Ginsenoside Rg1 Prevents Cognitive Impairment and Hippocampus Senescence in a Rat Model of D-Galactose-Induced Aging

All experiments were performed in accordance with institutional and national guidelines and regulations and were approved by the Chongqing Medical University Animal Care and Use Committee.

Three months old male Sprague-Dawley rats were purchased from the Medical and Laboratory Animal Center of Chongqing and housed in a temperature and light-controlled room with free access to water and food. All surgeries were performed under sodium pentobarbital anesthesia, and all the efforts were made to minimize suffering.

Sixty animals were randomly divided into 4 groups (control, D-gal-administration, Rg1 treatment, and D-gal-administration plus Rg1 treatment). In the D-gal-administration group, D-gal (120 mg/kg·d) was injected subcutaneously daily into rats for 42 days. In the D-gal-administration plus Rg1 treatment group, ginsenoside Rg1 (20 mg/kg·d) was injected peritoneally daily concomitantly for 28 days from day 15 of D-gal injection. All control animals were given saline in the same volume subcutaneously and peritoneally, respectively. In the Rg1 treatment group, saline at the same volumn with D-gal injection was injected subcutaneously for 42 days, and Rg1 (20 mg/kg·d) was injected peritoneally for 28 days from day 15 of saline injection. The body weights were measured every 3 days,there were no significant differences between the groups. The body weight data are provided in the supporting information (Figure S1 in). File S1

Ginsenoside Rg1 (RSZD-121106, Purity = 98.3%) was purchased from Xi’an Haoxuan Biological Technology Co., Ltd (Xi’an, China), dissolved in ddH₂O at the concentration of 20 mg/ml, and sterilized by ultrafiltration. GSH/GSH-px kit, SOD kit and MDA kit were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). IL-1β kit, IL-6 kit and goat anti-rabbit secondary antibody were purchased from Wuhan Boster Bio-engineering Co., Ltd. (Wuhan, China). TNF-α Kit was obtained from Uscn Life Science Inc. (Wuhan, China). BCA kit and SA-β-gal Staining kit were purchased from Beyotime Institute of Biotechnology (Shanghai, China). Anti-β-tubulin III antibody was obtained from Sigma Co. LLC. Anti-GFAP antibody was purchased from Wuhan Sanying Biotechnology Inc. (Wuhan, China).

After the 42-day treatment, spatial memory of the rats was assayed by Morris water maze task. The maze was a tank (80 cm in radius and 45 cm high) filled with water at approximately 24°C. The tank was divided into 4 quadrants, one of which contained a circular escape platform (8 cm in diameter) placed at a fixed position, 2.5 cm below the surface of the water. There were visual cues around the water maze. Oriented navigation trials were performed 4 times per day, for 7 consecutive days with a constant interval of 1 h. In each trial, the animals were gently placed in water in one of the four quadrants, and the starting quadrant was varied randomly over the trials. Rats were allowed a maximum of 90 sec to find the escape platform, where it remained for 30 sec. For all training trials, the time that it took the rat to reach the submerged platform (escape latency) was recorded to assess spatial learning ability. On the eighth day, another set of tests consisting of a 120 sec trial with the platform removed was conducted. Besides escape latency before reaching the platform, time spent in the target quadrant and the number of target crossings over the previous location of the target platform were recorded to assess spatial memory ability. The target quadrant was defined as the quadrant that previously contained the platform, the radius of which was limited to 70 cm in this assessment.

After the therapy, hippocampuses were collected and lysed in ice bath for 30 min. The supernatant was collected after centrifugation (12000 rpm, 4°C, 30 min). GSH-px activity and GSH content, SOD activity and MDA content were detected by chemical colorimetric analysis according to the manufacturer’s instructions.

To detect GSH-Px activity, the enzymatic reaction in the tube, which contained NADPH, reduced GSH, sodium azide and glutathione reductase, was initiated by addition of H₂O₂. And the change in absorbance at 340 nm was monitored. Activity is expressed as U/mg protein. To detect reduced GSH content in each group, 1% trichloroacetic acid was added to the lysates. After centrifugation at 10 000×g for 15 min, protein free lysates were obtained. The reaction mixture for determination of GSH content consisted of lysates and 5, 5′-dithiobis-(2-nitrobenzoic acid) (DTNB) 6 mmol/L. The absorbance at 405 nm was monitored for 6 min using a microtiter plate reader (Bio-Rad Ltd, Japan). The content of GSH was calculated as l mg GSH/g protein from the change in the rate of absorbance on the basis of a standard curve.

The assay for total superoxide dismutases (SOD) is based on the ability to inhibit the oxidation of oxymine by the xanthine-xanthine oxidase system. One unit (U) of SOD activity was defined as the amount that reduced the absorbance at 550 nm by 50%, and data were expressed as units per microgram of hippocaumpus protein. Thiobarbituric acid reaction (TBAR) method was used to determine the MDA which can be measured at the wave length of 532 nm by reacting with thiobarbituric acid (TBA) to form a stable chromophore production. MDA content was expressed as nmol per milligram of hippocampal protein. Protein concentration was measured using the method of Bradford. Bovine serum albumin was used as a standard.

The supernatant was collected as above, and the levels of proinflammatory cytokines IL-1β, IL-6 and TNF-α in the hippocampus in each group were measured by ELISA kit following the manufacture’s instructions. The level of both cytokines was determined by the competitive binding of the cytokines in the samples with ¹²⁵I-radiolabled IL-1β and TNF-a standards respectively. Data were shown as the actual content of the lysates per mg tissue lysates.

Animals were reanaesthetized and transcardially perfused with 4% buffered paraformaldehyde solution (pH 7.4), then the brains were separated and post-fixed for 4 hours, followed by dehydration in 20% sucrose at 4°C. Free floating sections of 20 µm were cut with a cryotome.

For immunofluorescence analysis, appropriate sections were recovered and washed with TBS with 0.3% Triton X-100 followed by blocking with 1.5% normal goat for 4 h at room temperature. Sections were then incubated with anti-β-tubulin III (a neuron marker), anti-GFAP (an astrocyte marker) and Gal-c (a mature oligodendrocyte marker) antibodies all diluted at 1∶200 for 24 h at 4°C. Later, after TBS washes, sections were incubated with secondary antibodies for 1 h at 37°C. DAPI was used for nuclear staining. Finally, 20 µl of glycerole was applied to each slide and a cover slip was sealed in place. All slides were viewed directly under a fluorescence microscope (Eclipse, Nikon). Controls included omitting the primary and secondary antibodies. The total numbers of cells were estimated on three randomly selected sections taken through the central extent of the dentate gyrus area.

The SA-β-gal (senescence-associated β-galactosidase) staining was carried out according to the manufacturer’s instructions. Frozen section preparation procedure described above was used for cytochemical staining. In brief, slides were washed twice by PBS, fixed by Fixative Solution for 15 min at room temperature, and stained by X-Gal Staining Solution (100 mM sodium phosphate, 2 mM MgCl2, 150 mM sodium chloride, 0.01% sodium deoxycholate, 0.02% NP-40, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, and 1 mg/ml X-gal, pH 6) for 24 hours at 37°C in dark without CO₂. After the incubation, sections were washed in PBS and viewed under bright field at 400×magnification. Quantitative image analysis was performed by a blinded observer counting at least 3 random fields. The intensity of SA-β-gal-positive cells was evaluated by means of a ROD (relative optical density) value. ROD of SA-β-gal-positive cells in CA3 area of the hippocampus was obtained after transformation of mean gray values into ROD using the formula: ROD = log (256/mean gray). Images were collected from at least three different sections per structure and per animal by Image Pro Plus software.

On the day 41 of treatment, BrdU (50 mg kg⁻¹ in saline) was administered intraperitoneally three times with an interval of 4 hours. 12 hours after the final administration, the rats (5–6 animals per group) were reanaesthetized and perfused transcardially with 4% PFA in PBS. Hippocampuses were collected and postfixed. Frozen sections and immunofluorescence were performed as previously described. The primary antibody was the mouse monoclonal anti-BrdU (1∶200; Sigma Co. LLC.). The total number of BrdU⁺ cells was estimated on three randomly selected sections taken through the central extent of the dentate gyrus area with a fluorescence microscope (Eclipse, Nikon).

Hippocampuses in each group were collected after the therapy. Total protein was extracted, and the concentrations were measured by a BCA procedure. Samples containing 50 µg protein were separated on SDS-PAGE and transferred to PVDF membranes. Membranes were incubated overnight at 4°C with the anti-SOX2 antibody diluted at 1 : 500. The secondary antibody was diluted at 1 : 5,000 in TBST. The membranes were visualized using the enhanced chemiluminescence detection system (Pierce, USA). The level of β-actin was used as an internal control. Relative intensities were quantified using Quantity One (Bio Rad).

Hippocampuses were collected after the therapy. Total mRNA was extracted using TRIZOL Reagent (TaKaRA, Japan), according to the manufacturer’s protocol. OD260/OD280 of RNA: 1.8∼2.0. First-strand cDNA was created by RT (TaKaRA, Japan). Real-time PCR was carried out using BIO-RAD sequence detection system (cfx96). DNA was amplified by an initial incubation at 94°C for 5 min followed by 40 cycles of 94°C denaturation for 15 sec, annealing at 60°C for 60 sec, 72°C extension for 1 min. mRNA expression levels were normalized against Gapdh mRNA level and analyzed by the comparative cycle threshold method. The PCR primers used are provided in the supporting information (Table S1 in File S1).

Telomere lengths were measured from the hippocampus according to the previously described method [10]. In brief, after extraction, DNA was inspected for integrity, digested, resolved by gel electrophoresis, transferred to a membrane, hybridized with labeled probes and exposed to X-ray film. The telomere lengths were measured by Western Biotechnology Corporation (Chongqing, China).

The supernatant was collected as above. The concentrations were measured by Coomassie brilliant blue. The PCR reaction mixture contained 5 µl 10× TRAP buffer, 1 µL dNTPs, 1 µl Taq polymerase,1 µl TS primer and 2 µl extract of telomerase, was incubated for 30 min at 23°C for telomerase-mediated extension of the TS primer. The reaction mixture was subjected to 35 cycles at 94°C for 30 sec, 50°C for 30 sec, and 72°C for 90 sec. TRAP reaction products were separated by 10% polyacrylamide gel electrophoresis and detected by SYBR green (Gene, Inc.) staining.

SPSS version 17.0 software was used for statistical analyses. One-way ANOVA was used for comparison of mean values across the groups and multiple comparisons were made by LSD test. Differences were considered significant at P<0.05.

The hidden-platform version of the Morris water maze, a hippocampus-dependent task, requires an animal to learn and remember the relationships between multiple distal cues and the platform location to escape the water [11]. As shown in Figure 1A, rats in the D-gal administration group had significant impairment in spatial learning ability during the seven-day place navigation training because of the longer escape latency compared to the control rats (P<0.05); while ginsenoside Rg1 treatment to D-gal administrated rats significantly shortened the escape latency to the similar levels of the control rats. Rats in the Rg1 treatment group showed similar spatial learning ability to that of the control rats during the navigation training.

To assess the spatial memory more directly, the rats were subjected to another trial in which the target platform was removed on the next day after the navigation training. As shown in Figure 1B, for the D-gal administered rats it took longer time to reach the location of the removed platform, and crossed the location fewer times compared to the control group. However, rats in both Rg1 treatment group and D-gal administration plus Rg1 treatment group showed no remarkable differences in the escape latency and target crossing times compared to the control group. In addition, rats in the control group, Rg1 treated group and D-gal administration plus Rg1 treatment group spent more time in the target quadrant than the D-gal administrated rats.

These results indicated that aging model rats had impairments in spatial learning and memory, while the treatment of ginsenoside Rg1 could restore the age-related cognitive impairment caused by D-gal administration.

Aging is known to be associated with a slow decline in brain functions and be accompanied by progressive memory loss, dementia and cognitive dysfunctions. As the hippocampus is closely related to learning and memory ability, we have speculated that the loss of cognitive capacity of the rats in the D-gal administration group is related to the hippocampus aging.

SA-β-gal is one of the most widely used biomarkers for aging cells [12], and aged cells are stained in blue by it in the cytoplasm. As shown in Figure 2, barely any SA-β-gal positive cells were observed in DG area among all the groups, while aged cells were observed in the CA3 area. This pattern was consistent with a previous study [13]. The intensity of SA-β-gal staining was evaluated by means of a ROD (relative optical density) value. The ROD of the SA-β-gal stainingwas not statistically significant between the control group and the Rg1-treated group. Remarkably, D-gal administration induced a remarkable increase in the ROD of the SA-β-gal staining, compared to the control group (Figure 2, Table 1). However, in the D-gal administration plus Rg1 treatment group, the ROD was significantly reduced (Figure 2, Table 1). It demonstrates that Rg1 can protect the hippocampus against senescence.

Telomeres become progressively shortened with each replication of cells and this feature is widely used to evaluate senescence. And the telomerase is responsible for telomere length maintenance. We detected both telomere lengths and telomerase activity in the hippocampus to evaluate the effect of ginsenoside Rg1 on brain senescence.

As we expected, both the telomere lengths and the telomerase activity were reduced in the D-gal-administration group, compared with the control, while ginsenoside Rg1 remarkably increased these two parameters in the D-gal administration plus Rg1 treatment group (Figure 3). Moreover, there were no differences between the control group and the Rg1 treatment group (Figure 3).

Although new neurons are generated in the adult hippocampus throughout life by NSCs/NPCs, the rate declines with the increasing age [14]–[17]. Here, we observed the NSCs/NPCs differentiation in hippocampus by immunofluorescence with anti-β-tubulin III (a neuron marker), anti-GFAP (glial fibrillary acid protein, an astrocyte marker) and Gal-c (galactocerebroside, a mature oligodendrocyte marker) antibodies. Though naïve NSCs/NPCs can be marked by GFAP as well, the astrocytes can be identified by the strong expression of GFAP and its morphological character of ramified branches.

Neurons positive for β-tubulin III had a large and irregularly shaped soma and an eccentrically-placed spherical nucleus in the dentate gyrus (DG) area of hippocampus, and oligodendrocytes positive for Gal-c had a few branches, while astrocytes showed activated characteristics by exhibiting hypertrophy, with very thick, highly ramified and intensely immunostained branches in the D-gal-administration group (Figure 4, panel 1). Furthermore, a remarkable decrease of the cells positive for β-tubulin III and a significant increase of the cells positive for Gal-c and GFAP were observed in the D-gal-administration group, compared with the control group and the Rg1 treated group (Figure 4, Table 2). However, in the D-gal-administration plus Rg1 treatment group, the number of cells positive for β-tubulin III increased remarkably, while the number of cells positive for Gal-c and GFAP decreased, compared with the D-gal-administration group (Figure 4, Table 2). This suggests that Rg1 promote NSCs/NPCs differentiation into neurons rather than glial cells in the aged hippocampus.

A continuous decrease in the number of NSCs/NPCs underlies the age-related decline in hippocampal neurogenesis [18]. Commonly used markers for neural stem cells include SOX2 and Nestin. The transcription factor SOX2 is involved in the proliferation and/or maintenance of NSCs/NPCs and in neurogenesis [19]. The intermediate filament protein, Nestin, is expressed predominantly in stem cells of the adult brain and is required for the proper self-renewal of NSCs [20]. We further detected the expression of SOX2 and Nestin to investigate the effect of Rg1 on NSCs/NPCs survival in aged hippocampus. In accordance with our expectation, the protein expression of SOX2 in the D-gal-administration group was significantly lower than that of the control group. Although Rg1 didn’t increase SOX2 expression of the Rg1 treated group relatively to the controls, Rg1 treatment partially rescued the reduction of SOX2 expression in the D-gal-administration plus Rg1 treatment group (Figure 5A). It suggests that the ginsenoside Rg1 can protect NSCs/NPCs in the hippocampus of aged rats.

However, the expression of Nestin mRNA was increased violently in the D-gal administration group, and the treatment of Rg1 reversed this D-gal induced increase (Figure 5B). As re-expression of Nestin also associated with the astroglial activation during neurodegeneration [21], and the GFAP positive cells in the D-gal administration group showed activated astrocyte appearance (Figure 4), we suppose that the enhanced expression of Nestin is due to the activation of astroglials. Therefore, we further explored the expression of Aeg1 (astrocyte elevated gene-1), a novel modulator of reactive astrogliosis. In accordance with the expression of Nestin, the expression of Aeg1 increased remarkably in the D-gal administration group, and decreased significantly in the D-gal-administration plus Rg1 treatment group (Figure 5C). Additionally, the expression levels of SOX2, Nestin and Aeg1 of the Rg1 treatment group weren’t significantly different from the control group. The results demonstrate that astrocytes are activated by D-gal administration as in early stage of neurodegenetive disease or brain damage; however, astrocytes activation is reduced by Rg1 treatment.

To explore whether ginsenoside Rg1 induced new cells production in the hippocampus, the total number of new cells was estimated in the dentate gyrus of all groups using immunofluorescence of BrdU (Figure 6). A student’s t-test confirmed that the total number of BrdU⁺ cells was lower in the dentate gyrus of D-gal administered rats relative to controls (Figure 6; Table 2). Although single treatment with Rg1 could not increase the total new cell number compared with the control, BrdU⁺ cells increased notably in the D-gal-administration plus Rg1 treatment group relatively to the D-gal administered rats (Figure 6; Table 2). It suggests that Rg1 promotes neurogenesis by increasing the number of new cells.

Oxidative stress of ROS (reactive oxygen species) is one of the main causes of cells’ senescence. SOD (Superoxide Dismutase) and GSH-px (glutathione peroxidase) are two important enzymes that participate in the removal of ROS from the cellular environment. MDA (Malondialdehyde) is an end-product of ROS-induced peroxidation, and it is widely used as an oxidative stress biomarker; while GSH is the substrate of GSH-Px and its consumption decreases with age. We evaluated the SOD activity and MDA contents, GSH-Px activity and GSH reduced level in the hippocampus to figure out whether the anti-aging effect of ginsenoside Rg1 was mediated by alleviating oxidative stress.

Compared to the control group, SOD activity, GSH-Px activity and GSH consumption decreased significantly and the MDA content increased remarkably in the hippocampus in D-gal-administration group (Table 3). Meanwhile, Rg1 rescued the reduction of SOD activity, GSH-Px activity and GSH consumed level significantly, and partially rescued the increase of MDA in the D-gal-administration plus Rg1 treatment group (Table 3). Interestingly, Rg1 single treatment also increased the activity of the two anti-oxdative enzymes and the reduced level of GSH, and reduced the MDA content. It shows that ginsenoside Rg1 exerts antioxidant effects against oxidative stress, by enhancing activity of endogenous anti-oxidative defense enzymes.

Chronic inflammation in the brain is associated with natural aging and neurodegeneration. Increased levels of proinflammatory cytokines as IL-1β, IL-6 and TNF-α have been found in inflammatory tissue and correlated well with aged brain.

The levels of IL-1β, IL-6 and TNF-α increased significantly in the hippocampus of the D-gal-administration group, compared with the control group. However, the levels of the proinflammatory cytokines were reduced notably in the D-gal-administration plus Rg1 treatment group, relatively to the D-gal-administration group (Table 4). Meanwhile, Rg1 single treatment didn’t reduce the levels of IL-1β, IL-6 and TNF-α in the Rg1 treatment group, compared with the controls. It suggests that inflammation in D-gal induced aged hippocampus is alleviated by ginsenoside Rg1 treatment.

P19^Arf-Mdm2-p53-p21^Cip1/Waf1 pathway is a main signal transduction pathway involved in cell aging processes. Therefore, we performed qRT-PCR to explore the mRNA expressions of p53, p19^Arf and p21^Cip1/Waf1, which are in the core position of the pathway. The expression of Gapdh was used as the inner control. As shown in Figure 7, the mRNA expression of p53, p19^Arf and p21^Cip1/Waf1 in the D-gal-administration group was violently higher than that of the control group. However, in the D-gal-administration plus Rg1 treatment group, the expression of the genes was significantly lower than that of the D-gal-administration group (Figure 7). Meanwhile, there were no differences between the control group and the Rg1 treatment group. It indicates that ginsenoside Rg1 can down-regulate the expression of senescence-associated genes in the hippocampus of brain-aged rats.