wrote the manuscript. All authors have read and
   agreed to the published version of the manuscript.

Institutional Review Board Statement

   The study was performed following the Guidance for the Care and Use of
   Laboratory Animals, the University of South China (protocol code
   SYXK2020-0002, approved on 10 January 2020).

Informed Consent Statement

   Not applicable.

Data Availability Statement

   Not applicable.

Conflicts of Interest

   No conflict of interest needs to be declared.

Funding Statement

   This work was partially supported by the Locus Scholarship Program of
   Hunan Province, China (2019-23 to X.S.), the TENOVUS Scotland (S20-02
   to X.S.), the Chief Scientist Office/the RS Macdonald Charitable Trust
   (SNRF2021 to X.S.), Hunan Provincial Natural Science Foundation of
   China (2022JJ50178 to J.H.), National Natural Science Foundation of
   China (82260226 to B.C., 82060213 to Y.Y.), National College Students
   Innovation and Entrepreneurship Training Program (202210547020 to
   H.W.), Hainan research and development project (ZDYF2022SHFZ108 to
   B.C., ZDYF2022SHFZ066 to Y.Y.), Educational and scientific research
   project of Hainan Medical University (HYYB202042 to B.C.).

Footnotes

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References