Experimental design and exercise protocol

A total of 45 Male SPF Sprague–Dawley rats (3-month-old) were bought from the Experimental Animal Centre of Chengdu Dashuo Biological Technology Co., Ltd. and were housed under SPF conditions at the center. The rats were given free access to standard feed and water and housed on a 12 h of light and dark cycle. After 1 week of acclimatization, the rats were randomly divided into three groups, 15 in each group: the saline control (DC) group, D-gal-induced aging (DA) group, and D-gal-induced aging + exercise (DE) group. Aging was induced in rats in the DA and DE group by intraperitoneal injection of D-gal at a dose of 100 mg/kg body weight/d for six weeks. Rats in the DC group were injected with the same volume of saline. Furthermore, swimming was selected as the aerobic exercise mode for the DE group in a transparent glass jar (160 cm*60 cm*110 cm) with a water depth of 80 cm and a temperature of 32 ± 2 °C (Li et al. 2019). The exercise protocol was performed for 60 min/day six days/week for six weeks. The Animal Ethics Committee (Batch No: 2021–13) of Chengdu Sport University approved all animal experiments.

Morris water maze (MWM) test

The MWM test was performed as previously reported (Chen et al. 2020). The MWM apparatus consisted of a water-filled circular pool filled with water. In one quadrant, a platform was placed 1 cm below the surface of the water. The MWM test was performed over six days, included the navigation phase (1st–5th days) and spatial exploration phase (6th day). For first five consecutive days, four trials per day were conducted and during each trail rats were placed in the water in different quadrants and allowed to find platform. If a rat failed to find the correct platform within 120 s, it was slowly directed to the platform and allowed to stay on it for 10 s. The escape latency of each rat within 120 s was recorded. The average escape latency to platform of the rats was been calculated. The platform was removed on the sixth day, and the swimming paths of the rats and the number of first platform crossings within 60 s were recorded. The animal behavior analysis system and the MWM video analysis system were provided by Anhui Huaibei Zhenghua Biologic Apparatus Facilities.

Enzyme-linked immunosorbent assay (ELISA)

The SOD activity in brain homogenates was measured by SOD-ELISA kit (mlbio, Shanghai, China). The expression of MDA was measured by MDA-ELISA kit (mlbio, Shanghai, China). The level of cAMP in the hippocampus was quantified by using an cAMP-ELISA kit (mlbio, Shanghai, China) according to the manufacturer’s protocol. After the color development, the absorbance was measured at 450 nm with a fluorescence reader (Thermo, USA).

Hematoxylin and eosin (H&E) and nissl staining

H&E staining was performed according to conventional methods (Chen et al. 2020). Rat brain tissues were perfused with normal saline followed by 4% paraformaldehyde (PFA) solution for 24 h. Then the tissues were paraffin-embedded, sectioned at 5-μm thickness (RM2016, Leica), and stained with H&E or toluidine blue. Coronal brain sections were used for H&E and Nissl staining, and the morphological structure of hippocampal neurons was observed under a light microscope. Individual cell number in the CA3 region was quantified using ImageJ software.

RT-qPCR

Total RNA was extracted from hippocampal tissue using TRIzol (Invitrogen) according to the manufacturer's instructions. The concentration and purity of total RNA were determined, and RNA integrity was examined by agarose gel electrophoresis. cDNA was synthesized by reverse transcription using the BIO-RAD iScriptTM cDNA Synthesis Kit. RT-qPCR was performed using the SYBR Green method. The primer sequences for PDE-4 and the internal reference gene β-actin (TaKaRa) are shown in Table 1. The relative gene expression was calculated using the 2−∆∆CT method (CT = threshold cycle).

Table 1 The primer sequences for PDE-4 and the internal reference gene β-actinWestern blot analysis

After carefully removing the brain, the hippocampus was quickly dissected on ice. Then tissue samples were lysed using RIPA lysis solution (containing PMSF), and the protein concentration was determined using BCA Protein Assay Kit (Beyotime, Shanghai, China). Equal amounts of total protein were mixed with loading buffer and boiled for 10 min. Then the proteins were separated on 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes. The membranes were then blocked for 2–3 h at room temperature (RT) with TBST buffer containing 5% skimmed milk. The blots were incubated overnight at 4 °C with the following antibodies: PDE-4 (ab14628, Abcam, 1:1000), PKA (ab75991, Abcam, 1:1000), CREB (ab32515, Abcam, 1:1000), PSD-95 (ab18258, Abcam, 1:1000), GAPDH (AF7021, Affinity, 1:3000) and β-actin (AF7018, Affinity, 1:3000). The membranes were further incubated for 2 h at RT with horseradish peroxidase-conjugated antibody, IgG (H + L) (S0001, Affinity, 1:5000) diluted in TBST containing 5% skimmed milk. After washing, the protein bands were finally visualized using an imaging system. The integrate gray value of each band was measured using ImageJ (National Institutes of Health, Bethesda, USA) to analyze the relative expression of each protein.

Immunofluorescence staining

Rat brain tissue sections (RM2016, Leica) were embedded in paraffin. The paraffin sections were dewaxed and subjected to antigen retrieval and blocked with 5% BSA at RT for one hour. Then the sections were incubated with primary antibodies synaptophysin (Syp; 36,406, CST, 1:200) overnight at 4 °C. Subsequently, the sections were washed with PBS and incubated with secondary antibody (BA1090, Boster, 1:400) at RT for two hours. Then again washed with PBS, sealed with a drop of mounting medium (containing DAPI) and placed in an oven at 37 °C for 20 min. The sections were photographed under a fluorescence microscope (Imager Z2, Zeiss, Germany). The average fluorescence intensity was analyzed using ImageJ software.

Massarray methylation assay

The PDE-4 gene methylation level in rat hippocampal tissues was measured by time-of-flight mass spectrometry. The CpG site was concentrated at residues 60–502 of PDE-4, and this fragment was selected as the target sequence. Primers were designed using EpiDesigner software. The primers for methylation analysis of the PDE-4 gene are shown in Table 1. Total DNA was extracted from hippocampal tissue, 200 µL of tissue lysis solution and 40 µL of proteinase K were added, and the samples were placed in a water bath at 55 °C to lyse them fully. Then, 200 µL of binding buffer was added, the samples were placed in a 70 °C water bath to allow full binding, 100 µL of isopropanol was added, the samples were shaken well, and the supernatant was collected. The DNA concentration and DNA purity were assessed by agar gel electrophoresis and measurement of optical density values, respectively. Amplified PCR reagents were detected by methylation using the Sequenom MassArray system, and then PCR was performed. The SAP reaction was then performed, and the T-cut reaction/RNase A precipitation was followed by desalting and resin purification. DNA methylation was quantified at the target site of the PDE-4 gene using MassARRAY® EpiTYPER™ software that came with the mass spectrometry methylation detection platform.

Statistical analysis

The one-way analysis of variance (ANOVA) and Tukey post hoc test were used to compare differences between two or more groups, respectively. Two-way ANOVA and Tukey post-hoc comparison were employed for time-dependent analysis. All of the results are shown as the mean ± standard deviation (SD). P < 0.05 was considered significant. All statistical analyses were performed with GraphPad Prism 8.0 (La Jolla, CA, USA). *, **, and ***indicate P < 0.05, P < 0.01, and P < 0.001, respectively.