Pediococcus acidilactici attenuates chronic stress-induced depression via generating metabolite indole-3-lactic acid and downregulating neuroinflammation | Journal of Neuroinflammation

Strain cultivation

The Pediococcus acidilactici (strain GOLDGUT-EZMind), isolated from traditionally fermented sauerkraut originating from the Qinghai-Tibet Plateau in China, was provided by WonderLab. This strain was deposited at the China General Microbiological Culture Collection Center (CGMCC), under the accession number CGMCC 35671. The bacteria were cultured in MRS broth under anaerobic conditions at 37 °C for 24 h. For subsequent oral gavage in mice, the cells were resuspended in phosphate-buffered saline (PBS) to a final concentration of 1 × 10⁹ CFU/100 µL.

Animals

Eight-week-old male C57BL/6J mice were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) and housed in the animal facility at Northwest A&F University under standard conditions. All animal procedures were approved by the Animal Ethics Committee of Northwest A&F University (Ethics No. IACUC2024-1110) and conducted in accordance with the Guide for the Care and Use of Laboratory Animals (Eighth Edition, ISBN-10: 0-309-15396-4).

Experiment 1 (Pediococcus acidilactici intervention)

24 mice were randomly divided into 3 groups, after one week of adaptive feeding, the mice were subjected to a six-week chronic unpredictable mild stress (CUMS) protocol. In brief, the CUMS regimen included a variety of mild stressors [25], such as forced swimming at 4 °C for 5 min, restraint for 3 h, water deprivation for 24 h, food deprivation for 24 h, wet bedding for 24 h, tilted cages for 24 h, and tail pinching for 5 min. These stressors were randomly scheduled throughout the week to reduce predictability. Control group animals were not exposed to any stress. Pediococcus acidilactici CGMCC 35671 or PBS was administered via oral gavage (100 µL/day) [26].

Experiment 2 (AHR intervention)

To explore the mechanisms of AHR signaling pathway, 16 mice were randomly divided into 2 groups, the mice received the AHR antagonist CH-223191 (Glpbio, Shanghai, China) or corn oil at a dose of 10 mg/kg/day through intraperitoneal injection for the last 4 weeks (100 µL/day) [27].

Behavioral experimentsOpen field test (OFT)

The open field test is employed to evaluate spontaneous exploratory activity and anxiety-like behavior in mice [28]. The experiment utilized an opaque, uncovered monitoring box (50 cm × 50 cm × 50 cm), with the bottom divided into 25 equal square regions. A camera positioned above the arena recorded the mice’s movement trajectories over a 5-minute period. Total distance traveled and time spent in central area were analyzed to assess spontaneous activity levels and anxiety. Prior to testing, the arena was cleaned to remove any feces from the previous mouse and wiped with 75% alcohol to eliminate odor interference from other mice.

Elevated plus maze test (EPMT)

The elevated plus maze (EPM) is commonly used to evaluate anxiety-like behavior in mice. The apparatus was elevated 70 cm above the ground and consisted of two open arms (30 cm × 8 cm), two closed arms (30 cm × 8 cm × 15 cm), and a central area (8 cm × 8 cm). Mice were placed in the central area, and their movement was recorded for 5 min. The time spent in the open arms was analyzed to assess anxiety levels. Prior to testing, the arena was cleaned to remove any feces from the previous mouse and wiped with 75% alcohol to eliminate odor interference from other mice.

Tail suspension test (TST)

Mice were suspended by the tail for 6 min by attaching them to a horizontal bar positioned 50 cm above the floor using adhesive tape applied 2 cm from the tip of the tail in a visually isolated area. A mouse was considered immobile when it floated in an upright position, making only minimal movements. The duration of immobility of tail-suspended mice during the final 4 min was recorded using timers. This procedure was conducted by a single observer who was blind to the specific experimental groups and strictly adhered to standard testing protocols [29].

Sucrose preference test (SPT)

The sucrose preference test serves as a key indicator for assessing anhedonia, which is characterized by a diminished interest in rewarding stimuli, a manifestation of emotional disturbances often used to evaluate depressive-like behaviors in mice. This experiment comprises two phases: acclimation training and testing [30]. During the acclimation training phase, two bottles containing a 1% (w/v) sucrose solution are provided in each cage for 24 h. Subsequently, one bottle is replaced with pure water for an additional 24 h, followed by a 12-hour fasting and water deprivation period. At this point, one bottle of 1% (w/v) sucrose solution and one bottle of pure water are prepared, and their initial volumes are recorded. After 12 h, the remaining volumes of both the sucrose solution and pure water are measured. The consumption of the sucrose solution and pure water, as well as the sucrose preference index, are calculated using the following formula:

$$\begin{aligned}&\mathrm{Sucrose\:Preference\:Index}=\mathrm{Sucrose\:Solution\:Consumption}/\\&(\mathrm{Sucrose\:Solution\:Consumption}+\mathrm{Pure\:Water\:Consumption})\end{aligned}$$

Three-chamber social test (TCST)

The three-chamber social test was employed to evaluate social preference and social novelty preference in mice [31]. The apparatus consisted of three interconnected chambers (left, center, right). During the first 10 min, the test mouse was permitted to freely explore all three chambers to acclimate to the environment, and the time spent in each chamber was recorded. Social preference was assessed in the subsequent 10 min: a wire cup containing an unfamiliar mouse (Mouse 1, matched for sex, age, and group) was placed in the right chamber, while an empty wire cup was positioned in the left chamber. The time the test mouse spent investigating (sniffing, climbing) and entering each chamber was recorded. The social preference index was calculated using the formula:

$$\begin{aligned}\mathrm{The}\;\mathrm{sociability}\;=\;&(\mathrm{time}\;\mathrm{spent}\;\mathrm{interacting}\;\mathrm{with}\;\mathrm{Mouse}\;1\;\\&-\;\mathrm{time}\;\mathrm{spent}\;\mathrm{interacting}\;\mathrm{with}\;\mathrm{the}\;\mathrm{empty}\;\mathrm{cup})\\&/(\mathrm{time}\;\mathrm{spent}\;\mathrm{interacting}\;\mathrm{with}\;\mathrm{Mouse}\;1\:\\&+\:\mathrm{time}\;\mathrm{spent}\;\mathrm{interacting}\;\mathrm{with}\;\mathrm{the}\;\mathrm{empty}\;\mathrm{cup})\end{aligned}$$

In the final 10 min, the social novelty preference test was conducted: a new unfamiliar mouse (Mouse 2, matched for sex, age, and group) was placed in the previously empty cup in the left chamber, while the right chamber remained unchanged. The time the test mouse spent interacting with both mice was recorded. The social novelty preference index was calculated as:

$$\begin{aligned}\mathrm{The}\;\mathrm{social}\;\mathrm{novelty}\;=\;&(\mathrm{time}\;\mathrm{spent}\;\mathrm{interacting}\;\mathrm{with}\;\mathrm{Mouse}\;2\;\\&-\;\mathrm{time}\;\mathrm{spent}\;\mathrm{interacting}\;\mathrm{with}\;\mathrm{Mouse}\;1)\\&/(\mathrm{time}\;\mathrm{spent}\;\mathrm{interacting}\;\mathrm{with}\;\mathrm{Mouse}\;2\:\\&+\:\mathrm{time}\;\mathrm{spent}\;\mathrm{interacting}\;\mathrm{with}\;\mathrm{Mouse}\;1)\end{aligned}$$

High-performance liquid chromatography (HPLC) analysisIndole-3-lactic acid (ILA) in strain culture medium

To prepare the samples, 200 µL of MRS culture medium (fermented for 24 h) was diluted with pre-chilled methanol (−20 °C) to a final volume of 1 mL. The diluted medium was incubated at −20 °C for 20 min before being centrifuged (12,000 rpm, 10 min, 4 °C). The resulting supernatant was filtered through a 0.22 μm microporous membrane to remove particulate matter.

The filtered supernatant was immediately analyzed using HPLC (LC-16; Shimadzu, Kyoto, Japan) [32]. For ILA quantification, 10 µL of the sample was injected into the chromatograph, equipped with a C18 column (Agilent, 4.6 × 250 mm, 5 μm) and a fluorescence detector set at an excitation wavelength of 282 nm and an emission wavelength of 352 nm. Chromatographic separation was performed at a column temperature of 30 °C using a flow rate of 1.0 mL/min and gradient elution with chromatographic-grade methanol (mobile phase A) and 15 mmol/L sodium dihydrogen phosphate (NaH₂PO₄, PH 2.8,mobile phase B). The gradient program was as follows: 0–12 min, 42% A; 12–28 min, 50% A; and 28–35 min, 85% A.

ILA in mouse serum

To prepare the samples, 60 µL of serum was diluted with pre-chilled methanol (−20 °C) to a final volume of 300 µL. The diluted serum was incubated at −20 °C for 20 min before being centrifuged (12,000 rpm, 10 min, 4 °C). The resulting supernatant was filtered through a 0.22 μm microporous membrane to remove particulate matter.

Subsequent analyses of HPLC were the same as ILA in strain culture medium.

5-Hydroxytryptamine (5-HT) and Dopamine (DA) in mouse cortex

Cortical tissues were homogenized in saline to create a 10% (w/v) homogenate, which was centrifuged (12,000 rpm, 15 min, 4 °C) to obtain the supernatant. A precipitating reagent (0.60 mol/L HClO4, 1.20 mol/L K₂HPO₄, and 2.00 mmol/L Na2EDTA) was then added to the supernatant in a 1:1 ratio. The mixture was incubated on ice for 10 min before undergoing a second centrifugation under the same conditions. The resulting supernatant was filtered through a 0.22 μm microporous membrane to remove particulate matter and prepare the sample for analysis.

Immediately after filtration, the supernatant was analyzed via HPLC (LC-16; Shimadzu, Kyoto, Japan) [33]. For quantification of 5-HT and DA, 20 µL of each sample was injected into the chromatograph, which was equipped with a C18 column (Agilent, 4.6 × 250 mm, 5 μm) and a fluorescence detector set at an excitation wavelength of 290 nm and an emission wavelength of 330 nm. The chromatographic separation was performed at 30 °C with a flow rate of 1.0 mL/min. The mobile phases used were chromatographic-grade methanol (mobile phase A) and a 0.1 mol/L sodium acetate buffer (pH 5.1, containing 0.1 mmol/L disodium EDTA, mobile phase B), with an elution ratio of 1:9.

Immunofluorescence staining

After the euthanasia of the mice, brain tissues were extracted, with one-half of each brain dissected and placed into vials containing 4% (v/v) paraformaldehyde (pH 7.4) for 24 h. The specimens were subsequently embedded in paraffin and sectioned into 5 μm slices. Deparaffinization was carried out with xylene, followed by sequential washes in 100%, 90%, 80%, and 70% ethanol, each for 5 min, along with three washes in PBS (pH 7.4) for 5 min each. The slices were then incubated overnight at 4 °C with primary antibodies targeting ionized calcium-binding adapter molecule 1 (IBA-1). After this incubation, the slides were washed with PBS and incubated with a secondary antibody for 2 h. Information regarding the antibodies is provided in Additional file 1. DAPI was utilized for nuclear staining to visualize the sections. Immunofluorescence images were obtained using an inverted fluorescent microscope (DM5000 B, Germany), and positive regions were quantified using Image J software [34].

Quantitative real-time PCR (qRT-PCR)

Tissue samples were accurately weighed and placed into 2.0 mL grinding tubes. To assess mRNA levels in mouse tissues, BIOZOL (Bioer Technology, Hangzhou, China) was added. Quantification was performed using a NanoDrop One (Thermo Fisher Scientific, Shanghai, China). The ueIris II qRT-PCR System First-Strand cDNA Synthesis Kit (US Everbright, Suzhou, China) enabled the reverse transcription of total RNA into cDNA. For quantitative PCR (qPCR), the 2× SYBR Green qPCR Master Mix Kit (US Everbright, Suzhou, China) was used to quantify mRNA levels. The primer sequences are provided in Additional file 2. Ct values were normalized to GAPDH, and relative gene expression levels were determined using the 2−ΔΔCt method.

Enzyme-linked immunosorbent assay (ELISA)

Serum and cortical tissues were processed to detect inflammatory cytokines and corticosterone, including tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), interleukin-6 (IL-6), interleukin-22 (IL-22), and corticosterone. All procedures followed the instructions provided with the ELISA kits (xl-Em0359, xl-Em0187, xl-Em0196, xl-Em0217, xl-Em1949, Xinle Biotechnology, Shanghai, China). A standard curve was generated to calculate the concentration, which was expressed as ng/g of protein.

Measurement of malondialdehyde (MDA) and superoxide dismutase (SOD)

Serum and cortical tissues were harvested from mice. Cortical tissues were homogenized in PBS to generate a 10% (w/v) homogenate, which was centrifuged to isolate the supernatant. Protein concentrations in the cortical homogenate were determined using the BCA protein assay kit. Subsequently, MDA levels and SOD activity in both serum and the cortical supernatant were quantified utilizing specific assay kits (Solarbio, Beijing, China), following the protocols provided by the manufacturer.

Statistical analysis

Data analysis was performed using GraphPad Prism 8.0 (GraphPad Software Inc., San Diego, CA, USA). One-way ANOVA was utilized to identify significant differences among the groups. Subsequently, Tukey’s multiple comparison test was applied as a post hoc analysis. Unpaired two-tailed t-test was used to compare two groups. Results are expressed as mean ± SEM, with p < 0.05 regarded as statistically significant.