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| ORIGINAL ARTICLE |
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| Year : 2023 | Volume
: 66
| Issue : 4 | Page : 209-219 |
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Sericin improves memory and sociability impairments evoked by transient global cerebral ischemia through suppression of hippocampal oxidative stress, inflammation, and apoptosis
Seyed Mehdi Vatandoust1, Javad Mahmoudi2, Shahrbanoo Oryan1, Fereshteh Farajdokht3, Saeed Sadigh-Eteghad2, Siamak Sandoghchian Shotorbani4, Huaxi Xu5, Delaram Eslimi Esfahani1
1 Department of Animal Biology, Faculty of Biological Sciences, Kharazmi University, Tehran, Iran
2 Neurosciences Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
3 Neurosciences Research Center; Department of Physiology, School of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran
4 Department of Immunology, Faculty of Medicine; Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
5 Department of Immunology, Jiangsu University of Medical Sciences, Zhenjiang, China
| Date of Submission | 13-Jan-2023 |
| Date of Decision | 08-Mar-2023 |
| Date of Acceptance | 27-Mar-2023 |
| Date of Web Publication | 01-Jun-2023 |
Correspondence Address:
Dr. Javad Mahmoudi
Neurosciences Research Center, Tabriz University of Medical Sciences, Tabriz
Iran
Dr. Shahrbanoo Oryan
Department of Animal Biology, Faculty of Biological Sciences, Kharazmi University, Tehran
Iran
 Source of Support: None, Conflict of Interest: None
DOI: 10.4103/cjop.CJOP-D-23-00006
Sericin (Ser) is a natural neuroactive macromolecule with diverse pharmacological properties, and our previous findings have shown its neuroprotective potentials. This study aimed to investigate the therapeutic potential of Ser on cognitive dysfunction induced by transient global cerebral ischemia/reperfusion (tGI/R) and its mechanism of action. The tGI/R was induced in BALB/c mice by bilateral occlusion of the common carotid arteries for two 5 min followed by a 10-min reperfusion period. After 24 h, mice were treated with normal saline or different doses of Ser (100, 200, and 300 mg/kg) for 10 days. Cognitive performances were assessed using the Barnes maze and social interaction tasks. Oxidative stress markers including superoxide dismutase (SOD), glutathione peroxidase (GPx), total antioxidant capacity (TAC), and malondialdehyde (MDA) as well as pro-inflammatory cytokines (interleukin (IL)-6 and tumor necrosis factor-alpha) and anti-inflammatory cytokine (IL-10) were assessed in the hippocampus. Markers of apoptosis (pro- and cleaved caspase-9 and 3, Bax, and Bcl-2) were assessed by Western blotting. Besides, transferase-mediated dUTP nick end-labeling assay was used to detect apoptotic cell death. We show here that Ser administration improved tGI/R-induced cognitive deficits, enhanced the activity of SOD and GPx, increased TAC levels, while reduced MDA levels. Notably, Ser decreased neuronal apoptotic cell death in the hippocampal dentate gyrus (DG) region, accompanied by suppression of neuroinflammation, downregulation of pro-apoptotic proteins (caspase-9, caspases-3, and Bax), and upregulation of anti-apoptotic protein, Bcl-2. Taken together, Ser administration protected hippocampal neurons from apoptotic cell death by impeding oxidative stress and inflammatory responses and, in turn, improved cognitive function in the tGI/R mice.
Keywords: Apoptosis, global cerebral ischemia, inflammation, oxidative stress, sericin, sociability, social novelty preference
How to cite this article:
Vatandoust SM, Mahmoudi J, Oryan S, Farajdokht F, Sadigh-Eteghad S, Shotorbani SS, Xu H, Esfahani DE. Sericin improves memory and sociability impairments evoked by transient global cerebral ischemia through suppression of hippocampal oxidative stress, inflammation, and apoptosis. Chin J Physiol 2023;66:209-19 |
How to cite this URL:
Vatandoust SM, Mahmoudi J, Oryan S, Farajdokht F, Sadigh-Eteghad S, Shotorbani SS, Xu H, Esfahani DE. Sericin improves memory and sociability impairments evoked by transient global cerebral ischemia through suppression of hippocampal oxidative stress, inflammation, and apoptosis. Chin J Physiol [serial online] 2023 [cited 2023 Dec 9];66:209-19. Available from: https://www.cjphysiology.org/text.asp?2023/66/4/209/378079 |
| Introduction | |  |
Global cerebral ischemia is a clinical situation resulting from reversible severe hypotension, cardiac arrest, or other conditions that disrupt brain oxygen and glucose levels.[1] Following ischemia, multiple events such as oxidative stress, inflammatory responses, apoptosis, and excitotoxicity are being activated to induce primary neuronal injury.[2],[3] Then, secondary neuronal injury is initiated following re-establishment of the blood flow (reperfusion phase) to the ischemic areas which worsens neuronal insults.[1],[2],[3]
Indeed, ischemic insults via activation of astroglial and microglial cells induce uncontrolled neuroinflammation and change the balance of oxidative stress molecules which this vicious cycle promotes apoptosis and delayed neuronal injury.[4],[5]
The delayed neuronal injury persists for several days and exacerbates neuronal cell death in susceptible areas such as cerebral cortex, striatum, and hippocampus resulting in behavioral abnormalities.[6],[7] There is a robust association between hippocampal neuronal loss and ischemia-induced cognitive dysfunction,[8] so that cognitive deficits commonly occur in 70% of survived patients nearly 3 months following a global cerebral ischemic attack, leading to disability and decreased quality of life.[9],[10] Importantly, delayed neuronal loss in the hippocampus may serve an opportunity for using effective neuroprotective regimens to restore its function and allow the survivors to return to their normal cognitive activities. Therefore, achieving a therapeutic option for treatment of ischemic insults and its associated cognitive impairments is of great interest.
Sericin (Ser), a hydrophilic protein obtained from the silkworm, makes up nearly 30% weight of the silkworm cocoon. It is formed of 18 amino acids and is rich in hydrophilic essential amino acids, such as serine (33.4%) and glycine (16.7%).[11],[12],[13] As studies reported, it showed cognitive enhancement, antidepressant, and anxiolytic properties via different mechanisms including anti-inflammatory, antioxidant, and anti-apoptotic activities.[13],[14] Ser has been shown to limit diabetes-induced hippocampal neuronal insults through modulation of Akt signal transduction and reduction of apoptotic cell death.[15] However, to date, there is no report of the effect of Ser on the transient global cerebral ischemia/reperfusion (tGI/R) injury.
Technically tGI/R model is a reliable platform with a low mortality rate to assess mechanisms underlying global ischemia insults and develop possible therapeutic approaches.[16],[17] This study sought to assess the influences of Ser on spatial learning and memory and social interaction parameters, as well as hippocampal oxidative stress, inflammatory cytokines, and apoptosis in the tGI/R-affected mice.
| Materials and Methods | | |
Animals and study design
Seventy-five adult male BALB/c mice (25–30 g) were purchased from Homa Teb Tabriz CO. (HTT Co., Tabriz, Iran) and were housed under conventional laboratory conditions, temperature: 25 ± 2°C, humidity: 50% ± 6%, and 12 h of light/dark cycle. Following 5 days of acclimation to the new condition, mice were randomly assigned into five groups (15 mice in each group), including control, normal saline (NS), Ser100, Ser200, and Ser300. The control mice were submitted to sham surgery and after 24 h received NS. The NS, Ser 100, 200, and 300 groups underwent tGI/R surgery and after 24 h received NS and or Ser at the doses of 100, 200, and 300 mg/kg, respectively [Figure 1]. All solutions were freshly prepared each day and administrated at constant volume of 10 ml/kg via gastric gavage for 10 consecutive days. All experimental processes were performed in agreement with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH; Publication No. 85-23, revised 1985), and the procedures were approved by the regional ethics committee (IR.KHU.REC.1399.013). | Figure 1: Study design. Transient global cerebral ischemia/reperfusion (tGI/R).
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Induction of transient global cerebral ischemia/reperfusion
The animal was anesthetized by a mixture of isoflurane (3% for induction and 1.5% for maintenance) in oxygen and subjected to the tGI/R procedure.[18] For this purpose, both common carotid arteries were exposed by a midline incision in the neck and sternum. The arteries were isolated from adjacent tissues, temporarily occluded for 5 min by aneurysm clips, and then reperfused for 10 min. Subsequently, the arteries were occluded for another 5 min. At the end of the second occlusion, the clips were removed and the surgical site was sutured with a 6-0 silk suture. The control mice had the identical surgical procedure as the tGI/R-submitted mice, but the arteries were not occluded, and the incision was subsequently closed.
Regional cerebral blood flow measurement
The induction of tGI/R was confirmed using laser Doppler flowmetry which measures cerebral microperfusion. Hemodynamic alterations were monitored from the beginning of anesthesia induction to 10 min after reperfusion using a probe at the intact skull (3.5 mm right from the bregma). The percentage of data recorded for 60 s after occlusion (that differed from baseline values) was used to determine the change in regional cerebral blood flow (RCBF).[19] Due to bilateral common carotid occlusion, RCBF was reduced, where the mean RCBF was found to be <10%–15% of the baseline. This returned to 80%–90% of baseline once the clips were removed, indicating accurate induction of tGI/R.
Neurological outcome
The Neurological Severity Score (NSS) test was performed 1 day after tGI/R induction to assess probable neurological alterations in the animals.[20] The 10 tasks in the test measure motor function, reflexes, balance, and alertness, among other elements of neurological function. The scale runs from 0 to 10, with 0 denoting no impairment and 10 denoting the most severe impairment. The NSS results revealed no significant differences between the study groups (data are not shown).
Behavioral tests
Barnes maze
A black circular polyvinyl chloride maze (15 mm thick, 100 cm in diameter) with 20 circular holes (diameter: 5 cm) evenly spaced around the maze's perimeter was used to evaluate spatial learning and memory. To keep the mouse from jumping down, an escape box (20 cm × 15 cm × 5 cm) was placed below the target hole, and the maze was set on top of a 50 cm high stool. The experiment was conducted in a room with simple visual cues on the walls that remained consistent throughout the experiment. A buzzer (producing 80 dB tones) was utilized as an unpleasant stimulus to boost motivation to flee.
The process lasted 6 days and consisted of three sessions. The mouse was placed inside a black cylindrical start chamber in the middle of the platform for 10 s on day 1 (habituation phase), then the start chamber was picked up, the buzzer was turned on, and the mouse was led toward the escape box. The buzzer was turned off as soon as the mouse entered the escape box. Accordingly, the animal was permitted to stay there for 60 s. Each mouse was subjected to four acquisition trials per day on days 2–5 (training phase). The mouse was allowed 3 min to walk through the maze and discover the escape box during the acquisition phase. If the animal was unable to locate the escape box during this time, it was manually guided to the target box and given 15 s to stay in it.
The animal's reference memory was tested for 3 min in the absence of the escape box on day 6 (probe test). Over 4 days of training, the latency to reach the escape box was assessed, as well as the elapsed time in the target quadrant during the probing session.[21]
Sociability and social novelty preference tests
To measure social cognitive abilities, a three-chambered acrylic box (60 cm × 45 cm × 50 cm) was employed.[22] Acrylic walls with sliding doors (6 cm) split the box into three compartments, allowing the animal to freely move between them. In each side chamber, two empty containers (11 cm L, 10 cm bottom diameter, 5 mm transparent polycarbonate bars spaced 1 cm apart) were inserted. The test was conducted over 2 days and consisted of three phases including habituation, sociability (social affiliation), and social novelty preference phases. The 1st day consisted of habituation and sociability test phase, and the 2nd day consisted of social novelty preference test phases.
At the beginning of the habituation phase, the doors between chambers were opened, and the subject mouse was placed in the central room and permitted to freely move in the box for 10 min. The mouse was then led to the center compartment once the lateral doors were closed. During this period, the distance traveled by the mouse was monitored.
For the second phase, a stimulus mouse (Stranger A) was previously habituated to the enclosure and placed in the upturned containment cup in one of the lateral chambers. In another side chamber, an empty container was placed. Then, the subject mouse was allowed 10 min to traverse through the compartments. Then, the subject mouse was guided to the central room and the doors were closed and returned to its home cage. The social affiliation ratio was calculated by dividing the total time spent in direct contact with a container holding Stranger A by the total time spent in direct contact with empty container and Stranger A. At the 2nd day, the Stranger A mouse was again placed in its side chamber container, while a new stimulus mouse (Stranger B) was placed in the previously vacant container in the opposite outer chamber. The side doors were opened, and the subject mouse was given another 10 min to investigate all parts of the box. The subject mouse's direct contact time with both containers (containing Strangers A and B) was recorded. The social novelty preference ratio was measured by dividing the amount of time spent in direct contact with Stranger B by the total amount of time spent in direct contact with Strangers A and B.
Behavioral analysis
Behavioral instruments were purchased from Arman Poshtiban Co. (Arman Medica® Co., Tabriz, Iran) and data were recorded by an experimenter who was blind to the nature of treatments and examined by Noldus EthoVision™ software (Noldus, The Netherlands). After each test, the maze and box of the social interaction test was cleaned with 70% alcohol, and residues were removed to limit olfactory cues.
Sampling
The animals were sacrificed under anesthesia by an intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg) mixture. Then, the brains were excised following decapitation. Hippocampal tissues were dissected on ice, placed into labeled microtubes, and stored in a −80°C freezer. Moreover, five brain samples from each group were fixed in 4% paraformaldehyde (pH 7.4) for histological assessments.
Oxidative stress parameters
The hippocampal samples were homogenized in chilled 1.15% KCl solution and then centrifuged at 10,000 g for 30 min at 4°C. The total protein concentration was measured by the Bradford method. The activity of superoxide dismutase (SOD) was measured using the RANSOD kit (Randox Labs; Crumlin, UK) according to the Delmas-Beauvieux et al. technique.[23] To launch the reaction, 1 mL of the reaction mixture (0.1 mL phosphate-buffered saline, 0.6 mL phosphate buffer (0.5 M), 0.1 mL nitroblue tetrazolium (57 mM), and 0.1 mL xanthine (1 mM) were introduced to xanthine oxidase (50 mU)). Then, by measuring the absorbance at 560 nm with a spectrophotometer, SOD activity was calculated as U per mg protein. The Paglia and Valentine technique was used to calculate the activity of glutathione peroxidase (GPx)[24] with the help of a RANSEL kit (Randox Labs; Crumlin, UK).
To assess tissue malondialdehyde (MDA) levels, the Draper and Hadley technique[25] was utilized. In brief, the presence of thiobarbituric acid-reactive compounds (TBARS) as MDA in hippocampus homogenates was spectrophotometrically evaluated at 532 and 520 nm. The results were expressed in nanomoles per milligram of protein.
The total antioxidant capacity (TAC) of hippocampal homogenates was measured by Ghiselli et al. method.[26] Antioxidants in the homogenate samples reduce Fe3+ to Fe2+, producing a colored complex. Briefly, the absorbance was read at 593 nm by a TAC kit (Randox, UK) and reported as nmol/mg protein.
Measurement of hippocampal pro- and anti-inflammatory cytokines
Interleukin (IL)-6, IL-10, and tumor necrosis factor (TNF)-alpha protein levels in the hippocampal tissue were determined using commercially available enzyme-linked immunosorbent assay kits (Elabscience, USA) according to the manufacturer's instructions.
Western blot analysis
To homogenize the frozen hippocampus samples, a lysis solution combined with a protease inhibitor cocktail from the radioimmunoprecipitation assay was used. The homogenized samples were then centrifuged at 13,000 g for 15 min at 4°C, and the protein content in the supernatant was measured using the Bradford technique. Following that, each sample was run through a 12.5% polyacrylamide gel and electrophoresis before being transferred to a polyvinylidene difluoride membrane (Roche, UK). At 4°C overnight, the membranes were probed with particular primary antibodies such as anti-Bax (1:500, sc-493), anti-Bcl-2 (1:500, sc-492), anti-caspase-3 (1:500, sc-7148), anti-caspase-9 (1:500, sc-81663), and anti-actin (internal reference control). Membranes were then rinsed three times with 0.1% TBST (Tris-buffered saline + 0.1% Tween 20) before being incubated for 1 h at room temperature with a horseradish peroxidase-conjugated secondary antibody (1:5000). Membranes were then immersed in enhanced chemiluminescence detection reagent (Amersham, UK), and signals were visualized using radiography film (Kodak, USA). Band densities were estimated using ImageJ version 1.62 software (US National Institutes of Health, Bethesda, MD, USA).
Terminal transferase-mediated dUTP nick end-labeling immunofluorescence
The terminal transferase-mediated dUTP nick end-labeling (TUNEL) staining assay was performed to detect apoptosis in the dentate gyrus (DG) subfield using a commercially available kit (Sigma-Aldrich, Germany). Briefly, 4 μm thick serial sections were obtained from paraffin-embedded samples and mounted on slides and then incubated with TUNEL assay mixture containing fluorescein-12-dUTP and recombinant terminal deoxynucleotidyl transferase (TdT) for 60 min at 37°C. Furthermore, nuclear DNA was labeled by the 4',6-diamidino-2-phenylindole staining. Then, bright green color TUNEL-positive cells on a blue background were counted for five fields per section using fluorescence microscopy (Optica, Italy) in a blinded manner.
Statistical analysis
The data were analyzed using GraphPad Prism 6.01 (GraphPad Software Inc., San Diego, CA, USA) and expressed as mean ± standard error of the mean (SEM). The results of the Barnes test were evaluated using a two-way ANOVA (group vs. day) and a Tukey's post hoc test. To find statistically significant differences between the groups, one-way ANOVA was used followed by a Tukey's post hoc test for additional variables. P < 0.05 was set as statistically significant.
| Results | | |
The effect of sericin on spatial learning and memory parameters
Two-way ANOVA of escape latency time in the training sessions revealed significant effects of group (F(4,240) = 6.419, P < 0.001) and day (F (3,240) = 56.39, P < 0.001), but not of their interaction (F (15,240) = 0.9018, P = 0.5459). On the 4th day of the training session, the induction of global ischemia increased latency time to detect the target box compared with the control group [P < 0.05, [Figure 2]a]. However, at doses of 200 (P < 0.05) and 300 mg/kg (P < 0.01), Ser reduced the latency time on the 3rd and 4th days. | Figure 2: Spatial learning and memory profiles in Barnes maze task in the study groups. (a) Mean escape latencies within 4 days of training and (b) time spent in the target quadrant. Data are presented as mean ± SEM (n = 13). Significant differences tested by one- or two-way ANOVA followed by Tukey's post hoc test. *P < 0.05, ***P < 0.001 compared to the control group. #P < 0.05, ##P < 0.01, ###P < 0.001 compared to the NS-received group. Ser: Sericin, NS: Normal saline, SEM: Standard error of the mean.
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The time spent in the target quadrant during the probe session was also substantially different in the study groups (F (4, 60) = 18.59, P < 0.0001). The tGI/R mice receiving NS spent less time in the target quadrant than the control group, according to intergroup comparisons [P < 0.001, [Figure 2]b]. The Ser300 group, on the other hand, spent significantly more time in the target quadrant than the NS group (P < 0.001).
The effect of sericin on the social interaction test parameters
The overall traveled distance between the experimental groups was not significantly different after the habituation phase of the sociability and social novelty preference tests [F (4, 60) = 0.3231, P = 0.12; [Figure 3]a]. Moreover, a significant difference in the social affiliation ratio was found (F (4, 60) = 8.841, P < 0.0001) among the groups. After post hoc analysis, it was found that tGI/R-affected animals had a lower social affiliation ratio than the control group [P < 0.01, [Figure 3]b]. Nonetheless, compared to the NS-treated ischemic mice, treatment with Ser at a dose of 300 mg/kg dramatically increased this ratio (P < 0.05). | Figure 3: Social interaction test parameters in the study groups. (a) The mean total distance traveled, (b) social affiliation ratio, and (c) social novelty preference ratio. Data are presented as mean ± SEM (n = 13). **P < 0.01, ***P < 0.001 versus control group, #P < 0.05, ##P < 0.01 versus NS group. Ser: Sericin, NS: Normal saline, SEM: Standard error of the mean.
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Furthermore, one-way ANOVA revealed that the novelty affiliation ratios among the groups differed significantly (F (4, 60) = 8.093, P < 0.0001). Multiple comparisons revealed that as compared to the control group, global ischemia dramatically lowered the novelty affiliation ratio [P < 0.001, [Figure 3]c]. Nonetheless, at a dose of 300 mg/kg, Ser considerably raised this ratio compared with NS mice (P < 0.01).
The effect of sericin on the hippocampal oxidative stress markers
In the hippocampus of the study groups, significant differences were found in MDA levels (F (4, 25) = 11.20, P < 0.001), SOD (F (4, 25) = 14.75, P < 0.001) and GPx activities (F (4, 25) = 10.62, P < 0.001), and TAC levels (F (4, 25) = 26.63, P < 0.0001).
As shown in [Figure 4]a, the induction of global ischemia considerably elevated MDA levels in the NS-received animals (P < 0.01) compared with the control group. Ser at the doses of 200 and 300 mg/kg, on the other hand, significantly lowered MDA levels in tGI/R-infected mice (P < 0.01 and P < 0.001, respectively). | Figure 4: Hippocampal oxidative stress marker levels in the experimental groups. (a) MDA levels, (b) SOD activity, (c) GPx activity, and (d) TAC. Data are presented as mean ± SEM (n = 6). **P < 0.01, ***P < 0.001 versus control group. ##P < 0.01, ###P < 0.001 versus NS group. Ser: Sericin, NS: Normal saline, MDA: Malondialdehyde, SOD: Superoxide dismutase, GPx: Glutathione peroxidase, TAC: Total antioxidant capacity, SEM: Standard error of the mean.
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Furthermore, when tGI/R-exposed animals were compared with the control group, hippocampal activities of SOD [P < 0.001, [Figure 4]b] and GPx [P < 0.001, [Figure 4]c] were significantly lower in the tGI/R-submitted mice. However, the Ser300 group showed substantially greater hippocampus SOD and GPx activities than the NS group (P < 0.01 for both).
As compared to the non-ischemic group, tGI/R significantly lowered hippocampus TAC levels [P < 0.001, [Figure 4]d. Nonetheless, in comparison with the NS group, Ser significantly enhanced hippocampus TAC levels in the Ser200 and Ser300 groups (P < 0.05 and P < 0.001, respectively).
The effect of sericin on the hippocampal interleukin-6, tumor necrosis factor-alpha, and interleukin-10 levels
The hippocampal levels of IL-6, TNF-α, and IL-10 differed significantly across the study groups, according to a one-way ANOVA (F (4, 25) = 57.80, P < 0.001; F (4, 10) = 47.02, P < 0.0001; and (F (4, 25) = 35.28, P < 0.0001, respectively). The induction of tGI/R considerably elevated hippocampal levels of IL-6 and TNF-α in the NS-received animals compared to the control group (P < 0.001 for both). When compared with the NS group, Ser at doses of 200 (P < 0.05) and 300 mg/kg (P < 0.001) significantly lowered hippocampal levels of IL-6 and TNF-α (P < 0.01 for Ser200 and P < 0.001 for Ser300) [Figure 5]a and [Figure 5]b. | Figure 5: Hippocampal inflammation marker levels in different groups. (a) IL-6, (b) TNF-α, and (c) IL-10 levels. Data are presented as mean ± SEM. (n = 6) ***P < 0.001 versus control group. #P < 0.05, ##P < 0.01, ###P < 0.001 versus NS group. Ser: Sericin, NS: Normal saline, IL-6: Interleukin-6, TNF-α: Tumor necrosis factor-alpha, SEM: Standard error of the mean.
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Furthermore, the induction of global ischemia significantly [P < 0.001, [Figure 5]c] reduced the concentration of IL-10, an anti-inflammatory cytokine, as compared to the control animals. However, Ser markedly increased IL-10 hippocampal levels in the Ser200 and Ser300 groups (P < 0.001, for both groups), compared to the mice in the NS group.
The effect of sericin on the hippocampal apoptosis proteins and TUNEL-positive cells in the dentate gyrus
As shown in [Figure 6]a, [Figure 6]b, [Figure 6]c, [Figure 6]d, [Figure 6]e, there were significant differences in Bax and Bcl-2 protein levels, as well as cleaved caspase-9/pro-caspase-9 and cleaved caspase-3/pro-caspase-3 ratios in the hippocampus of the study groups, according to the findings of one-way ANOVA (F (4, 20) = 30.13, P < 0.001; F (4, 20) = 21.24, P < 0.0001; F (4, 20) = 64.86, P < 0.001; and F (4, 20) = 15.41, P < 0.001, respectively). | Figure 6: Hippocampal apoptosis marker levels in different groups. Mean fold change of (a) Bax, (b) Bcl-2, (c) cleaved caspase-9 to pro-caspase-9 ratio, and (d) cleaved caspase-3 to pro-caspase-3. (e) Representative images of corresponding proteins in blot. Data are presented as mean ± SEM (n = 3). **P < 0.01, ***P < 0.001 versus control group. #P < 0.05, ##P < 0.01, ###P < 0.001 versus NS group. Ser: Sericin, NS: Normal saline, SEM: Standard error of the mean.
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Multiple comparisons demonstrated that tGI/R substantially increased Bax [Figure 6]a protein expression in the NS group while decreasing Bcl-2 [Figure 6]b protein expression in the control group (P < 0.001 for both). Nonetheless, Ser at the doses of 200 (P < 0.01) and 300 (P < 0.001) mg/kg significantly decreased Bax protein levels and elevated Bcl-2 levels at the 300 mg/kg dose (P < 0.01) in tGI/R-affected mice.
The cleavage of pro-caspase-9 [P < 0.001, [Figure 6]c] and pro-caspase-3 [P < 0.001, [Figure 6]d] was also considerably higher in the NS-treated group than the control group. However, as compared with the NS group, the Ser200 and Ser300 groups had substantially reduced cleaved caspase-9/pro-caspase-9 ratio (P < 0.001 for both doses) and cleaved caspase-3/pro-caspase-3 ratio (P < 0.05 for 200 and P < 0.001 for 300).
The result of one-way ANOVA also indicated a significant difference (F (4, 20) = 15.94, P < 0.001) [Figure 7] in the number of TUNEL-positive cells in the DG among the experimental groups. Post hoc analysis showed that tGI/R increased TUNEL-positive cells as compared to the control animals (P < 0.001). However, Ser at doses of 200 (P < 0.05) and 300 (P < 0.001) markedly decreased apoptotic cells in the DG region of the tGI/R-subjected mice. | Figure 7: The effects of Ser on apoptotic cell death in the hippocampal DG region of the study groups. (a) Representative image of TUNEL-positive cells. (b) Changes in number of apoptotic cells in the DG. Data are presented as mean ± SEM (n = 5). ***P < 0.001 versus control group. #P < 0.05, ###P < 0.001 versus NS group. Ser: Sericin, NS: Normal saline, DG: Dentate gyrus, TUNEL: Terminal transferase-mediated dUTP nick end-labeling, SEM: Standard error of the mean.
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| Discussion | | |
Through multiple diverse pathogenic processes, such as oxidative stress, inflammatory signaling pathways, and apoptosis, tGI/R injury results in permanent neuronal death and functional deficits.[27] Our study's main findings included improved spatial learning and memory, social interaction parameters (sociability and social novelty preference) via inhibition of oxidative stress, modulation of inflammatory cytokines, and downregulation of apoptosis markers in the hippocampus, all of which confirmed Ser-protective effects against ischemic insults in tGI/R-affected mice.
Increasing evidence shows that cerebral ischemia often results in progressive cognitive deterioration by prompting irreversible neuronal death through several multifaceted pathological processes, particularly oxidative stress, inflammatory signaling pathways, and apoptosis in the critical brain regions such as the hippocampus.[28],[29],[30] Since Ser has capability to inhibit these molecular pathways, it seems that it can also provide protection against the detrimental effects of ischemic stroke on the brain and cognitive function.[31]
Previous studies showed that Ser improved memory in Alzheimer's disease and aging mice models.[22],[32] Similarly, the present study demonstrated that Ser improved tGI/R-induced cognitive outcomes, as indicated by lower latency time and longer time spent in the target quadrant in the Barnes maze test.
Animal and human studies also demonstrated that cerebral ischemia disrupted social function and induced neuropsychiatric disorders, such as anxiety and depression.[33],[34],[35] Klarić et al. reported that focal cerebral ischemia reduced sociability but did not affect social novelty.[34] This study showed that tGI/R impaired sociability parameters, as indicated by lowered social affiliation and social novelty preference ratios. However, Ser at the dose of 300 mg/kg markedly improved sociability and social novelty preference.
Cerebral ischemic injuries also impair synaptic transmission accompanied by disruption of electrical activities of neurons.[36] Ser not only has an intrinsic neurotropic activity but also upregulated synaptic proteins which contribute to synaptic function.[37],[38] Besides, an in vitro study reported that Ser protected mouse primary cortical neurons from oxygen/glucose deprivation injury by reduction of lactate dehydrogenase (a marker of neuronal injury) and promotes axonal extension and branching.[37] Furthermore, Ser may improve memory performance by inhibiting acetylcholinesterase enzyme activity and increasing acetylcholine neurotransmitter levels in the brain.[32]
Oxidative stress is the core pathological step in global ischemia, which refers to an imbalance between oxidative and antioxidant defensive systems, resulting from excessive free radical production and reduced scavenging capacity of enzymatic and nonenzymatic systems.[27] On the one hand, it has been found that the hippocampus has less ischemic tolerance than other brain regions due to its low antioxidant capacity.[39],[40],[41] In line with that, our findings showed that the induction of global ischemia not only increased hippocampal content of MDA but also reduced activities of SOD and GPx and TAC levels, indicating reduced brain ability to neutralize reactive free radicals. However, Ser treatment diminished that hippocampal MDA levels enhanced the activity of SOD and GPx and increased TAC. The ability of Ser to reduce hippocampal oxidative stress has been reported in different experimental models of neuronal dysfunctions.[13],[22],[42] Emerging evidence shows that Ser inhibits lipid peroxidation, stabilizes free radicals, and suppresses tyrosinase activity. Mechanistically, the presence of high content of hydroxy amino acids (threonine and serine) with hydroxyl groups in this protein contributes to its reducing power and trace element chelating ability.[43],[44] Moreover, another potent antioxidant effect of Ser is attributable to the augmentation of enzymatic antioxidant activity.[13],[22]
Evidence also showed that cerebral ischemia markedly increases brain pro-inflammatory cytokines, such as TNF-α, IL-6, and IL-1β, which in turn provokes blood-brain barrier breakdown and secondary neuronal loss.[45],[46] Clinical studies also reported that the increased plasma levels of cytokines following ischemia were linked to stroke severity and long-term clinical outcomes.[47],[48] Likewise, we found that the tGI/R resulted in a substantial increase in hippocampal TNF-α and IL-6 levels accompanied by a drop in IL-10 level, a potent anti-inflammatory cytokine. On the other hand, the anti-inflammatory effects of Ser have been confirmed in previous studies.[13],[22] In tandem with that, we showed that Ser reversed ischemia-induced inflammation in the hippocampus of tGI/R-subjected mice.
Apoptosis is another pathophysiological process that is involved in neuronal damage during cerebral ischemia.[49],[50] Our result also confirmed that the tGI/R induced apoptosis in the hippocampus, as indicated by a marked increase of TUNEL-positive cells in the DG subfield. Accumulating evidence shows that activation of oxidative stress and neuroinflammation by global cerebral ischemia contribute to subsequent apoptotic or necrotic cell death.[50],[51],[52],[53],[54] Previous studies showed that Ser exerted neuroprotective properties in the hippocampus and prefrontal cortex of stressed and aged mice.[13],[22],[55] In that line, the present study demonstrated that Ser reduced the number of TUNEL-positive cells, diminished protein levels of Bax while increased Bcl-2 protein expression, and repressed the cleavage of pro-caspase-9 and pro-caspase-3 in the tGI/R-affected mice.
| Conclusion | | |
tGI/R contributed to hippocampal neuronal damage, partially through the induction of oxidative stress, neuroinflammation, and apoptosis, resulting in spatial learning and memory impairment, and social interaction deficits. Our results highlighted the beneficial effects of Ser in reducing cognitive impairment caused by tGI/R through activating antioxidant systems and inhibiting inflammatory and apoptotic signaling pathways in the hippocampus.
Acknowledgments
The authors thank the Neurosciences Research Center staff and the Department of Animal Biology of Kharazmi University for their technical support. This article was derived from a Ph.D. thesis of Mr. Seyed Mehdi Vatandoust.
Data availability
Derived data supporting the findings of this study are available from the corresponding author on request.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
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