In total, 45 male (weight, 25–30 g; age, 6 months) AD mice expressing human APP and PS1 genes and 15 C57BL/6 mice (age, 6 months) were obtained from the Experimental Animal Center of the China Medical University. The present study was approved by the Animal Welfare and Ethics Committee of the China Medical University Laboratory (Institutional Animal Care and Use Committees approval no. 2018236). All mice were offered food and water ad libitum and housed in pathogen-free facilities under a 12-h light/dark cycle in a controlled room temperature of 12–24°C and 60% relative humidity. The mice used in the present study exhibited amyloid plaques and NFT from age 9–12 months. Upon completion of the experiments, mice were euthanized by intraperitoneal injection of 50 mg/kg body weight sodium pentobarbital, followed by heart perfusion or dislocation of the cervical spine. Brain tissue samples were collected from wild-type or APP/PS1 transgenic mice. The pathology was primarily localized to the HC, amygdala and cerebral cortex. To reduce potential variations among the experimental mice, the mice used in the present study were first grouped by similar weight and at 12-months old. Then mice from each litter were equally distributed to the different study groups. Specifically, the mice were randomly divided into four groups as follows: i) Wild-type mice (control group; n=15); ii) APP/PS1 transgenic mice (AD group; n=15); iii) APP/PS1 transgenic mice were treated with the κ-opioid receptor agonist, U50488H for 28 days (1.25 mg/kg) using an osmotic pump (U50488H group; n=15); iv) APP/PS1 transgenic mice were treated with U50488H for 28 days (1.25 mg/kg) using an osmotic pump and were injected through an intracerebral pump injection with CaMKII antagonist, KN93 (5 µM/day) for 28 days (KN93 group; n=15). KN93 (Sigma-Aldrich; Merck KGaA) was dissolved in 0.9% saline containing 1% DMSO and diluted to a concentration of 1 mM.

Spatial memory was assessed using the Morris water maze test, which included the concealed platform test and the space exploration test (27,28). Before each trial period, the mice were brought to the room with the water maze to allow for acclimation. For spatial learning, the mice were trained for five consecutive days to find a hidden platform in the Morris water maze. During each trial, the mouse started from the middle of one of the four quadrants, facing the wall of the pool. The trial ended when the animal climbed onto the platform (diameter, 10 cm). The mice were not allowed to search for the platform for more than 60 sec, after which they were guided to the platform. For the space exploration test, the platform was removed after the end of the hidden platform test. Then, 24 h after the hidden platform test, the mice were placed in the same starting position as the hidden platform test and their swimming paths were recorded for 60 sec. The time each mouse spent in the original quadrant and the number of times each mouse crossed the original platform location were recorded. The Morris water maze video analysis system was used for data processing.

Upon completion of the water maze experiments, five mice from each group were anesthetized by intraperitoneal injection with 50 mg/kg sodium pentobarbital. Following anesthesia, mice were perfused through the heart with PBS and then with pre-cooled 4% (w/v) paraformaldehyde, then the mouse's brain was rapidly removed. The brain tissue was placed in an embedding box, 4% paraformaldehyde was added and the box was placed in a freezer at −80°C for 24–48 h for rapid freezing. A cold microtome was used to slice the tissue into 5-µm sections. The sections were deparaffinized and rehydrated through a graded series of alcohol. Hydrogen peroxide (3%) solution was used to inactivate endogenous peroxidases and the sections were washed once with PBS (pH 7.4). The sections were exposed to a 0.1 M sodium citrate solution for antigen retrieval. Next, the sections were incubated overnight at 4°C in Aβ antibody (cat. no. sc-28365; 1:1,000; Santa Cruz Biotechnology, Inc.). The next day the sections were washed three times in PBS, incubated with a biotin-labeled secondary antibody (cat. no. sc-525409; 1:1,000; Santa Cruz Biotechnology, Inc.) at 37°C for 30 min, then washed again in PBS. The 3,3′-diaminobenzidine, as a chromogen, was used to stain sections for 3 min at room temperature to visualize the Aβ-positive cell staining. The cell nuclei were counterstained with hematoxylin, a neutral resin was used to seal coverslips onto the microscope slides and the stained sections were observed with a light microscope (Olympus Corporation) with a magnification of ×40.

The sections that were mounted on glass microscope slides were heated to melt the paraffin. Then the sections were deparaffinized and rehydrated through a series of graded alcohol. The tissue sections were rinsed with tap water for 10 min. Hematoxylin staining was performed for 2 min at room temperature and the sections were rinsed with tap water for 10 min and immersed in 1% hydrochloric alcohol for 3 sec. Eosin staining was performed for 1 min at room temperature, then 95% ethanol followed by two changes of anhydrous ethanol were used to dehydrate the sections. The sections then were immersed in two changes of xylene for 5 min each. Finally, a neutral mounting medium was used to seal coverslips over the sections. Pathological changes in the tissue sections were observed using a light microscope (Olympus Corporation) with a magnification of ×40.

At the end of the water maze experiment, five mice in each group were sacrificed by dislocating the cervical spine. The brain of the mice was collected. The brain tissue was lysed in Laemmli buffer with homogenization and sonication three time for 10 sec/time on ice (QSonica LLC). Following tissue homogenization, the Glu (cat. no. CES122Ge; Wuhan USCN Business Co., Ltd.), collagen II (cat. no. CB85527920; Boswio; http://www.boswio.com), IL-18 (cat. no. SEA064Mu; Wuhan USCN Business Co., Ltd.) and IL-1β (cat. no. SEA563Mu; Wuhan USCN Business Co., Ltd.) content was detected using ELISA kits according to the manufacturer's instructions. The kit was equilibrated to room temperature and the required reaction plate was removed. Then the standards and diluted samples were added into the wells of the corresponding reaction plate and incubated at room temperature for 20 min. Subsequently, the reaction plate was washed, HRP-labeled secondary antibody provided in the kit was added to each well and incubated at 37°C for 30 min. The plate was rinsed, and the color developing solution was added in the dark. The plate was incubated for 15 min at room temperature, and then the stop solution was added to each well. The optical density (OD) value at 450 nm was read using a microplate reader. The OD value was used as the vertical coordinate and the standard concentrations were used as the horizontal coordinate. A standard curve was constructed and the curve equation and R-value were calculated to determine the corresponding concentration values for each sample.

The brain tissue samples were immersed in a 30% sucrose solution overnight followed by immersion in optimal cutting temperature embedding reagent at 4°C for 6 h, then stored at −80°C for 24 h and sectioned (thickness, 100 µm), according to a previous publication (27). The Golgi-Cox staining procedure was performed according to the instructions from the FD Rapid GolgiStain™ kit (FD Neurotechnologies, Inc.). The dendritic spine density of neurons was analyzed using ImageJ software (version 1.52a; National Institutes of Health).

The brain tissue samples were homogenized in iced-cold RIPA buffer containing protease inhibitor (Santa Cruz Biotechnology, Inc.) and protein concentration was estimated using BCA reagent. Then, 30 mg protein lysates per lane were loaded, separated by 6–12% SDS-PAGE gradient gels and transferred onto nitrocellulose membranes, then the membranes were blocked in a 5% skimmed milk solution at room temperature for 1 h. Tris-buffered saline with 0.1% Tween (TBST) was used to wash the membranes and then the membranes were incubated with the primary antibody including anti-NMDAR (1:1,000; cat. no. ab274377; Abcam), anti-p-CaMKII [1:1,000; cat. no. 12716S; Cell Signaling Technology, Inc. (CST)], anti-CaMKII (1:1,000; cat. no. 3362S; CST), anti-p-CREB (1:1,000; cat. no. 9198S; CST), anti-CREB (1:1,000; cat. no. 9197S; CST), anti-NCAM (1:1,000; cat. no. 99746S; CST), anti-NR2B (1:1,000; cat. no. ab254356; Abcam), anti-GluR1 (1:1,000; ab183797; Abcam), anti-SYN (1:1,000; cat. no. ab212184; Abcam), anti-PSD95 (1:1,000; cat. no. 3409S; CST), anti-GAPDH (1:1,000; cat. no. 2118S; CST), anti-pro-caspase-1 (1:1,000; cat. no. 24232S; CST), anti-pro-IL-1β (1:1,000; cat. no. 31202S; CST), anti-ASC (1:1,000; cat. no. 67824S; CST) and NLRP3 (1:1,000; cat. no. 15101S; CST) overnight at 4°C. The PVDF membranes were washed three times in PBST, incubated with anti-rabbit IgG, HRP-linked antibody (1:1,000; cat. no. 7074S; CST) at room temperature for 1 h and the PVDF membranes were washed in TBST. The labeled protein bands were visualized using an ECL kit (Amersham Biosciences) according to the manufacturer's instructions.

Paraffin-embedded tissues were sectioned (thickness, 4 µm) using a microtome and placed on glass microscope slides. The sections were deparaffinized at room temperature by immersion in two changes of xylene and two changes of absolute ethanol for 10 min each. Subsequently, the sections were rehydrated in a graded series of ethanol and washed in 10 mM PBS three times for 5 min each. Antigen retrieval was performed at 95°C for 3 min. After the sections were cooled to room temperature, they were washed in PBS, immersed in 5% normal goat serum (Invitrogen; Thermo Fisher Scientific, Inc.) in PBS and maintained at 37°C for 1 h. Sections were then incubated with anti-ionized calcium binding adaptor molecule 1 (IBA1; 1:100; cat. no. sc-32725; Santa Cruz Biotechnology) and NLRP3 (1:50; cat. no. NBP2-12446; Novus Biologicals, LLC) or anti-ASC (1:800; cat. no. 67824S; CST) and anti-pro-caspase-1 (1:100; cat. no. NBP2-15713; Novus Biologicals, LLC) antibodies at 4°C overnight. The next day, the sections were washed with PBS and incubated with goat anti-rabbit IgG antibody (1:1,000; cat. no. 8889S; CST) or goat anti-mouse IgG antibody (1:1,000; cat. no. 4408S; CST) at 37°C for 1 h, then washed with PBS. DAPI was added at room temperature for 7 min. Subsequently, the sections were sealed with neutral mounting medium and observed with a fluorescence microscope.

The data were analyzed using SPSS version 21.0 (IBM Corp.) and are presented as means ± standard deviation. Differences between two groups were analyzed using unpaired Student's t-test. Differences among multiple groups were analyzed using one-way ANOVA followed by Tukey's post hoc test for pairwise comparisons. P<0.05 was considered to indicate a statistically significant difference.

To investigate the effect of the KOR agonist, U50488H, on the spatial memory of APP/PS1 mice, U50488H was administered subcutaneously for 28 days, using an osmotic pump. The Morris water maze was used to evaluate the cognitive function of the mice. Although impaired learning and spatial memory in the Morris water maze was observed in APP/PS1 mice compared with control mice, the mice in the U50488H group showed a significant improvement in their cognitive abilities (Fig. 1A-C). Aβ plaque deposition was decreased in the prefrontal cortex and HC in the APP/PS1 mice treated with U50488H (Fig. 1D). The pathological changes in brain tissue samples were observed using H&E staining. The neurons in 12 month-old APP/PS1 mice were sparse and disorganized, with the loss of numerous neurons. However, the morphology of neurons in the group treated with U50488H was improved (Fig. 1E). Furthermore, the expression of PSD95 was increased in the HC of U50488H-treated mice. Taken together, these results demonstrated that U50488H reduced brain injury in APP/PS1 mice.

Glu is the primary neurotransmitter in the CNS. When the Glu concentration increases significantly, extensive pathological damage occurs in brain tissues (29,30). ELISA analysis revealed that Glu concentrations in the APP/PS1 mice were significantly higher than in the U50488H treatment group (Fig. 2A). As the Ca²⁺/CaMKII/CREB signaling pathway is involved in synaptic plasticity, the central components of the pathway, including NMDAR, CaMKII, p-CaMKII, CREB and p-CREB were analyzed. Based on the western blot results, the expression levels for NMDARs and the phosphorylation levels of CaMKII and CREB in the U50488H-treated group decreased significantly when compared to the APP/PS1 mice (Fig. 2B). Golgi-Cox staining was used to analyze the number of dendritic spines in the HC. The results demonstrated that the number of spines increased in the treated mice when compared to the APP/PS1 mice, which indicated that U50488H helped improve the growth of dendritic spines and improved synaptic plasticity in the treated mice (Fig. 2C). To investigate the underlying mechanism, western blot analysis was performed to detect the synaptic plasticity-related proteins, neural cell adhesion molecule, N-methyl D-aspartate receptor subtype 2B, Glu receptor 1, PSD95 and α-synuclein. The expression levels for these proteins were observed to be higher in the U50488H-treated group than in the APP/PS1 mice (Fig. 2D). These results demonstrated that treatment with U50488H improved synaptic plasticity in APP/PS1 mice.

It has previously been reported that cell pyroptosis depends on cysteine aspartic acid-specific protease to promote the release of the inflammatory mediators, IL-1β and IL-18 (31). Notably, the levels of collagen II antibody, IL-18 and IL-1β in the serum of mice treated with U50488H decreased significantly (Fig. 3A). This indicated that U50488H played a critical role in inhibiting microglial pyroptosis in APP/PS1 mice. Therefore, immunofluorescence was performed to detect cell pyroptosis-related proteins. The results showed that the expression levels of NLRP3 and pro-caspase-1 in the U50488H-treated mice decreased significantly (Fig. 3B and C). To confirm the fluorescence results, pro-caspase-1, pro-IL-1β, apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC) and NLRP3 were analyzed using western blot analysis. The expression levels of ASC, pro-IL-1β, pro-caspase-1 and NLRP3 in the U50488H-treated group were significantly lower when compared with those of the APP/PS1 mice (Fig. 3D). These results indicated that the KOR agonist inhibited microglial pyroptosis in APP/PS1 mice.

CaMKII is an important member of the calmodulin regulatory protein family and plays a role in the pathophysiological processes of numerous diseases, such as neuropsychological disorders (32). Therefore, the regulatory mechanisms of the Ca²⁺/CaMKII/CREB signaling pathway in synaptic plasticity were investigated. KN93 was injected intraperitoneally into APP/PS1 mice for 28 days. The mice also received U50488H, which was administered subcutaneously for 28 days using an osmotic pump. KN93 is a CaMKII specific inhibitor, which inhibits its phosphorylation activity. Learning and memory was assessed in the treated and control mice using the Morris water maze. The learning and memory abilities of the KN93-treated group was observed to decrease (Fig. 4A). In addition, the density of the dendritic spines in the CA1 area of the HC was evaluated using Golgi-Cox staining. The dendritic spine density in the KN93-treated group was decreased when compared to that of the untreated group (Fig. 4B). Furthermore, the mechanisms by which the CaMKII antagonist blocked changes in synaptic plasticity induced by the KOR agonist were evaluated. Western blot analysis showed that the expression of synaptic plasticity-related proteins decreased in the KN93-treated group (Fig. 4C). These observations demonstrated that the CaMKII inhibitor blocked the effects of U50488H, which had improved synaptic plasticity in the APP/PS1 mice.

Based on the abovementioned results, it was hypothesized that the CaMKII inhibitor blocked inhibition of the KOR agonist on pyroptosis. The ELISA assay revealed a significant increase in the expression levels of pyroptosis-related proteins (Fig. 5A). Treatment with KN93 prevented the decrease in NLRP3 and pro-caspase-1 protein expression when compared to mice in the U50488H-treated group (Fig. 5B and C). In addition, changes in the expression of pyroptosis-related proteins were examined. The results consistently demonstrated that the expression levels of NLRP3, ASC, pro-caspase-1 and pro-IL-1β proteins increased in the KN93-treated mice (Fig. 5D). These observations demonstrated that the inhibition of pyroptosis in APP/PS1 mice microglia by the KOR agonist could be eliminated by KN93 treatment.