Hippocampal Mrp8/14 signaling plays a critical role in the manifestation of depressive-like behaviors in mice

Male BALB/c mice (6–8 weeks of age, 20–23 g of weight) were purchased from the Animal Center (Second Military Medical University, Shanghai, China). All animals were given 1–2 weeks to acclimate to colony conditions before the experiments began. The mice were maintained under standard conditions (humidity 52 ± 2%, temperature 22 ± 1 °C) with a 12-h light/dark cycle and ad libitum access to food and water unless otherwise stated. After acclimation, mice were randomly assigned to the control and experiment groups using random numbers generated in Microsoft Excel (Additional file 1: Figure S1). All experimental protocols were approved by the Second Military Medical University Animal Care Committee. All experiments were conducted in accordance with related guidelines and laws.

ABR-215757 (quinoline-3-carboxamide) was provided by Active Biotech AB (Lund, Sweden). Recombinant mouse Mrp8 (E.coli derived, purity > 95%, cat. no. ab108120) and Mrp14 (E.coli derived, purity > 90%, cat. no. ab109951) were purchased from Abcam (Abcam, Cambridge, UK). Recombinant mouse Mrp8/14 heterodimer (E.coli derived, purity > 95%, cat. no. 8916-S8) was obtained from R & D SYSTEMS (R & D SYSTEMS, Minneapolis, USA). The endotoxin level is less than 0.10 EU per 1 μg in the recombinant proteins. TAK-242 (C15H17CIFNO4S, cat. no. HY-11109) was obtained from MedChem Express (MedChem Express, New Jersey, USA). Anti-Mrp8 goat polyclonal antibody (cat. no. sc-8113), anti-Mrp14 goat polyclonal antibody (cat. no. sc-8115), anti-TLR4 mouse monoclonal antibody (cat. no. sc-293072), and anti-RAGE mouse monoclonal antibody (cat. no. sc-365154) were purchased from Santa Cruz. Anti-MyD88 rabbit monoclonal antibody (cat. no. 4283) and Anti- NF-κB p65 rabbit monoclonal antibody (cat. no. 8242) were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-Phospho-NF-κB p65 rabbit polyclonal antibody (cat. no. AB11011) was purchased from AbSci (Nanjing, China). Anti-iNOS rabbit monoclonal antibody (cat. no. ab205529) was purchased from Abcam. Anti-ACTB rabbit polyclonal antibody (cat. no. D110001–0100) was obtained from BBI Life Science. IRDye 800CW goat anti-mouse antibody (cat. no. 926–68070), goat anti-rabbit antibody (cat. no. 926–32211), and IRDye 680RD donkey anti-goat antibody (cat. no. 925–68074) were provided by LI-COR Biosciences.

As a classic animal model of depressive symptoms, CUMS model has been widely used for more than 20 years [27]. The CUMS protocol was performed as described previously [28]. Briefly, on a daily basis, mice were exposed to specific unpredictable stressors, including restraint for 2 h, cage shaking for 30 min, 45° cage tilt for 12 h, 4 °C swimming for 5 min, 45 °C oven for 10 min, damp bedding for 12 h, and food and water deprivation for 24 h. For the intervention experiment, ABR-215757 or normal saline was injected (IP) daily for the whole CUMS period. Generally, animals were housed in group cages (4–5 mice/cage) except during some of the manipulations (e.g., restraint stress) and tests (e.g., weighing and the sucrose preference test). The CUMS procedure generally lasts for 4 weeks. Body weight was assessed every week. Sucrose preference test and tail suspension test were adopted to identify the depressive-like behaviors.

Sucrose preference test (SPT) was performed as described in a previous study [29]. To determine sucrose preference, mice were provided with two bottles (randomized placement), filled with either tap water or 1% sucrose solution (w/v). Mice were acclimated to 1% sucrose for 48 h. The bottles were weighed both before and after the experiment, and consumption was quantified following overnight bottle choice. The sucrose preference was defined as the ratio of the weight of sucrose solution consumption to the total water intake, i.e., sucrose preference = sucrose consumption / (sucrose consumption + water consumption) × 100%. The test was performed in dark phase (6–10 p.m.).

Tail suspension test (TST) was carried out according to our previous report [30]. The mouse was suspended by the tail using adhesive tape for 1 min for acclimation and another 5 min for detection. The tail climbing behaviors were prevented by passing mouse tails through a small plastic cylinder prior to suspension. The immobility time during the latter 5-min-long suspension was recorded and analyzed by Tail Suspension SOF-821 (Med Associates, Inc., St. Albans, VT, USA). The experiment was conducted in the dark without interruption. To eliminate olfactory interference, the hooks and chambers were cleaned with 75% ethanol between two separated test sessions. Experimental grouping was blinded to the tester assessing immobility.

After weighing and behavioral tests, mice under general anesthesia were fixed on a heated pad. Blood was collected from left ventricle. Then, mice were perfused transcardially with ice-cold saline for about 3 min. Hippocampi were isolated on ice, temporarily frozen in liquid nitrogen, and stored at − 80 °C. Blood samples were allowed to stand for 30 min at room temperature and centrifuged at 4000 rpm for 15 min. The supernatant serum was collected and stored at − 80 °C. Cell samples were prepared as stated below.

Mouse serum Mrp8 and Mrp14 were determined using enzyme-linked immunosorbent assay kits (ELISAs, cat. no. CSB-EL020641MO and CSB-EL020642MO) from CUSABIO and CusAb (Wuhan, China). All the assays were performed according to the manufacturer’s instructions. The detection range was 0.625–40 ng/mL for Mrp8, 0.45–30 ng/mL for Mrp14. The ELISA kits also have high precision, including high intra-assay precision (CV < 8%) and inter-assay precision (CV < 10%).

The frozen mouse hippocampi and BV2 microglia were homogenized in ice-cold RIPA buffer (Beyotime Institute of Biotechnology, Nantong, Jiangsu, China) containing 1 mM protease inhibitor PMSF (Beyotime Institute of Biotechnology) and 10% PhosSTOP phosphatase inhibitor (Roche, Indianapolis, IN, USA). Protein concentration in the lysate was determined with the BCA assay (Beyotime Institute of Biotechnology). Then, samples were mixed with 5× loading buffer and heated at 100 °C for 10 min. Protein samples were separated by 10–15% sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, USA). After two-hour blocking with 5% nonfat milk at room temperature, membranes were incubated with primary antibodies overnight at 4 °C. After washing, membranes were further incubated with fluorescent second antibodies for 1 h at room temperature. Bands were visualized using Odyssey Infrared Imaging System (LI-COR, Inc., Lincoln, NE, USA) and quantified with National Institutes of Health (NIH) Image J software.

Intracerebroventricular (ICV) cannulation was operated for ICV injection as described previously [6]. Briefly, after anesthesia, the dorsal aspect of the skull was shaved and swabbed with 75% ethanol. The mice were subsequently fixed on a stereotaxic apparatus (RWD Life Science, Shenzhen, China). A guide cannula was vertically implanted in the right ventricle according to the following stereotaxic coordinates: anteroposterior − 0.6 mm; mediolateral − 1.1 mm; and dorsal ventricular − 2 mm. Then, a matched syringe needle was placed into the cannula, and the syringe was removed at 3 min after injection. To verify entry into the right ventricle before our main experiments, 5 μL of trypan blue dye was injected into the cannula, and the mouse brain was cut into slices for observing. Before the initiation of following experiments, animals were allowed 2 weeks to recover after operations.

Mrp14 inhibitor ABR-215757 was dissolved and prepared as recommended by Active Biotech. Other drugs were dissolved or diluted using sterile endotoxin-free isotonic saline. ABR-215757 (10 mg/kg body weight [31]) was administrated through intraperitoneal (IP) injection once a day during CUMS period. Recombinant mouse Mrp8 (3 μg, 6 μL [32]), Mrp14 (3 μg, 6 μL), and Mrp8/14 heterodimer (3 μg, 6 μL) were injected via ICV administration. Recombinant proteins were diluted using normal saline for ICV injection. The control mice were treated with 6 μL normal saline (Additional file 1: Figure S1). This dose was selected based on a previous report [32], which showed recombinant Mrp8 promoted TLR4 activation in PBMCs and microglia. Behavioral tests were conducted 24 h after the recombinant protein treatment. TLR4 inhibitor TAK-242 was injected (IP, 3 mg/kg body weight [6]) 30 min prior to rMrp8/14 administration (Additional file 1: Figure S1). These inhibitors (ABR-215757 and TAK-242) can permeate the blood–brain barrier [33].

The levels of gene expression of inflammatory cytokines were determined by real-time RT-PCR. Brain tissue was stored in liquid nitrogen, and BV2 microglia were washed twice with ice cold PBS and then processed for RNA extraction. Total RNA was extracted from the hippocampus or BV2 microglia utilizing a standard method of TRIzol reagent (Invitrogen, Carlsbad, CA, USA). After quantification, 2 μg of total RNA was used for cDNA synthesis using PrimeScript™ RT Master Mix (TaKaRa, Shiga, Japan). Primer sequences were tested for sequence specificity using Primer-BLAST in NCBI. As tabulated in Additional file 2: Table S1, the primers used in this study were obtained commercially from Sangon Biotechnology (Shanghai, China). The RT-PCR amplification was performed using 2 μL of cDNA and MaximaTM SYBR Green/ROX qPCR Master Mix (Fermentas, Waltham, MA, USA) on an Applied Biosystems 7500 (Life Technologies Corporation., Carlsbad, CA, USA). Melting curve analysis was utilized to verify primer specificity, and a comparative threshold cycle method was adopted to determine the fold changes of each gene expression relative to β-actin.

A murine microglial cell line BV2 was obtained from American Type Culture Collection (ATCC, Rockville, MD, USA). Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies/Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS, Life Technologies/Gibco) at 37 °C in an incubator with 95% air and 5% CO₂. For nitric oxide (NO) assay and Western Blot, cells were seeded onto 6-well culture plates. For real-time RT-PCR and reactive oxygen species (ROS) assay, cells were seeded onto 12-well culture plates. After treatment with TAK-242 (1 μM) for 30 min, cells were administrated with rMrp8/14 (0.5 μg/mL) for 24 h. Then, the supernatants were collected for NO assay, and cells were used for Western Blot, real-time RT-PCR, and ROS assay.

NO of cell supernatant was detected using Griess Reagent (Beyotime Institute of Biotechnology, Haimen, China) according to the manufacturer’s instructions. Samples and standards were added into a 96-well plate, and then Griess Reagent I and II were added successively. The absorbance was determined at a wavelength of 540 nm using a microplate reader. ROS was determined by a Reactive Oxygen Species Assay Kit (Beyotime Institute of Biotechnology). In brief, after washing, cells were incubated with fluorescent probe DCFH-DA (10 μM, Beyotime Institute of Biotechnology) for 20 min at 37 °C in an incubator. Then, the extracellular DCFH-DA was cleared, and images were obtained using a fluorescence microscope (Carl Zeiss) at the 488 nm excitation wavelength and 525 nm emission wavelength.

All data were presented as mean ± standard error of the mean (SEM). Normality and homogeneity of variance were assessed before comparisons using Kolmogorov–Smirnov test and Levene’s test. Statistical analyses were carried out with Student’s t test when comparing between two variables, and one-way or two-way ANOVA followed by LSD post hoc tests when comparing among multiple variables. Omnibus F values with degrees of freedom were reported for each ANOVA. Differences were considered statistically significant only when p < 0.05. All the analyses were performed with IBM SPSS 21.0 (IBM Corp., Armonk, N.Y.).

Behavioral tests and body weight were measured after CUMS period (Additional file 1: Figure S1a). Coinciding with previous studies [6, 27], 4-week CUMS could induce the depressive-like behaviors in rodents. Specifically, the body weight was significantly attenuated in CUMS-exposed mice compared with control mice (Fig. 1a; t₍₂₇₎ = 3.63, p < 0.01, 29.18 ± 0.53 vs. 25.78 ± 0.41). CUMS significantly decreased the sucrose preference, indicating the impaired sensitivity to reward and anhedonia, which is a core symptom of major depression (Fig. 1b; t₍₁₈₎ = 2.51, p = 0.02, 81.65 ± 5.57 vs. 57.87 ± 7.66). The stressed mice also showed longer immobility duration in the TST (Fig. 1c; t₍₁₈₎ = 2.22, p = 0.04, 44.87 ± 3.05 vs. 57.98 ± 5.05). On the other hand, the results of ELISA showed that relative to the control group, serum Mrp8 (Fig. 1d; t₍₂₀₎ = 2.15, p = 0.04, 4.93 ± 0.35 vs. 6.04 ± 0.37) and Mrp14 (Fig. 1e; t₍₂₀₎ = 2.96, p < 0.01, 4.49 ± 0.08 vs. 4.77 ± 0.06) were slightly upregulated after CUMS intervention. Western blot revealed that hippocampal Mrp8 (Fig. 1f and g; t₍₄₎ = 3.00, p = 0.01, 0.91 ± 0.26 vs. 2.14 ± 0.31) and Mrp14 (Fig. 1f and h; t₍₄₎ = 5.41, p < 0.01, 1.10 ± 0.03 vs. 1.46 ± 0.08) were also increased. Collectively, these data demonstrate that CUMS elicited depressive-like behaviors and increased Mrp8 and Mrp14 protein in both serum and hippocampus.

TLR4 signaling pathway has been shown to have a pivotal role in stress-induced neuroinflammation [34] and major depressive disorder [35]. To validate whether TLR4 signaling pathway is activated in the hippocampus of stressed mice, we detected the protein level of TLR4, MyD88, and the NF-κB p65 phosphorylation. Stress exposure increased TLR4 (Fig. 2a and d; t₍₄₎ = 3.18, p = 0.03, 1.00 ± 0.04 vs. 1.22 ± 0.06) but not MyD88 (Fig. 2a and e; t₍₄₎ = 0.53, p = 0.62, 1.00 ± 0.11 vs. 1.06 ± 0.04) at the protein level. CUMS also activated the phosphorylation of NF-κB p65 (Fig. 2b and f; t₍₄₎ = 3.74, p = 0.02, 1.00 ± 0.14 vs. 1.66 ± 0.11). Conversely, RAGE expression remained unaffected (Fig. 2c and g; t₍₄₎ = 0.92, p = 0.41, 1.00 ± 0.06 vs. 1.06 ± 0.03). Taken together, CUMS activated TLR4/NF-κB signaling pathway but did not affect the expression of RAGE.

To further investigate the functional role of Mrp14 in the development of depressive symptoms, the inhibitor ABR-215757 was injected daily during the CUMS period (Additional file 1: Figure S1b). As shown in Fig. 3a, the body weight of mice in CUMS group was significantly lower than control or vehicle mice (F_(1,29) = 4.54, p = 0.01; post hoc: vehicle vs. CUMS, p = 0.03, 28.44 ± 1.29 vs. 26.84 ± 1.78), while CUMS could not significantly decrease body weight of ABR-215757-treated mice (post hoc: ABR-215757 vs. CUMS + ABR-215757, p = 0.06, 28.44 ± 1.29 vs. 27.16 ± 1.08). However, ABR-215757 did not significantly rescue body weight of stressed mice (post hoc: CUMS vs. CUMS + ABR-215757, p = 0.66, 26.84 ± 1.78 vs. 27.16 ± 1.08). Besides, ABR-215757 increased the sucrose preference (Fig. 3b; F_(1,36) = 5.67, p = 0.02; post hoc: CUMS vs. CUMS + ABR-215757, p < 0.05, 57.28 ± 4.79 vs. 76.31 ± 4.26) and decreased the immobility duration in TST (Fig. 3c; F_(1,26) = 6.91, p < 0.01; post hoc: CUMS vs. CUMS + ABR-215757, p < 0.001, 62.21 ± 6.70 vs. 32.12 ± 5.15). We also found that ABR-215757 alleviated TLR4 expression (Fig. 3d and g; F_(1,12) = 7.85, p < 0.01; post hoc: CUMS vs. CUMS + ABR-215757, p = 0.02, 1.36 ± 0.05 vs. 1.14 ± 0.04) and p65 phosphorylation (Fig. 3e and h; F_(1,12) = 9.68, p < 0.01; post hoc: CUMS vs. CUMS + ABR-215757, p = 0.03, 1.33 ± 0.06 vs. 1.13 ± 0.07) after CUMS exposure. The RAGE expression did not change significantly among these groups (Fig. 3f and i; F_(1,12) = 0.72, p = 0.56, 0.91 ± 0.03 vs. 1.07 ± 0.09). Thus, ABR-215757 could alleviate CUMS-induced depressive-like behaviors and TLR4/NF-κB signaling activation.

To determine whether Mrp8, Mrp14, and Mrp8/14 affected the development of depressive-like behaviors, recombinant Mrp8, Mrp14, Mrp8/14, and vehicle reagent were injected (ICV) to mice (Additional file 1: Figure S1c). Mice administrated with Mrp14 and Mrp8/14 presented lower sucrose preference (Fig. 4a; F_(3,36) = 3.44, p = 0.03; post hoc: vehicle vs. Mrp8, Mrp14, and Mrp8/14, all p < 0.05, 83.17 ± 7.02 vs. 59.90 ± 6.83, 55.03 ± 7.08, 53.09 ± 8.82). All of the recombinant Mrp8, Mrp14, and Mrp8/14 treatment could result in a longer immobility duration (Fig. 4b; F_(3,18) = 3.16, p = 0.05; post hoc: vehicle vs. Mrp8, p = 0.03; vehicle vs. Mrp14, p = 0.02; vehicle vs. Mrp8/14, p = 0.02, 45.02 ± 3.11 vs. 65.40 ± 6.08, 65.91 ± 5.45, 68.61 ± 7.92). Besides, we found that the mRNA levels of inflammatory cytokines (TNF-α, IL-1β, IL-6) were elevated in the hippocampus after being challenged by any of these recombinant proteins (Fig 4c; F_(3,14) = 20.54, p < 0.001 for TNF-α, 1.00 ± 0.10 vs. 6.17 ± 0.47, 2.70 ± 0.31, 3.76 ± 0.71; F_(3,14) = 24.70, p < 0.001 for IL-1β, 1.00 ± 0.26 vs. 13.60 ± 0.97, 4.54 ± 1.14, 4.67 ± 1.93; F_(3,14) = 7.59, p < 0.01 for IL-6, 1.00 ± 0.22 vs. 1.13 ± 0.08, 1.72 ± 0.14, 2.09 ± 0.23). Microglia activation has been suggested to be mainly responsible for depression [36, 37]. The results of RT-PCR indicated that recombinant proteins could upregulate IBA-1 gene expression (Fig. 4c; F_(3,14) = 7.79, p < 0.01; post hoc: vehicle vs. Mrp8, p = 0.03; vehicle vs. Mrp14, p < 0.01; vehicle vs. Mrp8/14, p < 0.001, 1.00 ± 0.25 vs. 2.76 ± 0.31, 3.30 ± 0.57, 4.85 ± 0.79). After these recombinant proteins treatment, the hippocampal TLR4 level (Fig. 4d and g; F_(3,12) = 9.13, p < 0.01; post hoc: vehicle vs. Mrp8, p = 0.02; vehicle vs. Mrp14, p < 0.01; vehicle vs. Mrp8/14, p < 0.001, 1.00 ± 0.04 vs. 1.23 ± 0.05, 1.34 ± 0.03, 1.41 ± 0.09) and p65 phosphorylation (Fig. 4e and h; F_(3,12) = 10.29, p < 0.01; post hoc: vehicle vs. Mrp8, p = 0.03; vehicle vs. Mrp14, p < 0.01; vehicle vs. Mrp8/14, p < 0.001, 1.00 ± 0.03 vs. 1.60 ± 0.13, 1.90 ± 0.22, 2.34 ± 0.24) were elevated. No significant differences were found in RAGE expression among the four groups (Fig. 4f and i; F_(3,12) = 1.51, p = 0.26, p < 0.001, 1.00 ± 0.09 vs. 1.12 ± 0.12, 1.25 ± 0.07, 1.01 ± 0.10). Collectively, central administration of Mrp8, Mrp14, and Mrp8/14 induced depressive-like behaviors, enhanced TLR4/NF-κB signaling pathway, and promoted the generation of proinflammatory cytokines.

Since Mrp8/14 heterodimer is the most abundant form [11, 12], we conducted further studies in vivo and ex vivo using Mrp8/14 instead of Mrp8 and Mrp14. To elucidate whether TLR4 was a mediator in Mrp8/14-evoked depressive-like behaviors, mice were pretreated with TLR4 inhibitor TAK-242 before the ICV injection of recombinant Mrp8/14 (Additional file 1: Figure S1d). As expected, compared with the recombinant Mrp8/14-group mice, the recombinant Mrp8/14 + TAK-242-group mice had significantly higher sucrose preference (Fig. 5a; F_(2,23) = 4.99, p = 0.01; post hoc: Mrp8/14 vs. Mrp8/14 + TAK-242, p < 0.01, 53.30 ± 8.19 vs. 73.40 ± 6.48) and shorter immobility time in the tail suspension test (Fig. 5b; F_(2,23) = 5.24, p = 0.01; post hoc: Mrp8/14 vs. Mrp8/14 + TAK-242, p < 0.01, 94.91 ± 8.56 vs. 66.88 ± 3.69). The NF-κB p65 phosphorylation evoked by recombinant Mrp8/14 was inhibited by TAK-242 treatment (Fig. 5c and d; F_(2,9) = 18.21, p < 0.001; post hoc: Mrp8/14 vs. Mrp8/14 + TAK-242, p < 0.001, 1.45 ± 0.09 vs. 0.95 ± 0.06). We further detected the changes of the expression of proinflammatory cytokines. The results showed that relative to the recombinant Mrp8/14 group, the mRNA levels of proinflammatory cytokines in the hippocampus of recombinant Mrp8/14 + TAK-242 were reduced significantly (Fig. 5e; post hoc: Mrp8/14 vs. Mrp8/14 + TAK-242, all p < 0.05; 10.16 ± 2.06 vs. 5.77 ± 1.14 for TNF-α, 10.16 ± 1.15 vs. 4.83 ± 1.30 for IL-1β, 1.65 ± 0.24 vs. 1.08 ± 0.13 for IL-6). Moreover, Mrp8/14-induced hippocampal IBA-1 overexpression was attenuated by TAK-242, which was shown by the RT-PCR assay of IBA-1 (Fig. 5e; F_(2,20) = 43.71, p < 0.001; post hoc: Mrp8/14 vs. Mrp8/14 + TAK-242, p < 0.01, 3.42 ± 0.27 vs. 2.40 ± 0.19). Taken together, pretreatment of TAK-242 could inhibit the Mrp8/14-induced depressive-like behaviors and neuroinflammation.

To further verify the effects of TAK-242 on recombinant Mrp8/14-induced microglia activation, we performed experiments using BV2 microglia. Two of the most important molecules released by activated microglia are the bioactive free radical NO [38] and ROS [39]. The increase of iNOS expression in both protein level and mRNA level was attenuated by pretreatment of TAK-242 in BV2 cells (Fig. 6a; F_(1,12) = 26.66, p < 0.001; post hoc: Mrp8/14 vs. Mrp8/14 + TAK-242, p < 0.001, 1.60 ± 0.12 vs. 0.78 ± 0.04; Fig. 6b; F_(1,11) = 14.19, p < 0.001; post hoc: Mrp8/14 vs. Mrp8/14 + TAK-242, p < 0.001, 1.34 ± 0.13 vs. 0.63 ± 0.04). The production of NO was also significantly decreased after TAK-242 administration (Fig. 6c; F_(1,11) = 17.81, p < 0.01; post hoc: Mrp8/14 vs. Mrp8/14 + TAK-242, p = 0.03, 5.17 ± 0.36 vs. 4.00 ± 0.30). Moreover, our data showed that TAK-242 reduced the generation of ROS induced by recombinant Mrp8/14 (Fig. 6d). Thus, Mrp8/14-induced microglia activation was mediated by TLR4.