Abstract

   Olfactory bulb (OB) plays an important role in protecting against
   harmful substances via the secretion of antioxidant and detoxifying
   enzymes. Environmental enrichment (EE) is a common rehabilitation
   method and known to have beneficial effects in the central nervous
   system. However, the effects of EE in the OB still remain unclear. At 6
   weeks of age, CD-1® (ICR) mice were assigned to standard cages or EE
   cages. After 2 months, we performed proteomic analysis. Forty-four
   up-regulated proteins were identified in EE mice compared to the
   control mice. Gene Ontology analysis and Kyoto Encyclopedia of Genes
   and Genomes Pathway demonstrated that the upregulated proteins were
   mainly involved in metabolic pathways against xenobiotics. Among those
   upregulated proteins, 9 proteins, which participate in phase I or II of
   the xenobiotic metabolizing process and are known to be responsible for
   ROS detoxification, were validated by qRT-PCR. To explore the effect of
   ROS detoxification mediated by EE, glutathione activity was measured by
   an ELISA assay. The ratio of reduced glutathione to oxidized
   glutathione was significantly increased in EE mice. Based on a linear
   regression analysis, GSTM2 and UGT2A1 were found to be the most
   influential genes in ROS detoxification. For further analysis of
   neuroprotection, the level of iNOS and the ratio of Bax to Bcl-2 were
   significantly decreased in EE mice. While TUNEL^+ cells were
   significantly decreased, Ki67^+ cells were significantly increased in
   EE mice, implicating that EE creates an optimal state for xenobiotic
   metabolism and antioxidant activity. Taken together, our results
   suggested that EE protects olfactory layers via the upregulation of
   glutathione-related antioxidant and xenobiotic metabolizing enzymes,
   eventually lowering ROS-mediated inflammation and apoptosis and
   increasing neurogenesis. This study may provide an opportunity for a
   better understanding of the beneficial effects of EE in the OB.

   Keywords: environmental enrichment, olfactory bulb, metabolizing
   enzymes, antioxidant, detoxification, glutathione

Introduction

   The olfactory bulb (OB) is a region of brain that plays a critical role
   in health and pathological behaviors such as the detection of hazards
   in the environment, food selection, olfactory memory formation, mating,
   and maintenance of mood ([37]1–[38]4). Tight communication between the
   OB and other olfactory systems can transfer xenobiotics to the brain by
   the active axonal transport of olfactory neurons, bypassing the blood
   brain barrier ([39]5). To maintain cellular integrity and homeostasis
   of the brain from environmental chemicals, it is necessary to have
   active xenobiotic metabolism during the olfactory transport ([40]6).

   Xenobiotic metabolism is a metabolism pathway that modifies the
   chemical structure of foreign, either exogenous or endogenous,
   substances so that maintain homeostasis and cellular integrity.
   Xenobiotic metabolism process includes three steps; Phase I modifies
   xenobiotics via hydrolysis, oxidation, and reduction and thereby
   produces the active metabolites. Phase II further conjugates the phase
   I product with transferase enzymes to yield hydrophilic products.
   Lastly, Phase III excretes the final modified product out of the cells
   via various transporters ([41]6). Namely, metabolizing enzymes such as
   the cytochrome P450 (CYPs) family, the UDP-glucuronosyltransferases
   (UGT) family, glutathione-transferases (GST) family and the aldehyde
   dehydrogenase (ADH) family are involved in the detoxification process
   ([42]6, [43]7). Especially, the phase II enzymes such as UGT and GST
   family plays an important role in determining the rate of xenobiotic
   metabolism and ROS detoxification ([44]8).

   Previous studies have shown olfactory bulbectomy in rodents leads to
   cortical and hippocampal cell death ([45]9, [46]10), suggesting that
   the OB is related to other brain regions. Moreover, the OB is a special
   nervous structure that changes its neural cells over lifetime. Several
   studies have shown that newly generated cells in the subventricular
   zone (SVZ) of adult mouse migrate into the OB through the rostral
   migratory stream (RMS), forming functional granule cells in the OB and
   constituting the network of the OB to other brain regions ([47]11,
   [48]12). Since the OB constantly interacts with external environment
   and encounters harmful air-borne chemicals, the above ongoing process
   is important for proper OB function ([49]13).

   Environmental enrichment (EE) is a rehabilitation strategy for rodents
   and the enhancement of the brain by surroundings that include various
   objects such as running wheels, novel toys and allow large social
   interactions to experience the effects of a complex combination of
   physical, cognitive, and social stimulation in the brain
   ([50]14–[51]16). In many studies, EE has been implicated in improvement
   of the brain functions such as neuroprotection and neurogenesis in an
   animal model of aging ([52]17–[53]20), ischemic brain injury ([54]14),
   and neurodegenerative diseases ([55]14, [56]21). These diseases and
   brain injuries, accompanied with an increase in oxidative stress and
   olfactory impairment, eventually lead to a clinically significant
   problem ([57]22, [58]23). However, this event can be reversed by
   exercise via the increased expression of antioxidant enzymes ([59]17,
   [60]24).

   Previous studies have investigated the effect of EE, odor enrichment,
   and xenobiotic inhalant on the OB of normal mice ([61]25–[62]27).
   Allowing xenobiotic inhalants or various odorants in a timely fashion,
   it could induce changes in gene expressions and odor perception
   ([63]25, [64]26). Several studies have reported the beneficial effect
   of EE on the OB ([65]25, [66]27). In contrast to the previous studies,
   our study focuses on the effect of EE on the OB of normal mice without
   utilizing any inhalant or timely-exposure odorants.

   The aim of this study was to determine whether there are alterations in
   physiological responses after long-term exposure to EE in the intact OB
   of normal mice. Here, we showed the expression of metabolizing enzymes,
   which were predominated in the OB of EE mice. Moreover, we found that
   EE enhances neurogenesis and ROS detoxification, resulting in the
   reduction of inflammation and apoptotic process. These results imply
   that EE has beneficial effects on the OB through setting an optimal
   status for antioxidant and xenobiotic metabolism.

Materials and methods

Experimental procedures

Animals and housing conditions

   At 6 weeks of age, a total of CD-1® (ICR) mice were assigned randomly
   to either environmental enrichment (EE) or standard cage (SC) group.
   For 2 months, EE mice were housed in a huge cage (86 × 76 × 31 cm^3)
   containing novel objects, such as tunnels, shelters, toys, and running
   wheels for voluntary exercise, and allowing for large social
   interaction (12–15 mice/cage) (Figure [67]1A) while control mice were
   housed in standard cages (27 × 22.5 × 14 cm^3) without any novel
   objects (4–5 mice/cage) (Figures [68]1B,C) ([69]14). All animals were
   housed in a facility accredited by the Association for Assessment and
   Accreditation of Laboratory Animal Care (AAALAC) and were given ad
   libitum access to food and water under alternating 12-h light/dark
   cycles. Used mice of this study were all female mice and purchased from
   the Jackson Laboratory.

Figure 1.

   [70]Figure 1
   [71]Open in a new tab

   Experimental design for environmental enrichment. (A) Mice in an
   enriched environment accessed freely not only to water and food but
   also to tunnels, shelters, toys, running wheels, and the large space of
   social interaction. (B,C) Mice in a standard cage freely accessed to
   water and food. (D) A schematic time schedule for the experiment. Mice
   of 6 weeks of age were randomly assigned and reared in either a control
   cage or EE cage for 2 months. After the housing conditions, brain
   tissues were harvested for proteomic analysis to see differentially
   expressed proteins. Long-term exposure to EE during adulthood is known
   to induce the beneficial effects on brain health with cellular and
   molecular alterations.

Ethic statement

   All procedures were reviewed and approved by the Animal Care and Use
   Committee of the Yonsei University College of Medicine (Identification
   code: 2014-0125-3). All procedures were in accordance with the
   guidelines of the National Institutes of Health's Guide for the Care
   and Use of Laboratory Animals. These regulations, notifications, and
   guidelines originated and were modified from the Animal Protection Law
   (2008), the Laboratory Animal Act (2008), and the Eighth Edition of the
   Guide for the Care and Use of Laboratory Animals (NRC 2011). They were
   sacrificed at 8 weeks after the housing conditions under ketamine (100
   mg/kg) and xylazine (10 mg/kg) anesthesia by intraperitoneal injection.
   Thereafter, they were given an intracardial perfusion of 4%
   paraformaldehyde, and the brain tissues were harvested for histological
   assessments. All efforts were made to minimize animal suffering.

Experimental grouping

   In this study, a total of 28 CD-1® (ICR) female mice at 6 weeks of age
   were randomly housed either in environmental enrichment (N = 14) or
   standard cages (N = 14). Among the subjects, 3 mice per group were
   recruited for the proteomics analysis after housing for 2 months (N =
   6). To validate the results of proteomics and measure iNOS levels, 4
   mice per group were further recruited at 8 weeks post-conditions (N =
   8). Moreover, 3 mice per group were recruited for an ELISA assay to
   measure glutathione activity and apoptosis process (N = 6). Finally, 4
   mice per group were further recruited for histological assessments (N =
   8). A schematic timeline for this study is described in Figure [72]1D.

Proteomic analysis

Protein sample preparation

   Olfactory bulb of mouse brain tissues was homogenized in 200 μl of cold
   RIPA buffer (50 mM Tris-HCl, pH 7.5, 1% Triton X-100, 150 mM NaCl, 0.1%
   sodium dodecyl sulfate (SDS), 1% sodium deoxycholate) with a protease
   inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA) in the ice. The
   tissue lysates were centrifugated for 20 min at 13,000 g at 4°C. The
   supernatant containing the protein extracts were stored at −80°C until
   use. The protein concentration of the supernatant was determined using
   Bradford assay, according to the manufacturer's instructions.

In-solution digestion

   Protein samples were digested by in-solution method after quantified
   using a bicinchoninic acid (BCA) assay kit (Thermo Scientific, Foster
   City, CA, USA). Briefly, samples were resolved in a digestion solution
   of 6 M urea (Sigma-Aldrich) and 50 mM tris-HCl in water. Protein
   reduction was performed with 10 mM DTT (Sigma-Aldrich) for 1 h at 37°C.
   Additionally, 55 mM iodoacetamide (Sigma-Aldrich) were treated for 30
   min at room temperature in darkness for alkylation of cysteine
   residues. The resulting samples were digested by sequencing-grade
   modified trypsin (Promega, Madison, WI, USA) overnight at 37°C. The
   digested peptides were desalted using C18 Spin-Column (Thermo
   Scientific).

TMT labeling and OFF-GEL fractionation

   For TMT labeling, digested peptides were resuspended in 200 mM triethyl
   ammonium bicarbonate (TEAB, Sigma-Aldrich). TMT™ Isobaric Mass Tagging
   reagent (Thermo Scientific) were dissolved in anhydrous acetonitrile
   and added to each sample. After incubation for 1 h at room temperature,
   the reaction was quenched with 5% hydroxylamine. The labeled samples
   were pooled into one tubes and separated with an Agilent 3100 OFF-GEL
   fractionator system (Agilent Technologies, Santa Clara, CA, USA)
   according to manufacturer's instructions. Finally, resulting samples
   were desalted using C18 Spin-Column (Thermo Scietific).

LC-MS analysis and data acquisition

   Prepared samples were analyzed using Q Exactive mass spectrometer
   (Thermo Finnigan, San Jose, CA, USA) coupled with EASY-nLC 1000 (Thermo
   Finnigan). The tryptic peptides were loaded into a trap column (3 μm
   sized C18 resin into 75 μm × 2 cm) and separated by the analytical
   column (3 μm sized C18 resin into 75 μm × 50 cm) at a flow rate of 300
   nL/min. The separated peptides were eluted by solvent A (0.1% formic
   acid in water) and solvent B (0.1% formic acid in acetonitrile) with a
   linear gradient for 60 min. Each MS scan was followed by 8 MS/MS scans
   of the most abundant peak to the eighth-most abundant peak of the MS
   scan in data-dependent method with dynamic exclusion options (repeat
   duration was 30 s and repeat count was set to 1). The normalized
   collision energy for MS/MS was 27%. To identify peptides, TurboSEQUEST
   (Thermo Electron, San Jose, CA, USA) was used to search MS/MS spectra
   against the Uniprot database for forward sequences (17098 Mus musculus
   protein sequence entries as of July 15, 2014). The mass tolerance was
   set to 10 ppm for precursor ions, 0.8 Da for fragment ions.
   Carbamidomethylation of cysteine (+57.0215 Da) and oxidation of
   methionine (+15.9949 Da) were set as the static modifications and
   variable modifications, respectively. Scaffold Q+S (version 4.3.4,
   Proteome Software Inc., OR, USA) was used to compute probability of
   proteins and peptides ([73]28). Search results were validated using the
   protein and peptide cut-off probabilities with >99% and 95%
   probability, respectively. Spectral counts were calculated by Scaffold
   software after normalization by total spectral counts of each MS run
   for an estimate of expression fold change between the experimental
   groups ([74]29). An absorbance spectra peak for each interested protein
   is provided in Figure [75]S1.

Bioinformatics and pathway analysis

   To identify protein function of the differentially expressed genes,
   gene ontology (GO) was used for further analysis. GO annotation
   analysis with the Database for Annotation, Visualization and Integrated
   Discovery (DAVID) annotation tool describes protein function with three
   basic categories: associated biological process (BP), molecular
   function (MF) and cellular components (CC). This categorization gives
   an overview of the interested protein functions. Moreover, pathway
   enrichment analysis was performed using the Kyoto Encyclopedia of Genes
   and Genomes (KEGG) pathway. Pathways that are up-regulated in EE mice
   compared to control mice were identified. Within the identified
   pathways, genes that are involved in metabolism, drug metabolism, and
   xenobiotic metabolism were sorted out for further validation (Tables
   [76]1– 3).

Table 1.

   List of proteins found to be up-regulated proteins in OB of mice
   treated with EE.
   Accession number Accession name Protein name Gene symbol Fold change
   P-value
   [77]Q80ZU7 BPIB3_MOUSE BPI fold-containing family B member 3 Bpifb3 4.4
   0.0001
   [78]P08074 CBR2_MOUSE Carbonyl reductase [NADPH] 2 Cbr2 3.9 0.0001
   [79]Q5SQ27 Q5SQ27_MOUSE MCG140354 Sec14l3 3.1 0.0001
   [80]Q8VCT4 CES1D_MOUSE Carboxylesterase 1D Ces1d 3.1 0.0001
   [81]P11679 K2C8_MOUSE Keratin, type II cytoskeletal 8 Krt8 3.1 0.0001
   [82]P97872 FMO5_MOUSE Dimethylaniline monooxygenase [N-oxide-forming] 5
   Fmo5 2.9 0.0001
   [83]P33267 CP2F2_MOUSE Cytochrome P450 2F2 Cyp2f2 2.9 0.0001
   [84]Q80X89 UD2A1_MOUSE UDP-glucuronosyltransferase 2A1 Ugt2a1 2.8
   0.0001
   [85]Q91X75 Q91X75_MOUSE Cyp2a4 protein Cyp2a5 2.7 0.0001
   [86]P05784 K1C18_MOUSE Keratin, type I cytoskeletal 18 Krt18 2.7 0.0001
   [87]Q8K157 GALM_MOUSE Aldose 1-epimerase Galm 2.6 0.0001
   [88]Q8K1G6 Q8K1G6_MOUSE Protein Muc5b Muc5b 2.6 0.0001
   [89]P52430 PON1_MOUSE Serum paraoxonase/arylesterase 1 Pon1 2.5 0.0001
   [90]Q8C6B0 Q8C6B0_MOUSE MCG20149, isoform CRA_a Mettl7a1 2.5 0.0001
   [91]Q8BHB9 CLIC6_MOUSE Chloride intracellular channel protein 6 Clic6
   2.5 0.0008
   [92]P40936 INMT_MOUSE Indolethylamine N-methyltransferase Inmt 2.3
   0.0001
   [93]P15626 GSTM2_MOUSE Glutathione S-transferase Mu 2 Gstm2 2.3 0.0001
   [94]P00329 ADH1_MOUSE Alcohol dehydrogenase 1 Adh1 2.3 0.0001
   [95]P00186 CP1A2_MOUSE Cytochrome P450 1A2 Cyp1a2 2.3 0.0001
   [96]Q9WV19 Q9WV19_MOUSE Cytochrome P450, family 2, subfamily g,
   polypeptide 1 Cyp2g1 2.2 0.0001
   [97]A2BGH0 BPIB4_MOUSE BPI fold-containing family B member 4 Bpifb4 2.1
   0.0001
   [98]Q91WU0 CES1F_MOUSE Carboxylesterase 1F Ces1f 2.1 0.0001
   [99]O88312 AGR2_MOUSE Anterior gradient protein 2 homolog Agr2 2.1
   0.0001
   [100]E9QN99 E9QN99_MOUSE Alpha/beta hydrolase domain-containing protein
   14B Abhd14b 2.1 0.0001
   [101]P12265 BGLR_MOUSE Beta-glucuronidase Gusb 2.1 0.0001
   [102]Q9EQF5 DPYS_MOUSE Dihydropyrimidinase Dpys 2.1 0.0023
   [103]P30115 GSTA3_MOUSE Glutathione S-transferase A3 Gsta3 2.1 0.0001
   [104]Q9D379 HYEP_MOUSE Epoxide hydrolase 1 Ephx1 2.0 0.0001
   [105]Q9R092 H17B6_MOUSE 17-beta-hydroxysteroid dehydrogenase type 6
   Hsd17b6 2.0 0.0015
   [106]Q9D8W7 OCAD2_MOUSE OCIA domain-containing protein 2 Ociad2 1.9
   0.0001
   [107]J3QMN6 J3QMN6_MOUSE Dimethylaniline monooxygenase
   [N-oxide-forming] Fmo6 1.9 0.0001
   [108]P56395 CYB5_MOUSE Cytochrome b5 Cyb5a 1.9 0.0001
   [109]Q99KP3 CRYL1_MOUSE Lambda-crystallin homolog Cryl1 1.8 0.0001
   [110]Q5SGK3 AOXB_MOUSE Aldehyde oxidase 3 Aox3l1 1.8 0.0001
   [111]O70475 UGDH_MOUSE UDP-glucose 6-dehydrogenase Ugdh 1.7 0.0001
   [112]Q7TNG8 LDHD_MOUSE Probable D-lactate dehydrogenase, mitochondrial
   Ldhd 1.7 0.0001
   [113]B2RX12 MRP3_MOUSE Isoform 2 of Canalicular multispecific organic
   anion transporter 2 Abcc3 1.7 0.0011
   [114]O35945 AL1A7_MOUSE Aldehyde dehydrogenase, cytosolic 1 Aldh1a7 1.7
   0.0001
   [115]Q5SXG7 VMO1_MOUSE Vitelline membrane outer layer protein 1 homolog
   Vmo1 1.6 0.0022
   [116]Q91WR8 GPX6_MOUSE Glutathione peroxidase 6 Gpx6 1.6 0.0001
   [117]Q9JII6 AK1A1_MOUSE Alcohol dehydrogenase [NADP(+)] Akr1a1 1.6
   0.0001
   [118]P52196 THTR_MOUSE Thiosulfate sulfurtransferase Tst 1.6 0.0001
   [119]P59242 CING_MOUSE Cingulin Cgn 1.6 0.0014
   [120]P37040 NCPR_MOUSE NADPH–cytochrome P450 reductase Por 1.5 0.0001
   [121]Open in a new tab

Molecular analysis

RNA extraction

   Total RNA was extracted from tissue samples as previously described
   ([122]30). Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto,
   CA, USA) with the A260/A280 ratio for quality control analysis was used
   to evaluate purity of RNA extracted from olfactory bulb tissues, and
   visual valuation of the 28S:18S rRNA ratio using gel electrophoresis
   was used to evaluate the RNA integrity.

Quantitative real-time PCR (qRT-PCR)

   To further validate proteomic analysis, qRT-PCR for interested
   differentially expressed genes was performed in triplicate. One
   microgram of purified total RNA was used for reverse transcription into
   cDNA using a ReverTra Ace® qPCR TR master Mix with gDNA remover
   (TOYOBO). The expression of each gene of interest was profiled using a
   LightCycler 480 (Roche Applied Science, Mannheim, Germany) using the
   LightCycler 480 SYBR Green master mix (Roche Applied Science). The
   validated genes are as follows: subfamily A7 (ALDH1A7);
   Carboxylesterase 1D (CES1D); Cytochrome P450, family 2, subfamily a,
   polypeptide 5 (CYP2A5); Serum paraoxonase 1 (PON1); Cytochrome P450,
   family 1, subfamily a, polypeptide 2 (CYP1A2); Dihydropyrimidinase
   (DPYS); Alcohol dehydrogenase 1 (ADH1); Alcohol dehydrogenase [NADP(+)]
   (AKR1A1); Crystalline, lambda1 (CRYL1), forward 5′-3′ and reverse
   5′-3′; UDP-glucuronosyltransferase 2 family, polypeptide A1 (UGT2A1);
   Glutathione S-transferase, alpha3 (GSTA3); Glutathione S-transferase Mu
   2 (GSTM2); Epoxide hydrolase 1 (EPHX1); Thiosulfate sulfurtransferase
   (TST). To confirm detoxifying activity by EE, we also identified iNOS
   level. And glyceraldehyde-3-phosphate dehydrogenase (GADPH) was used as
   the loading control. The expression level of each gene of interest was
   obtained using the 2^−ΔΔCt method ([123]31). Target-gene expression was
   normalized relative to the expression of GAPDH and represented as fold
   change relative to the control. The primer sequences are described in
   Table [124]S1.

Glutathione activity assay (ELISA)

   The concentration of total glutathione, oxidized glutathione, ratio of
   reduced glutathione to oxidized glutathione were measured in OB
   extracts using the glutathione detection kit from Enzo Life Sciences
   (Enzo Life Sciences, East Farmingdale, NY, USA, ADI-900-160) following
   the manufacturer's instructions. The values were obtained at 1:40
   dilution and expressed as pmol/well and each well contained the total
   volume of 200 μl.

Western blot

   To measure the expression of apoptotic neuronal cells, the protein was
   extracted as described above. The equal amounts of protein samples (40
   μg) were denatured and separated by 4–12% Bis-Tris gels in 1× NuPage
   MES SDS Running Buffer (Invitrogen, Eugene, OR, USA). Proteins were
   transferred at 4°C onto a polyvinylidene difluoride membrane
   (Invitrogen) in NuPage Transfer Buffer (Invitrogen) with 20% (vol/vol)
   methanol. Membranes were blocked and then incubated overnight at 4°C
   with the following antibodies: Anti-Bax (1: 1,000 dilution, Santa Cruz
   Biotechnology), Anti-Bcl-2 (1:1,000 dilution, Santa Cruz
   Biotechnology), and Anti-Actin (1:1,000 dilution, Santa Cruz
   Biotechnology). The next day, blots were washed three times with TBS
   plus Tween 20 and incubated at room temperature for 1 hr with
   horseradish peroxidase-conjugated secondary antibodies (1:3,000
   dilution, Santa Cruz). An enhanced chemiluminescence detection system
   (Amersham Pharmacia Biotech, Little Chalfont, UK) was used to visualize
   the protein. The intensity of the protein bands was recorded with a
   Fujifilm LAS-3000 imager.

Histological analysis

Immunohistochemistry

   Immunohistochemistry was performed as previously described ([125]32).
   The animals were sacrificed and perfused with 4% paraformaldehyde. The
   harvested brain tissues were cryosectioned at 16-μm thickness along the
   sagittal plane, and immunohistochemistry staining was performed on four
   sections over a range of >128 μm. Sections were stained with primary
   antibodies against Ki67 (1:200; Abcam), and secondary antibodies such
   as Alexa Fluor 488 goat anti-Rabbit (1:400; Invitrogen) and mounted on
   glass slides with fluorescent mounting medium containing
   4′,6-diamidino-2-phenylindole (Vectorshield, Vector, Burlingame, CA).
   The stained sections were analyzed using confocal microscopy (LSM700;
   Zeiss, Gottingen, Germany). Ki67^+ cell area (μm^2) with respect to
   DAPI area (/μm^2) was evaluated using ZEN Imaging Software (Blue
   edition; Zeiss).

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay

   For analysis of apoptosis, the selected areas of the RMS were analyzed
   for each group. Fluorometric TUNEL assay (Promega, Madison, WI, USA)
   was conducted according to the manufacturer's protocol. Images of
   apoptotic cells were taken using a fluorescent microscopy (LSM700), and
   positive apoptotic cells (μm^2) with respect to DAPI area (/μm^2) were
   quantified using ZEN Imaging Software (Blue edition; Zeiss).

Statistical analysis

   Statistical analyses were performed using Statistical Package for
   Social Sciences (SPSS) software (IBM Corporation, Armonk, NY, USA;
   version 23.0). All data were expressed as median and interquartile
   range (25th−75th percentiles). Mann–Whitney U-tests were used to
   compare the difference between groups. Linear regression analysis was
   conducted to identify the relationship between xenobiotic metabolizing
   proteins and ROS detoxification (glutathione activity) and a univariate
   analysis was also used identify relationships between EE and xenobiotic
   metabolizing proteins and glutathione activity. A P < 0.05 was
   considered statistically significant.

Results

Environmental enrichment significantly increased the proteins associated with
metabolic pathways and xenobiotic metabolism

   After placing mice in either SC cage or EE cage for 2 months, the mice
   were sacrificed according to the time schedule (Figure [126]1D). The
   proteomic analysis revealed that 44 up-regulated proteins were
   identified in EE mice compared with control mice with a cut-off fold
   change >1.5 (p < 0.05) (Table [127]1). The up-regulated proteins in the
   OB of mice exposed to EE were categorized based on biological processes
   (BP), molecular functions (MF), and cellular components (CC) (Table
   [128]2). BP based GO analysis identified the up-regulated proteins were
   related to “oxidation-reduction process” (GO:0055114), “response to
   toxic substance” (GO:0009636), “epoxygenase P450 pathway” (GO:0019373),
   “metabolic process” (GO:0008152), “short-chain fatty acid catabolic
   process” (GO:0019626), “aromatic compound catabolic process”
   (GO:0019439), “ethanol catabolic process” (GO:0006068), “cellular
   aromatic compound metabolic process” (GO:0006725), “cellular response
   to cadmium ion” (GO:0071276), “retinoic acid metabolic process”
   (GO:0042573), and “drug metabolic process” (GO:0017144). Moreover, in
   Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, the most
   highlighted cluster in the up-regulated genes contains overall themes
   for metabolic pathways and xenobiotic metabolism (Table [129]3).

Table 2.

   Gene ontology with Database for Annotation, Visualization, and
   Integrated Discovery (DAVID) generated by EE up-regulated genes.
   Category ID Term Count % P-value Genes
   GOTERM_BP_DIRECT GO:0055114 Oxidation-reduction process 15 34.88
   <0.0001 CYP2F2, LDHD, UGDH, CYB5A, CYP1A, POR, FMO5, CRYL1, FMO6, CBR2,
   AKR1A1, GPX6, ADH1, HSD17B6, ALDH1A7
   GOTERM_BP_DIRECT GO:0009636 Response to toxic substance 5 11.63 <0.0001
   CYP2F2, PON1, EPHX1, CES1D, INMT
   GOTERM_BP_DIRECT GO:0019373 Epoxygenase P450 pathway 3 6.98 0.0019
   CYP2G1, CYP2F2, CYP2A5
   GOTERM_BP_DIRECT GO:0008152 Metabolic process 6 13.95 0.0030 GSTM2,
   GSTA3, GUSB, UGDH, UGT2A1, ALDH1A7
   GOTERM_BP_DIRECT GO:0019626 Short-chain fatty acid catabolic process 2
   4.65 0.0043 CES1F, CES1D
   GOTERM_BP_DIRECT GO:0019439 Aromatic compound catabolic process 2 4.65
   0.0129 PON1, EPHX1
   GOTERM_BP_DIRECT GO:0006068 Ethanol catabolic process 2 4.65 0.0150
   ADH1, ALDH1A7
   GOTERM_BP_DIRECT GO:0006725 Cellular aromatic compound metabolic
   process 2 4.65 0.0150 EPHX1, CYP1A2
   GOTERM_BP_DIRECT GO:0071276 Cellular response to cadmium ion 2 4.65
   0.0256 CYP2A5, CYP1A2
   GOTERM_BP_DIRECT GO:0042573 Retinoic acid metabolic process 2 4.65
   0.0319 ADH1, ALDH1A7
   GOTERM_BP_DIRECT GO:0017144 Drug metabolic process 2 4.65 0.0381 FMO5,
   CYP1A2
   GOTERM_MF_DIRECT GO:0016491 Oxidoreductase activity 13 30.23 <0.0001
   FMO5, CRYL1, CBR2, GPX6, CYP2F2, AKR1A1, ADH1, LDHD, UGDH, HSD17B6,
   ALDH1A7, CYP1A2, POR
   GOTERM_MF_DIRECT GO:0016712 Oxidoreductase activity, acting on paired
   donors, with incorporation or reduction of molecular oxygen, reduced
   flavin or flavoprotein as one donor, and incorporation of one atom of
   oxygen 4 9.30 0.0001 CYP2G1, CYP2F2, CYP2A5, CYP1A2
   GOTERM_MF_DIRECT GO:0050660 Flavin adenine dinucleotide binding 4 9.30
   0.0006 FMO5, FMO6, LDHD, POR
   GOTERM_MF_DIRECT GO:0020037 Heme binding 5 11.63 0.0006 CYP2G1, CYP2F2,
   CYP2A5, CYB5A, CYP1A2
   GOTERM_MF_DIRECT GO:0019899 Enzyme binding 6 13.95 0.0016 GSTM2,
   CYP2A5, EPHX1, CYB5A, CYP1A2, POR
   GOTERM_MF_DIRECT GO:0004497 Monooxygenase activity 4 9.30 0.0018 FMO5,
   FMO6, CYP2F2, CYP1A2
   GOTERM_MF_DIRECT GO:0050661 NADP binding 3 6.98 0.0034 FMO5, FMO6, POR
   GOTERM_MF_DIRECT GO:0008392 Arachidonic acid epoxygenase activity 3
   6.98 0.0049 CYP2G1, CYP2F2, CYP2A5
   GOTERM_MF_DIRECT GO:0008395 Steroid hydroxylase activity 3 6.98 0.0065
   CYP2G1, CYP2F2, CYP2A5
   GOTERM_MF_DIRECT GO:0042803 Protein homodimerization activity 7 16.28
   0.0081 GSTM2, CRYL1, GALM, ADH1, CLIC6, PON1, AGR2
   GOTERM_MF_DIRECT GO:0005506 Iron ion binding 4 9.30 0.0117 CYP2G1,
   CYP2F2, CYP2A5, CYP1A2
   GOTERM_MF_DIRECT GO:0004499 N,N-dimethylaniline monooxygenase activity
   2 4.65 0.0199 FMO5, FMO6
   GOTERM_MF_DIRECT GO:0016705 Oxidoreductase activity, acting on paired
   donors, with incorporation or reduction of molecular oxygen 3 6.98
   0.0206 CYP2F2, CYP2A5, CYP1A2
   GOTERM_MF_DIRECT GO:0004745 Retinol dehydrogenase activity 2 4.65
   0.0459 ADH1, HSD17B6
   GOTERM_CC_DIRECT GO:0031090 Organelle membrane 7 16.28 <0.0001 FMO5,
   FMO6, CYP2F2, CYP2A5, EPHX1, CYB5A, CYP1A2
   GOTERM_CC_DIRECT GO:0070062 Extracellular exosome 18 41.86 <0.0001
   GSTA3, GUSB, UGDH, DPYS, ABHD14B, CYB5A, SEC14L3, TST, CRYL1, GALM,
   KRT18, AKR1A1, KRT8, CLIC6, PON1, METTL7A1, VMO1, MUC5B
   GOTERM_CC_DIRECT GO:0043231 Intracellular membrane-bounded organelle 10
   23.26 <0.0001 FMO5, CYP2F2, GUSB, PON1, HSD17B6, EPHX1, CYB5A, CYP1A2,
   POR, MUC5B
   GOTERM_CC_DIRECT GO:0005783 Endoplasmic reticulum 11 25.58 0.0004 FMO5,
   CYP2F2, GUSB, CES1F, HSD17B6, EPHX1, CYB5A, CES1D, CYP1A2, AGR2, POR
   GOTERM_CC_DIRECT GO:0005789 Endoplasmic reticulum membrane 8 18.60
   0.0007 FMO5, FMO6, CYP2F2, CYP2A5, EPHX1, CYB5A, CYP1A2, POR
   GOTERM_CC_DIRECT GO:0005829 Cytosol 10 23.26 0.0116 GSTM2, CRYL1,
   AKR1A1, ADH1, UGDH, CES1F, DPYS, CES1D, ABHD14B, INMT
   GOTERM_CC_DIRECT GO:0005615 Extracellular space 8 18.60 0.0385 TST,
   AKR1A1, GUSB, CES1F, PON1, CES1D, AGR2, MUC5B
   [130]Open in a new tab

Table 3.

   Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways identified to
   be significantly up-regulated from EE using Database for Annotation,
   Visualization, and Integrated Discovery (DAVID) software.
   Category Term Count % P-value Genes
   KEGG_PATHWAY Metabolic pathways 16 37.20930233 <0.0001 GUSB, UGDH,
   DPYS, CYP1A2, TST, CRYL1, GALM, CBR2, AKR1A1, ADH1, CYP2A5, PON1,
   HSD17B6, UGT2A1, CES1D, ALDH1A7
   KEGG_PATHWAY Metabolism of xenobiotics by cytochrome P450 8 18.60465116
   <0.0001 GSTM2, GSTA3, CBR2, CYP2F2, ADH1, EPHX1, UGT2A1, CYP1A2
   KEGG_PATHWAY Drug metabolism—cytochrome P450 7 16.27906977 <0.0001
   GSTM2, FMO5, GSTA3, FMO6, ADH1, UGT2A1, CYP1A2
   KEGG_PATHWAY Pentose and glucuronate interconversions 5 11.62790698
   <0.0001 CRYL1, AKR1A1, GUSB, UGDH, UGT2A1
   KEGG_PATHWAY Retinol metabolism 6 13.95348837 <0.0001 ADH1, CYP2A5,
   HSD17B6, UGT2A1, ALDH1A7, CYP1A2
   KEGG_PATHWAY Drug metabolism—other enzymes 4 9.302325581 0.0007 GUSB,
   DPYS, UGT2A1, CES1D
   KEGG_PATHWAY Glutathione metabolism 3 6.976744186 0.0156 GSTM2, GSTA3,
   GPX6
   KEGG_PATHWAY Glycolysis / Gluconeogenesis 3 6.976744186 0.0220 GALM,
   AKR1A1, ADH1
   KEGG_PATHWAY Steroid hormone biosynthesis 3 6.976744186 0.0367 HSD17B6,
   UGT2A1, CYP1A2
   KEGG_PATHWAY Ascorbate and aldarate metabolism 2 4.651162791 0.0904
   UGDH, UGT2A1
   [131]Open in a new tab

Differentially expressed proteins (DEPs) were validated by qRT-PCR

   After 8 weeks of the exposure to EE or SC, mice were sacrificed, and
   the olfactory bulbs were extracted and lysed for proteomic and qPCR
   analysis. The expression level of 44 identified proteins were
   significantly different in terms of the expression level between
   control group and EE group. The up-regulated proteins via pathway
   analysis were validated using qRT-PCR analysis. Xenobiotic metabolizing
   phase I enzymes, such as ALDH1A7, DPYS, PON1, CRYL1, AKR1A1, CYP1A2,
   and xenobiotic metabolizing phase II enzymes, such as UGT2A1, GSTM2,
   GSTA3 were significantly increased in EE mice compared with control
   mice (Figure [132]2; p < 0.05).

Figure 2.

   [133]Figure 2
   [134]Open in a new tab

   Validation of the up-regulated proteins associated with xenobiotic
   metabolism. Among the 44 up-regulated proteins, which were associated
   with xenobiotic metabolism and detoxifying enzymes, a total of 9 genes,
   phase I (ALDH1A7, DPYS, PON1, CRYL1, AKR1A1, CYP1A2) (A) and phase II
   (UGT2A1, GSTIM2, GSTA3) (B), were significantly increased in EE mice
   compared to control mice (^*p < 0.05, by Mann-Whitney U test). Values
   are median (interquartile range) and a red tick line indicates the
   relative expression of control mice.

EE enhanced glutathione activity and induced ROS detoxification

   Glutathione activity is important for the removal of ROS, which
   generated from endogenous environment and during xenobiotic metabolism,
   and glutathione redox status can be a marker for oxidative stress. The
   number of oxidized glutathione was significantly lower in EE mice
   compared to control mice (Figure [135]3A; p < 0.05), and the ratio of
   reduced glutathione (GSH) to oxidized glutathione (GSSG) was
   significantly higher in EE mice compared to control mice (Figure
   [136]3B; p < 0.05), and no significant difference in the number of
   total glutathione was observed in between EE mice and control mice
   (Data not shown).

Figure 3.

   [137]Figure 3
   [138]Open in a new tab

   Glutathione activity. The number of oxidized glutathione in EE mice was
   significantly decreased compared to control mice (A), and GSH/GSSG
   ratio was significantly increased in EE mice compared with control mice
   (B). Values are median (interquartile range). An asterisk indicates
   significant difference (^*p < 0.05, by Mann–Whitney U-test).

The significant association between xenobiotic metabolizing enzymes and
glutathione activity via EE was founded

   Linear regression analysis was conducted to identify which xenobiotic
   metabolizing enzymes mostly contribute to glutathione activity (Table
   [139]4). In the analysis of oxidized glutathione, GSTM2 (p = 0.008),
   GSTA3 (p = 0.022), CYP2A5 (p = 0.004), ALDH1A7 (p = 0.009), and UGT2A1
   (p = 0.002) were found to be significantly correlated. In the analysis
   of GSH/GSSG, GSTM2 (p = 0.003), GSTA3 (p = 0.008), CYP2A5 (p = 0.029),
   ALDH1A7 (p = 0.004), UGT2A1 (p = 0.003), CRYL1 (p = 0.049), AKR1A1 (p =
   0.024), and DPYS (p = 0.016) were found to be significantly correlated.
   The final stepwise multiple regression model revealed a significant
   association between oxidized glutathione and UGT2A1 (p = 0.002), and
   significant associations between GSH/GSSG and GSTM2 (p = 0.024) and
   GSH/GSSG and UGT2A1 (p = 0.029). EE was significantly associated with
   GSTM2 (Coefficient (SE) = 0.625 (0.072); reference = SC; p < 0.001 and
   UGT2A1 (Coefficient (SE) = 2.688 (0.685); p < 0.001), individually.
   Also, EE was significantly correlated with glutathione activity-related
   factors including oxidized glutathione [Coefficient (SE) = −1.186
   (0.319); p = 0.002] and the ratio of GSH to GSSG [Coefficient (SE) =
   26.218 (5.403); p < 0.001], respectively.

Table 4.

   Linear regression analysis of xenobiotic metabolism-related genes for
   predicting glutathione activity.
   Factor Oxidized Glutathione GSH/GSSG
   Model Univariate analysis Multivariate analysis Univariate analysis
   Multivariate analysis
   Genes Coefficient (SE) p-value Coefficient (SE) p-value Coefficient
   (SE) p-value Coefficient (SE) p-value
   GSTM2 −1.526 (0.505) 0.008[140]^* – – 32.762 (9.241) 0.003[141]^*
   22.895 0.024[142]^*
   GSTA3 −0.881 (0.346) 0.022[143]^* – – 19.249 (6.399) 0.008[144]^* – –
   CYP2A5 −0.587 (0.176) 0.004[145]^* – – 9.231 (3.837) 0.029[146]^* – –
   ALDH1A7 −0.432 (0.145) 0.009[147]^* – – 8.993 (2.719) 0.004[148]^* – –
   UGT2A1 −0.307 (0.083) 0.002[149]^* −0.307 0.002[150]^* 5.768 (1.678)
   0.003[151]^* 3.916 0.029[152]^*
   CRYL1 −1.863 (1.052) 0.096 NA 42.259 (19.818) 0.049[153]^* – –
   AKR1A1 −1.333 (0.699) 0.075 32.038 (12.823) 0.024[154]^* – –
   DPYS −0.517 (0.267) 0.071 12.925 (4.817) 0.016[155]^* – –
   PON1 −0.111 (0.214) 0.611 5.318 (3.994) 0.202 NA
   [156]Open in a new tab
   ^*

   p < 0.05; SE, standard error; NA, not associated. Linear regression
   analysis was conducted to identify significantly xenobiotic
   metabolism-related genes affecting oxidized glutathione (OG) and the
   ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) in
   olfactory bulb of mouse brain. The stepwise multiple linear regressions
   included UGT2A1 and GSTM2 in the final models.

EE mediated neuroprotection and preserved newly generated cells

   The expression of iNOS is highly up-regulated when the cells are in
   oxidative stress, and the relationship between apoptotic markers (Bax
   and Bcl-2) is important for regulating cell apoptosis. To measure the
   expression of iNOS, RT-PCR was performed with the referenced primers
   (Table [157]S1) ([158]33). The representative figures of RT-PCR for the
   iNOS expression between EE and control group were shown in Figure
   [159]4A. The expression of iNOS in EE mice was significantly lower than
   that of control mice (Figure [160]4B; p < 0.05). To further evaluate
   the apoptotic process and neuroprotection in the OB, western blot was
   conducted. The representative figures of WB for Bax, Bcl-2, and Actin
   between EE and control group were shown in Figure [161]4C. The ratio of
   Bax, a pro-apoptosis marker to Bcl-2, an anti-apoptosis marker was
   significantly decreased in EE mice compared to control mice (Figure
   [162]4D; p < 0.05). Taken together, the upregulation of xenobiotic
   metabolizing enzymes mediated by EE induces glutathione-mediated ROS
   detoxification, eventually reducing oxidative stress and apoptosis
   process. IHC was performed to evaluate neuroprotection and cell
   proliferation in the RMS. The RMS is a structure that have a full of
   neuroblasts, which originated from the SVZ. These neuroblasts further
   migrate along the RMS into the OB, resulting in the replacement of OB
   interneurons. In the analysis of TUNEL assay, apoptotic cells were
   significantly decreased in the RMS of EE mice compared to control mice
   (Figures [163]5A–G; p < 0.05) In the analysis of newly generated cells,
   Ki67^+ cells in the RMS were significantly increased in EE mice
   compared with control mice (Figures [164]5H–N; p < 0.05).

Figure 4.

   [165]Figure 4
   [166]Open in a new tab

   iNOS expression and apoptotic process. The expression of iNOS in the OB
   of EE mice were significantly decreased compared to control mice (A,B).
   Moreover, the ratio of Bax to Bcl-2 in EE mice was significantly
   decreased compared to control mice (C,D). Values are median
   (interquartile range). An asterisk indicates significant difference
   (^*p < 0.05, by Mann–Whitney U-test).

Figure 5.

   [167]Figure 5
   [168]Open in a new tab

   Histological assessments for neuroprotection and cell proliferation in
   the RMS. TUNEL^+ cells were indicated in green (A–F). TUNEL^+ cells in
   EE mice were significantly decreased in the RMS compared to control
   mice (G; p < 0.05). Ki67^+ cells were indicated in green (H–M). Ki67^+
   cells in EE mice were significantly increased in the RMS compared to
   control mice (N; p < 0.05). Values are median (interquartile range). An
   asterisk indicates significant difference (^*p < 0.05, by Mann–Whitney
   U-test). Scale bars for (A,C,H,L) represent 100 μm, scale bars for
   (B,D,I,M) represent 20 μm, and scale bars for (E,F,J,K) represent 10
   μm.

Discussion

   In this study, we provided evidences that EE has the protective effects
   on the OB through setting an optimal status for antioxidant and
   xenobiotic metabolism. The proteomic analysis revealed the DEPs in the
   OB of EE mice compared with control mice. GO analysis was conducted to
   identify protein function with DAVID 6.8 beta annotation tool and KEGG
   pathways. In the KEGG pathways, the most highlighted themes were
   general metabolism and xenobiotic metabolism within several biological
   processes.

   To validate the expression of the up-regulated proteins, qRT-PCR was
   performed. Among the phase I metabolizing enzymes, ALDH1A7, DPYS, PON1,
   CRYL1, AKR1A1, CYP1A2 were found to be significantly increased in EE
   mice compared with control mice and among the phase II metabolizing
   enzymes, UGT2A1, GSTM2, GSTA3 were found to be significantly increased
   in EE mice compared with control mice. The activation ratio of phase I
   and phase II xenobiotic metabolism is important because the product of
   phase I could be more deleterious than the original form of
   xenobiotics. The phase-II genes encode for a battery of enzymes that
   are essential in the antioxidation and detoxification of xenobiotics
   and endogenous reactive electrophilic compounds ([169]34). The
   transcriptional regulation of the phase II genes is achieved by the
   binding of nuclear factor erythroid2- related factor (Nrf2) to the
   consensus antioxidant response element (ARE) sequence in the promoter
   regions.

   Nrf2 has been shown to regulate the expression of a group of genes
   encoding cytoprotective enzymes in response to cell exposure to certain
   toxicants, including reactive oxygen species (ROS), electrophiles, as
   well as dithiolethiones, isothiocyanates, and triterpenoids ([170]35).
   The target genes include ones encoding such enzymes as antioxidative,
   catalyzing electrophile conjugation, glutathione homeostasis, producing
   reducing equivalents, proteasome function and other. Among others,
   glutathione reductase catalyzes the reduction of glutathione disulfide
   (GSSG) to the sulfhydryl form glutathione (GSH), which is a critical
   molecule in resisting oxidative stress and maintaining the reducing
   environment of the cell ([171]36).

   To further analyze the rate of phase II enzyme activity, one of the
   phase II enzymes, glutathione, the GSH/GSSG detection assay was
   performed. The GSH/GSSG ratio in the OB of EE mice was significantly
   higher than in the OB of control mice, and oxidized glutathione was
   significantly decreased in EE mice compared to control mice. However,
   the level of total glutathione was not significantly different between
   the groups. These results suggest that EE has beneficial effects on the
   OB of the mouse brain by setting an optimal status for getting rid of
   ROS via enhancement of antioxidant and detoxifying enzyme activity.

   To further investigate the relationship between glutathione-mediated
   ROS detoxification and the validated up-regulated metabolizing enzymes,
   the regression analyses were conducted to identify whether the genes
   predict the glutathione activity. While GSTM2, GSTA3, CYP2A5, ALDH1A7,
   UGT2A1 were correlated in oxidized glutathione, GSTM2, GSTA3, CYP2A5,
   ALDH1A7, UGT2A1, CRYL1, AKR1A1, DPYS were correlated in GSH/GSSG ratio.
   The stepwise multiple linear regression indicated that UGT2A1 was the
   most influential gene in oxidized glutathione, and GSTM2 and UGT2A1
   were the most influential genes in the GSH/GSSG ratio.

   GSTM2 stabilizes reactive metabolites formed by CYPs, UGT, and ST, and
   the proper activity of GST isoenzymes is important for olfactory acuity
   ([172]37). A fluctuation in the GST activity was observed in striatal
   neurons of Huntington's diseases, reflecting its protective role for
   oxidative damage to DNA, proteins, and lipids ([173]38). The expression
   of GST activity is highly expressed in rat olfactory bulb compared to
   that of the cerebral cortex ([174]37). Interestingly, the
   transcriptional level of Nrf2 is positively correlated to GST
   activities in most cases ([175]39). GSTM2 also takes an important role
   in glutathione-mediated antioxidant activity and clearance of odorants
   ([176]40).

   UGTs, consisting of genetically variable family of enzymes, detoxify
   various exogenous xenobiotics from air pollutants to food and
   endogenous lipids ([177]41). These enzymes involved in all three phases
   of xenobiotic metabolism ([178]42) and are detectable in olfactive
   mucosa and olfactory bulb ([179]43). Various isoforms of UGTs are
   highly expressed in the olfactory area. Their expression is highly
   related to the transfer rate of xenobiotics via the nasal pathway to
   the brain, the modulation of olfactory perception, and the
   detoxification of exogenous or endogenous compounds, which are
   important for physiological olfactory processing in the system
   ([180]44).

   During xenobiotic metabolism, the numerous amounts of ROS and reactive
   nitrogen species generate in the membrane of the cell and mitochondria.
   These reactive species can exert deleterious effects on DNA, protein,
   and membrane of the cell ([181]44). Furthermore, excessive ROS
   generation or oxidative stress activates stress-mediated MAPKs,
   ultimately leading to cell death ([182]37). The accumulation of
   endogenous ROS can be alleviated by either enzymatic or non-enzymatic
   antioxidants defense ([183]45). In RT-PCR and WB analysis, the
   significant reductions of the iNOS expression and apoptotic process
   were observed in EE mice compared with control mice. This
   neuroprotective effect may be due to the up-regulation of glutathione
   activity and the reduction of ROS.

   The OB relays various information from the olfactory epithelium, which
   has a direct contact with external environment, to the olfactory cortex
   ([184]43, [185]46). When encountering and metabolizing xenobiotics,
   continuous neural regeneration and synaptic plasticity occur in sensory
   cell axons and mitral cells of the OB ([186]6). Most of the progenitor
   cells from the subventricular zone that migrate along the migratory
   stream to the OB differentiate into granule cells (GC) ([187]47). These
   cells are inhibitory interneurons, and their activity is extremely
   important to modulate mitral cell activity and to accurately shape the
   output from the OB to higher olfactory centers ([188]38, [189]40).
   Notably, olfactory interneurons are continuously generated throughout
   adulthood and their integration in the OB circuits is regulated by
   sensory experience ([190]48). Several studies have shown that exposing
   animals to an olfactory enrichment increases survival of adult-born
   neurons in the OB ([191]49, [192]50). In the presence of powerful
   neurogenic stimulus, such as voluntary exercise, the proliferative
   capability of SVZ stem cells turns out to be more plastic ([193]51).
   Moreover, EE increases neurogenesis in the granular cell layer (GCL) of
   the adult OB ([194]52). Consistent with the previous studies, our
   results showed that EE significantly reduced the apoptosis of newly
   regenerated cells in the RMS.

   Oxidative stress in brain is related with many factors such as
   environmental toxin, aging, and diseases. Previous studies have shown
   oxidative stress could be alleviated by environmental enrichment
   ([195]17, [196]24, [197]53), and the dramatic effect can be shown in
   the aging rat brain ([198]17, [199]24). EE also mediates various
   functional improvements via the increased expression of growth factors
   such as FGF-2 in the hypoxic ischemic mice ([200]14). Moreover, EE also
   induces behavioral and functional improvement associated with synaptic
   plasticity and neurogenesis ([201]54–[202]57). However, the mechanisms
   underlying the effect of EE on xenobiotic metabolism in the OB are less
   clear. As indicated in the study of Yokota et al., EE during the
   perinatal period can significantly modulate the gene expression related
   to inflammation in the OB via the proper activation of xenobiotic
   metabolism, eventually resulting in neuroprotection ([203]25).
   Consistent with the previous study, our results showed the beneficial
   effects of EE that can alleviate endogenous oxidative stress via the
   glutathione-mediated ROS detoxification in the adult mouse brain and
   can also mediate neuroprotection, resulting in more newly regenerated
   cells in the RMS and modulation of the OB circuits.

   Previous studies suggested that the complexity of the environment is
   the key feature of EE that can provide a wide range of opportunities
   for stimulation in various brain regions ([204]21, [205]58–[206]60). In
   our study, we provided larger space with the items varying in size,
   shape, smell, and texture in EE. The active interaction between the
   mice and items available in the environment may lead to dramatic
   results in endogenous ROS detoxification of normal adult mice. However,
   since the activity response of antioxidant enzymes in the brain is
   probably dependent on various factors such as the duration of EE, sex,
   and strain of mice, further prospective studies to evaluate the effect
   of EE based on these factors are needed.

   Since there is little information available for the effect of EE on the
   intact OB of normal mice, our study may provide a possible protective
   mechanism of EE. In the present study, the proteomic analysis revealed
   the DEPs, which are associated with metabolic pathways and xenobiotic
   metabolism, in EE mice compared with control mice. EE can induce the
   neuroprotective effects via the up-regulation of glutathione-associated
   antioxidant enzymes in the OB. EE also creates the optimal state
   against ROS by the up-regulation of GSH/GSSG ratio, down-regulation of
   iNOS expression and apoptotic process, and up-regulation of cell
   proliferation.

Author contributions

   JHS and SP equally contributed to this study. JHS performed most of the
   experiments, analyzed data, wrote the manuscript, and contributed to
   the study concept and design. SP performed molecular study, analyzed
   data, wrote the manuscript, and contributed to English editing. Y-KS
   conducted statistical analysis. B-GN managed animals. JWK and KPK
   conducted proteomic analysis and LC-MS analysis. HYL and S-RC wrote the
   manuscript, developed the study concept, and supervised the project.
   All authors read and approved the manuscript.

Conflict of interest statement

   The authors declare that the research was conducted in the absence of
   any commercial or financial relationships that could be construed as a
   potential conflict of interest.

Acknowledgments