ACR and curcumin were purchased from Amresco Co. (Solon, OH, USA) and Sigma chemicals Co.(St. Louis, MO, USA), respectively.
Male Sprague-Dawley rats, weighing 200–220 g, were obtained from Hubei Experimental Animal Research Center (Hubei, China). Rats were housed in standard translucent cages (5 animals/cage) under controlled standard conditions (23 ± 2 °C, 55 ± 5% relative humidity, 12 h light/dark cycle) with restricted access to standard rat chow and free access to tap water. After acclimation for 1-week, healthy animals were randomly assigned into 4 groups (10 rats per group): normal control group; ACR-intoxicated control group; low-dose (50 mg/kg) curcumin treatment group and high-dose (100 mg/kg) curcumin treatment group. A dose of 40 mg/kg ACR (dissolved in normal saline) was intraperitoneally injected every other day for 4 weeks in all animals except the normal control group. The normal rats received saline as control. Meanwhile, rats in the curcumin treatment groups were daily administered with curcumin at the corresponding oral administration dose for 4 weeks. The doses of ACR and curcumin were chosen based on the previous study  and preliminary experiments. The normal and ACR-intoxicated control animals were orally administered with the same volume of distilled water. Body weight and behavioral alterations were monitored once a week. At 24 h after the last administration, all animals were euthanized by CO2 asphyxiation, brain tissues were quickly collected.
All rats were subjected to behavioral analysis to assess their motor functions.
In the hind limb splay examination [3, 5], the hind paws of rats were inked, then the rats were placed in a horizontal position of 30 cm high and dropped onto a white paper. The distance between the center points of right and left heels were recorded as the landing foot spread distance.
In the movement initiation test [5, 24], the rat was held by its hind limbs and its torso, one forelimb was lifted above a table in order that the body weight was supported by the other forelimb alone. Then, rat was allowed to initiate stepping movements for one forelimb, and then the other. The averaged time period to initiate one step was recorded as the response latency for each forelimb.
In the gait score test [3, 5], animals were placed on the table and were observed for 3 min. Gait was scored as follow: 1: normal gait; 2, slightly abnormal gait characterized by slight ataxia, weakness and foot splay; 3, moderately abnormal gait characterized by obvious ataxia and foot splay with limb spread during ambulation; 4, severely abnormal gait characterized by a combination of all the above symptoms, dragging hind limbs and inability to support body weight.
The collected brain tissues were fixed with 10% neutral-buffered formalin followed by dehydrating and paraffin-embedding. Then, embedded brain sections (5-μm thickness) were stained with hematoxylin and eosin (HE) for histopathological observation. The histopathological changes in cerebral cortex, hippocampal CA1, CA3, and dentate gyrus regions were analyzed.
The apoptotic neurons in the brain sections were detected using the terminal deoxynucleotidyl transferase mediated dUTP nick end labelling (TUNEL) assay. After deparaffinization and rehydration, the brain sections were permeabilized with proteinase K solution, then exposed to the mixture of biotinylated nucleotide dUTP and recombinant terminal deoxynucleotidyl transferase (TdT) following the instruction manual of TUNEL Apoptosis Assay Kit (Servicebio, Wuhan, China). Staining with 4,6-diamino-2-phenyl indole (DAPI) (Sigma, St. Louis, USA) was performed to visualize nuclei. Images were obtained under a fluorescent microscope (Olympus, Center Valley, USA).
Total RNA of brain cerebral cortex tissues was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The expression levels of TERT mRNA were measured by real-time PCR using all-in-OneTM qPCR master mix AOPR-1200 (GeneCopoeia, Rockville, MD, USA). The sequences of primer sets for TERT were 5′-TGTTCCTGTTCTGGCTAATGG- 3′(forward) and 5′-CCTCTTGTGACAGTTCCCGT-3′ (reverse). β-actin gene was applied as a reference.
Paraffin-embedded brain sections of 5-μm thickness were incubated with a rabbit anti-TERT antibody (Servicebio, Wuhan, China), then a biotinylated goat anti-rabbit secondary antibody (Servicebio, Wuhan, China). Immune complexes were visualized by incubation with 3,3′-diaminobenzidine tetrachloride (DAB) and hematoxylin.
Measurement of parameters related to oxidative stress in cerebral homogenates
The brain tissue were homogenized with 9 times the volume of PBS on ice and then centrifuged to prepare homogenates. The contents of malondialdehyde (MDA) and glutathione (GSH) as well as the activities of SOD and GSH-Px in the cerebral homogenates were measured following the respective manufacturer’s protocols (Nanjing Jiancheng Bio-Engineering Co., Ltd., Nanjing, China). Protein contents in the cerebral homogenates were determined using the bicinchoninic acid assay kit (Nanjing Jiancheng Bio-Engineering Co., Ltd., Nanjing, China).
Measurement of IL-1β and TNF-ɑ levels in cerebral homogenates
The concentrations of IL-1β and TNF-ɑ in cerebral homogenates were determined using using ELISA kits according to the manufacturer’s instructions (IL-1β: PeproTech Inc., NJ, USA; TNF-ɑ: R&D Systems, Minneapolis, MN, USA).
All experiments were conducted with two technical replicates. Data were expressed as the mean ± SD, and analyzed using one-way analysis of variance (ANOVA) with post hoc Tukey test by SPSS 22.0 software. P < 0.05 or P < 0.01 was considered statistically significant.