Experimental animals

Sprague Dawley male rats (2 months of age,180–220 g) were purchased from Vitong Lihua Experimental Animal Co., Ltd. (Zhejiang, China; Animal certificate number: SCXK-(Zhejiang) 2019–0001). All rats were acclimated for 1 week under 12-h light/dark conditions at 23–25 °C, 60 ± 10% humidity, and were provided with free water and food. All procedures were approved and carried out under the guidance of the Animal Ethics Committee of Guangzhou University of Traditional Chinese Medicine (approval number 20200717003).

Experimental design and drug administration

The experimental rats were randomly divided into the following five groups (n = 10 rat/group): Control (con) group (0.9% saline s.c. + 0.9% saline i.g.), D-galactose (D-gal) group (150 mg/kg/day s.c. + 0.9% saline i.g.), low dose Bushen-Yizhi Formula (BSYZ-L) group (D-gal 150 mg/kg/day s.c. + BSYZ 3 g/kg/day i.g.), high dose Bushen-Yizhi Formula (BSYZ-H) group (D-gal 150 mg/kg/day s.c. + BSYZ 6 g/kg/day i.g.), Donepezil (DNP) group (D-gal 150 mg/kg/day s.c. + DNP 2 mg/kg/day i.g.). After one week of adaptive feeding, all rats treated with D-gal s.c., while the con group was given 0.9% saline i.g. Next, starting in the third week of the trial, rats received 0.9% saline, BSYZ, or DNP orally for 8 consecutive weeks. The experimental protocol is showed in Fig. 1.

Fig. 1

Experimental protocol of the study. Rats were randomly divided into 5 groups after one week of adaptive feeding. Expect for the control group, all animals received subcutaneous injection of D-gal (150 mg/kg) for 10 weeks. The rats in the BSYZ-L, BSYZ-H and DNP groups received oral gavage every day in the third week, and last into the tenth week. The behavioral tests, including morris water maze, open field test and novel object recognition task) were performed during the ninth and tenth weeks. After the behavioral tests, the animals were sacrificed and brain tissue was collected for experimental analysis

Animal behavioral testMorris water maze test (MWM)

The Morris water maze pool is 150 cm in diameter and 60 cm high. The tank was filled with 40 cm high water, and an appropriate amount of non-toxic food stain (Mixol, Germany) was added to the water so that the platform and the bottom of the pool were invisible to rats. A 10-cm-diameter platform was placed on the target quadrant. An automated camera was fixed above the center of the maze to monitor and record the animal's route during behavioral tests. The day before training, the rats underwent an acclimation period, allowing them to swim in the pool and rest on the platform for 30 s. During training, the rats were placed in one of the quadrants and allowed to search for underwater platforms. Animals that did not find the platform within the allowed time were physically guided and placed on the platform for 10 s before being removed. On day 6 after training, a probe test was performed without a platform in the pool to check memory retention. The animals were allowed to swim from one of the quadrants for 60 s, and the number of times the rats crossed the quadrant and the time they spent in the formal platform area were measured.

Open field test (OFT)

The rats were tested of locomotor activity in OFT, which is a common measure of general activity and exploratory behavior in mice and rats. The OFT instrument (rectangular) interior walls are painted black, the dimensions are 100 × 100 cm, and the walls are 40 cm. The bottom is divided into 25 smaller rectangular units. The animals were acclimated for 30 min in a quiet, dark laboratory before the experiment began. Each rat was placed in the center and allowed to explore freely for 5 min. At the end of each measurement, the OFT apparatus was cleaned with 70% alcohol and dried after each test to avoid the effect of odor on the next rat.

Novel object recognition task (NOR)

Using the field test box, the non-spatial memory ability was tested by taking advantage of the rat's instinct to touch and explore novelties. On the first day of the experiment, the animals were placed in the experimental box and familiarized with the environment for 5 min. The next day, two identical objects were placed symmetrically in the experimental box. The distance between the two objects is 10 cm from the side and back walls of the box. Record the total time the rat spends exploring both objects within 5 min. After each probe, the secretions should be cleaned with 75% alcohol. On the third day, one of the familiar objects was replaced by another new object of similar size but different shape and color. Time spent exploring novel and familiar objects was recorded. Object detection is defined as placing the mouse's nose within 3 cm of the object, or sniffing, licking, touching the object with its front paws, and sniffing at the same time.

Brain tissue collection and sample preparation

After assessment of cognitive ability, rats were anesthetized with pentobarbital sodium (30 mg/kg). For biochemical analysis, they immediately removed the brain, carefully dissected the hippocampus and cerebral cortex regions, and snap-frozen in liquid nitrogen. For immunostaining and morphological analysis, saline was perfused transcardially, followed by 4% paraformaldehyde (PFA). Brain tissue was carefully removed, immersed in water, fixed in ice-cold PFA at 4 °C for 72 h, then rinsed with phosphate-buffered saline (PBS), placed in 30% sucrose solution for 48 h, and frozen in OCT complexes. Serial 20 um coronal tissue sections were cut on a cryostat (Leica, Nussloch, Germany) and then thawed on gelatin-coated slides.

Sample collection and preparation for 1H-NMR spectroscopy

After cortical samples were thawed at 4 °C, 200 mg of cortical samples were homogenized by adding 600 μL methanol and 300 μL distilled water in an ice bath. After centrifugation at 4 °C and 13,000 r/min for 20 min, we pipet 500 μL of the supernatant into a 1.5 mL EP tube and use a vacuum drying concentrator to dry the samples. Then, the dried samples were dissolved in 600 μL of mixed phosphate buffer (0.2 mol/L Na2HPO4, NaH2PO4, 0.005% TSP, pH 7.4), centrifuged at 4 °C, 14,000 r/min for 10 min, and 550 μL of supernatant was precisely collected, and transferred into a 5 mm NMR tube for NMR analysis.

NMR analysis and data preprocessing

The 1H-NMR spectra were collected on a Bruker 500 MHz Avance III NMR spectrometer (Bruker, Germany). The spectra were recorded using a NOSEY pulse sequence at 298.15 K (25 °C), and the following parameters were used: FID resolution of 0.188 Hz; pulse time 14 μs; sampling time 2.654 s; delay time 1.0 s; sampling interval 40.5 μs and sampling point 65,536.

All acquired data were Fourier transformed using MestReNova software (Mestrelab Research, Santiago de Compostella, Spain). The obtained spectra were manually phased and the baseline was corrected to calibrate peak positions with a TSP of zero. Subsequently, the spectra were integrated by chemical shift interval δ 0.50—9.00 segmented with δ 0.01 as integration area, water peaks removed (δ 4.72—δ 5.2), and data normalized for further analysis.

Betaine assay

To verify the result of nontarget metabolomics, the content of betaine was determined. As a methyl donor, betaine promotes a variety of biological processes. The betaine assay kit (Heifei Lai Er Bio-Tech Hefei, China) was used to measure the level of betaine in the brain. According to the manufacturer’s instruction, we weighed about 0.2 g of the oven dried sample, added 1 ml of extraction solution, and placed at 60 °C extraction for 30 min, during constant shaking. The supernatant was obtained after centrifuging for 15 min at 25 °C after adding 3 mg of the reagent 4 for shocking thoroughly. The methanol was volatilized clean in a 70 °C oven, then diluted with double distilled water to a volume of one ml. By adding the sample and reagent in accordance with the operating procedures, mix thoroughly. The microplate reader was preheated for more than 30 min, the wavelength was adjusted to 525 nm, and the OD value of each tube was measured. Finally, the concentration is calculated by the formula.

SOD, MDA and GSH assays

Firstly, the tissue was rapidly homogenized in ice-cold PBS (9 times of tissue weight) using a homogenizer. After centrifugation at 4500 g for 15 min at 4 °C, the supernatant was collected to determine the protein concentration using a protein assay kit (Beyotime, China). For measuring SOD, the Superoxide Dismutase Detection Kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) was used. According to the manufacturer's instruction [26], the assay was carried out. GSH serves as the body's most significant non-enzymatic antioxidant and scavenges free radicals. As directed by the manufacturer's instruction, we employed a Reduced glutathione assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) to ascertain the level of GSH present in brain tissue. GSH may form a yellow molecule when it reacts with dithiodinitrobenzoic acid (DTNB), which enables colorimetric quantitative measurement of GSH content at 405 nm. MDA is a marker of lipid peroxidation. In accordance with the manufacturer's instruction from the Malondialdehyde assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), we employed thiobarbituric acid (TBA) to measure the MDA concentration [26, 27]. Finally, a colorimetric test at 532 nm was performed on the supernatant in each tube.

ATP assay

The ATP assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) measures the level of adenosine 5'-triphosphate (ATP), the most fundamental carrier of energy conversion in living things. The tissue was precisely weighed, and a 10% homogenate was created by adding 9 L of cold, double-distilled water to an ice water bath (portions were removed and centrifuged at 3500 rpm for 10 min to measure protein concentration of the supernatant). The homogenate was submerged in boiling water for 10 min, extracted, and subjected to a 1-min mixed extraction procedure. Following a 10-min centrifugation period at 3500 g, the supernatant was collected and loaded as manufacturer's instruction. Finally, it was allowed to stand for 5 min at room temperature after mixing, and the absorbance value of each tube at 636 nm wavelength was measured.

NAD+/NADH Assay

The NAD+/NADH assay kits (WST-8, Abbkine Scientific Co., Ltd, California, America) were provided to measure the NAD+ and NADH contents following the manufacturer’s instructions. We weighed 20 mg of rat brain tissue and added 100 µL of NAD Extraction Buffer or NADH Extraction Buffer to the homogenized samples, which were used to extract NAD and NADH for further analysis. Briefly, the absorbance at 450 nm was measured immediately after adding the sample to the 96-well plate according to the instructions, followed by incubation at room temperature for 30 min, and then measuring the absorbance at 450 nm. Finally, The NAD+/NADH ratio was calculated according to the formula.

TNF-α, IL-1β and IL-6 ELISA kit assays

Before detection, cortical and hippocampal tissues were homogenized as before. TNF-α, IL-1β and IL-6 ELISA Kit Assays (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) were detected according to the manufacturer’s recommendations.

Nissl staining

After preparing coronal tissue sections of brain tissue as described above, which were fixed with 4% paraformaldehyde for 10 min, and then rinsed with distilled water 3 times for 2 min each time. The sections were stained with Nissl's stain solution (Beyotime Biotechnology, Shanghai, China) for 10 min in a 37 °C oven. After 10 min, rinse both sides with distilled water twice and then perform the following operations: Dehydrated in 95% ethanol for 2 min, then switch to fresh 95% ethanol for another 2 min. Dimerized with xylene for 5 min, then dimerized with xylene for another 5 min with fresh xylene. Finally, the sections were covered with rhamsan gum. Images were acquired with microscope (NICON Eclipse 80i, Nicon, Japan).

TUNEL apoptosis assay kit

The apoptosis of nerve cells in D-gal-induced rats was saaessed by TUNEL staining (Beyotime Biotechnology, Shanghai, China). In summary, the frozen sections were fixed with 4% paraformaldehyde and then conducted with 0.25% Triton-X 100. Then, the sections were washed with PBS, and incubated in the TUNEL reaction solution for 1 h at 37 °C in humidified chamber in the dark, after which DAPI was used to stain the nucleus for 10 min. Finally, images were recorded under an fluorescence microscope (DMI8, Leica, Germany).

Immunoblotting and immunofluorescence

Western blot analysis was performed according to the previous method in our laboratory. The bicinchoninic acid (BCA) method (Beyotime, China) was used to quantify the protein in cerebral infarct hemispheres or astrocytes. Protein samples were separated by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA). The membrane was blocked with 5% milk for 2 h at room temperature, incubated with the target protein-specific primary antibody overnight at 4 °C, and then incubated with the secondary antibody for 2 h at room temperature. Digital images of protein bands were acquired using Chemidoc XRS (Bio-Rad, Hercules)., CA, USA) and Bio-Rad Image Lab 5.2.1 software (Bio-Rad Labs, California, USA) for quantification.

For immunofluorescence, the fixed brain slices were rinsed 3 times with PBS (5 min/time). Treated with proteinase K for 5 min and rinsed twice with f-PBS for 5 min each. It was then blocked with 5% goat serum (0.3% Triton X-100 diluted in PBS) for 30 min at room temperature and then rinsed with PBS. Slides were incubated with primary antibodies overnight at 4 °C, followed by secondary antibodies with TRITC- or FITC-conjugated antibodies for 2 h at room temperature. After incubation, the secondary antibody solution was carefully removed, washed with PBS for 10 min, and counterstained with DAPI. Finally, the slides containing brain sections were covered with glass coverslips by using mounting media. Images were acquired with fluorescence microscope (DMI8, Leica, Germany).

Statistical analysis

Combining published literature and NMR databases, including HMDB and BMRB, metabolites were identified by matching the signal peaks of the spectrum to individual metabolites. Multivariate analysis of NMR data was performed using SIMCA-P 13.0 (Umetrics, Switzerland), including principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), and orthogonal partial least squares discriminant analysis (OPLS-DA). The visualization of the model was implemented with SIMCA-P 13.0 and the quality of the model was assessed using the correlation coefficients R2X and Q2Y. The differential metabolites were obtained by s-plot plot combining VIP > 1, independent samples t-test p < 0.05, and analysis of variance to compare metabolite differences among multiple groups. Heatmap of differential metabolites among 3 groups by using the complete clustering method. The relative peak areas of differential metabolites among 3 groups were calculated using one-way ANOVA followed by post-hoc Tukey’s test.

For statistical analysis, all data are expressed as the mean ± standard error of the mean (M ± SEM). Statistical significance was determined by one-way ANOVA followed by the Tukey’s test. A value of P < 0.05 was considered to be statistically significant.