DL0410 (purity ≥98% according to HPLC) was obtained from the Institute of Materia Medica, Chinese Academy of Medical Sciences (Beijing, China). Donepezil was purchased from Shandong Jinan Dexinjia Biotechnology (Jinan, China). Memantine was from Shanghai Jingchun Chemical Industry (Shanghai, China). D-galactose (V900922), adenosine 5′-diphosphate sodium salt (ADP, A2754) and succinic acid sodium (224731) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Bovine serum albumin (BSA, 0332) and Tris-HCl (T22980) were obtained from Ameresco (Solon, OH, USA). L-glutamate, sucrose, ethylenediaminetetraacetic acid disodium salt dehydrate (EDTA·2Na), KH₂PO₄, Na₂HPO₄, KCl, NaCl and MgCl₂·6H₂O were purchased from Beijing Chemical Reagents Company (Beijing, China). L-malate (TM002601) was obtained from Sinopharm Chemical Reagents (Beijing, China). RIPA Buffer (#9806) and primary antibodies against cleaved caspase 3 (#9662), cyclo-oxygenase-2 (COX2, #12282) and inducible nitric oxide synthase (iNOS, #13120) were purchased from Cell Signaling Technology (Danvers, MA, USA). An Amplex Red Acetylcholine/Acetylcholinesterase Assay kit (A12217) was obtained from Invitrogen (Carlsbad, CA, USA). Primary antibodies against GAPDH (sc25778), receptor for advanced glycosylation end products (RAGE, sc365154), phosphorylated (p)-NF-κB P65 (sc33020), glial fibrillary acidic protein (GFAP, sc33673), Ionized calcium-binding adapter molecule 1 (Iba-1, sc98468), phosphorylated-c-Jun N-terminal kinase (p-JNK, sc6254) and poly(ADP-ribose) polymerase (PARP, sc7150) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). A protease inhibitor cocktail (CW2200S), phosphatase inhibitor cocktail (CW2383S), bicinchoninic acid (BCA) Protein Assay kit (CW0014S), horseradish peroxidase (HRP)-conjugated secondary antibodies (anti-rabbit CW0234S, anti-mouse CW0221S), and ChemiGlow Western Blotting Detection Reagents (CW0049M) were purchased from Kangwei Biotechnology (Beijing, China). An advanced glycation end product (AGE)-competitive enzyme-linked immunosorbent assay (ELISA) kit (STA317) and thiobarbituric acid-reactive substances (TBARS) assay kit (for malondialdehyde (MDA) quantitation) (STA332) were obtained from Cell Biolabs (San Diego, CA, USA). Kits for total antioxidant capability (TAOC) assays (S0121), catalase assay (S0051), total glutathione peroxidase (GPx) assay (S0058) and total superoxide dismutase (SOD) assay with WST-8 (S0101) were purchased from Beyotime Biotechnology (Beijing, China).

All treatment and maintenance of animals was undertaken according to the Guide for the care and use of laboratory animals (National Institutes of Health, Bethesda, MD, USA) and approved by the Animal Care Committee of Peking Union Medical College and Chinese Academy of Medical Sciences (Beijing, China).

Male ICR mice (18–20 g, 8 weeks) were purchased from Vital River Laboratory Animal Technology (Beijing, China). Mice were raised in an environment at 23 ± 1°C and humidity of 50 ± 10%. Five mice per cage had free access to food and water under a controlled 12-h light–dark cycle (light on from 08:00 to 20:00). Mice were divided randomly into seven groups with 20 mice each group: control group, model group, DL0410-1 mg/kg group (DL-1 mg/kg group), DL0410-3 mg/kg group (DL-3 mg/kg group), DL0410-10 mg/kg group (DL-10 mg/kg group), Donepezil-3 mg/kg group (Don-3 mg/kg group), Memantine-3 mg/kg group (Mem-3 mg/kg group).

D-galactose was dissolved in sterile saline. DL0410, donepezil and memantine were dissolved in distilled water. They were given at 0.1 mL/10 g. In the first 6 weeks, mice in the control group were administered (s.c.) saline in the neck, and mice in the other six groups were administered D-galactose (180 mg/kg, s.c.) in the neck. At the end of the 6th week, the step-down test was used to confirm impairment of memory and recognition in mice treated with D-galactose (Figures 1B,C). From the 7th week, mice in the control group were given water (p.o.) and saline (s.c.) in the neck, mice in the model group were given water (p.o.) and D-galactose (s.c.) in the neck, and mice in the other five groups were given the corresponding drugs (p.o.) and D-galactose (s.c.) in the neck. Four weeks later, behavioral tests were conducted, and all tests were undertaken 30–40 min after drug administration. By the end of the behavioral tests, two mice in the control group, two mice in the model group, three mice in the Mem-3 mg/kg group, one mouse in the DL-1 mg/kg group, two mice in the DL-3 mg/kg group and two mice in the DL-10 mg/kg group had died.

The separated cortex and hippocampus were homogenized immediately in cold separation medium (250 mM sucrose, 1 mM EDTA, 10 mM Tris, 0.1% BSA, pH 7.4) with a glass homogenizer on ice. The homogenate was centrifuged at 1000× g for 10 min at 4°C, and the supernatant was removed to a new Eppendorf tube. Then, the supernatant was centrifuged at 10,000× g for 10 min at 4°C. The supernatant was discarded, and the pellet was resuspended in 100 μL of cold separation medium. The protein concentration of an individual mitochondrion was determined using a BCA protein assay kit (Avetisyan et al., 2016).

A Clark oxygen electrode was used to evaluate mitochondrial respiration. First, 40 μL of an individual mitochondrion and 460 μL of respiratory medium were added into the reactor, and the baseline recorded. After the baseline had been stable for 1 min, 3 μL of L-glutamate (2 mol/L) and 3 μL of L-malate (1 mol/L) or 6 μL of succinate (1 mol/L) was added to induce a decrease in oxygen concentration. Then, 3 μL of ADP (50 mmol/L) was added to induce stage-3 respiration. When ADP was exhausted, stage-4 respiration appeared. The following indices were calculated: respiratory velocity of stage 3 (V3), V4, respiration control rate (RCR = V3/V4), and oxidative phosphorylation rate (OPR = V3 × ADP/O) (Zhang et al., 2017).

Thirty minutes after drug administration, mice were sacrificed. The cortex and hippocampus were collected and stored at −80°C.

In an assay to measure the level of ACh and activity of AChE, AGE, MDA and anti-oxidative enzymes, the cortex and hippocampus were homogenized together by Omni bead ruptor in 5 volumes of 0.01 M phosphate-buffered saline (PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄). Then, the homogenates were centrifuged at 12,000× g for 30 min at 4°C. The supernatant was collected and stored at −80°C for measurement.

Before western blotting, the cortex and hippocampus were homogenized, respectively, in five volumes of RIPA buffer supplemented with a protease inhibitor cocktail and phosphatase inhibitor cocktail. The supernatant was collected after centrifugation at 12,000× g for 30 min at 4°C. Loading buffer was added to the supernatant, which was then boiled for 10 min. The denatured protein was stored at −80°C. The protein concentration was determined by the BCA protein assay kit.

Measurement of AChE activity and Ach level was conducted in 96-well plates following the instructions of the Amplex Red Acetylcholine/Acetylcholinesterase Assay kit. For the assay of AChE activity, the reaction involved 100 μL of sample, 50 μM of Ach, 200 μM of Amplex Red Reagent, 0.1 U/mL of choline oxidase and 1 U/mL of HRP. The reaction was undertaken at room temperature for 20 min in the dark. Fluorescence intensity was measured at an excitation wavelength of 560 nm and emission wavelength of 580 nm.

The reaction system for ACh measurement was 100 μL of sample, 0.5 U/mL of AChE, 200 μM of Amplex Red Reagent, 0.1 U/mL of choline oxidase and 1 U/mL of HRP. The reaction system was incubated at room temperature for 30 min in the dark. Fluorescence intensity was measured at an excitation wavelength of 560 nm and emission wavelength of 580 nm (Park et al., 2016).

Ten percent SDS-PAGE was used to separate protein samples (about 80 μg protein), which were then transferred to PVDF membranes (Millipore, Billerica, MA, USA). PVDF membranes were then blocked by 5% BSA for 1 h at 37°C. PVDF membranes were cut off in accordance with the molecular weight, and incubated with different primary antibodies (rabbit anti-GAPDH, 1:1000 dilution; mouse anti-RAGE, 1:800; rabbit anti-p-NF-κB P65, 1:800; mouse anti-GFAP, 1:1000; rabbit anti-Iba-1, 1:800; mouse anti-p-JNK, 1:800; rabbit anti-PARP, 1:1000 from Santa Cruz Biotechnology; rabbit anti-COX2, 1:1000; rabbit anti-iNOS, 1:1000 from Cell Signaling Technology) at 4°C overnight. On the second day, PVDF membranes were incubated with HRP-conjugated secondary antibody (anti-mouse and anti-rabbit, 1:2000) for 2 h at 37°C. Protein bands were detected by an ECL western blotting kit using a ChemiDoc-It™ imaging system (UVP, Upland, CA, USA). GAPDH was selected as a control. The gray value was analyzed by Gel-pro 32 (Media Cybernetics, Rockville, MD, USA).

Three mice per group were selected at random and anesthetized by CO₂ exposure. Perfusion was conducted with saline solution and 4% paraformaldehyde. Right brains were removed and fixed with 4% paraformaldehyde overnight. Brains were embedded with paraffin, cut into pieces of thickness 30 μm, and used for IHC staining.

Slides were dewaxed with xylene and a graded series of ethanol solutions. They were incubated with primary antibody (anti-GFAP and anti-Iba-1) overnight at 4°C. After rinsing with PBS, slides were incubated with secondary antibody for 1 h at room temperature. Then, they were visualized with diaminobenzidine using an Elite ABC kit (Vector, Burlingame, CA, USA). Hematoxylin was used for nuclear staining. For quantification of the mean integral optical density (IOD), three fields of the hippocampus or cortex were chosen for each slide. Each individual field was imaged at 400× magnification using a microscope and calculated using Image-Pro Plus 6.0 (Media Cybernetics, Rockville, MD, USA).

After perfusion with saline solution and 4% paraformaldehyde, CA1 areas of the hippocampus in left brains were fixed with 4% paraformaldehyde for 2 h. Then, they were placed in 2.5% glutaraldehyde overnight at 4°C, followed by washing with 0.01 M PBS and preservation in 0.01 M PBS at 4°C. They were dehydrated with a graded series of alcohol solutions, embedded in Epon resin, and cut into pieces of thickness 50–60 nm. Slides were stained with uranyl acetate and lead citrate and observed under a transmission electron microscope (H-7650; Hitachi, Tokyo, Japan). For the ultrastructure of mitochondria, images were observed at 150,000× magnification. For the quantification of synapses, 10–15 fields were chosen randomly for each slide at 50,000× magnification.

Data are expressed as the mean ± SEM. Statistical analysis were conducted in GraphPad Prism software (San Diego, CA, USA). Data for the navigation trial of the Morris water maze was analyzed with two-way ANOVA with repeated measures, with treatment and time taken as two factors. Other data were evaluated with one-way ANOVA following Dunnett’s multiple comparisons test. p < 0.05 was considered significant.

DL0410 is a potent inhibitor of AChE in vitro. In the present study, the effect of DL0410 on the cholinergic system in the hippocampus and cortex was investigated. There were no differences between the control group and model group in terms of ACh level or AChE activity (Figure 3). DL0410 (10 mg/kg) and donepezil tended to increase the ACh level and inhibit AChE activity, but memantine had no effect on these two indices.

In healthy individuals, there is a balance between oxidative and anti-oxidative systems. However, this system is broken if oxidative stress becomes too strong for the anti-oxidative system to overcome, and cellar components (e.g., lipids, proteins) would be subject to damage. The anti-oxidative system consists of reducing substances and anti-oxidative enzymes (catalase, GPx and SOD). ROS, the main oxidant, arises from the damage to the electron-transfer chain in mitochondria. We explored the ameliorating effect of DL0410 on oxidative stress from the aspects of antioxidative defense and mitochondrial protection.

Accumulation of AGEs and MDA (hallmarks of oxidative damage) in the model group was significantly greater than that in the control group (p < 0.01 for both; Figures 4A,B). DL0410 could reduce levels of AGEs and MDA (p < 0.01 for AGE at 1, 3 and 10 mg/kg, p < 0.05 for MDA at 1 mg/kg, p < 0.001 for MDA at 3 mg/kg, p < 0.01 for MDA at 10 mg/kg), whereas donepezil and memantine reduced only the MDA level (p < 0.001 for donepezil and p < 0.01 for memantine).

The TAOC of the hippocampus and cortex was investigated further using TEAC as the control (Figure 4C). The TEAC of the model group was lower than that of the control group (p < 0.01). DL0410, donepezil and memantine could increase the TEAC level in the hippocampus and cortex (p < 0.0001 for DL0410 at 3 mg/kg, p < 0.01 for DL0410 at 1 mg/kg, and p < 0.05 for DL0410 at 10 mg/kg, donepezil and memantine).

Moreover, we tested the activities of catalase, GPx and SOD in the hippocampus and cortex (Figures 4D–F). The activity of catalase in the model group was significantly lower than that of the control group (p < 0.05), whereas the activities of GPx, and SOD in the model group were not affected. In comparison with model mice, DL0410 could improve the activities of catalase, GPx and SOD (p < 0.05 for catalase; p < 0.05 for GPx at 1 and 3 mg/kg, p < 0.001 for GPx at 10 mg/kg; p < 0.05 for SOD at 3 mg/kg). Donepezil only improved the SOD notably (p < 0.05), and memantine increased the activity of GPx (p < 0.01).

The mitochondrial respiration of the hippocampus and cortex was evaluated using L-glutamate and L-malate (NADH chain) or succinate (FADH₂ chain) as substrates. The improvement of DL0410 on the NADH chain is shown in Figures 4G,H. Long-term administration of D-galactose could decrease the RCR (p < 0.0001) and OPR (p < 0.0001) in comparison with the control group. DL0410 could improve the RCR (p < 0.05 at 1, 3 and 10 mg/kg) and increase OPR (p < 0.0001 at 1, 3 and 10 mg/kg). The effect of DL0410 on the FADH₂ chain is shown in Figures 4I,J. Compared with the control group, D-galactose administration did not affect the RCR, whereas D-galactose could decrease the OPR (p < 0.01). DL0410 could increase OPR significantly (p < 0.0001 at 1, 3 and 10 mg/kg). Donepezil and memantine could also increase the RCR in the NADH chain and OPR in both chains.

The ultrastructure of mitochondria in the hippocampus was explored further by TEM. Mitochondria in the control group were in an electron-lucent state, with integrated mitochondrial membranes and clearly visible mitochondrial cristae (Figure 4K). In contrast, mitochondria in the model group displayed blurred cristae in electron-dense matrix, and non-integrated mitochondrial membranes. DL0410 (10 mg/kg) could improve the impaired structure of mitochondria remarkably, and the mitochondria showed integrated membranes and clear mitochondrial cristae in electron-lucent matrices.

We have showed that D-galactose could induce AGEs accumulation in the hippocampus and cortex. RAGE (a multi-ligand receptor of AGEs expressed in tissue with high levels of glycol-oxidation) has been reported to be co-localized with activated astrocytes and microglia in the brain. Interaction of AGE with RAGE could activate NF-κB, which would further regulate the expression of pro-inflammatory mediators. In this part of the study, we further investigated the inflammation level in the hippocampus and cortex.

First, we investigated the status of astrocytes and microglia. The specific markers GFAP and Iba-1 were used to label activated astrocytes and microglia, respectively. In the resting state, astrocytes exhibited a small soma and 3–6 processes, but were activated by D-galactose and resulted in hypertrophy and more processes. The swelling of microglia was found in the activated state. Compared with the control group, the expression of GFAP and Iba-1 in the model group was upregulated significantly (p < 0.01 for GFAP in the cortex and hippocampus; p < 0.01 for Iba-1 in cortex, p < 0.05 for Iba-1 in the hippocampus; Figure 5), denoting activation of astrocytes and microglia in model mice. DL0410 not only inhibited the expression of GFAP in the hippocampus and cortex (p < 0.01 for the cortex at 3 mg/kg, p < 0.001 for the cortex at 10 mg/kg; p < 0.01 for the hippocampus at 3 and 10 mg/kg), but also reduced the expression of Iba-1 in the hippocampus and cortex (p < 0.01 for the cortex at 1 mg/kg, p < 0.001 for the cortex at 3 mg/kg, p < 0.0001 for the cortex at 10 mg/kg; p < 0.05 for the hippocampus at 1 mg/kg, p < 0.01 for the hippocampus at 3 and 10 mg/kg). Donepezil and memantine inhibited GFAP expression, and donepezil affected Iba-1 expression.

Western blotting showed that RAGE expression (Figures 6A,B) in the hippocampus and cortex was upregulated by D-galactose (p < 0.01 for the cortex, p < 0.001 for the hippocampus). DL0410 reversed the high level of RAGE in the hippocampus and cortex (p < 0.05 for the cortex at 10 mg/kg; p < 0.05 for the hippocampus at 1 mg/kg, p < 0.01 for the cortex at 3 and 10 mg/kg). Western blotting for nuclear transcription factors are shown in Figures 6A,C. p-P65 in the model group was induced to a higher level in the hippocampus and cortex (p < 0.001 for the cortex, p < 0.0001 for the hippocampus), and DL0410 could notably inhibit the phosphorylation of subunit P65 in the hippocampus and cortex (p < 0.01 for the cortex; p < 0.001 for the hippocampus at 1 and 3 mg/kg, p < 0.0001 for the hippocampus at 10 mg/kg). Donepezil and memantine also decreased the expression of RAGE and p-P65 in the hippocampus and cortex.

Inflammatory mediators were explored further, including COX2 and iNOS (Figures 6A,D,E). D-galactose could induce a higher level of COX2 expression in the hippocampus and cortex (p < 0.001 for the cortex; p < 0.01 for the hippocampus). DL0410 could decrease COX2 expression significantly (p < 0.01 for the cortex at 3 mg/kg, p < 0.05 for the cortex at 10 mg/kg; p < 0.05 for the hippocampus at 1 and 3 mg/kg, p < 0.01 for the hippocampus at 10 mg/kg). Expression of iNOS was also increased (p < 0.05 for the cortex, p < 0.001 for the hippocampus), and decreased significantly by DL0410 (p < 0.05 for the cortex at 3 and 10 mg/kg; p < 0.05 for the hippocampus at 1 and 3 mg/kg, p < 0.01 for the hippocampus at 10 mg/kg). Donepezil and memantine also inhibited expression of COX2 and iNOS.

When oxidative stress and inflammation are increased, JNK known as stress activated protein kinase, would be phosphorylated and involved in apoptosis. Caspase 3, a key executor of apoptosis, is cleaved to an activated form and involved in cell death. We further investigated the level of p-JNK, cleaved caspase 3 and cleaved PARP (Figures 7A–D). Compared with the control group, D-galactose could induce a higher level of p-JNK (p < 0.001 for the hippocampus and cortex), cleaved caspase 3 (p < 0.001 for the cortex; p < 0.01 for the hippocampus) and cleaved PARP (p < 0.01 for the cortex; p < 0.0001 for the hippocampus) in the hippocampus and cortex. DL0410 was able to inhibit the expression of p-JNK, cleaved caspase 3 and cleaved PARP in the hippocampus and cortex. Donepezil and memantine could inhibit the expression of p-JNK, cleaved caspase3 and cleaved PARP in hippocampus and cortex as well.

Synapses are the basic units for the acquisition and retention of information. Synapses might be subjected to damage when if apoptosis occurs in the brain. TEM was utilized to observe changes in synapses. In comparison with the control group, the number of synapses in the model group was reduced significantly (p < 0.01), accompanied by swollen dendritic spines and blurred membranes in presynaptic and postsynaptic ends (Figure 7E). DL0410 could alleviate the swelling of dendritic spines and injured membranes, and increase the number of synapses (p < 0.001). The protection afforded by DL0410 to synapses contributes directly to the process of learning.