Akebia saponin D acts via the PPAR‐gamma pathway to reprogramme a pro‐neurogenic microglia that can restore hippocampal neurogenesis in mice exposed to chronic mild stress

Abstract Background Using drugs to modulate microglial function may be an effective way to treat disorders, such as depression, that involve impaired neurogenesis. Akebia saponin D (ASD) can cross the blood–brain barrier and exert anti‐inflammatory and neuroprotective effects, so we wondered whether it might influence adult hippocampal neurogenesis to treat depression. Methods We exposed C57BL/6 mice to chronic mild stress (CMS) as a model of depression and then gave them ASD intraperitoneally once daily for 3 weeks. We investigated the effects of ASD on microglial phenotype, hippocampal neurogenesis, and animal behavior. The potential role of the peroxisome proliferator‐activated receptor‐gamma (PPAR‐γ) or BDNF–TrkB pathway in the pro‐neurogenesis and anti‐depressant of ASD was identified using there inhibitors GW9662 and K252a, respectively. The neurogenic effects of ASD‐treated microglia were evaluated using conditioned culture methods. Results We found that CMS upregulated pro‐inflammatory factors and inhibited hippocampal neurogenesis in dentate gyrus of mice, while inducing depressive‐like behaviors. Dramatically, ASD (40 mg/kg) treatment reprogrammed an arginase (Arg)‐1+ microglial phenotype in dentate gyrus, which increased brain‐derived neurotrophic factor (BDNF) expression and restored the hippocampal neurogenesis, and partially ameliorated the depressive‐like behaviors of the CMS‐exposed mice. K252a or neurogenesis inhibitor blocked the pro‐neurogenic, anti‐depressant effects of ASD. Furthermore, ASD activated PPAR‐γ in dentate gyrus of CMS mice as well as in primary microglial cultures treated with lipopolysaccharide. Blocking the PPAR‐γ using GW9962 suppressed the ASD‐reprogrammed Arg‐1+ microglia and BDNF expression in dentate gyrus of CMS mice. Such blockade abolished the promoted effects of ASD‐treated microglia on NSPC proliferation, survival, and neurogenesis. The pro‐neurogenic and anti‐depressant effects of ASD were blocked by GW9962. Conclusion These results suggested that ASD acts via the PPAR‐γ pathway to induce a pro‐neurogenic microglia in dentate gyrus of CMS mice that can increase BDNF expression and promote NSPC proliferation, survival, and neurogenesis.


| INTRODUC TI ON
Major depressive disorder affects millions of people worldwide and has multifactorial causes, leading to heterogeneous clinical presentations. 1,2 Current anti-depressant medications often lack efficacy, 3,4 highlighting the need for further studies of how depression and related diseases can be treated.
The occurrence and development of depression have been linked to decreased neurogenesis in the adult hippocampus. [5][6][7] Restoring hippocampal neurogenesis can enhance stress resistance and mitigate depressive symptoms, 8,9 so this may be an effective strategy for treating depression. 10,11 During neurogenesis in adult hippocampus, neural stem/progenitor cells (NSPCs) proliferate and differentiate into neurons. 12 In neurogenic niches, NSPCs produce neurons that support learning, memory, and behavior, and this production continues through adulthood. 13 Efficient neurogenesis depends on a supportive "neurogenic niche" in the hippocampus. 9 An important modulator of neurogenesis in this niche is microglia. [14][15][16] Pro-inflammatory microglia suppress adult hippocampal neurogenesis, while anti-inflammatory microglia do the opposite. 9,17,18 Chronic stress hyperactivates microglial cells, which secrete inflammatory mediators that impair neuroplasticity and neurogenesis. [19][20][21] Nevertheless, microglia are endowed with phenotypic plasticity to regulate physiological responses and behavioral outcomes during stress. [22][23][24][25] In particular, the Arg-1 + microglia were considered a neuroprotective microglial phenotype. 26,27 Our previous research found that this subgroup of microglia can secrete brain-derived neurotrophic factor (BDNF) to promote hippocampal neurogenesis in responding to chronic stress, helping protect against depressive-like symptoms. 9 Thus, pharmacological modulation of the microglial phenotype may allow control of NSPC proliferation and differentiation to treat disorders associated with neurogenic dysfunction.
Several natural products show promise for modulating microglial phenotype. 21 For example, the akebia saponin D (ASD), a triterpenoid saponin, is abundant in the traditional Chinese medicine Radix Dipsaci, which exerts anti-osteoporotic, anti-inflammatory, and neuroprotective effects. [28][29][30] Studies have shown that ASD can efficiently cross the blood-brain barrier 28 and exerts neuroprotective effects. 31 We have shown that ASD regulates microglial function to ameliorate neuroinflammation and depression-like behaviors of mice exposed to lipopolysaccharide (LPS). 32,33 However, we are unaware of studies exploring whether ASD influences hippocampal neurogenesis, which may thereby help explain its antidepressant effects.
Thus, here we investigated the effects of ASD on microglial phenotypes, hippocampal neurogenesis, and depressive-like behaviors in mice exposed to chronic mild stress (CMS). To gain greater mechanistic insights, we also examined the effects of conditioned medium from ASD-induced microglia on NSPC proliferation, survival, and neuronal differentiation.

| Animals
Male C57BL/6J mice (7-8 weeks old) were purchased from Changsha Tianqin Biotechnology, caged individually, and assigned unique numbers. The mice were habituated to their new environment for 1 week. The mice were then habituated to a 1% sucrose solution for 48 h. Sucrose preference and body weight were determined weekly as described in Section 2.4.1. Body weight and sucrose preference during the first 3 weeks served as the pretreatment baseline, and animals were allocated into seven groups as described in Section 2.3.1.

| Chronic mild stress (CMS)
Animals were exposed to CMS for 6 weeks as described. 9 Everyday animals were exposed to two to three of the following stressors in random order: empty water bottles (12 h), food deprivation (12 h

| Treatment with ASD and imipramine
Akebia saponin D (99.92% pure) was purchased from Chengdu Alfa Biotechnology and dissolved to a concentration of 4 mg/mL in 0.9% saline. After 4 weeks of CMS, the mice were allocated into seven Conclusion: These results suggested that ASD acts via the PPARγ pathway to induce a pro-neurogenic microglia in dentate gyrus of CMS mice that can increase BDNF expression and promote NSPC proliferation, survival, and neurogenesis.

| Treatment with K252a or GW9662
To investigate the role of peroxisome proliferator-activated receptorgamma (PPARγ) pathway in ASD regulation of microglia phenotype, we used the PPARγ inhibitor GW9662 (Sigma-Aldrich). To investigate the potential role of the BDNF-tropomyosin receptor kinase B (TrkB) pathway in pro-neurogenic effects of ASD, the K252a (Sigma-Aldrich) was used to block the TrkB. GW9662 or K252a was dissolved in 0.9% saline containing 5% dimethyl sulfoxide (DMSO) at a concentration of 1 mg/mL and 2.5 μg/mL, respectively. After  The sucrose preference test was performed as described. 36 Mice were individually housed, deprived of food and water for 12 h, and then given access to 1% sucrose solution (A) and water (B) for 2 h.
The bottle positions were switched daily to avoid a side bias. The sucrose preference was calculated each week for each mouse using the formula: 100 × [VolA/(VolA + VolB)]. The sucrose consumption was normalized to body weight for each mouse.

| Forced swimming test (FST)
At 24 h before the test, each mouse was placed individually for 10 min in a glass cylinder (height, 25 cm; diameter, 15 cm) that was filled with water to a depth of 15 cm at 26°C. The next day, the mice were placed again in the same situation for 6 min. An observer masked to treatment conditions recorded the latency between suspension and first abandonment of struggle as well as the time spent immobile during the last 4-min period.

| Open field test (OFT)
Mice were placed into an open field (50 × 50 cm 2 ) and allowed to explore freely for 15 min. Total distance and time spent in the center (25 × 25 cm 2 ) were quantified using video tracking software (OFT100, Taimeng Tech).

| Culture of NSPCs with conditioned medium from pre-treated microglia
NSPCs were obtained from the hippocampus of 8-week-old male C57BL/6J mice as described. 38 Microglia were plated at a density of
Following the immunocytochemistry, RT-PCR analysis, Western blot analysis, and conditional culture of NSPC were performed.
In some experiments involving blockade of BDNF-TrkB signaling pathway in NSPCS, BDNF receptor antagonist K252a (100 ng/mL) 9 were added to the conditioned medium from the microglia treated by ASD and LPS. Then, the conditioned medium was used for proliferation culture or differentiation culture of NSPC.

| BrdU incorporation
To determine NSPC proliferation and differentiation in the brain, mice received intraperitoneal injections of 5′-bromo-2′deoxyuridine (BrdU; Sigma-Aldrich; 50 mg/kg/day) for 7 days. 9 Mice were euthanized at 7 days after the last injection. To examination neuronal survival in the granular layer, animals were injected with a double dose of BrdU and euthanized at 7 weeks after injection.
To determine the NSPCs and newborn neurons survival during culture in conditioned medium from microglia activated with LPS in the presence or absence of ASD, the NSPCs were incubated with BrdU (100 ng/mL) for 24 h in proliferation medium. 9 After that, these NSPCs were allowed to grow for 7 days in differentiation medium.
Half of the volume of culture medium for induced differentiation was replaced with the microglia-conditioned medium (M-CM). After 3 days, the survival of the newborn neurons was measured using immunofluorescence as described in Section 2.13.
Brains were removed, fixed in 4% paraformaldehyde for 48 h, and washed with PBS, and we used the 30% sucrose to provide cryoprotection against the damaging water crystals formed around 0°C. 21 Coronal sections containing the hippocampus were obtained using a cryostat slicer (CM1900; Leica Microsystems). Six sequential slices were collected into each well of a 12-well plate containing PBS with 0.02% sodium azide and stored at 4°C. The 20μm-thick slices were used for immunofluorescence, and the 100μm-thick slices were used for protein and RNA extraction.

| RNA extraction and real-time PCR (RT-PCR)
The dentate gyrus was isolated form slices containing the hippocampus. Total RNA was isolated from dentate gyrus or cultured cells using Trizol (Invitrogen Life Technologies) according to the manufacturer's instructions. RT-PCR was performed using the First Strand cDNA Synthesis Kit (TaKaRa) according to the manufacturer's instructions. RT-PCR amplification was performed using a Bio-Rad CFX 96 system (Bio-Rad Laboratories) and the primers in Table S1.
Each sample was tested in triplicate. The threshold cycle (Ct) number was determined from the linear phase of the amplification plot using the −ΔΔC t method, and values were normalized against the housekeeping gene β-actin.

| Enzyme-linked immunosorbent assay (ELISA)
The dentate gyrus was dissociated from slices containing the hippocampus, flash-frozen in liquid nitrogen, and homogenized.
Primary microglia were cultured in six-well plates at 5 × 10 5 cells/cm 2 and then treated for 24 or 48 h with LPS or PBS in the presence or absence of ASD. The culture medium was collected, microglia were lysed in cell lysis buffer (Solarbio), and the lysates were centrifuged at 1000 g for 30 min. The concentration of total protein in the supernatant was determined using the BCA kit (BOSTER), and each sample was diluted to 1 g/mL. Then, samples were assayed using commercial ELISAs against the following signaling factors: interleukin (IL)-1β, tumor necrosis factor (TNF)α, IL-10, IL-4, insulin-like growth factor (IGF)-1, and brain-derived neurotrophic factor (BDNF; BOSTER); inducible nitric oxide synthase (iNOS) and arginase (Arg-1; Elabscience); transforming growth factor (TGF)β (4A Biotech); and basic fibroblast growth factor (bFGF), epidermal growth factor (EGF; ColorfulGene Biotech), and nerve growth factor (NGF; BOSTER). The manufacturer-specified detection limit of all kits was 1-4 pg/mL.

| Western blotting (WB)
Mice were anesthetized with 10% pentobarbital and transcardially perfused with 0.9% NaCl. Hippocampi were removed and homogenized. Cultures of proliferative NSPCs were sonicated in RIPA buffer containing protease and phosphorylase inhibitors (Solarbio), lysates were centrifuged at 1000 g for 30 min, and equal amounts of soluble protein were fractionated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred to PVDF membranes. Membranes  Table S2.
Membranes were washed three times with TBST, incubated for 30 min with secondary antibody (1:10,000; Abcam), washed three times with TBST, and then developed for 1-2 min using enhanced chemiluminescence (Millipore, MMAS). Blots were visualized using the ChemiDoc Touch system (Bio-Rad), and band intensity was quantified using Alpha software (version 1.45 J; National Institutes of Health).

| Immunohistochemistry and immunocytochemistry
The following types of cells were plated separately at a density of

| Molecular docking
The potential binding of ASD to peroxisome proliferator-activated receptor (PPAR)γ was explored in molecular docking studies using the Surflex-Dock module in Sybyl-X2.1 software (Tripos Associates), based on previous work. 39

| Imaging and statistical analyses
Images were analyzed as described. 9 Statistical analyses were performed using GraphPad Prism (version 8.0, SPSS Inc.). Experimental data were expressed as mean ± SEM. Kolmogorov-Smirnov test was used to assess data distribution. For normally distributed data, one-way ANOVA was used to analyze differences between multiple groups. Data that did not exhibit a normal distribution were analyzed using Kruskal-Wallis tests between multiple groups. Differences were considered statistically significant if p < 0.05.

| Akebia saponin D ameliorates depressive-like behaviors of CMS mice
We first evaluated the anti-depressant efficacy of ASD in mice ( Figure 1A,B). The anhedonia of mice was evaluated by sucrose preference test ( Figure 1C). We found that CMS mice displayed lower sucrose preference than control animals. Both ASD (40 or 80 mg/kg) and IMI (10 mg/kg) administration markedly increased sucrose preference in the CMS-exposed mice ( Figure 1C). The behavioral despair of mice was evaluated by forced swimming test ( Figure 1D). The CMS mice displayed a shorter latency and longer immobility time in the forced swimming test. Both ASD (40 or 80 mg/kg) and IMI (10 mg/kg) treatments increased the latency and decreased the total duration of immobility in forced swimming test in the CMS-exposed mice ( Figure 1D).
CMS decreased the time spent in center in open field test, which was reversed by 3-week treatment with ASD (40 mg/kg; Figure 1E). In contrast, neither ASD nor IMI affected the distance traveled or immobility time in the open field test ( Figure 1E). These results suggest that ASD ameliorates depressive-like behaviors in CMS mice.

| Akebia saponin D rescues CMS-induced deficits in hippocampal neurogenesis
NSPCs in the dentate gyrus (DG) of the hippocampus proliferate and differentiate into neurons even in adulthood, and this neurogenesis is negatively associated with depression and positively associated with the efficacy of anti-depressants. 9,21 Consistent with this, CMS strikingly reduced the number of newborn neurons (DCX + ) in the hippocampus of our mice (Figure 2A,B). In fact, CMS reduced the numbers of BrdU + cells and DCX + -BrdU + cells and the rate of neuronal F I G U R E 2 Akebia saponin D promotes hippocampal neurogenesis of CMS-exposed mice. (A) Immunofluorescence micrographs of hippocampal newborn neurons (BrdU + -DCX + cells), marked with arrowheads. Proliferating neural stem/precursor cells were labeled with 5′-bromo-2′deoxyuridine (BrdU, green), and immature neurons were labeled with doublecortin (DCX, red

| The anti-depressant effects of akebia saponin D depend in part on promoting hippocampal neurogenesis
To investigate the role of neurogenesis in antidepressant effects of ASD, we used the temozolomide (TMZ) to ablate neurogenesis in ASD-treated CMS mice ( Figure 3A). TMZ treatment significantly reduced the number of DCX + , BrdU + , and DCX + -BrdU + cells, as well as the rate of neuronal differentiation in SGZ of hippocampus of CMS + ASD mice ( Figure 3B-F). Ablation of neurogenesis using TMZ abolished the anti-depressant effects of ASD in the sucrose preference test ( Figure 3G) and forced swimming test ( Figure 3H), but did not affect the traveled distances in open filed test ( Figure 3I). These results suggest that the anti-depressant effects of ASD depend in part on promoting hippocampal neurogenesis.

| Akebia saponin D reprogrammes a proneurogenic microglia in dentate gyrus of CMS mice
Microglia control the neurogenic microenvironment, and the Arg-1 + microglia contribute to hippocampal neurogenesis. 9 Therefore, we examined the effects of ASD on Arg-1 + microglia in dentate gyrus of CMS-exposed mice. The results showed that ASD significantly increased the percentage of Arg-1 + microglia in dentate gyrus of CMS-exposed mice ( Figure 4A,B). The immobility time in forced swimming test was negatively correlated with Arg-1 + microglia in dentate gyrus of mice ( Figure 4C). ASD also reversed the CMSinduced increases in the pro-inflammatory factors TNFα, iNOS,

and IL-1β and decreases in the anti-inflammatory factors IL-4 and
Arg-1 in dentate gyrus of mice ( Figure 4D). Analogously, CMS substantially reduced the levels of IGF-1, TGFβ, and BDNF, while ASD significantly increased BDNF in dentate gyrus of mice ( Figure 4D).
The BDNF levels were positively correlated with Arg-1 levels in dentate gyrus of mice ( Figure 4E). The results from immunofluorescent staining showed that ASD upregulated the BDNF in Arg-1 + microglia in dentate gyrus ( Figure 4F). Considering that ASD increases the microglial secretion of BDNF, which in turn promotes neurogenesis from NSPCs, we examined the levels of phosphorylation of the BDNF-specific receptor TrkB in hippocampus of mice. The results showed that CMS reduced the levels of p-TrkB in the SGZ of hippocampus, which ASD reversed ( Figure 4G).
To confirm that ASD directly regulates microglial function, we examined the effects of ASD on primary cultures of microglia that were treated with LPS as a model of neuroinflammation ( Figure S1).
LPS shifts microglia toward a pro-inflammatory phenotype that inhibits NSPC proliferation, survival, and differentiation. 21 Pretreatment with ASD at 50 or 100 μM, but not 10 μM, prevented LPS from upregulating iNOS and TNFα and increased the expression of IL-10, Arg-1, and BDNF at 24 and 48 h (Figures S1 and S2).
To confirm that the ASD-induced changes in microglia in turn influence NSPCs, we treated primary cultures of microglia in different ways, then transferred the culture medium to NSPC cultures, and observed their proliferation, survival, and neuronal differentiation ( Figure 4H and Data are mean ± standard error of the mean (SEM). *p < 0.05, **p < 0.01, ***p < 0.001 versus the control (Ctrl) group; # p < 0.05, ## p < 0.01, ### p < 0.001 versus the CMS group; & p < 0.05, && p < 0.01, &&& p < 0.001 versus the CMS + ASD group based on one-way ANOVA with Tukey's multiple-comparisons test.  Figure 4K). Overall, the effects of ASD were similar to those of pioglitazone, an agonist of PPARγ pathway, which reprogrammes a pro-neurogenic microglial phenotype.

| The PPARγ plays a critical role in reprogramming of pro-neurogenic microglia by akebia saponin D in dentate gyrus of CMS mice
Since mammalian target of PPARγ signaling plays a key role in induction of anti-inflammatory microglial phenotypes, 40 we asked whether ASD acts via such signaling to exert its "microglial reprogramming" effect. Therefore, we explored the potential binding between ASD and PPARγ ( Figure 5A). Docking studies predicted the ligand ASD bound to the PPARγ with a stability of −7.34 ± 0.34 kJ/ mol ( Figure 5A). Indeed, after treatment with ASD, p-PPARγ was significantly increased in hippocampal dentate gyrus of CMSexposed mice ( Figure 5B). The results from immunofluorescent staining showed that PPARγ localized in cytoplasm and nucleus of Arg-1 + microglia in the dentate gyrus of mice that were exposed to CMS and then treated with ASD ( Figure 5C,D).
To confirm the role of PPARγ in induction of the pro-neurogenic microglia in dentate gyrus of ASD-treated mice, we repeated the above experiments in the presence of the PPARγ antagonist GW9662 ( Figure 5E), which effectively blocked the PPARγ pathway in dentate gyrus ( Figure 5G). Such blockade abolished the ability of ASD to increase the numbers of Arg-1 + microglia in the dentate gyrus of CMS mice ( Figure 5F). Blockade of PPARγ signaling in ASD-treated primary microglia also abolished the ability of ASD-M-CM to stimulate NSPC proliferation and neuronal differentiation ( Figure 5H-J). These results suggest that PPARγ plays a critical role in reprogramming of pro-neurogenic microglia by akebia saponin D.
Activation of PPARγ with ASD reversed the CMS-induced decrease in BDNF and p-TrkB levels in dentate gyrus of mice, which also abolished by GW9662 treatment ( Figure 5G). Interestingly, the TrkB inhibitor K252a treatment effectively blocked the p-TrkB, but did not affect the p-PPARγ and BDNF levels in dentate gyrus of CMS + ASD mice ( Figure 5G

| DISCUSS ION
Our previous research revealed that modulation of microglial phenotype and function may be an effective neurotherapy for depression. 9,41 Consistent with reports that natural products can be effective modulators of microglial phenotype and promoters of neurogenesis, 21 here we demonstrate in vivo and in vitro that ASD, the major active ingredient in the traditional Chinese medicine Dipsacus asper Wall., can induce a pro-neurogenic microglial phenotype in a PPARγ-dependent manner, which activates the BDNF-TrkB pathway in NSPCs to promote their proliferation and neuronal differentiation. The resulting neurogenesis can ameliorate depressive-like behaviors in CMS mice. F I G U R E 4 Akebia saponin D reprogrammes a pro-neurogenic microglia phenotype in the dentate gyrus of CMS mice. (A) Immunofluorescence micrographs of Arg-1 + microglia in the dentate gyrus of CMS mice after treatment with ASD. Scale bar, 100 μm. (B) Immunofluorescence micrographs and quantification of Arg-1 + microglia in the dentate gyrus of control (Ctrl) or CMS mice after treatment with saline or ASD. Scale bar, 5 μm. (C) Correlation of immobility time in the forced swimming test with the percentage of Arg-1 + microglia in dentate gyrus of Ctrl, ASD, CMS, and CMS + ASD mice. Each circle represents one mouse (n = 5). (D) Levels of pro-and anti-inflammatory cytokines and neurotrophic factors in the dentate gyrus of Ctrl and CMS mice following ASD treatment. (E) Correlation of BDNF with Arg-1 levels in dentate gyrus of Ctrl, ASD, CMS, and CMS + ASD mice. Each circle represents one mouse (n = 4). (F) Immunofluorescence micrographs of BDNF in Arg-1 + microglia of the dentate gyrus of CMS + ASD mice. Scale bar, 50  We previously reported that ASD at 40 mg/kg significantly ameliorated depressive-like behaviors in LPS-treated mice. 32 Here we found similar effects of ASD in mice exposed to CMS, a classical model of depression. 42  Consistent with that study, we found here that the PPARγ agonist pioglitazone, like ASD, induced an anti-inflammatory microglial phenotype. Our findings here may help explain how ASD can attenuate microglia-mediated inflammation in animal models of depression. 32,33 The strong ability of NSPCs to proliferate and differentiate makes them promising targets for repairing nerve injury. 54 However, adverse changes in the microenvironment of the CNS, including signals from microglia, 55 can induce NSPCs to differentiate into astrocytes at the expense of neurons, which increases the risk of glial scar formation. 21 A pro-inflammatory phenotype of microglia, which can be induced in animal models using CMS or LPS, 56 is thought to suppress adult NSPC proliferation. 21 Here, we also found that factors secreted by microglia exposed to LPS inhibited differentiation of adult NSPCs into neurons. Pretreating microglia with ASD, in contrast, enabled microglia to promote NSPC proliferation, survival, and neuronal neurogenesis. Thus, ASD appears to induce secretion of neurogenic factors from microglia, which then influence NSPCs.
This mechanism may explain how ASD can exert anti-depressant effects and mitigate cognitive impairment in animal models of depression. 30,32 One of the neurogenic factors secreted by ASD-treated microglia appears to be BDNF, which plays a neuroprotective and neurotrophic role. [57][58][59] We found that ASD treatment upregulated BDNF in hippocampal microglia of CMS mice. Pretreating microglia with ASD before LPS increased the secretion of BDNF into the medium, such that this conditioned medium promoted NSPC proliferation and neuronal differentiation. ASD also activated the BDNF receptor, TrkB, in the SGZ of CMS mice. Consistent with a role of BDNF as mediator of ASD-induced neurogenesis, we found that NSPC proliferation and differentiation correlated with an increase in levels of F I G U R E 5 Blocking the PPARγ signaling pathway abolished the pro-neurogenic microglia induced by akebia saponin D in dentate gyrus of CMS mice. (A) Molecular docking of akebia saponin D (green) to peroxisome proliferator-activated receptor (PPAR)γ. Key residues predicted to be involved in the complexes are shown in green for akebia saponin D and in blue for the binding partners. (B) Western blotting of PPARγ and phosphorylated PPARγ (p-PPARγ) in the dentate gyrus (DG) of hippocampus of control (Ctrl) or CMS mice after treatment with saline or ASD. Levels of p-PPARγ were normalized to those of PPARγ (n = 3, each sample in triplicate). (C) Fluorescence micrographs showing PPARγ expression in Iba1 + microglia of dentate gyrus in CMS mice after treatment with ASD. PPARγ was stained with antibody (green), microglia were stained with an anti-Iba1 antibody (pink), and nuclei were stained with DAPI (blue). The nuclear translocation of PPARγ was indicated by arrowhead. Scale bar, 10 μm. (D) Fluorescence micrographs showing PPARγ expression in Arg-1 + microglia of dentate gyrus in CMS mice after treatment with ASD. PPARγ was stained with antibody (green), Arg-1 + microglia were stained with an anti-Arg-1 antibody (red), and nuclei were stained with DAPI (blue). The nuclear translocation of PPARγ was indicated by white arrowhead. Scale bar, 10 μm. (E) Scheme of the experimental procedure detailing the blocking of PPARγ or BDNF-TrkB signaling pathway in ASD + CMS mice. ASD, akebia saponin D; CMS, chronic mild stress; GW9662, PPARγ inhibitor; FST, forced swimming test; K252a, TrkB inhibitor; OFT, open field test; SPT, sucrose preference test. (F) Effects of GW9662 or K252a treatment on the levels of Arg-1 + microglia in dentate gyrus of ASD + CMS mice. (G) Effects of GW9662 or K252a treatment on levels of p-PPARγ, PPARγ, BDNF, TrkB, and p-TrkB in dentate gyrus of ASD + CMS mice (n = 3, each sample in triplicate). BDNF levels were normalized to those of β-actin, while levels of p-PPARγ and p-TrkB were normalized to those of PPARγ and TrkB, respectively. We found that the ability of ASD to reprogramme proneurogenic microglial phenotype is mediated by PPARγ, a liganddependent transcription factor belonging to the nuclear hormone receptor superfamily. 60 PPARγ regulates the expression of antiinflammatory cytokines, 61 and the PPARγ agonists pioglitazone or rosiglitazone can switch activated microglia cells from a proinflammatory to anti-inflammatory state. 62 Our previous research showed that ASD acts via PPARγ to switch activated microglia from a pro-inflammatory to anti-inflammatory phenotype in vitro. 33 In present study, we further demonstrated that ASD acts via PPARγ to induce a pro-neurogenic microglial phenotype in dentate gyrus of CMS-exposed mice and mitigate depressive-like mouse behaviors. Conversely, blocking the PPARγ signaling pathway abolished the Arg-1 + microglia and BDNF expression induced by ASD in dentate gyrus of CMS-exposed mice, as well as the proneurogenic and antidepressant effects of ASD. Drugs may have potential effects on NGF receptor TrkA, 63 and subsequent exploration of BND-TrkB targeting inhibitors is needed.
Taken together, these experiments strongly suggest that ASD acts via the PPARγ pathway to reprogramme a pro-neurogenic microglia in dentate gyrus of CMS mice that can increase BDNF expression and promote NSPC proliferation, survival, and neuronal differentiation (Figure 7).

F I G U R E 6
The pro-neurogenic and anti-depressant effects of akebia saponin D depend on the PPARγ pathway. (A-E) Effects of K252a or GW9662 on the pro-neurogenic effects of akebia saponin D in CMS-exposed mice. Proliferating neural stem/precursor cells were labeled with 5′-bromo-2′deoxyuridine (BrdU, green), and immature neurons were labeled with doublecortin (DCX, red). Scale bar, 100 μm. The BrdU + -DCX + cells were indicated by white arrowheads. The hippocampal immature neurons (DCX + cells), proliferating neural stem/ precursor cells (BrdU + cells), newborn neurons (BrdU + -DCX + cells), and percentage of BrdU + -DCX + cells out of BrdU + cells were quantized. Results for each group were obtained from four mice, for each of which five hippocampal slices were examined at 40× magnification. Each dot in the bar graph represents the average of all micrographs for one mouse. (F and G) Effects of K252a or GW9662 on the maturation of hippocampal newborn neurons in ASD + CMS mice. Proliferating neural stem/precursor cells were labeled with 5′-bromo-2′deoxyuridine (BrdU, green), and mature neurons were labeled with neuron-specific nucleoprotein (NeuN, pink). The BrdU + -NeuN + cells were marked with arrowheads. Scale bar, 100 μm. (H-J) Effects of K252a or GW9662 on the anti-depressant effects of akebia saponin D (n = 8-10). Anhedonia, behavioral despair, and spontaneous activity were assessed separately in sucrose preference test, forced swimming test, and open field test. Data are mean ± standard error of the mean (SEM). *p < 0.05, **p < 0.01, ***p < 0.001 versus the control (Ctrl) group; # p < 0.05, ## p < 0.01, ### p < 0.001 versus the CMS group; & p < 0.05, && p < 0.01, &&& p < 0.001 versus the CMS + ASD group based on one-way ANOVA with Tukey's multiple-comparisons test.
F I G U R E 7 Schematic diagram of how akebia saponin D may restore hippocampal neurogenesis. Akebia saponin D acts via the PPARγ pathway to reprogramme a pro-neurogenic microglial phenotype in dentate gyrus that can increase BDNF expression which to promote NSPC proliferation, survival, and neural differentiation, thus restores hippocampal neurogenesis and ameliorates depression-like behavior in CMS-exposed mice.
This neurogenesis then mitigates CMS-induced deficits in hippocampal neurogenesis and depressive-like behaviors. Our results justify further studies of ASD as a potential treatment for depression and may inspire new lines of research targeting the PPARγ pathway in disorders involving impaired neurogenesis.

AUTH O R CO NTR I B UTI O N S
JZ, TZ, and ZY conceived and designed the study. JZ and QL wrote the manuscript, which was revised by TZ and ZY, approved by all the authors. QL, CX, and HH performed behavioral tests and immunostaining. LL cultured NSPCs and performed immunofluorescence and cytokine assays. DS performed statistical analyses of the data.

ACK N OWLED G M ENTS
We are grateful to Creaducate Consulting GmbH for help in revising the manuscript.

CO N FLI C T O F I NTER E S T S TATEM ENT
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.