Chronic stress has been recognized to induce the alterations of neuronal and glial cells in the hippocampus, and is thus implicated in cognitive dysfunction. There is increasing evidence to indicate that natural compounds capable of exerting neuroprotective and antioxidant activities, may function as potential therapeutic agents for cognitive impairment. The present study examined the neuroprotective effects of pinostrobin from
Chronic stress affects brain function and induces long-term alterations in various neural systems related to anxiety, depression, cognition and insomnia (
Herbal medicinal substances exerting neuroprotective effects have been considered to be pharmacological agents for the treatment of several neurodegenerative diseases. Pinostrobin, a natural bioflavonoid compound isolated from the rhizomes of
A total of 28 adult male Wistar rats (weighing 200-220 g, at the commencement of the experiment) were obtained from the Nomura Siam International (Bangkok, Thailand). The animals were allowed a week for acclimatization prior to the commencement of the experiments. All experimental procedures were conducted during the dark phase of the light cycle with standard chow and water available
Fresh rhizomes of
All animals were randomly divided into four experimental groups (7 rats per group) as follows: i) The control group, no stress; ii) the vehicle + CRS group; iii) the pinostrobin 20 mg/kg + CRS goup; and iv) the pinostrobin 40 mg/kg + CRS group. The rats were exposed to restraint stress using a wire mesh restraint secured with butterfly clips that closely fit the body of the rats, as described in a previous study (
All animals were deeply anesthetized via an intraperitoneal injection of thiopental sodium (70 mg/kg body weight) and perfused with ice-cold 0.1 M PBS. The brains (n=28) were immediately removed and cut at the midline, dividing the brain into two hemispheres. The hippocampus from the left hemisphere was separated and stored at -80˚C for biochemical determination. The right hemisphere was fixed with ice-cold 4% paraformaldehyde and cryopreserved in 12.5% sucrose for immunohistochemistry. The brains were sectioned into 30-µm-thick sections using a cryostat microtome (AST500, Amos Scientific Pty Ltd.) and stored in an anti-freeze solution (4˚C). The coronal sections were rinsed and incubated in 3% H2O2 and followed by 3% normal horse serum (cat. no. A9647, Sigma-Aldrich; Merck KGaA) for non-specific blocking. Subsequently, sections were incubated with the primary antibodies, mouse anti-glial fibrillary acidic protein (GFAP; 1:500, cat. no. MAB5628, MilliporeSigma), or rabbit anti-excitatory amino acid transporter 2 (EAAT2; (1:200, cat. no. ab41621, Abcam) at 4˚C overnight. The sections were washed in 0.1 M PBS for 30 min and incubated for 2 h at room temperature with biotinylated donkey anti-mouse secondary antibody (1:500, cat. no. 715-065-150, Jackson ImmunoResearch Europe, Ltd.) or anti-rabbit (1:500, cat. no. 711-065-152, Jackson ImmunoResearch Europe, Ltd.). The sections were rinsed in 0.1 M PBS followed by 1 h of incubation in 0.1% extravidin peroxidase (1:1,000, cat. no. E2886, Sigma-Aldrich; Merck KGaA) at room temperature, and then rinsed again. Immunolabeling was developed using a nickel-enhanced 3,3'-diaminobenzidine (DAB) reaction (cat. no. D12384, Sigma-Aldrich; Merck KGaA). Finally, the sections were washed in 0.1 M PBS and, then mounted on positive charged slides, dehydrated using graded alcohols, cleared in xylene, and cover-slipped using mounting medium (cat. no. 107961, Sigma-Aldrich; Merck KGaA). For Nissl staining, the sections were stained with 0.1% cresyl violet (cat. no. 1.05235.0025, Sigma-Aldrich; Merck KGaA) for 8 min at 60˚C, dehydrated with ethanol, cleared in xylene, and coverslipped using mounting medium.
The level of MDA was evaluated as an indicator of lipid peroxidation. A total of seven hippocampi from each group were homogenized in 0.1 M PBS (pH 7.4), centrifuged at 9,279 x g at 4˚C for 15 min, and the supernatant was subjected to the thiobarbituric acid reaction according to the protocol previously described by Nakmareong
SOD activity was determined using a colorimetric assay kit (cat. no. S19160, Sigma-Aldrich; Merck KGaA) according to the manufacturer's instructions. The results were presented as the inhibition rate (%).
Catalase activity was measured based on the enzyme degradation of H2O2 according to previously published study with some modifications (
To determine the density of neurons, hippocampal images were captured at x40 magnification using a bright-field microscope (Nikon Corporation). Images of subregions of the hippocampus (at x40 magnification), including CA1, CA2 and CA3 were subjected to exhaustive manual counts using NIS Elements imaging software version 5 (Nikon Corporation). For thresholding function, the immunoreactivity of the astrocytes within the hippocampus was determined using the thresholding function of Image J software (Version 1.53, National Institutes of Health). Image thresholding is the frequently used technique to quantitatively determine the alterations in immunolabelled material as previously described (
The Y-maze test was used to measure working memory in animal by recording a spontaneous alternation (
The NOR test was performed in a black open field box (45x65x45 cm, Laboratory Animal Research Center, University of Phayao, Thailand) to determine the recognition memory. The test composed of three sessions (habituation phase, training phase and test phase) using a previously described method with minor modifications (
All data were analyzed using GraphPad Prism 9 (GraphPad Software, Inc.). Data are expressed as the mean ± SEM. Statistical analysis was performed using one-way ANOVA, followed by Tukey's post hoc test for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.
The antioxidant activities of pinostrobin were determined by measuring the levels of MDA, which is an indicator of lipid peroxidation, and the activities of some of the main scavenging enzymes, such as SOD and CAT in the hippocampus (
The Y-maze and NOR tests were used to determine whether pinostrobin could reverse CRS-induced cognitive impairment (
Chronic stress is considered an important risk factor that can induce neuronal damage (
To examine whether CRS affects astrocytes, the alteration of GFAP-immunoreactive astrocytes in the hippocampus was further evaluated using immunohistochemistry and threshold analysis. As shown by the results illustrated in
To determine the mechanisms through which pinostrobin altered the level of glutamate transporter expressed in astrocytes, the immunoreactivity of EAAT2 was further investigated using immunohistochemical analysis. As illustrated in
The present study investigated the neuroprotective effects of pinostrobin on cognitive performance in rats exposed to CRS. The results suggested that the administration of pinostrobin ameliorated chronic stress-induced cognitive impairment by exerting antioxidant effects, reducing neuronal cell damage, and enhancing the expression of astrocytic GFAP and EAAT2 in the hippocampus. Previous studies have revealed that chronic stress is an important regulatory factor for the development of cognitive dysfunction (
According to previous findings, the supplementation of natural compounds can ameliorate learning and memory deficits involved in the CRS-induced production of oxidative stress (
Moreover, the ameliorative effects of pinostrobin on cognitive deficits were determined using the Y-maze test. Working memory is one of the short-term memories that can be decreased in Alzheimer's disease. The Y-maze test was extensively used to evaluate the function of hippocampal spatial working memory by counting the number of arm entries and calculating the percentage of spontaneous alternation (
There is accumulating scientific evidence to demonstrate that chronic stress can provoke hippocampal neurodegeneration, including neuronal apoptosis, decreased synaptic plasticity and the reduction of dendritic spine density, which results in learning and memory impairment (
To further elucidate the possible neuroprotective mechanisms of pinostrobin, the disruptions of astrocytes involved in CRS-induced memory deficits were determined. Previous studies have suggested that CRS-induced astrocyte dysfunction may be associated with learning and memory deficits (
The results demonstrated herein revealed the dose-dependent effects of pinostrobin in treating CRS-induced cognitive deficits. In a previous study on the toxicity of pinostrobin, it was reported that pinostrobin was non-toxic and was not mutagenic to male rats within the 1-100 mg/kg dose range (
In conclusion, the present study indicated that treatment with pinostrobin significantly attenuated chronic stress-induced cognitive impairment by decreasing the levels of oxidative stress, reducing neuronal damage, and by enhancing the function of astrocytes and EAAT2 in the hippocampus of rats. Therefore, these findings suggest that pinostrobin may have potential medicinal value as a neuroprotective agent for the prevention and treatment of chronic stress-induced cognitive deficits and other cognitive disorders.
Not applicable.
The database used and/or analyzed during the present study are available from the corresponding author on reasonable request.
RK designed the study, performed all the experiments and wrote the manuscript. ST was involved in the study methodology and wrote the manuscript. SS was involved in the preparation of the extract. TP and JJ were involved in the data analysis and in editing the manuscript. All authors have read and approved the final manuscript. RK and ST confirmed the authenticity of all the raw data.
The experimental protocol was approved by the Ethics Committee of the Laboratory Animal Research Center, University of Phayao, Phayao, Thailand (approval no. 640104006).
Not applicable.
The authors declare that they have no competing interests.
Chemical structure of pinostrobin.
Effects of pinostrobin on oxidative stress markers in the hippocampus. Graphs demonstrate the (A) MDA level, (B) SOD activity, and (C) CAT activity. Data are expressed as the mean ± SEM. **P<0.01 vs. the control group; #P<0.05 and ##P<0.01 vs. the vehicle-CRS group. CRS, chronic restraint stress; PSB, pinostrobin; MDA, malondialdehyde; SOD, superoxide dismutase; CAT, catalase.
Effects of pinostrobin on learning and memory performance. Graphs demonstrate the percentage of spontaneous alternation using (A) the Y-maze test, and (B) the recognition index using the novel object recognition test. Data are expressed as the mean ± SEM. **P<0.01 vs. the control group; #P<0.05 and ##P<0.01 vs. the vehicle-CRS group. CRS, chronic restraint stress; PSB, pinostrobin.
Effects of pinostrobin on neuronal damage in the hippocampus. (A-C) Graphs demonstrate neuronal density in the hippocampal CA1, CA2 and CA3 regions. (D) Representative images of Nissl staining of the hippocampal CA1, CA2 and CA3 regions at x40 magnification. Black squares illustrate the changes in neuronal density. Scale bar, 50 µm. Data are expressed as the mean ± SEM. **P<0.01 vs. the control group; #P<0.05 and ##P<0.01 vs. the vehicle-CRS group. CRS, chronic restraint stress; PSB, pinostrobin.
Effects of pinostrobin on the alteration of GFAP-labeled astrocytes in the hippocampus. (A) Immunohistochemical staining for GFAP in the hippocampus and threshold images of GFAP in CA1. (B-D) Graphs demonstrate the GFAP expression in subregions of the hippocampus. Black arrows illustrate morphological changes of astrocytes. Scale bar, 100 µm. Data are expressed as the mean ± SEM. **P<0.01 vs. the control group; ##P<0.01 vs. the vehicle-CRS group. CRS, chronic restraint stress; PSB, pinostrobin; GFAP, glial fibrillary acidic protein.
Effects of pinostrobin on the alteration of astroglial EAAT2 immunoreactivity in the hippocampus. Graphs demonstrate EAAT2 expression in the hippocampal (A) CA1, (B) CA2, and (C) CA3 subregions. (D) Representative images of EAAT2 immunoreactivity in the hippocampal CA1, CA2 and CA3 subregions. Scale bar, 100 µm. Data are expressed as the mean ± SEM. **P<0.01 vs. the control group; #P<0.05 and ##P<0.01 vs. the vehicle-CRS group. CRS, chronic restraint stress; PSB, pinostrobin; EAAT2, excitatory amino acid transporter 2.