30 male Sprague–Dawley rats (8 weeks old, 215 ± 20 g) were purchased from Guangdong Medical Experimental Animal Center (http://www.gdmlac.com.cn/). All rats were kept in an environment where the ambient temperature is maintained at 22ཞ23℃, the relative humidity is 45%ཞ50%, and the light cycle is 12/12 hours. All rats were fed adaptively for one week, and they were free to eat and drink. Those with normal drinking and eating were included in the experiment.
The construction of the PTU rat model referred to the previous literature . Rats were randomly divided into control group (n = 10) and model group (n = 20). The rats in the control group drank water normally, while the rats in the PTU group drank tap water containing 0.05% PTU (IP0420, Solarbio, China). After 28 days, the rats in the model group were randomly divided into PTU group (n = 10) and PTU + DMF25 group (n = 10). The rats in the control group and the PTU group were given saline once a day, while the rats in the PTU + DMF25 group were given 25 mg/kg DMF (ID0320, Solarbio, China) once a day for 14 days. The body weight of each group of rats was measured weekly.
Morris water maze test
The learning and memory abilities of rats are tested through the Morris water maze test. The diameter of the maze was 1.6 m and the height was 50 cm. The pool water was dyed black with food coloring. The water depth was 30 cm, and the water temperature was 22–23℃. The experiment was divided into two parts: positioning navigation and space search. In the positioning navigation experiment, the rats received 4 days of training, 4 times a day. The specific training was as follows: each time before entering the water, put the rat on the underwater platform to adapt for 30 seconds, record the swimming distance of the rat from the four quadrants and different entry points to the platform within 60 seconds, and the average of the 4 results was the final grade. On the fifth day, the space search experiment was carried out, the platform was removed, and the rats were placed into the water from the entry point in the opposite quadrant of the platform, facing the wall of the pool, and the swimming trajectory of the rats within 60 seconds was recorded and analyzed.
Specimen collection and preparation
After the water maze experiment, the rats were fasted for 12 hours but were allowed to drink water freely. After the rats were anesthetized, blood was taken from the abdominal aorta, and the collected blood was centrifuged to obtain serum. The content of T3 and T4 in serum was measured by an automatic biochemical analyzer (C1600, Abbott, USA) within 48 hours. The rats were euthanized with an overdose of sodium pentobarbital, the brain tissues of the rats were separated, a part of the brain tissue was fixed in 4% paraformaldehyde, paraffin embedded and sectioned, and the rest was stored in a refrigerator at -80 ℃ for Western blot and quantitative reverse transcription polymerase chain reaction (qRT-PCR).
After the paraffin sections were conventionally deparaffinized and hydrated, they were reacted with Nissl staining solution (G1036, Wuhan Google Biotechnology Co., Ltd., China) in an oven at 60°C for 20 min. After washing the sections with distilled water, the sections were dried in an oven at 60°C. Finally, the sections were routinely dehydrated, transparent and sealed.
Total RNA from rat brain tissue was extracted by total RNA extraction kit (R1200, Solarbio, China). Total RNA was reverse transcribed into cDNA with the help of reverse transcription kit (CW2569, cwbiotech, China). Then the SYBR Green qPCR kit (CW2601, cwbiotech, China) was used for qPCR. β-actin was employed as an internal control. The primers were listed as follows: BDNF, forward: 5’- GGCAGGCTTTGATGAGACCG-3’ and reverse: 5’-TCACCTGGTGGAACTCAGGGT-3’; β-actin, forward: 5’-AACCTTCTTGCAGCTCCTCC-3’ and reverse: 5’-TACCCACCATCACACCCTGG-3’. Relative expressions of BDNF were analyzed by Real-Time PCR Detection system (CFX96, Bio-rad, USA) with 2−ΔΔCt method.
Western blot was performed as previously described. The protein sample was collected from rat brain tissue by RIPA lysate (P0013D, Beyotime, China), PMSF (ST506, Beyotime, China) and protease inhibitors (60237, Beyotime, China). Then the protein was sequentially quantified by BCA kit (pc0020, Solarbio, China). The protein was separated with 10% separating gel, and then the protein was transferred to the PVDF membrane (10600023, GE Healthcare Life, USA). Before incubating the primary antibody, the membrane needed to be blocked with 5% skimmed milk. Primary antibodies include anti-BDNF antibody (ab108319, Abcam, UK) and anti-β-actin antibody (ab8226, Abcam, UK). After incubating with the primary antibody overnight at 4°C, the membrane reacted with the secondary antibody goat anti rabbit (ab205718, Abcam, UK) or goat anti mouse (ab6789, Abcam, UK) at room temperature for 2 hours. The membrane was developed on a chemiluminescence instrument (610020-9Q, Qinxiang, China) with ECL luminescence reagent (C510045, Sangon, China). β-actin was used as internal control.
Data were analyzed by SPSS 16.0 (SPSS, Chicago, USA) and represented as mean ± standard deviation. One-way analysis of variance was used for measurement data among multiple groups, and SNK analysis was used for comparison between groups. Kruskal-Wallis H test was used for results of uneven variance. P < 0.05 was accepted to be statistically significant.