Probucol, a “non-statin” cholesterol-lowering drug, ameliorates D-galactose induced cognitive deficits by alleviating oxidative stress via Keap1/Nrf2 signaling pathway in mice

Effect of Prob on plasma cholesterol, organ index and general appearance in mice

As shown in Figure 1A, the levels of total cholesterol (TC) in Prob-treated groups significantly decreased compared to Prob-untreated groups, indicating the hypocholesterolemic effect of Prob. The body weight and food consumption of each mouse were also observed during Prob treatment. The results showed that Prob treatment did not affect body weight and food consumption of mice, suggesting that Prob treatment did not affect the general appearance of mice (Figure 1B and 1C). Additionally, the organ indices of liver, spleen and kidney in D-gal group were significantly reduced compared to the control group, whereas, Prob treatment markedly reversed D-gal-induced decrease in organ indices, implying that Prob may prevent D-gal-triggered organ atrophy (Figure 1D–1F).

Figure 1. Effect of Prob on plasma cholesterol, organ index and general appearance in mice. (A) Effect of Prob on plasma cholesterol level. (B, C) Effect of Prob on body weight and food consumption of mice, respectively. (D) The representative picture of liver, kidney and spleen of mice. (E–G) Effect of Prob on liver, kidney and splenic organ index, respectively. Data in each experiment represent mean ± SEM from 10 independent samples. Statistically significant differences were calculated by one-way ANOVA using the SPSS 20.0 software. *P<0.05 and ** P<0.01, versus D-gal group.

Prob treatment improves the cognitive deficits in D-gal-induced aging model mice

MWM test

The spatial learning and memory was detected using MWM test. After four-day training, escape latency and percentage of time in the target quadrant on day 5 were recorded. As illustrated in Figure 2A and 2B, escape latency in D-gal was markedly longer than that in the control group, while the percentage of quadrant time significantly increased in the control group than that in the D-gal group, indicating the impaired cognitive function was induced by D-gal. Notably, Prob could remarkably shortened escape latency and prolong the quadrant time in the targeted quadrant of mice treated with D-gal (Figure 2A and 2B), implying that Prob holds the potential to improve learning and memory deficits induced by D-gal.

Figure 2. Prob treatment improves the cognitive deficits in D-gal-induced aging model mice. (A) The representative traces during the probe test. (B, C) Effect of Prob on escape latency and percentage of quadrant time in the MWM test, respectively. (D, E) Effect of Prob on step-through latency and errors in training in passive avoidance test. Data in each experiment represent mean ± SEM from 10 independent samples. Statistically significant differences were calculated by one-way ANOVA using the SPSS 20.0 software. *P<0.05 and ** P<0.01, versus D-gal group.

Passive avoidance test

The retention memory was evaluated using the 24h retention trial of the passive avoidance test. The results showed that higher memory errors and poorer retention latency in D-gal group than that in the control group was observed (Figure 2A and 2B), suggesting that D-gal induces retention memory impairments. Importantly, Prob treatment significantly reversed D-gal-induced increased memory errors and declined retention latency (Figure 2A and 2B), implying that Prob possess the capacity to ameliorate D-gal-triggered retention memory loss.

Prob treatment alleviates hippocampal neuronal loss in D-gal-induced aging mice

It has been documented that neurons in hippocampal CA1 region are highly susceptible to oxidative stress, which may cause neuronal loss and morphological change. Thus, in the present study, the hippocampal neurons in the CA1 region were chosen to be stained by H&E and morphology of neurons in the CA1 region was observed using 200×light microscopy. The results showed that the hippocampal neurons in the control group were arranged in an organized pattern with clear boundaries, whereas the hippocampal neurons of D-gal group exhibited loose and irregular arrangements and condensed nucleus was observed (Figure 3A). Importantly, the neurons in hippocampal CA1 region of Prob group displayed regularly and densely and fewer karyopyknotic neurons were found (Figure 3A). Additionally, using Imaging-Pro-Plus software, the number of neurons in hippocampal CA1 region was calculated. The results that the number of neurons in D-gal group was significantly lower than control group, whereas, Prob treatment markedly increased neuronal numbers as compared to D-gal group (Figure 3A).

Figure 3. Prob treatment alleviates hippocampal neuronal loss in D-gal-induced aging mice. (A) H&E staining. (B) Nissl staining. (C) Cell apoptosis was detected by the TUNEL assay. The representative sections of hippocampal CA1 region were chosen and used to quantify positive cell numbers by a blinded observer. Data in each experiment represent mean ± SEM from 4-5 independent samples. Statistically significant differences were calculated by one-way ANOVA using the SPSS 20.0 software. *P<0.05 and ** P<0.01, versus D-gal group.

Neuronal numbers in the CA1 region were also evaluated using Nissl staining. The results revealed that the number of Nissl positive neurons in D-gal group was remarkably decreased compared with the control group, while Prob treatment inhibited the reduction of neuronal numbers induced by D-gal (Figure 3B), indicating that Prob could rescue neuronal loss in the CA1 region. Additionally, cell apoptosis of CA1 region in D-gal group was higher than that in the control group, while Prob treatment restrained D-gal-induced apoptosis (Figure 3C), suggesting the inhibitory role of Prob in neuronal apoptosis.

Collectively, these results suggest that Prob treatment has an ability to alleviate hippocampal neuronal loss in D-gal-induced aging mice.

Prob treatment ameliorates D-gal-induced oxidative stress in D-gal-induced aging mice

As shown in Figure 4, the cerebral ROS and MDA levels in D-gal group were significantly higher than those in the control group, indicating that D-gal triggers oxidative stress in the brains of aging mice. Notably, Prob treatment remarkably inhibited the increased levels of ROS and MDA in D-gal-treated mice (Figure 4A, 4D and 4E), suggesting that Prob can ameliorate D-gal-induced cerebral oxidative stress. However, Prob did not affect MDA and ROS levels in normal mice.

Figure 4. Effect of Prob on ROS and MDA level, and the activities of SOD and GSH-PX. (A–C) MDA level and the activities of SOD and GSH-PX in plasma, respectively. (D–G) Level of ROS and MDA, and the activities of SOD and GSH-PX in plasma, respectively. Data in each experiment represent mean ± SEM from 10 independent samples. Statistically significant differences were calculated by one-way ANOVA using the SPSS 20.0 software. *P<0.05 and ** P<0.01, versus D-gal group.

Prob improves the activity of SOD and GSH-PX in D-gal-induced aging mice

The amelioration of oxidative stress by Prob in D-gal-induced aging mice suggests that Prob may influence the activities of antioxidant enzymes. The results showed that the activities of SOD and GSH-PX in the plasma and brains of D-gal group was significantly reduced compared to control group (Figure 4B, 4C, 4F and 4G). Importantly, Prob treatment improved the activities of SOD and GSH-PX in the plasma and brains of D-gal-induced aging mice (Figure 4B, 4C, 4F and 4G). Nevertheless, Prob did not impact on the activities of SOD and GSH-PX in the plasma and brains of normal mice (Figure 4B, 4C, 4F and 4G).

Effect of Prob on the expression of SOD, GSH-PX and HO-1 in D-gal-induced aging mice

As illustrated in Figure 5, the mRNA levels of cerebral SOD, GSH-PX and HO-1 in D-gal group were markedly higher than those in the control group. However, the protein levels and activities of SOD, GSH-PX and HO-1 in the brain were remarkably declined in D-gal group as compared to control group (Figure 5). Importantly, Prob treatment elevated mRNA and protein levels of cerebral SOD, GSH-PX and Ho-1 of D-gal-induced aging mice (Figure 5).

Figure 5. Effect of Prob on SOD, GSH-PX and HO-1 expression. (A–C) SOD, GSH-PX and HO-1 mRNA expression, respectively. (D–F) SOD, GSH-PX and HO-1 protein expression, respectively. Data in each experiment represent mean ± SEM from 10 independent samples. Statistically significant differences were calculated by one-way ANOVA using the SPSS 20.0 software. ** P<0.01, versus D-gal group.

Effect of Prob on the expression of Keap1/Nrf2 pathway

Since the expression of SOD, GSH-PX and Ho-1 gene is regulated by Keap1/Nrf2 pathway, we speculated that Prob might have impacts on Keap1/Nrf2 signaling expression. The results of Western blot assay showed that the levels of Keap1 and Nrf2 in D-gal group did not exhibit significant difference compared to control group (Figure 6). Notably, Prob could enhance Nrf2 expression in the brains of D-gal-induced aging mice (Figure 6). However, Prob had no effect on cerebral Keap1 expression (Figure 6). These results indicate that Keap1/Nrf2 pathway is involved in the enhanced expression of antioxidant enzymes by Prob.

Figure 6. Effect of Prob on the expression and dissociation of Keap1/Nrf2 complex. (A) The dissociation of Keap1/Nrf2 complex was assessed by Co-IP assay. (B) The protein expression of Nrf2 in nucleus was assessed by Western blot assay. Data in each experiment represent mean ± SEM from 4-5 independent samples. Statistically significant differences were calculated by one-way ANOVA using the SPSS 20.0 software. ** P<0.01, versus D-gal group.

Prob promotes dissociation of Keap1/Nrf2 complex leading to the accumulation of Nrf2 in nucleus

It has been well established that oxidative stress leads to the dissociation of Keap1/Nrf2 complex promoting Nrf2 to accumulate in the nucleus and activate gene expression of antioxidant enzymes, which in turn, ameliorates cellular oxidative stress. Thus, we investigated whether Prob has an impact on the dissociation of Keap1/Nrf2 complex. The results of Co-IP analysis showed that the ratio of ip/input of Nrf2 in D-gal group remarkably reduced as compared to control group (Figure 6A), indicating that D-gal promotes the uncoupling of Keap1/Nrf2 complex. Importantly, Prob treatment facilitated the dissociation of Keap1/Nrf2 complex as represented by a significant decrease in the ratio of ip/input of Prob group than D-gal group (Figure 6A). Additionally, the nuclear expression of Nrf2 in Prob group was markedly increased than that in D-gal group (Figure 6B), implying that Prob promotes dissociation of Keap1/Nrf2 complex leading to the accumulation of Nrf2 in nucleus.

Drugs and reagents

Prob was obtained from Shanghai Aladdin Bio-chem Technology Co., Ltd (Shanghai, China). SOD, GSH-PX activity kits, MDA and ROS detection kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Keap1, Nrf2, SOD and GSH-PX antibodies were from Santa Cruz Biotechnology, Inc. (Texas, USA). Ho-1 antibody was from Merck Millipore (Massachusetts, USA). Other reagents were all provided from Sigma-Aldrich unless otherwise noted.

Animals, drug treatment, and tissue preparation

Six- to eight-week-old adult male Kunming strain mice obtained from Xuzhou Medical University were randomly divided into five groups: Control group, Control+ Prob high-dosage group, D-gal group, D-gal+ Prob high-dosage group and D-gal+ Prob low-dosage group. The low- and high-dosage Prob-treated groups were intraperitoneally injected with 10 and 20mg/kg Prob daily, respectively; D-gal-treated groups were given subcutaneously with D-gal (150mg/kg) for six weeks. The treatment regimens of Prob and D-gal were according to previous studies [22, 35]. Control and D-gal groups were administrated intraperitoneally with the same volume of distilled water. The mice were euthanatized while the brains were excised for H&E staining, activity detection of antioxidant enzymes, western blotting, and real-time PCR assay. Moreover, liver, kidney and spleen of mice were excised for calculating liver, kidney and splenic organ index respectively. The organ index was calculated using the following equation: organ index = organ weight (g)/ body weight (g) × 100%. Animal care and experimental procedures were implemented in accordance with the document “Guidance Suggestions for Caring for Laboratory Animals” produced by the Ministry of Science and Technology of China in 2006.

Morris water maze test

Cognitive function was detected by Morris Water Maze (MWM) test as previously described [36, 37]. The mice were subjected to four days of training trials. In each trial, the mice were trained to locate the hidden platform. On day 5, the capacity of spatial memory was detected using a spatial probe test. In the test, the percentage of time spent in the quadrant where the former platform was placed was recorded.

Passive avoidance test

Passive avoidance test was performed for measuring memory retention in mice according to a previous study [38]. Briefly, the mice were placed individually in the illuminated compartment of automated passive avoidance system. By pressing the pedal, the apparatus was activated. The guillotine door was raised after 10 seconds allowing the mice to enter the dark compartment. The door was then closed and mice were given a foot-shock (0.2mA), for 2 seconds on training day. 24h after the training session, memory errors and step-through latency were recorded. If mice did not enter the dark compartment within 5min, the mice were assigned a score of 300 seconds.

Haematoxylin and eosin (H&E) staining

The H&E staining assay was performed according to previous studies [39, 40]. Briefly, after brains were excised and fixed with formaldehyde for 24h, samples were rehydrated and embedded in paraffin. Sections of brain were prepared and stained by haematoxylin and eosin. The histological changes were observed by a light microscope (Olympus BX-50, Japan).

Nissl staining

Nissl staining was performed according to the manufacturer’s instructions (Beyotime Biotechnology, China). In brief, ethanol-dehydrated samples were prepared and stained with Nissl staining solution for 10min at 37 °C. The total number of cells at CA1 region in each section was counted.

TUNEL staining

TUNEL staining assay was used to determine the degree of cell apoptosis in hippocampal CA1 region according to the manufacturer’s instructions (Meilun Biotechnology, China). Sections were also stained for neuronal marker microtubule-associated protein (MAP2) (1:200, Proteintech, USA), with nuclei stained with DAPI, to identify neurons in hippocampal CA1 region. The percentages of neurons with apoptotic nuclei (both MAP2 and TUNEL positive) were then quantified.

Real-time PCR

RT-PCR assay was performed as previously described [41, 42]. After extraction of mRNA and reverse transcription, samples were mixed with SYBR Green Master Mix and primers, and then, the reactions were performed. The primer sequences were as follows: SOD (105bp), sense (5′- GGCAAAGGTGGAAATGAAGA-3′), antisense (5′- CTCAGACCACACAGGGAATG-3′); GSH (184bp), sense (5′- CGAGCGAGTGGTGACGTA TG-3′), antisense (5′- ACGGGCAGTATAGTCGTCCT-3′); HO-1 (117bp), sense (5′- GAACCCAGTCTATGCC CCAC -3′), antisense (5′- GGCGTGCAAGGGATGATT TC -3′); surviving (105bp), sense (5′-CGTCACTTTGT CTGCCACTC-3′), antisense (5′-TCAGCCCTTAGCAC TCACCT-3′); ACTB (130bp), sense (5′-GTGCTATGT TGCTCTAGACTTCG-3′), antisense (5′- ATGCCACAG GATTCCATACC-3′).

Western blot assay

Western blot assay was conducted as previously described [37, 43]. The protein samples were extracted and separated by SDS-polyacrylamide gel electrophoresis in 10% Tris-glycine gels and transferred to a nitrocellulose membrane. Primary antibodies including Keap1 (dilution: 1:500), Nrf2(dilution: 1:500), SOD (dilution: 1:500), GSH-PX (dilution: 1:500) and Ho-1 (dilution: 1:500) were incubated at 4 °C overnight, followed by incubation with IRDye purified secondary antibody (dilution: 1:10000). The immunopositive bands were visualized at Ex/Em=778 nm/795 nm.

Cholesterol level detection

Total cholesterol level was measured in plasma using an enzymatic method according to the oxidase/peroxidase system using commercial kit reagents (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

SOD, GSH-PX activity and MDA detection

SOD, GSH-PX activity and MDA level were detected according to SOD, GSH activity and MDA detection assay kit instructions, respectively. In brief, action buffers were added to the supernatants of tissue samples. The mixtures were incubated at 37 °C and the optical densities were measured at 550nm, 412nm, and 532nm using visible spectrophotometer for SOD, GSH-PX activities and MDA, respectively. The activities of SOD and GSH-PX were expressed as the μmoL per g of protein sample, and the level of MDA was expressed as nmoL/mL.

ROS detection

ROS level was detected according to ROS detection assay kit instruction. Briefly, 100nM DCFH-DA was added to single-cell suspension for 30min. Then cells were collected, washed and detected at 485nm excitation wavelength and 525nm emission wavelength. The fluorescence intensity of ROS was expressed as fluorescence intensity per μg protein.

Co-Immunoprecipitation (Co-IP) assay

Co-IP assay was performed according to a previous study [44]. Briefly, brain samples were lysed in iced cold PIPA buffer and the supernatants were collected by centrifuging at 14000rpm. The lysates were incubated with Keap1 antibody and protein A/G agarose gel overnight. The gel was washed with RIPA buffer, boiled with SDS loading buffer and subjected to SDS-PAGE resolution and Western blot assay. Nrf2 antibody was used to detect the interaction of these two proteins.

Quantification analysis

For H&E, Nissl and TUNEL/MAP double staining assays, cells were analyzed according to a previous study [45]. Briefly, the sections approximately at the center of the hippocampal CA1 region were selected at 100 μm intervals from 3.2 mm anterior to the bregma to 6.8 mm posterior to the bregma, and the neuronal numbers of 5 sections of each sample per group were analyzed by a blinded observer using Imaging-Pro-Plus 6.0 software (Media Cybernetics Inc., America).

Statistical analysis

The data of each group were presented as mean±SEM. One-way analysis of variance (ANOVA) was used to analyze the variance using SPSS software for Windows 20.0. P<0.05 was considered to be statistically significant.