Male C57BL/6J mice (weight, 18–20 g, n=20) were purchased from the Jinan Pengyue Laboratory Animal Breeding Co., Ltd. Mice were housed 2 mice per cage under a 12 h light/dark cycle at 18–22°C. The present study complied with the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals (24) and was approved by the Ethics Committee of Qingdao Mental Health Center (Qingdao, China; approval no. 2022061). The mice were randomly divided into four groups as follows: i) control group (PBS; n=5); ii) LPS group (n=5); iii) LPS + NC group (n=5); and iv) LPS + MCPIP1 group (n=5). An adenovirus-associated vector (AAV) containing MCPIP1 (Shanghai GenePharma Co., Ltd.) was used. After induction using 4% isoflurane and maintenance using 2% isoflurane, 2 µl of negative control (NC) AAV or MCPIP1 AAV were injected into the hippocampus regions (−1.6 mm anteroposterior of bregma, ±1.8 mm lateral and −1.6 mm dorsoventral of bregma) of mice (age, 6 weeks, n=5) using a brain location microinjection pump (200 nl/min; Stoelting Co.). The mice in control and LPS groups were subjected to a sham procedure. At 4 weeks after injection, mice were intraperitoneally injected with PBS or LPS (1 mg/kg) for 2 days (25). If improper injection operation caused the death of the mouse, the mouse was excluded and replaced. All data derived from animal studies were analyzed by an experimenter blind to experimental conditions. The order of the animals was randomized prior to behavioral tests. Sucrose preference test (SPT), open field test (OFT), tail suspension test (TST) and forced swimming test (FST) were performed as described previously (26). After testing depression-like behavior, the mice were sacrificed by anesthesia with isoflurane (4% for induction and 2% for maintenance) and transcardial perfusion using ice-cold PBS and then 4% paraformaldehyde in PBS overnight at 4°C. Following perfusion, the brains were collected for subsequent analysis. The left hippocampus was frozen and the right hippocampus was treated with paraffin.

During the adaptation phase, the mice were housed in cages with two bottles of 1% sucrose solution for two days. On the following day, one bottle of 1% sucrose solution and one bottle of tap water were placed in each cage. On day 4 mice were deprived of water and food for 24 h. Mice were allowed access to the 1% sucrose solution and tap water bottles for 3 h, to avoid bottle side preference, the positions of the two bottles were swapped. Sucrose preference during the 3 h test was calculated as: Sucrose consumption/(water consumption + sucrose consumption) ×100.

Briefly, mice were placed in a square wooden box (40×40×40 cm) for 5 min. The mice were gently placed in the center of the square facing in the same direction each time. Anilab software (AVTAS version 5.0, Anilab Scientific Instruments Ltd. Co.) was used to analyze the travel distance of the mice.

Mice were individually suspended from a retort stand with adhesive tape placed 1 cm from the tail tip and placed 50 cm above the floor. The duration of immobility was recorded during a 6 min test and analyzed using Anilab software (ver 6.50, Anilab Scientific Instruments Ltd. Co.).

Mice were placed in a glass beaker (5,000 ml) containing 4,000 ml of water (25±1°C) for 6 min, and the struggling time was recorded.

Mouse BV2 transformed microglial cells (cat. no. CL-0493; Procell Life Science & Technology Co., Ltd.) were incubated at 37°C in MEM-non-essential amino acid solution containing 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin. Cells were transfected with 50 nM pcDNA-MCPIP1, empty vector (Shanghai GenePharma Co., Ltd.), short interfering RNA (si)-MCPIP1 or si-NC (Shanghai GenePharma Co., Ltd.) at 37°C for 24 h using Lipofectamine 3000. LPS (1 µg/ml; cat. no. L8880; Beijing Solarbio Science & Technology Co., Ltd.) was added to the transfected BV2 cells at 37°C for 12 h. LPS + si-MCPIP1 + TAK-242 group cells were pretreated with TAK-242, a selective TLR4 inhibitor (cat. no. HY-11109; MedChemExpress), at 37°C for 30 min. The sequences were as follows: si-MCPIP1 Sense: 5′-CCGAGAUCCUCUCCUACAAGU-3′, Anti-sense: 5′-UUGUAGGAGAGGAUCUCGGCA-3′. Si-NC Sense: 5′-UUCUCCGAACGUGUCACGUTT-3′; Anti-sense: 5′-ACGUGACACGUUCGGAGAATT-3′.

Total RNA was extracted from tissues and cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. RNA (1.0 µg) was reverse transcribed using HiScript^® II Q RT SuperMix for qPCR (cat. no. R223-01; Vazyme Biotech Co., Ltd.) according to the manufacturer's protocols. mRNA levels were quantified using a ChamQ SYBR qPCR Master Mix kit (cat. no. Q311-02; Vazyme Biotech Co., Ltd.) according to the manufacturer's protocols. Thermocycling conditions were as follows: Initial denaturation at 94°C for 30 sec, followed by 35 cycles of denaturation at 94°C for 5 sec and extension at 60°C for 30 sec. GAPDH was used as the endogenous control. Relative mRNA expression levels were assessed using the 2^−ΔΔCq method (27). Specific primer pairs are listed in Table I.

Proteins were extracted from tissues and cells using RIPA buffer (cat. no. P0013C; Beyotime Institute of Biotechnology). The protein concentration was determined using a BCA protein assay kit (P0012S; Beyotime Institute of Biotechnology). Proteins (30 µg/line) were separated using 10% SDS-PAGE (cat. no. P0012A; Beyotime Institute of Biotechnology) and transferred onto a PVDF membrane (cat. no. FFP24; Beyotime Institute of Biotechnology). After blocking with 5% non-fat dry milk in Tris-buffers saline for 2 h at 25°C, the membrane was incubated with primary antibodies against MCPIP1 (1:2,000; cat. no. 25009-1-AP; Proteintech Group Inc.), Iba-1 (1:2,000; cat. no. ab178846; Abcam), TLR4 (1:1,000; cat. no. A5258; Abclonal Biotech Co., Ltd.), MyD88 (1:1,000; cat. no. A21905; Abclonal Biotech Co., Ltd.), TRAF6 (1:2,000 cat. no. ab33915; Abcam), phosphorylated (p)-IκBα (1:1,000; cat. no. ab133462; Abcam), IκBα (1:2,000; cat. no. 4814T; CST Biological Reagents Co., Ltd.), p-p65 NF-κB (1:1,000; cat. no. 3033T; CST Biological Reagents Co., Ltd.), p65 NF-κB (1:2,000; cat. no. ab16502; Abcam), IL-6 (1:1,000; cat. no. 21865-1-AP; Proteintech Group Inc.), TNF-α (1:2,000; cat. no. A0277; Abclonal Biotech Co., Ltd.), IL-1β (1:2,000; cat. no. 16806-1-AP; Proteintech Group Inc.), iNOS (1:2,000; cat. no. ab178945; Abcam), Arg-1 (1:2,000; cat. no. 16001-1-AP; Proteintech Group Inc.) and GAPDH (1:10,000, cat. no. 10494-1-AP; Proteintech Group Inc.) at 4°C overnight. GAPDH was used as an internal reference protein. Following washing with TBS with 0.1% Tween20, membranes were then incubated with the HRP-conjugated Goat Anti-Rabbit IgG (H+L) secondary antibodies (1:10,000; SA00001-2; Proteintech Group Inc.) for 1 h at 25°C. ECL solution (cat. no. E412-02; Vazyme Biotech Co., Ltd.) was used to detect the bands. Analysis was performed using Image-Pro plus 6.0 software (Media Cybernetics).

Frozen brain sections (thickness, 30 µm) were blocked using 10% bovine serum albumin (Thermo Fisher Scientific Inc.) for 1 h at 25°C. Sections were incubated with antibodies against MCPIP1 (1:400; cat. no. 25009-1-AP; Proteintech Group Inc.) at 4°C overnight, followed by incubation with Alexa Fluor^® 488-conjugated secondary antibody (1:1,000; ab150077, Abcam) at room temperature in the dark for 1 h and DAPI at 25°C for 5 min. Sections were imaged using a Leica DMI 4000 B fluorescence microscope (Leica Microsystems GmbH). Images were taken from each rat for analysis by Image-Pro plus 6.0 software (Media Cybernetics).

Paraffin-embedded sections were deparaffinized in xylene at 25°C for 5 min followed by rehydration using 95, 70 and 50% ethanol solution 3 min in each. The sections were stained with H&E at 25°C for 8 min and imaged using a Leica DMI 4000 B fluorescence microscope (Leica Microsystems GmbH). Proportion of damaged neuronal cell was calculated by the number of cells exhibiting shrinkage.

The serum levels of IL-6, TNF-α, and IL-1β in mice were assessed using Mouse IL-6 ELISA Kit, Mouse TNF-α ELISA Kit, and Mouse IL-1β ELISA Kit (cat. nos. KE10007, KE10002 and KE10003, respectively; Proteintech Group Inc.).

BV2 cells were collected using trypsin, washed, and suspended in PBS. The cells were then incubated with FITC-conjugated CD16/CD32 antibodies (1:100; cat. no. 561728; BD Biosciences) and PE-conjugated CD206 antibodies (1:100; cat. no. 568273; BD Biosciences) at 4°C for 20 min. The expression of CD16/CD32 and CD206 was analyzed using an Accuri C6 Plus flow cytometer (BD Biosciences) and FlowJo v10.9.0 software (Tree Star Inc.).

pcDNA3.1-Flag-TRAF6, pcDNA3.1-His-MCPIP1, and pcDNA3.1-HA-Ubiquitin plasmids were purchased from Beijing Syngentech Co., Ltd. (Chian). BV2 cells were transfected with Flag-TRAF6, His-MCPIP1 and HA-ubiquitin at 37°C for 48 h. Then, cells (with the exception of control cells) were stimulated with 1 µg/ml LPS for 12 h and then treated with or without MG132 (20 µM) for an additional 4 h to block proteasomal degradation. The cells were lysed with buffer (50 mM Tris, 140 mM NaCl, 1% SDS), followed by clearing of cell lysates through centrifugation at 10,000 × g for 10 min. For IP assays, the anti-Flag (5 µg, cat. no. 20543-1-AP, Proteintech Group Inc.) were added at 4°C overnight. After incubation, 50 µl protein A/G Plus-Agarose (Santa Cruz) was added to the protein-antibody complexes and incubated at 4°C on a rotating device overnight. Immunoprecipitates were washed five times with immunoprecipitation buffer, centrifuged at 10,000 × g for 1 min, and a 2× sample loading buffer was added to the beads before boiling for 5 min. Pull-down samples were subjected to immunoblotting with an HA-tagged antibody (1:5,000; cat. no. 51064-2-AP; Proteintech Group Inc.), Flag-tagged antibody (1:20,000; cat. no. 20543-1-AP; Proteintech Group Inc.), His-tagged antibody (1:2,000; cat. no. 10,001-0-AP; Proteintech Group Inc.), and GAPDH (1:10,000; cat. no. 10494-1-AP; Proteintech Group Inc.). Following washing with TBS with 0.1% Tween20, membranes were then incubated with the HRP-conjugated Goat Anti-Rabbit IgG (H+L) secondary antibodies (1:10,000; SA00001-2; Proteintech Group Inc.) for 1 h at 25°C. ECL solution (cat. no. E412-02; Vazyme Biotech Co., Ltd.) was used to detect the bands. Analysis was performed using Image-Pro plus 6.0 software (Media Cybernetics).

Data are presented as mean ± SD. Statistical significance was evaluated using unpaired Student's t-test and one-way analysis of variance with Tukey's post hoc test using GraphPad Prism version 8 (Dotmatics). P<0.05 was considered to indicate a statistically significant difference.

A significant increase in MCPIP1 mRNA and protein expression levels was demonstrated in the hippocampus of LPS-treated mice compared with the control (Fig. 1A and B). Immunofluorescence results also indicated that the intensity of MCPIP1 staining increased significantly in LPS-treated mice compared with the control (Fig. 1C).

To further evaluate the potential effect of MCPIP1 on depression, an AAV that overexpressed MCPIP1 was injected into the hippocampus of mice. MCPIP1 mRNA and protein expression levels increased significantly after AAV infusion compared with the AAV-NC (Fig. 2A and B). The SPT provides a measurement of anhedonia, lack of interest in reward stimuli, and depression. The results indicated that LPS treated MCPIP1 overexpression mice exhibited a significantly higher preference for sucrose solution compared with the LPS + NC mice (Fig. 2C), which indicated decreased anhedonia behavior in MCPIP1 overexpression mice. There was no significant difference in the distance moved between mice in the LPS + NC and LPS + MCPIP1 groups in the OFT (Fig. 2D), which indicated that MCPIP1 overexpression did not significantly affect locomotor activity. The TST was then performed as an acute stress assay to measure the immobility time that was associated with the induction of depression using LPS. MCPIP1 overexpression resulted in less despair behavior, as indicated by the shorter time of immobility, compared with that in the LPS + NC mice (Fig. 2E), and this was also confirmed by the FST (Fig. 2F) that serves as an alternative acute stress assay. MCPIP1 overexpression decreased time of immobility, compared with the LPS + NC group (Fig. 2F). H&E staining demonstrated that the hippocampal neurons were normal in the control group and shrunken after LPS treatment. MCPIP1 overexpression also significantly decreased the number of injured neurons compared with the LPS + NC group (Fig. 2G).

MCPIP1 significantly reduced Iba-1 protein expression levels in LPS-treated mice compared with the LPS + NC group (Fig. 3A). RT-qPCR analysis demonstrated that MCPIP1 overexpression significantly inhibited the LPS-induced elevation of the mRNA expression levels of CD16, CD32 and iNOS, compared with the LPS + NC group (Fig. 3B). MCPIP1 overexpression significantly increased IL-4 and Arg-1 mRNA expression levels compared with the LPS + NC; mRNA expression levels of IL-4 and Arg-1 were significantly reduced by LPS treatment compared with the control. IL-10 and CD206 mRNA expression levels were significantly increased by MCPIP1 overexpression compared with the LPS + NC group; however, LPS treatment did not demonstrate a significant effect on their expression compared with the control (Fig. 3C). ELISA results demonstrated that MCPIP1 overexpression significantly decreased IL-6, TNF-α and IL-1β expression levels in LPS-treated mice compared with the LPS + NC group (Fig. 3D-F). The aforementioned results indicated that MCPIP1 served a role in the M2-polarization of microglia. Western blotting was performed to examine the TLR4/TRAF6/NF-κB signaling pathway. LPS-triggered significantly increased TLR4, MyD88, TRAF6, p-IκBα and p-p65 NF-κB protein expression levels compared with the control, which were significantly reduced by MCPIP1 overexpression compared with the LPS + NC group (Fig. 3G).

LPS treatment significantly upregulated MCPIP1 mRNA and protein expression levels in BV2 cells compared with the control (Fig. 4A and B). MCPIP1 overexpression and knockdown plasmids were transfected into BV2 cells. MCPIP1 protein expression levels significantly increased and Iba-1 protein expression levels significantly decreased after transfection with the MCPIP1 overexpression vector compared with the control (Fig. 4C). MCPIP1 knockdown decreased MCPIP1 protein expression levels and significantly increased Iba-1 protein expression levels (Fig. 5A). LPS treatment significantly increased the protein expression levels of IL-6, TNF-α, IL-1β and iNOS, and significantly decreased the protein expression levels of Arg-1, all compared with the control. MCPIP1 overexpression decreased protein expression levels of IL-6, TNF-α, IL-1β and iNOS, and increased the protein expression levels of Arg-1, all compared with the LPS + NC group (Fig. 4D). MCPIP1 knockdown aggravated PS-induced the protein expression levels of IL-6, TNF-α, IL-1β and iNOS, and further decreased arg-1 expression reduced by LPS (Fig. 5B). MCPIP1 overexpression significantly inhibited the LPS-induced expression of CD16/32 compared with the LPS + NC group. CD206 expression was significantly increased by MCPIP1 overexpression compared with the LPS + NC group; however, LPS treatment did not significantly affect CD206 expression levels compared with the control (Fig. 4E). MCPIP1 knockdown decreased CD206 expression levels compared with the LPS + si-NC group (Fig. 5C).

MCPIP1 overexpression significantly reduced the LPS-induced increase in the protein expression levels of TLR4, MyD88, TRAF6, p-IκBα and p-p65 NF-κB compared with the LPS + NC group. Moreover, MCPIP1 knockdown significantly increased the protein expression levels of TLR4, MyD88, TRAF6, p-IκBα and p-p65 NF-κB compared with the LPS + NC group, indicating that LPS-induced NF-κB activation was increased by MCPIP1 knockdown (Fig. 6A). LPS treatment markedly promoted the ubiquitination of TRAF6 and MCPIP1 notably inhibited LPS-induced TRAF6 ubiquitination in BV2 cells with or without MG132 treatment (Fig. 6B). TAK-242 was used to evaluate the function of MCPIP1 in the LPS-induced inflammatory response via the TLR4//TRAF6/NF-κB signaling pathway in BV2 cells. Western blotting demonstrated that TAK-242 treatment significantly reversed the upregulated protein expression levels of IL-6, TNF-α, IL-1β and iNOS and the downregulated protein expression levels of Arg-1 induced by MCPIP1 knockdown in BV2 cells, compared with the LPS + si-MCPIP1 group (Fig. 6C). Treatment with TAK-242 significantly reversed the upregulated protein expression levels of TLR4, MyD88, TRAF6, p-IκBα and p-NF-κB p65 which were induced by MCPIP1 knockdown (Fig. 6D). These data indicated that MCPIP1 knockdown promoted the LPS-induced inflammatory response via activation of the TLR4/TRAF6/NF-kB signaling pathway in BV2 cells.