Cyanidin-3-O-Glucoside Protects Against Cognitive Impairment in D-Galactose-Induced Aging Mice by Regulating Nrf2 and NF-κB Pathways

D-galactose (D-gal) was purchased from Beijing Biotopped Technology Co., Ltd. (Beijing, China). Cyanidin-3-O-glucoside (C3G) (purity = 94.59% as determined by HPLC) was obtained from Nanjing Jingzhu Biotechnology Co., Ltd. (Nanjing, China). Commercial kits for the determination of malondialdehyde (MDA), total antioxidant capacity (T-AOC), superoxide dismutase (SOD), Mn-SOD, reduced glutathione (GSH), and glutathione peroxidase (GSH-Px) in serum and brain tissue homogenates were all purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Commercial kits for detecting inflammatory cytokines including interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) in serum were acquired from MultiSciences Biotech Co., Ltd. (Hangzhou, China). Antibodies against Nrf2 (Cat No. 66504-1-Ig) and Keap1 (Cat No. 60027-1-Ig) were purchased from Proteintech Group, Inc. (Wuhan, China), while antibodies against IKKβ (Cat No. R1706-13) and NF-κB p65 (Cat No. ET1603-12) were obtained from Hangzhou Huabio Co., Ltd. (Hangzhou, China). All other chemical reagents used in this study were of analytical grade.

Male C57BL/6J mice aged 6–8 weeks were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) (Laboratory Animal Production License No.: SCXK (Jing) 2021-0006). All mice were housed under standard conditions (25 °C, 12 h light/dark cycle) with free access to food and water. All experimental protocols were approved by the Animal Experiment Ethics Committee of Beijing Technology and Business University (Approval No. Lunshen 2024 No. 163). After a 7-day acclimatization period, the mice were sorted by body weight and then allocated into different experimental groups using a snake-like randomization method to ensure comparable group mean weights. Ultimately, the mice were formally divided into four groups (n = 10 per group): normal control (NC) group, model group (500 mg/kg D-gal), low-dose C3G (C3G-L) intervention group, and high-dose C3G (C3G-H) intervention group. Mice in the NC group received a daily subcutaneous injection of normal saline (10 mL/kg), while those in the other groups received a daily subcutaneous injection of 5% (w/v) D-gal (500 mg/kg). Additionally, mice in the C3G-L and C3G-H groups were administered C3G via gavage at doses of 50 and 100 mg/kg, respectively. All treatments were performed once daily for 13 consecutive weeks.

The water maze experimental method referenced the research protocol by Qin et al. [20], employing the Morris Water Maze (MWM) test to evaluate spatial memory and long-term memory capabilities in mice. The maze (Model XR-XM101, Shanghai Xinruan Information Technology Co., Ltd., Shanghai, China) consisted of a circular pool (120 cm in diameter, 50 cm in height) filled with water, with a hidden platform (10 cm in diameter, 30 cm in height) submerged 1–2 cm under the surface. Experimental parameters were set as follows: 60 s swimming duration, 15 s platform residence time, “mouse experiment mode” selected, and the red boundary box adjusted to accurately frame the pool and platform. Distinct black-and-white markers of different shapes were placed on the four walls of the pool (North, South, East, West) to provide visual cues for spatial navigation. Notably, rodents achieve learning saturation with minimal individual variability on day 5 of consecutive training [21]. Accordingly, the MWM test was initiated at week 12 post-treatment and lasted 6 days, with days 1–5 devoted to acquisition training and day 6 to the spatial probe test. Acquisition training was conducted 1 h after daily gavage administration, with 4 training trials per mouse per day. Blinding was adopted throughout the experiment, and the personnel responsible for behavioral testing and data recording were unaware of the animal grouping information. For each trial, the mouse was placed into the water facing the pool wall from a predetermined quadrant, and the time taken to locate and mount the hidden platform (escape latency) was recorded. If a mouse found and remained on the platform for 15 s within the 60 s trial, its actual latency was recorded. Otherwise, the escape latency was recorded as 60 s, and the mouse was guided to the platform and allowed to remain there for 15 s to reinforce spatial memory. The interval between consecutive trials was 30–60 s. On day 6, the hidden platform was removed for the spatial probe test, which serves as an independent assessment of memory consolidation ability. Each mouse was released into the quadrant opposite the original platform location and allowed to swim freely for 60 s. The crossing frequency over the former platform location and the time spent exploring the target quadrant were recorded.

After the intervention period, mice were fasted for 12 h and then anesthetized via isoflurane inhalation. Following blood collection, mice were euthanized by cervical dislocation, and brain tissues were immediately dissected. Blood samples were centrifuged at 3500 rpm and 4 °C, for 15 min to separate serum, which was stored at −20 °C, for subsequent detection of inflammatory cytokines and antioxidant indices. The isolated brain tissues were rinsed with pre-cooled normal saline (0.9%, w/v), and surface moisture was blotted dry with sterile filter paper before being divided into three parts. The hippocampal region was rapidly dissected. One was fixed in 10% neutral buffered formalin for histopathological analysis, and the other was wrapped in aluminum foil and stored at −80 °C for subsequent qRT-PCR and Western blot analyses. The remaining brain tissue was homogenized in ice-cold saline (1:9, w/v) to prepare a 10% (w/v) tissue homogenate. The homogenate was centrifuged at 5000× g for 10 min at 4 °C, and the supernatant was collected and stored at −80 °C for antioxidant index determination.

Hippocampal tissue was fixed in 10% neutral buffered formalin solution for 24 h, followed by sequential tissue dehydration, xylene clearing, and paraffin embedding. Serial sections (5 μm thick) were cut and stained with Hematoxylin–eosin (H&E). Pathomorphological changes in the hippocampal tissue were observed and analyzed using a PANNORAMIC DESK/MIDI/250/1000 whole-slide scanner (3DHISTECH Ltd., Budapest, Hungary). The operators and analysts were unaware of the grouping information of the experimental animals to eliminate subjective bias in the evaluation process.

Quantitative determination of malondialdehyde (MDA), total antioxidant capacity (T-AOC), superoxide dismutase (SOD), manganese superoxide dismutase (Mn-SOD), reduced glutathione (GSH), and glutathione peroxidase (GSH-Px) levels was performed in serum and brain tissue homogenates. All aforementioned indices were measured using corresponding commercial kits, strictly following the manufacturers’ instructions. For brain tissue homogenate samples, all indices were calibrated based on the content per milligram of protein to standardize the results.

The serum concentrations of interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) were quantified using mouse-specific sandwich enzyme-linked immunosorbent assay (ELISA) kits, strictly following the manufacturer’s protocols.

Total RNA was extracted from hippocampal tissue using the High-Purity Total RNA Rapid Extraction Kit (Tiangen Biotech, Beijing, China), strictly following the manufacturer’s instructions. All RNA samples were quantified using a Thermo Scientific NanoDrop^TM 2000 spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA), and RNA integrity was verified via 1.5% agarose gel electrophoresis. Subsequently, 1 μg of the extracted RNA was reverse-transcribed into cDNA using the ReverTra Ace^® qPCR RT Master Mix (Toyobo Co. Ltd., Life Science Department, Osaka, Japan). qRT-PCR amplification and detection were conducted in Low-Profile PCR tubes using a Bio-Rad CFX96 Real-Time PCR System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Each 20 μL reaction system contained: 10 μL of SYBR^® Green Real-Time PCR Master Mix (Toyobo Co. Ltd., Life Science Department, Osaka, Japan), 10 μM of each forward and reverse primer, 1 μL of cDNA, and PCR-grade water to bring the volume to 20 μL. The thermal cycling protocol was set as follows: an initial polymerase activation step at 95 °C for 3 min, followed by 40 cycles of denaturation (94 °C for 30 s), annealing (60 °C for 40 s), and extension (72 °C for 1 min). Fluorescence was measured at the end of each annealing step to monitor amplification in real time. The specificity of the PCR products was confirmed by analyzing dissociation curves. Gene expression levels were normalized to the reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Relative mRNA expression was calculated using the 2^−ΔΔCt method with 6 independent biological replicates performed for each experimental group. The primer sequences are provided in Table 1. The amplification efficiency for each primer pair was determined using a standard curve and was confirmed to be between 90% and 110%. The specificity of amplification was verified by sequencing the PCR products, which matched the expected target sequences in GenBank.

Approximately 20 mg of hippocampal tissue was homogenized thoroughly on ice in RIPA lysis buffer containing 1% protease inhibitor. After homogenization, the mixture was centrifuged at 4 °C, and the supernatant was collected. Protein concentration was determined using the BCA assay. Samples were mixed with 4× loading buffer at a 3:1 ratio, boiled for 5 min, and then cooled to room temperature for immediate use or stored at −80 °C. Proteins were separated by SDS-PAGE using a 10% separating gel and a 5% stacking gel, followed by transfer onto a PVDF membrane. The membrane was blocked with 5% non-fat milk at room temperature for 1 h and then incubated overnight at 4 °C with the following primary antibodies: Nrf2 (1:2000), Keap1 (1:2000), IKKβ (1:1000), NF-κB p65 (1:1000), and rabbit anti-β-actin (1:50,000). Subsequently, the membranes were incubated with species-appropriate HRP-conjugated secondary antibodies at a dilution of 1:20,000 for 1 h at room temperature. Protein bands were visualized using ECL chemiluminescent substrate and detected with a chemiluminescence imaging system. β-actin was used as the internal reference, and protein expression levels were normalized to the band intensity of β-actin. Quantitative analysis of band optical density was performed using ImageJ software (version 1.8.0).

The inclusion criteria required animals to be in good health, well-acclimated to the environment, and in a stable condition, with 10 animals initially allocated per group. Animals were excluded if they died unexpectedly during model establishment or exhibited significant abnormal behavioral responses during testing (e.g., frequent floating, immobility against the wall, or motor impairments). Consequently, behavioral and certain biochemical analyses were performed with n = 8 per group. For qRT-PCR, sample selection was based on predefined RNA quality control criteria. Only samples with A₂₆₀/A₂₈₀ ratios between 1.8–2.1, A₂₆₀/A₂₃₀ ratios > 2.0, and no evident degradation upon agarose gel electrophoresis were included, resulting in n = 6 per group for this analysis. For Western blot, due to the constraints of gel electrophoresis lanes and sample processing capacity, a stratified random sampling method was employed to select n = 3 representative samples per group. All sample exclusions and selections were conducted based on objective, pre-established criteria, independently of subsequent experimental outcomes, thereby avoiding selection bias. Sample sizes for each analysis are clearly indicated in the corresponding figure legends.

All data are presented as mean ± standard deviation (SD). Normality and homogeneity of variance were confirmed via the Shapiro–Wilk and Levene’s tests, respectively, prior to parametric analyses. Specifically, repeated-measures ANOVA with Bonferroni post hoc multiple comparisons was used to assess time-course data (escape latency and swimming speed) during Morris water maze acquisition training. One-way ANOVA was performed, followed by Dunnett’s test to compare Western blot band gray values, while Duncan’s multiple range test was used for all other datasets. All statistical analyses were performed using SPSS 27.0, with statistical significance defined as p < 0.05.

The Morris water maze was used to evaluate the effects of C3G on spatial learning and long-term memory in D-gal-induced aging mice. As shown in Figure 1A, escape latency decreased across training days in all groups, indicating progressive learning. After five days of acquisition training, the escape latency of mice in the model group on the 5th day was longer (47.07 ± 6.23 s) compared with the NC group (30.41 ± 5.79 s, p < 0.001). The total swimming distance was also increased in the model group (637.42 ± 162.67 cm) relative to the NC group (385.15 ± 117.98 cm, p < 0.05) (Figure 1C). These results indicated that subcutaneous injection of D-gal accelerated aging and significantly impaired learning and memory abilities in mice, confirming the successful establishment of the aging model. Importantly, no significant difference in swimming speed was observed among the groups across all training days (p > 0.05), ruling out the influence of motor function on the results (Figure 1B). Compared with the model group, C3G intervention significantly shortened the escape latency on the 5th day, with the C3G-H group exhibiting the most prominent effect (p < 0.001). Its escape latency was reduced to 27.24 ± 8.93 s, representing a 42.07% reduction and comparable to the NC group. The total distance to reach the platform was also reduced in the C3G-H group (415.35 ± 125.27 cm, p < 0.05).

Results of the spatial probe test (Figure 1D,E) revealed that compared with the NC group, the model group exhibited a decrease in both the percentage of time spent in the target quadrant and the number of platform crossings (p < 0.05). In contrast, C3G intervention increased these two indices (p < 0.05), and the values in the C3G-H group were fully restored to the levels of the NC group (p > 0.05). Consistent with the acquisition training results, the spatial probe test further demonstrated that C3G exerted an protective effect on memory function in mice, with a more pronounced effect at the high dose. Representative swimming trajectory plots (Figure 1F) intuitively reflected the behavioral differences among groups. Mice in the model group mainly displayed thigmotactic and aimless swimming, while those in the C3G-H group exhibited an ordered search strategy centered on the target quadrant, similar to that of the NC group. Collectively, these findings confirmed that C3G significantly alleviates spatial memory impairment in D-gal-induced aging mice.

Hematoxylin–eosin (H&E) staining results of hippocampal tissue from each group are shown in Figure 2. In the NC group, neurons in the hippocampal Cornu Ammonis (CA) subregions CA1, CA2, CA3, and Dentate Gyrus (DG) subregions were neatly and densely arranged, with clear cell layer structure, plump cell morphology, distinct nuclear structure, and uniform chromatin distribution. No obvious pathological damage was observed. In contrast, representative sections of the model group exhibited sparse and disorganized arrangement of cell layers in the hippocampal CA1 and CA3 subregions, with increased intercellular spacing. Surviving neurons showed prominent apoptotic or necrotic features such as cellular shrinkage, pyknosis, and hyperchromatism. The granular cell layer of the DG subregion also displayed decreased cell density and disorganized arrangement. After C3G-L intervention, the pathological state of hippocampal tissue noticeable improvement. Neuronal arrangement exhibited a more orderly appearance. Although sporadic pyknosis remained observable, its occurrence was notably less frequent and severe compared to the model group, suggesting a potential neuroprotective effect of low-dose C3G. Notably, the improvement in pathological features was more pronounced in the C3G-H group. In representative sections of this group, the cellular morphology and structure of all hippocampal subregions (CA1, CA3, DG) were close to the NC group, with neurons arranged densely and intact in morphology. Pathological changes such as pyknosis were extremely rare. These observations from representative H&E-stained sections demonstrate that C3G treatment may alleviate hippocampal neuronal damage and improve the morphological integrity of neurons and tissue structure in D-gal-induced aging mice.

To investigate the impact of C3G on oxidative stress status in aging mice, key antioxidant enzyme activities and lipid peroxidation levels were measured in serum and non-hippocampal brain tissue. As shown in Figure 3, compared with the NC group, the model group exhibited decreased activities of T-AOC, GSH, SOD, Mn-SOD and GSH-Px in serum (p < 0.05), while the MDA content was significantly increased (p < 0.05). These results indicate that the endogenous antioxidant capacity of mice in the model group was impaired, lipid peroxidation damage was exacerbated, and the aging model was successfully established. After intervention with different doses of C3G, both C3G-L and C3G-H groups showed significantly increased activities of SOD, GSH, and Mn-SOD compared with the model group (p < 0.05). In addition to these indices, the C3G-H group also exhibited elevated T-AOC and GSH-Px activities (p < 0.05), with all detected indicators restored to the levels of the NC group (p > 0.05). Meanwhile, the MDA content was reduced in both C3G-treated groups (p < 0.05). These findings demonstrate that the protective effect of C3G on oxidative stress in aging mice is dose-dependent, with the C3G-H group showing the most prominent regulatory effect, fully restoring antioxidant-related indices to normal levels.

As an organ with high oxygen consumption and high lipid content, the brain is highly sensitive to oxidative stress and serves as a key target region for age-related cognitive decline. Non-hippocampal brain tissues were used to determine the following oxidative stress parameters. Compared with the NC group, the model group showed distinct oxidative brain damage (Figure 4), as indicated by an increase in MDA content and simultaneous reductions in T-AOC, GSH, SOD, and GSH-Px activities (p < 0.05). C3G intervention alleviated the abnormal oxidative stress in the model group. The C3G-L group exhibited higher T-AOC and lower MDA content (p < 0.05). Meanwhile, the C3G-H group showed reduced MDA content and elevated T-AOC, GSH, SOD, and GSH-Px activities (p < 0.05). More importantly, all its oxidative stress indices were restored to NC group levels (p > 0.05). These results further illustrate that C3G can effectively improve redox imbalance, inhibit lipid peroxidation, and thereby provide a mechanistic basis for its neuroprotective effect in ameliorating cognitive function in aging mice.

As shown in Figure 5, the serum levels of key pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) were measured in each group of mice. Compared with the NC group, the model group exhibited increased levels of all tested cytokines (p < 0.05). This confirms that the D-gal-induced aging model successfully induced a systemic inflammatory state in mice. C3G intervention significantly reduced serum pro-inflammatory cytokine levels in aging mice in a dose-dependent manner. Specifically, while the C3G-H group showed suppression of TNF-α, IL-1β, and IL-6 levels (p < 0.05), only IL-6 was reduced in the C3G-L group (p < 0.05). These results demonstrate that C3G can effectively lower serum pro-inflammatory cytokine levels and inhibit systemic inflammatory responses in D-gal-induced aging mice. This may be one of the key mechanisms underlying its protective effects on age-related phenotypes in mice.

As shown in Figure 6, compared with the NC group, the mRNA expression of Nfe2l2 (encoding nuclear factor E2-related factor 2, Nrf2) was downregulated in the hippocampal tissue of the model group (p < 0.05). Meanwhile, the mRNA expression of its negative regulatory gene Keap1 (encoding Kelch-like ECH-associated protein 1, Keap1) was upregulated (p < 0.05). Concurrently, the mRNA expression of Nrf2 downstream target genes, including Nqo1 (encoding the phase II detoxifying enzyme NAD(P)H: quinone oxidoreductase 1) and Hmox1 (encoding the antioxidant protein heme oxygenase-1), was also downregulated (p < 0.05). These results indicate that D-gal-induced aging can significantly inhibit the transcriptional expression of the Nrf2/Keap1 antioxidant pathway in the hippocampal tissue of mice. C3G intervention effectively attenuated this transcriptional suppression in both the C3G-L and C3G-H groups. Specifically, Keap1 mRNA expression was reduced (p < 0.05), while the mRNA expressions of Nfe2l2 and its downstream target genes Nqo1 and Hmox1 were increased (p < 0.05). These findings demonstrate that C3G may enhance endogenous antioxidant defense in hippocampal tissue by potentially activating Nrf2 signaling at the transcriptional level, and subsequently upregulating the expression of downstream antioxidant-related target genes Nqo1 and Hmox1.

As shown in Figure 7, compared with the NC group, the model group exhibited upregulated mRNA expression levels of NF-κB signaling pathway key genes Ikbkb (inhibitor of nuclear factor kappa-B kinase subunit beta, IKKβ), Nfkb1 (nuclear factor kappa-B subunit 1, p50) and RelA (v-rel avian reticuloendotheliosis viral oncogene homolog A, p65) in the hippocampal tissue (p < 0.05). In contrast, the mRNA expression level of its inhibitory gene Ikbα (nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha) was downregulated (p < 0.05). These results indicate that the NF-κB inflammatory pathway exhibited marked transcriptional upregulation in the hippocampal tissue of D-gal-induced aging model mice, suggesting a state of chronic inflammation. After C3G intervention, the mRNA expression levels of Ikbkb, Nfkb1 and RelA were all reduced compared with the model group (p < 0.05). This transcriptional regulatory effect showed a clear dose dependence. Notably, all aforementioned mRNA indicators in the C3G-H group were restored to levels that did not differ significantly from the NC group (p > 0.05). The findings suggest that C3G may attenuate the transcriptional upregulation of the NF-κB pathway in the hippocampal tissue of mice by potentially modulating the IKKβ/IκBα/p65 signaling axis at the transcriptional level.

To further validate the regulatory effects of C3G on the Nrf2 and NF-κB pathways in the hippocampus of D-gal-induced aging mice, this study selected the high-dose C3G (C3G-H) intervention group for subsequent analysis, focusing on key targets of both pathways based on the qRT-PCR findings. As shown in Figure 8, compared with the NC group, the protein expression of Nrf2 was downregulated in the brain of the model group (p < 0.001). Meanwhile, the expressions of Keap1 (p < 0.001), IKKβ (p < 0.001) and the NF-κB transcription factor p65 (p < 0.01) were upregulated. These alterations collectively indicate a state of chronic inflammation associated with impaired antioxidative capacity in D-gal-induced aging mice. In the C3G-H group, protein levels of IKKβ (p < 0.001), p65 (p < 0.05) and Keap1 (p < 0.01) were all decreased, while Nrf2 protein expression (p < 0.01) was increased relative to the model group. Notably, the protein expression levels of IKKβ and p65 were even restored to those of the normal control group (p > 0.05). Collectively, these results support that C3G treatment was associated with enhanced endogenous antioxidant defenses in hippocampal tissue via changes consistent with inhibition of Keap1 and activation of Nrf2, while also reducing chronic inflammation through modulation of key proteins involved in the IKKβ/p65 signaling. These observations may provide a potential molecular mechanism underlying the neuroprotective and anti-brain aging effects of C3G.