Experimental Animals.
Forty-eight healthy male Kunming mice, weighing 20 ± 2g, were provided by SPF (Beijing) biotechnology Company Limited [animal qualification certificate No. SCXK (Jing)-2016-0002] and were allowed to acclimatize for 3 days before the experiment. The mice were housed 4 per cage and maintained under a 12:12h light/dark cycle at 55% humidity and 22 ± 2 ℃ with free access to food and water. Procedures involving mice were approved by the Institutional Animal Care and Use Committee of Beijing University of Chinese Medicine. (BUCM-4-20190102-3008)
Drugs, Reagents and Instruments.
The dry powder of noni fruit juice aqueous extract was provided by Morinda Inc. The Noni powder was dissolved in water and prepared to a concentration of 0.5mg/10mL. Hydrocortisone injection was provided by Tianjin Kingyork Ltd.(lot.1801181). Total superoxide dismutase, Copper/Zinc superoxide dismutase, Manganese superoxide dismutase (T-SOD, CuZn-SOD, Mn-SOD), Catalase (CAT), Lipid peroxidation (LPO) and Malondialdehyde (MDA) kits were obtained from Nanjing Jiancheng Bioengineering Institute (lot.20200724,20200806,20200822,20200729). Anti-Nrf2, anti-KEAP1, anti-HO-1 and anti-β-Actin were all purchased from Bioss (Beijing, China) (lot.BJ07138310, BJ07012178, BJ02265489, AH11286487).
Grouping, Modeling and Experimental Design.
Forty-eight male Kunming mice were divided into 6 groups using a random number table, with 8 in each group: the control group, the model group (double distilled water 10mL/kg), the Ginseng group (1g/kg), the high-dose Noni group (1.33g/kg, H-Noni), the middle-dose Noni group (0.67g/kg, M-Noni), and the low-dose Noni group (0.33g/kg, L-Noni). The 40 rats were given hydrocortisone (15mg/kg) by gastric gavage for 14 consecutive days to establish the memory impairment model[14] except for those in the control group. At the same day, the corresponding drugs were administered were given by gastric gavage. The weight, brain index and behavior of the mice in each group were observed. There were no accidental deaths among the mice during the experiment. After the experiment, mice were killed by cervical dislocation and brain tissues were separated and stored at − 80℃ for analysis.
Measurement of Mouse Body Weight and Brain Index.
Each mouse was weighed weekly. The wet weight of the brain tissue was measured using a balance and the brain index (BI) was calculated as follows: BI = brain wet weight/mouse weight×100%.
Ethology Assessments.
Ethology was assessed with the step-down test and passive avoidance test. Incubation period is the time from the mice which were placed on the insulated platform in the box to the first time they jumped off the platform. Training phase is after 3min of acclimatization in a dark box, the mice were stimulated with immediate 36V alternating current and then placed on the platform for an acclimatization period. The incubation period and the number of errors in jumping off the platform within 5min were recorded. After 5d of cessation of training, the vanish test was performed. The incubation period was the time from the time each mouse was placed in the open chamber to the time when it was first shocked in the dark chamber. Since mice are nocturnal animals and instinctively flee to the dark, the electric shock was used to train their spatial learning and memory. The training phase: each mouse was put into the open chamber, the door was raised after 5s, the door was closed as soon as the animal entered the dark chamber completely, and an unavoidable electric shock of 36V was delivered to the mouse's feet for 2s. Then the mouse was moved to the open chamber and the process was repeated after 5min. The training was terminated when the mice remained in the light room for 5min. 24h later, the incubation period and the number of errors in entering the dark room within 5min were recorded for each mouse. The training was stopped for 5d, and the vanish test was performed.
Brain histology.
All brain tissues were fixed in 4% paraformaldehyde buffer for 36h and embedded in paraffin. Sections were cut at 4.0 µm and stained with hematoxylin and eosin (HE staining) for histopathologic examinations using standard protocols, all of the images were acquired with a Digital Pathology system Axio Scan.Z1 (Zeiss, Germany) for assessment the nerve cell.
Biochemical Assays.
The measurement of activities of T-SOD, CuZn-SOD, Mn-SOD, CAT, LPO and content of MDA tested in strict accordance with the instructions of the kit.
Western Blot Analysis.
After adding protease inhibitor, brain tissues were prepared on ice. The supernatants were centrifuged at 4000g and the tests were carried out strictly according to the instructions of the kit. Total protein was extracted from brain tissues in each group, and protein was quantified according to the bicinchoninic acid protein quantification kit instructions (Nanjing Jiancheng, China). Proteins were separated by 10% SDS-PAGE and transferred by electrophoresis onto polyvinylidene fluoride membranes, then incubated with TBST containing 5% no-fat milk at room temperature for 1 h, and then washed 5 times with TBST for 5 min each. Diluted Nrf2 (1:500), KEAP1(1:1000), HO-1(1:1000) and β-Actin antibodies (1:5000) were added separately, and the membranes were incubated overnight at 4℃. The next day, the membranes were rinsed 5 times with TBST for 5 min each, and the corresponding horseradish peroxidase-labelled secondary antibody (1:5000) was added. The membranes were incubated for 1 h at room temperature and then rinsed 5 times in TBST for 5 min each. The films were rapidly developed according to the electron chemiluminescence kit instructions (New Cell & Molecular Biotech, China). Image J software was utilized for the quantitative analysis of images. Each experiment for each protein was repeated 3 times.
Statistical Analysis.
Analysis was performed using SPSS (version 22.0, SPSS Inc., Chicago, IL, USA). The measurement data are represented as mean ± standard deviation (\(\stackrel{-}{x}\)±s). One-way ANOVA analysis followed by the Bonferoni method was adopted to compare the sample mean pairs. Values of p < 0.05 were considered statistically significant.