Edited by: Mohammad Badruzzaman Khan, Augusta University, United States
Reviewed by: Md Suhail Alam, University of Notre Dame, United States; Murali Vijayan, Texas Tech University Health Sciences Center, United States; Mohammad Farhan, Hamad Bin Khalifa University, Qatar
This article was submitted to Neurodegeneration, a section of the journal Frontiers in Neuroscience
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Chronic cerebral hypoperfusion (CCH) is a main cause of vascular dementia and is also an etiological factor of neurological diseases and mental disorders. However, few treatments are available for CCH, and new medications are needed. In the present study, we employed a rat model of CCH that was based on bilateral common carotid artery occlusion and investigated the therapeutic effects of resveratrol and its detailed mechanism of action. We evaluated neurological deficit scores and performed the Morris water maze test, hematoxylin and eosin staining, TUNEL staining, enzyme-linked immunosorbent assays, and Western blot. Resveratrol reduced neurological deficit scores in CCH rats and reduced pathological damage in the frontal cortex and hippocampus. Resveratrol activated autophagy and inhibited the expression of AKT/mechanistic target of rapamycin (mTOR) signaling pathway-related proteins. Treatment with a phosphoinositide-3 kinase inhibitor reversed the protective effect of resveratrol. These findings suggest that resveratrol improves cognitive function in a rat model of CCH and reduces oxidative stress-induced neuronal damage in the frontal cortex and hippocampus by activating autophagy and inhibiting neuronal apoptosis. These effects may be regulated by the AKT/mTOR signaling pathway.
Chronic cerebral hypoperfusion (CCH) can contribute to the development of various neurological diseases and mental disorders, including vascular dementia, Alzheimer’s disease, and Binswanger disease (
In states of oxidative stress, cells undergo autophagy to remove damaged mitochondria, the endoplasmic reticulum, and proteins to slow the cell death process (
Resveratrol is a natural polyphenolic phytoalexin with a range of protective physiological functions, including antioxidative and anti-inflammatory effects. It is used for the management of diabetes, neuroprotection and myocardium protection. Several studies reported that resveratrol induced autophagy (
A total of 96 male Sprague-Dawley rats, weighing 260–300 g, were purchased from the Beijing Vital River Laboratory Animal Technology Co., Ltd. The animal experiments were performed in the experimental animal department of China Medical University [experimental animal license no. SYXK (Liao) 20150001] with a 12 h/12 h light/dark cycle, controlled temperature (22°C ± 3°C), and controlled humidity (60% ± 5%). The animals had
The rats were randomly divided into four groups using the random number table method (
The animals in each group were fasted for 12 h with water that was provided before surgery. The rats were anesthetized by an intraperitoneal injection of 2% sodium pentobarbital (3 mg/kg) and placed in the supine position. The skin on the neck was prepared, and a midline incision was made to separate the bilateral common carotid arteries and vagus nerve. In the groups that were subjected to CCH, the bilateral common carotid arteries were permanently ligated at the distal end of the telecentric end with No. 4 surgical thread. The incision was then closed by layer-by-layer sutures. Gentamicin was injected subcutaneously to prevent infection, and the rats were returned to their home cages for feeding. The Res group was intragastrically administered resveratrol (50 mg/kg/day) after surgery (
Schematic diagram of the rat groups and treatment regimens. Because the scores were zero in the Sham group, there is no bar-plot for this group.
We used the Bederson neurological deficit scoring method (
Spatial memory function was assessed using the Morris water maze (Institute of Medicine, Chinese Academy of Medical Sciences, Beijing, China). The time spent locating the platform was recorded. The rats were placed in the water from four water inlet points on the pool wall twice daily for 5 days. The latency to find and climb on the platform (escape latency) in a 3 min session and the swimming path during this period (swimming distance) were recorded. The escape latency was used as a memory score for each rat, and average scores were calculated for four consecutive days. Afterward, the platform was removed, a water inlet point was selected to place the rat in the pool, and the number of times the rat crossed the original platform position during a 3 min session was recorded.
Hematoxylin and eosin (HE) staining was used to evaluate neuronal damage in the frontal cortex and hippocampus. After behavioral testing, the rats were euthanized by deep anesthesia and bloodletting. Brain tissue was quickly placed on ice, and longitudinal sections of the brain were fixed with 4% paraformaldehyde. The H&E staining kit was purchased from Beyotime (Jiangsu, China), and the experimental procedure was performed according to the manufacturer’s instructions.
Neuronal apoptosis in the frontal cortex and hippocampus was examined using a TUNEL staining kit (catalog no. 11684817910, Roche, Basel, Switzerland) according to the manufacturer’s instructions. Samples of the hippocampus and cerebral cortex were collected, fixed in paraformaldehyde, routinely dehydrated, paraffin embedding, and sectioned. Based on the number of slides and size of the tissue samples, the appropriate amounts of reagent 1 (TdT) and reagent 2 (dUTP) in the TUNEL kit were mixed in a 1:9 ratio and then added to the tissues. The sections were placed in a humidified 37°C incubator for 2 h and then evenly covered and blocked with 3% bovine serum albumin at room temperature for 30 min. Biotinylated nucleotides and terminal deoxynucleotidyl transferase were added to the sections (1:500 dilution) and incubated overnight at 4°C. The sections were then washed with phosphate-buffered saline (PBS) and covered with horseradish peroxidase (HRP)-labeled streptavidin in the dark for 50 min. DAPI staining solution was added, and the mixture was incubated at room temperature for 10 min in a darkroom, sealed with an anti-fluorescence quenching tablet, and photographed under a fluorescence microscope.
The oxidative stress markers superoxide dismutase (SOD; catalog no. SES134Ra, USCN, Wuhan, China), malondialdehyde (MDA; catalog no. CEA597Ge, USCN), and reduced glutathione (GSH; catalog no. CEA294Ge, USCN) and brain damage markers S-100β (catalog no. SEA012Ra, USCN) and neuron-specific enolase (NSE; catalog no. SEA537Ra, USCN) were detected by enzyme-linked immunosorbent assay (ELISA) kits in brain tissues according to the manufacturer’s instructions.
The immunofluorescence assay (IFA) was performed as previously described (
Tissue samples of the cortex and hippocampus were lysed together in RIPA buffer (Beyotime, Shanghai, China). Supernatants were collected, and proteins were quantified using the BCA Protein Assay kit (Beyotime, Shanghai, China). Denatured protein samples were separated on 10% sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) gels and then transferred to polyvinyl difluoridine (PVDF) membranes. Membranes were blocked with 5% non-fat dry milk for 2 h and incubated with the following primary antibodies overnight at 4°C: Bax (diluted 1:1000, catalog no. ab32503, Abcam, Cambridge, United Kingdom), Bcl2 (diluted 1:1000, catalog no. ab32124, Abcam, Cambridge, United Kingdom), cleaved caspase-3 (diluted 1:500, catalog no. ab13847, Abcam, Cambridge, United Kingdom), LC3B (diluted 1:1000, catalog no. ab48394, Abcam, Cambridge, United Kingdom), Beclin 1 (diluted 1:2000, catalog no. ab207612, Abcam, Cambridge, United Kingdom), AKT (diluted 1:500, catalog no. ab8805, Abcam, Cambridge, United Kingdom), mTOR (diluted 1:2000, catalog no. ab2732, Abcam, Cambridge, United Kingdom), S6K1 (diluted 1:5000, catalog no. ab32529, Abcam, Cambridge, United Kingdom), 4E-BP1 (diluted 1:1000, catalog no. ab2606, Abcam, Cambridge, United Kingdom), or GAPDH (diluted 1:5000, catalog no. ab8245, Abcam, Cambridge, United Kingdom). The membranes were then incubated with goat anti-rabbit IgG H&L (HRP) (diluted 1:5000, catalog no. ab6721, Abcam, Cambridge, United Kingdom) or goat anti-mouse IgG H&L (HRP) (diluted 1:5000, catalog no. ab205719, Abcam, Cambridge, United Kingdom) secondary antibody for 2 h at room temperature. Protein bands were visualized using an enhanced chemiluminescence (ECL) kit (catalog no. 35050, Pierce, MMAS, United States) and quantified by scanning densitometry using ImageJ software.
All of the statistical analyses were performed using SPSS 19.0 software. The data are expressed as mean ± standard deviation. Comparisons between groups were performed using one-way analysis of variance. Intra-group comparisons were performed using a repeated measures design. Values of
We first examined the effect of CCH on untreated rat brains. Neurological deficit scores increased after BCCAO, and both spatial cognitive ability and spatial memory decreased in CCH rats compared with the Sham group (
Resveratrol improved neurological damage and cognitive function in CCH rats. Neurological impairment was measured by the Bederson scoring method. Memory performance was measured in the Morris water maze test.
The retinal cortex and hippocampal CA1 area were monitored in CCH rats that were treated with resveratrol (Res group) at 3, 6, and 9 weeks of ischemia. Ischemia that was induced by CCH gradually improved, and nuclei were large and round with clear nucleoli, indicating that resveratrol improved brain damage that was caused by CCH (
Resveratrol alleviated brain damage in CCH rats. Neuronal pathological changes were detected by HE staining in the frontal cortex and hippocampal CA1 area in each group. ELISA was used to observe the brain damage markers S-100β and NSE.
To evaluate oxidative stress between experimental groups, we performed ELISAs for several known markers of oxidative stress. After resveratrol treatment, MDA levels gradually decreased in the frontal cortex and hippocampal CA1 area in the Res group, whereas SOD and GSH levels gradually increased (
Resveratrol inhibited oxidative stress in CCH rats. ELISA was used to detect oxidative stress factors.
Resveratrol inhibited neuronal apoptosis in CCH rats. TUNEL staining was used to detect neuronal apoptosis in the frontal cortex and hippocampal CA1 area in each group. Apoptosis-related proteins were detected by Western blot.
To explore the possible mechanism by which these physiological changes occurred, we examined the AKT/mTOR signaling pathway in each experimental group. The expression of AKT, mTOR, S6K1, and 4E-BP1 in the frontal cortex and hippocampal CA1 area increased over time in the CCH group compared with the Sham group (
Resveratrol reduced brain damage in CCH rats via the AKT/mTOR signaling pathway. Immunofluorescence staining and Western blot were used to detect the expression of AKT/mTOR signaling pathway-related proteins.
Resveratrol regulated autophagy in CCH rats. Immunofluorescence staining and Western blot were used to detect the expression of LC3B and Beclin 1.
To evaluate whether the protective effects of resveratrol against CCH depend on the PI3K/AKT/mTOR pathway, we treated CCH rats with both the PI3K inhibitor LY294002 and resveratrol. Neurological deficit scores in the PI3K group gradually increased, and their cognitive and spatial memory abilities were poor compared with the Res group (
PI3K inhibition reversed the therapeutic effects of resveratrol in CCH rats. Neurological impairment was measured by the Bederson scoring method. Memory performance was measured in the Morris water maze test. Neuronal pathological changes were detected by HE staining in the frontal cortex and hippocampal CA1 area in each group. ELISA was used to observe markers of brain damage and oxidative stress.
PI3K inhibition reversed the therapeutic effects of resveratrol in CCH rats. TUNEL assays were performed to evaluate neuronal apoptosis in the frontal cortex and hippocampal CA1 area in each group. Apoptosis-related proteins were detected by Western blot.
PI3K inhibition reversed the therapeutic effects of resveratrol in CCH rats. Immunofluorescence staining and Western blot were used to detect expression of the AKT/mTOR signaling pathway-related proteins LC3B and Beclin 1.
Chronic cerebral hypoperfusion is a pathological disorder that is known to lead to cognitive dysfunction. To investigate the effect of resveratrol on cognitive function in CCH, a rat model of CCH was established by permanently ligating the bilateral common carotid arteries. After establishing the CCH model, resveratrol was administered for 21 consecutive days. Cognitive function was evaluated in rats in the Morris water maze at 3, 6, and 9 weeks of ischemia. Cognitive function improved in CCH rats after resveratrol administration. We examined pathological damage to the frontal cortex and hippocampus using HE staining and found decreases in markers of oxidative stress and brain injury following resveratrol treatment. The Western blot data showed that resveratrol activated autophagy, decreased LC3B and Beclin 1 expression, and inhibited the expression of AKT/mTOR signaling pathway-related proteins. These results were the most significant at 9 weeks. After the administration of a PI3K inhibitor, the expression of AKT/mTOR signaling pathway-related proteins increased, and autophagy decreased, indicating that the neuroprotective effects of resveratrol were inhibited. However, we did not use quantitative polymerase chain reaction to detect the transcription levels of mTOR-related genes. Thus, we were unable to determine whether resveratrol exerts its functions through transcriptional regulation or an increase in the half-life of mTOR-related proteins. Future studies will seek to unveil the molecular mechanism of action of resveratrol. Previous studies have shown that neuronal cells in the frontal cortex and hippocampus play important roles in maintaining normal learning and cognitive ability. Therefore, blocking neuronal apoptosis in the frontal cortex and hippocampus is important for improving cognitive dysfunction. The present study showed that resveratrol improved cognitive function in CCH rats and improved neuronal damage in the frontal cortex and hippocampus.
Resveratrol is a non-flavonoid polyphenolic compound that is produced by plants (
The AKT/mTOR signaling pathway is a classic anti-apoptosis pathway that is involved in various cellular activities and closely related to cell survival, the cell cycle, cell proliferation, and apoptosis (
Resveratrol improved pathological damage and cognitive function in a rat model of CCH by activating autophagy to reduce oxidative stress and inhibiting neuronal apoptosis in the frontal cortex and hippocampus. These beneficial effects of resveratrol may be regulated by the AKT/mTOR signaling pathway.
The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.
All of the animal experiments conformed to internationally accepted standards and were approved by the Experimental Animal Welfare and Ethics Committee of China Medical University (IACUC No. 2018097).
NW and ZX conceived and designed the experiments. NW, JH, CP, JW, MM, and XS performed the experiments. JW, MM, and XS analyzed the data. NW and ZX wrote the manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.