Chia seeds oil ameliorate chronic immobilization stress-induced neurodisturbance in rat brains via activation of the antioxidant/anti-inflammatory/antiapoptotic signaling pathways

doi:10.1038/s41598-023-49061-w

. 2023 Dec 16;13(1):22409.

doi: 10.1038/s41598-023-49061-w.

Chia seeds oil ameliorate chronic immobilization stress-induced neurodisturbance in rat brains via activation of the antioxidant/anti-inflammatory/antiapoptotic signaling pathways

Norhan E Khalifa¹, Ahmed E Noreldin², Asmaa F Khafaga³, Mohamed El-Beskawy⁴, Eman Khalifa⁵, Ali H El-Far⁶, Abdel-Hasseb A Fayed⁷, Abdeldayem Zakaria⁷

Affiliations

¹ Department of Physiology, Faculty of Veterinary Medicine, Matrouh University, Matrouh, 51511, Egypt. norhan.elsayed@mau.edu.eg.
² Department of Histology and Cytology, Faculty of Veterinary Medicine, Damanhour University, Damanhour, 22511, Egypt. ahmed.elsayed@damanhour.edu.eg.
³ Department of Pathology, Faculty of Veterinary Medicine, Alexandria University, Edfina, 22758, Egypt.
⁴ Department of Animal Medicine, Faculty of Veterinary Medicine, Matrouh University, Matrouh, 51511, Egypt.
⁵ Department of Microbiology, Faculty of Veterinary Medicine, Matrouh University, Matrouh, 51511, Egypt.
⁶ Department of Biochemistry, Faculty of Veterinary Medicine, Damanhour University, Damanhour, 22511, Egypt.
⁷ Department of Physiology, Faculty of Veterinary Medicine, Alexandria University, Edfina, 22758, Egypt.

PMID: 38104182
PMCID: PMC10725506
DOI: 10.1038/s41598-023-49061-w

Chia seeds oil ameliorate chronic immobilization stress-induced neurodisturbance in rat brains via activation of the antioxidant/anti-inflammatory/antiapoptotic signaling pathways

Norhan E Khalifa et al. Sci Rep. 2023.

. 2023 Dec 16;13(1):22409.

doi: 10.1038/s41598-023-49061-w.

Authors

Norhan E Khalifa¹, Ahmed E Noreldin², Asmaa F Khafaga³, Mohamed El-Beskawy⁴, Eman Khalifa⁵, Ali H El-Far⁶, Abdel-Hasseb A Fayed⁷, Abdeldayem Zakaria⁷

Affiliations

¹ Department of Physiology, Faculty of Veterinary Medicine, Matrouh University, Matrouh, 51511, Egypt. norhan.elsayed@mau.edu.eg.
² Department of Histology and Cytology, Faculty of Veterinary Medicine, Damanhour University, Damanhour, 22511, Egypt. ahmed.elsayed@damanhour.edu.eg.
³ Department of Pathology, Faculty of Veterinary Medicine, Alexandria University, Edfina, 22758, Egypt.
⁴ Department of Animal Medicine, Faculty of Veterinary Medicine, Matrouh University, Matrouh, 51511, Egypt.
⁵ Department of Microbiology, Faculty of Veterinary Medicine, Matrouh University, Matrouh, 51511, Egypt.
⁶ Department of Biochemistry, Faculty of Veterinary Medicine, Damanhour University, Damanhour, 22511, Egypt.
⁷ Department of Physiology, Faculty of Veterinary Medicine, Alexandria University, Edfina, 22758, Egypt.

PMID: 38104182
PMCID: PMC10725506
DOI: 10.1038/s41598-023-49061-w

Abstract

Chronic immobilization stress plays a key role in several neuropsychiatric disorders. This investigation assessed the possible ameliorative effect of chia seed oil (CSO) against the neurodisturbance-induced in rats by chronic immobilization. Rats were randomly allocated into control, CSO (1 ml/kg b.wt./orally), restrained (6 h/day), CSO pre-restraint, and CSO post-restraint for 60 days. Results revealed a significant reduction in serum corticosterone level, gene expression of corticotrophin-releasing factor, pro-inflammatory cytokines, and oxidative biomarkers in restrained rats treated with CSO. The histopathological findings revealed restoring necrosis and neuronal loss in CSO-treated-restraint rats. The immunohistochemical evaluation revealed a significant reduction in the immuno-expression of caspase-3, nuclear factor kappa B, interleukin-6, and cyclooxygenase-2 (COX-2), and an elevation of calbindin-28k and synaptophysin expression compared to non-treated restraint rats. The molecular docking showed the CSO high affinity for several target proteins, including caspase-3, COX-2, corticotropin-releasing hormone binding protein, corticotropin-releasing factor receptors 1 and 2, interleukin-1 receptor types 1 and 2, interleukin-6 receptor subunits alpha and beta. In conclusion, CSO emerges as a promising candidate against stress-induced brain disruptions by suppressing inflammatory/oxidative/apoptotic signaling pathways due to its numerous antioxidant and anti-inflammatory components, mainly α-linolenic acid. Future studies are necessary to evaluate the CSO therapeutic impacts in human neurodisturbances.

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Conflict of interest statement

The authors declare no competing interests.

Figures

**Figure 1**
Diagram illustrates all the experimental groups and treatments.

**Figure 2**
Representative photomicrograph (HE, scale bar = 50 µm) of cerebrum tissues from rats subjected to chronic immobilization stress and/or chia seeds oil (CSO) pre and post-restraint: (A) Control, and (B) CSO-treated rats displaying normal neuroarchitecture. (C) Restraint-exposed rats showing darkly necrotic shrunken neurons (arrow). (D) CSO pre-restraint rats reveal the nearly normal architecture of neuropil and neurons. (E) CSO post-restraint rats showing apparently normal cerebral architecture with some necrotic neurons (arrow). (F) Semiquantitative histomorphometric evaluation of neuronal necrosis. All values are expressed as the mean ± SEM.

**Figure 3**
Representative photomicrograph (HE, scale bar = 50 µm) of hippocampal tissues from rats exposed to chronic immobilization stress and/or chia seeds oil (CSO) pre and post-restraint: (A) Control, and (B) CSO-treated rats revealed a normal histologic picture of the dentate gyrus. (C) restraint-exposed rats exposing congested blood vessels (arrowhead), shrunken neurons (thick arrow), and hyperchromatic neurons (thin arrow). CSO pre-restraint rats (D) and CSO post-restraint rats (E) revealed apparently normal hippocampal architecture with mild neuronal degeneration. (F) Semiquantitative histomorphometric evaluation of neuronal necrosis. All values are expressed as the mean ± SEM.

**Figure 4**
Representative photomicrograph (HE, scale bar = 50 µm) of cerebellum tissues from rats exposed to chronic immobilization stress and/or chia seeds oil (CSO) pre and post-restraint: (A) control, and (B) CSO-treated rats showing a normal cerebellar neuroarchitecture. (C) Restraint-exposed rats displaying shrunken neurons (thick arrow) in the molecular layer (ML), hypertrophic neurons in the granule cell layer (thin arrow) in the granular layer (GL), and shrunken Purkinje cells (arrowhead) distributed within Purkinje cells layer (PCL). (D) CSO pre-restraint rats exposing semi-normal histoarchitecture with a pyknotic (arrowhead) or few lost (arrow) Purkinje cells. (E) CSO post-restraint rats showed normal hippocampal histoarchitecture with more degenerated (arrowhead) Purkinje cells than CSO post-restraint rats. (F) Semiquantitative histomorphometric evaluation of neuronal necrosis. All values are expressed as the mean ± SEM.

**Figure 5**
Representative photomicrograph (scale bar = 50 µm) for immunoreactivity of Caspase-3 in cerebral (**A1–A5**), hippocampal (**B1–B5**), and cerebellar (**C1–C5**) from control (**A1, B1, C1**), CSO (**A2, B2, C2**), restraint (**A3, B3, C3**), CSO pre-restraint (**A4, B4, C4**), and CSO post-restraint (**A5, B5, C5**). Arrowheads indicate positive immune expression in control and treated groups. Statistical analysis for area % of caspase-3 immunoexpression in the cerebral (A6), hippocampal (B6), and cerebellar (C6) of control and treated groups. All values are expressed as the mean ± SEM. *CSO* chia seeds oil.

**Figure 6**
Representative photomicrograph (scale bar = 50 µm) for immunoreactivity of Cyclooxgynase-2 (COX-2) in cerebral (**A1–A5**), hippocampal (**B1–B5**), and cerebellar (**C1–C5**) from control (**A1, B1, C1**), CSO (**A2, B2, C2**), restraint (**A3, B3, C3**), CSO pre-restraint (**A4, B4, C4**), and CSO post-restraint (**A5, B5, C5**). Arrowheads indicate positive immune expression in control and treated groups. Statistical analysis for area % of COX-2 immunoexpression in the cerebral (A6), hippocampal (B6), and cerebellar (C6) of control and treated groups. All values are expressed as the mean ± SEM. *CSO* chia seeds oil.

**Figure 7**
Representative photomicrograph (scale bar = 50 µm) for immunoreactivity of nuclear factor kappa B (NFkB) in the cerebral (**A1–A5**), hippocampal (**B1–B5**), and cerebellar (**C1–C5**) from control (**A1, B1, C1**), CSO (**A2, B2, C2**), restraint (**A3, B3, C3**), CSO pre-restraint (**A4, B4, C4**), and CSO post-restraint (**A5, B5, C5**). Arrowheads indicate positive immune expression in control and treated groups. Statistical analysis for area % of NFkB immunoexpression in the cerebral (A6), hippocampal (B6), and cerebellar (C6) of control and treated groups. All values are expressed as the mean ± SEM. *CSO* chia seeds oil.

**Figure 8**
Representative photomicrograph (scale bar = 50 µm) for immunoreactivity of interleulin-6 (IL-6) in cerebral (**A1–A5**), hippocampal (**B1–B5**), and cerebellar (**C1–C5**) from control (**A1, B1, C1**), CSO (**A2, B2, C2**), restraint (**A3, B3, C3**), CSO pre-restraint (**A4, B4, C4**), and CSO post-restraint (**A5, B5, C5**). Arrowheads indicate positive immune expression in control and treated groups. Statistical analysis for area % of IL-6 immunoexpression in the cerebral (A6), hippocampal (B6), and cerebellar (C6) of control and treated groups. All values are expressed as the mean ± SEM. *CSO* chia seeds oil.

**Figure 9**
Representative photomicrograph (scale bar = 50 µm) for immunoreactivity of Calbindin-28K in hippocampal (**A1–A5**) and cerebellar (**B1–B5**) from control (**A1, B1**), CSO (**A2, B2**), restraint (**A3, B3**), CSO pre-restraint (**A4, B4**), and CSO post-restraint (**A5, B5**). Arrowheads indicate positive immune expression in control and treated groups. Statistical analysis for area % of calbindin-28K immunoexpression in the hippocampal (A6) and cerebellar (B6) of control and treated groups. All values are expressed as the mean ± SEM. *CSO* chia seeds oil.

**Figure 10**
Representative photomicrograph (scale bar = 50 µm) for microglia distribution using immunoreactivity for IBA1 in cerebral (**A1–A5**), hippocampal (**B1–B5**), and cerebellar (**C1–C5**) from control (**A1, B1, C1**), CSO (**A2, B2, C2**), restraint (**A3, B3, C3**), CSO pre-restraint (**A4, B4, C4**), and CSO post- restraint (**A5, B5, C5**). Arrowheads indicate positive immune expression in control and treated groups. Statistical analysis for area % of IBA1 immunoexpression in the cerebral (A6), hippocampal (B6), and cerebellar (C6) of all control and treated groups. All values are expressed as the mean ± SEM. *CSO* chia seeds oil.

**Figure 11**
Representative photomicrograph (scale bar = 50 µm) for immunoreactivity of synaptophysin (SYP) in the cerebral (**A1–A5**), hippocampal (**B1–B5**), and cerebellar (**C1–C5**) from control (**A1, B1, C1**), CSO (**A2, B2, C2**), restraint (**A3, B3, C3**), CSO pre-restraint (**A4, B4, C4**), and CSO post-restraint (**A5, B5, C5**). Arrowheads indicate positive immune expression in control and treated groups. Statistical analysis for area % of SYP immunoexpression in the cerebral (A6), hippocampal (B6), and cerebellar (C6) of control and treated groups. All values are expressed as the mean ± SEM. *CSO* chia seeds oil.

**Figure 12**
Representative photomicrograph (scale bar = 50 µm) for astrocytes distribution using immunoreactivity for GFAP in cerebral (**A1–A5**), hippocampal (**B1–B5**), and cerebellar (**C1–C5**) from control (**A1, B1, C1**), CSO (**A2, B2, C2**), restraint (**A3, B3, C3**), CSO pre-restraint (**A4, B4, C4**), and CSO post-restraint (**A5, B5, C5**). Arrowheads indicate positive immune expression in control and treated groups. Statistical analysis for area % of GFAB immunoexpression in the cerebral (A6), hippocampal (B6), and cerebellar (C6) of all control and treated rats. All values are expressed as the mean ± SEM. *CSO* chia seeds oil.

**Figure 13**
Molecular interaction of chia seed oil active ingredients (Lutein) with caspase-3 (A), corticotropin-releasing factor receptor 2 (CRHR2) (B), interleukin-1 receptor type 1 (IL-1R1) (C).

**Figure 14**
Molecular interaction of chia seed oil active ingredients (Alpha-tocopherol) with corticotropin-releasing hormone binding protein (CRH-BP) (A), interleukin-1 receptor type 2 (IL-1R2) (B), interleukin 6 receptor, alpha (IL6R) (C).

**Figure 15**
Molecular interaction of chia seed oil active ingredients (Delta-tocopherol) with cyclooxygenase-2 (COX-2) (A), (Sitostanol) with corticotropin-releasing factor receptor 1 (CRHR1) (B), (Beta-carotene) with interleukin-6 receptor subunit beta (IL6ST) (C), and (Squalene) with tumor necrosis factor receptor superfamily member 1A (TNFRSF1A) (D).

**Figure 16**
The correlation matrix heatmap demonstrates the Pearson correlation coefficient values between brain CRF gene expression, serum corticosterone, proinflammatory cytokines, brain oxidative stress markers, and immunoexpression. The positive values are in blue, and the negative values are in purple. It ranges from 1 to −1, whereby 1 means a strong positive correlation between the tested variables, −1 indicates a strong negative correlation between the tested variables, and 0 indicates that there is no correlation. *GPx* glutathione peroxidase, *CAT* catalase, *SOD* superoxide dismutase, *MDA* malondialdehyde, *IL-6* interleukin 6, *TNF-α* tumor necrosis factor-alpha, *CRF* corticotrophin-releasing factor, *IBA1* ionized calcium binding adaptor molecule, *GFAP* glial fibrillary acidic protein, *COX-2* cyclooxygenase-2, *SYP* synaptophysin, *NF-κB* nuclear factor kappa light chain enhancer of activated B cells (the reader is directed to the online version of this article for the color references interpretation of in this legend).

**Figure 17**
Diagram illustrating the protective influences of chia seed oil (CSO) against chronic immobilization stress and the detected mechanisms.

See this image and copyright information in PMC

References

1. O'Connor DB, Thayer JF, Vedhara K. Stress and health: A review of psychobiological processes. Annu. Rev. Psychol. 2021;72:663–688. doi: 10.1146/annurev-psych-062520-122331. - DOI - PubMed
1. Lowery-Gionta EG, et al. Chronic stress dysregulates amygdalar output to the prefrontal cortex. Neuropharmacology. 2018;139:68–75. doi: 10.1016/j.neuropharm.2018.06.032. - DOI - PMC - PubMed
1. Sathyanesan M, Haiar JM, Watt MJ, Newton SS. Restraint stress differentially regulates inflammation and glutamate receptor gene expression in the hippocampus of C57BL/6 and BALB/c mice. Stress. 2017;20:197–204. doi: 10.1080/10253890.2017.1298587. - DOI - PMC - PubMed
1. Willner P. The chronic mild stress (CMS) model of depression: History, evaluation and usage. Neurobiol. Stress. 2017;6:78–93. doi: 10.1016/j.ynstr.2016.08.002. - DOI - PMC - PubMed
1. Ali, W. B. et al. in Biology and Life Sciences Forum. Vol. 32 (MDPI).

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[1] O'Connor DB, Thayer JF, Vedhara K. Stress and health: A review of psychobiological processes. Annu. Rev. Psychol. 2021;72:663–688. doi: 10.1146/annurev-psych-062520-122331. - DOI - PubMed

[2] O'Connor DB, Thayer JF, Vedhara K. Stress and health: A review of psychobiological processes. Annu. Rev. Psychol. 2021;72:663–688. doi: 10.1146/annurev-psych-062520-122331. - DOI - PubMed

[3] Lowery-Gionta EG, et al. Chronic stress dysregulates amygdalar output to the prefrontal cortex. Neuropharmacology. 2018;139:68–75. doi: 10.1016/j.neuropharm.2018.06.032. - DOI - PMC - PubMed

[4] Lowery-Gionta EG, et al. Chronic stress dysregulates amygdalar output to the prefrontal cortex. Neuropharmacology. 2018;139:68–75. doi: 10.1016/j.neuropharm.2018.06.032. - DOI - PMC - PubMed

[5] Sathyanesan M, Haiar JM, Watt MJ, Newton SS. Restraint stress differentially regulates inflammation and glutamate receptor gene expression in the hippocampus of C57BL/6 and BALB/c mice. Stress. 2017;20:197–204. doi: 10.1080/10253890.2017.1298587. - DOI - PMC - PubMed

[6] Sathyanesan M, Haiar JM, Watt MJ, Newton SS. Restraint stress differentially regulates inflammation and glutamate receptor gene expression in the hippocampus of C57BL/6 and BALB/c mice. Stress. 2017;20:197–204. doi: 10.1080/10253890.2017.1298587. - DOI - PMC - PubMed

[7] Willner P. The chronic mild stress (CMS) model of depression: History, evaluation and usage. Neurobiol. Stress. 2017;6:78–93. doi: 10.1016/j.ynstr.2016.08.002. - DOI - PMC - PubMed

[8] Willner P. The chronic mild stress (CMS) model of depression: History, evaluation and usage. Neurobiol. Stress. 2017;6:78–93. doi: 10.1016/j.ynstr.2016.08.002. - DOI - PMC - PubMed

[9] Ali, W. B. et al. in Biology and Life Sciences Forum. Vol. 32 (MDPI).

[10] Ali, W. B. et al. in Biology and Life Sciences Forum. Vol. 32 (MDPI).

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Chia seeds oil ameliorate chronic immobilization stress-induced neurodisturbance in rat brains via activation of the antioxidant/anti-inflammatory/antiapoptotic signaling pathways

Affiliations

Chia seeds oil ameliorate chronic immobilization stress-induced neurodisturbance in rat brains via activation of the antioxidant/anti-inflammatory/antiapoptotic signaling pathways

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Conflict of interest statement

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