Male ICR mice (about 20 g) were obtained from Vital River Laboratory Animal Technology Co., Ltd., Shanghai, China. The mice were randomly divided into control, alcohol exposure (AE), and alcohol exposure and L. plantarum ST-III culture supernatant (AE/LP-cs) groups (n = 10–14). Mice were maintained on a 12-h light/12-h dark cycle and under standard temperature and humidity conditions. The LP-cs were mixed with drinking water (1:20) at a ratio ensuring one mouse consumed about 8–9 ml per day. Control group mice and AE group mice had free access to sterile water, and AE/LP-cs groups mice had free access to LP-cs. The AE group was given an intraperitoneal injection of alcohol (2 g/kg body weight, once a day, i.p.) for two consecutive days, with two gaps of 2 days without injections. LP-cs and sterile water were replaced every 2 days. Mice were maintained on treatment for 28 days. After treatment, the Morris water maze test was performed to evaluate mice’s spatial learning and memory abilities. Then they were anesthetized with Avertin. For histomorphological analysis, the animals were perfused with 4% paraformaldehyde in 0.1 M phosphate-buffered saline following the saline solution perfusion. The brains were rapidly detached and post-fixed by immersion in 4% paraformaldehyde. For molecular biological analysis, the hippocampus was separated from the brain after perfusion with 0.9% saline solution and rapidly stored at −80°C. The Laboratory Animal Ethics Committee of Wenzhou Medical University and the Laboratory Animal Center of Wenzhou Medical University approved the animal protocols.

The test was performed in a circular pool with a diameter of 120 cm and a height of 40 cm (Jiliang, Shanghai, China). The pool was filled with opaque water colored with milk powder and maintained at 26 ± 1°C. The training was performed using a hidden circular platform with six blocks of three 60 s trials separated by 20 min inter-block intervals as previously described (Nicholas et al., 2006; Wu et al., 2020a). During the training, the platform remained in the same location relative to the distal cues in the room. For each trial, mice were placed in the water at different start locations (E, S, W, and N) equally spaced and offset from the goal location by 45°. One hour following the sixth block, the hidden platform was removed, and the mice were scored during a 60 s probe trial. They were scored for latency to reach the goal and memory recall, determined by crossing over the previous platform location. Another probe trial was performed 24 h after training to assess memory consolidation and retrieval (Wu et al., 2020b).

For hematoxylin-eosin (H&E) staining, 50 μm brain sections were deparaffinized and hydrated, then stained with H&E solution. For Nissl staining, the tissue sections were deparaffinized and hydrated according to the manufacturer’s instructions (Beyotime) and stained with cresyl violet and Nissl differentiation solution. Finally, the hippocampal cell morphology was observed and photographed under a Nikon ECLIPSE 80i microscope (Nikon, Tokyo, Japan).

The hippocampus was homogenized in a lysis buffer containing a protease inhibitor cocktail (10 μL/ml, GE Healthcare Biosciences, PA, United States). The complex was centrifuged at 12,000 rpm/min at 4°C, and supernatants were removed to determine protein concentrations, calculated according to the standard curve. Proteins were separated on 10 or 12% SDS-polyacrylamide gels at 80 V for 2 h. After separation at 300 mA for 1.5 h, the protein bands were transferred to PVDF membranes. The membranes were blocked with 5% skimmed milk for 1.5 h. After washing three times with Tris-buffered saline with 0.05% Tween 20, the membranes were incubated with the primary antibodies overnight: JNK1/2, p-JNK1/2, GAPDH, superoxide dismutase 2 (SOD2), IL-6 (Proteintech, Chicago, IL, United States), Bax, Bcl-2, C-caspase3, postsynaptic dense protein 95 (PSD95), Nrf2, p-CREB133, BDNF, IL-1β, synapsin-1 synaptophysin, GRP78, ATF, protein kinase R-like endoplasmic reticulum kinase (PERK), p-PERK, p-IRE1α, CREB, p-CREB129, PKA, and IL-10 (Abcam, Cambridge, United Kingdom). The following day, membranes were incubated in corresponding secondary antibodies for 1 h at room temperature. Signals were visualized using the ChemiDocXRS + Imaging System (Bio-Rad). Blot bands were quantified using ImageJ software densitometry and expressed as relative density to GAPDH.

After dewaxing and hydration, brain sections were incubated in 3% H₂O₂ for 15 min, then in blocking solution for 45 min. Subsequently, sections were incubated with primary antibodies at 4°C overnight: SOD2 (Proteintech, Chicago, IL, United States), BDNF (Abcam, Cambridge, United Kingdom). After washing three times in phosphate-buffered saline, the sections were incubated with horseradish peroxidase-conjugated secondary antibodies for 4 h at 37°C. Then, the sections were reacted with 3,3-diaminobenzidine and imaged using a Nikon ECLIPSE 80i microscope (Nikon, Tokyo, Japan).

Experiments were repeated at least three times, and the tissues from each replicate were from different mice. The data results were expressed as mean ± SEM. The experimental data were analyzed using one-way ANOVA and post hoc Tukey or Tukey–Kramer tests. All statistical tests were conducted using GraphPad Prism 8.0 (San Diego, CA, United States). The optical density value of Nissl bodies was quantified by ImageJ 6.0 software. Differences with p < 0.05 were considered statistically significant. All figures were typeset using PowerPoint (Redmond, WA, United States) or Adobe Photoshop (San Jose, CA, United States) software.

The Morris water maze test reflects mice’s spatial and long-term spatial memory (Bromley-Brits et al., 2011). The time for the AE group to reach the platform from blocks four to six was longer than that of the control and AE/LP-cs groups. After training, the time to reach the platform was significantly reduced for all mice. Furthermore, there was significant difference in swimming speed between AE/LP-cs and AE groups (Figures 1A–C). At 24 h after water maze training, each group’s learning and memory function was tested again (Figures 1C–E). After 24 h, we found that AE mice had fewer crossings over the platform position and had longer latencies to the platform than those in control mice. Compared with the alcohol-induced group, the first time to the platform in the AE/LP-cs group was significantly shorter, and the number of crossings of the platform was significantly more significant (Figures 1D,E). The swimming track during the memory test further suggested that the memory of the AE mice was worse than the control and AE/LP-cs groups (Figure 1F). We also found that mice in the AE group had significantly lower percentages of time in the TQ quadrant than the control and AE/LP-cs groups during trial (Figure 1G). These results suggest that LP-cs improves alcohol-induced decline in learning and memory.

We investigated whether the hippocampal structure and cellular morphology were altered based on behavioral findings of impaired learning and memory in mice by H&E and Nissl staining (Figure 2). In the control group, the DG, CA1, and CA3 regions of the hippocampus of the mice had normal morphology, the cell layers were arranged tightly, uniformly, and neatly and the staining was uniform. The morphology of DG, CA1, and CA3 in the hippocampus of mice in the AE group changed significantly, including loose, sparse, and disordered cell arrangements, evident cell degeneration, necrosis and shedding, and indistinct cell membranes. The structure of hippocampal neurons in the brain of the AE/LP-cs group tended to be normal, and the pyramidal cells layer were arranged tightly and uniformly, significantly different from the AE group. In addition, we quantified the average optical density values of Nissl bodies in the DG, CA1, and CA3 of hippocampal neurons (Figures 2C–E). Which were significantly lower in the AE group compared with the control group. After administration with LP-cs, the DG and CA3 neurons damage were significantly mitigated as presented with higher average optical density of Nissl bodies. However, there was no significant difference of average optical density of Nissl bodies in the CA1 between the AE and AE/LP-cs group.

We measured hippocampal cell apoptosis. C-Jun N-terminal kinase (JNK) is associated with apoptosis and plays an essential role in regulating apoptosis. We measured protein expression levels of JNK and p-JNK in the hippocampus and found that phosphorylation levels of JNK in the AE group were significantly higher than in the other two groups (Figures 3A,B). The expression levels of the pro-apoptotic protein Bax and Cleaved-caspase 3 protein in the AE/LP-cs group were significantly lower than those in the AE group. Expression levels of the anti-apoptotic protein Bcl-2 were significantly higher than in the AE group and were close to the control group (Figures 3A,C–E). These findings suggest that LP-cs significantly alleviates alcohol-induced increases in JNK phosphorylation levels, decreases Bcl-2 expression, and upregulates Bax expression. All results indicates that alcohol causes hippocampal cell apoptosis, and LP-cs protects hippocampal cell apoptosis induced by alcohol.

Synapses are specialized regions of the contact portion of neurons specifically labeled with synaptic proteins. PSD95 is located beneath the postsynaptic membrane, is pressure-sensitive, and enhances synaptic remodeling. Synapsin-1 and synaptophysin promoted the regulated release of neurotransmitters, which is conducive to the recovery of nerve function (Kacířová et al., 2021; Patzke et al., 2021). These synapse-related proteins can be used to reflect hippocampal function. Western blot results showed that PSD95 and synapsin-1 in the AE group were lower compared with the control group. The expression level of PSD95, synapsin-1, and synaptophysin in the AE/LP-cs group was more robust than that in the AE group and was similar to the control group (Figures 4A–D). These results suggest that alcohol reduces the expression of synapse-related proteins, and LP-cs improves it.

Endoplasmic reticulum stress is a process in which the protein folding function of the ER becomes disordered when subjected to endogenous or exogenous stimuli. Many unfolded or misfolded proteins accumulate in the ER cavity and cause subsequent reactions such as induction of GRP78 expression (Oakes and Papa, 2015; Ibrahim et al., 2019). The ER stress sensor pathways, including IRE1/sXBP1, PERK/EIf2, and ATF6, orchestrated the primary regulatory circuits to ensure ER homeostasis (Di Conza and Ho, 2020). We analyzed these pathway-related proteins and found that expression level of GRP78 and ATF6 in the AE group was higher than that in the control group, and these proteins in the AE/LP-cs group were significantly lower than that in the AE group. We also found that phosphorylation levels of IRE1α and PERK were significantly higher in the AE group than in control and AE/LP-cs groups (Figures 5A–E). These findings suggest that alcohol induces ER stress and LP-cs alleviate alcohol-induced ER stress in the hippocampus.

Alcohol causes oxidative stress in the hippocampus (Zhao et al., 2021). When the antioxidant defense system is turned on, Nrf2 activates the transcription of antioxidant genes such as SOD2 (Hoang et al., 2019; Durymanov et al., 2020). Our western blot findings suggested that protein expression level of Nrf2 and SOD2 in the AE group was significantly lower than that in the control group, and protein expression level of Nrf2 and SOD2 in the AE/LP-cs group was close to that in the control group (Figures 5F–H). These findings suggest that alcohol reduces the antioxidant capacity of the hippocampus while LP-cs improves its antioxidant capacity.

CREB is essential for long-term synaptic plasticity changes, and synaptic plasticity mediates the conversion of short-term memory to long-term memory (Saura and Valero, 2011). Ser133 of the N-terminal kinase-inducible domain of CREB is the phosphorylation site of PKA, and p-CREB is the initiating factor for the expression of downstream gene BNDF, which affects synaptic transmission (Jagasia et al., 2009; Wu et al., 2020a). BDNF promotes the repair and survival of nerve cells, reduces cell apoptosis, and improves learning and memory (Michalski and Fahnestock, 2003; Lu et al., 2014). In the present study, phosphorylation levels of Ser133 protein of CREB in the AE group were significantly higher than those of Ser129 in the other two groups. Protein expression level of PKA and BNDF in the AE group was significantly lower than that in control and AE/LP-cs groups (Figures 6A–F). Consistent with western blot results, co-immunofluorescence staining showed that the fluorescence intensities of BDNF and SOD2 in the AE group were significantly lower than those in the control group, consistent with western blot results (Figure 6F). The results suggest that alcohol induces suppression of CREB activity and BDNF expression in the hippocampus, affecting learning and memory function, and LP-cs reverses these effects.

The NF-κB pathway is a canonical pro-inflammatory signaling pathway based on the role of NF-κB in pro-inflammatory gene expression (Lawrence, 2009). Studies showed that alcohol induces increased expression of inflammatory factors, including NF-κB, in the hippocampus (Wang et al., 2020). In the present study, expression level of NF-κB, IL-1β, IL-6, and IL-10 in the AE group were higher than that in control and AE/LP-cs groups. The expression level of NF-κB, IL-1β, IL-6, and IL-10 in the/LP-cs group were similar to that in the control group (Figures 7A–E). Moreover, we found that TNF-α was deposited in the CA1 region of the hippocampus from the AE group compared with the control group and was partially reversed by LP-cs administration (Figure 7F). These results suggest that LP-cs reduces alcohol-induced inflammation.