Pterostilbene mediates glial and immune responses to alleviate chronic intermittent hypoxia-induced oxidative stress in nerve cells

Abstract

Chronic intermittent hypoxia (CIH) induces oxidative stress in the brain, causing sleep disorders. Herein, we investigated the role of pterostilbene (Pte) in CIH-mediated oxidative stress in the brain tissue. A CIH mouse model was constructed by alternately reducing and increasing oxygen concentration in a sealed box containing the mouse; brain tissue and serum were then collected after intragastric administration of Pte. Neurological function was evaluated through field experiments. The trajectory of the CIH mice to the central region initially decreased and then increased after Pte intervention. Pte increased the number of neuronal Nissl bodies in the hippocampus of CIH mice, upregulated the protein levels of Bcl-2, occludin, and ZO-1 as well as the mRNA and protein levels of cAMP-response element binding protein (CREB) and p-BDNF, and reduced the number of neuronal apoptotic cells, Bax protein levels, IBA-1, and GFAP levels. Simultaneously, Pte reversed the decreased levels of superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), and BDNF and increased levels of malondialdehyde (MDA) in the serum of CIH mice. Pte increased Th2 cells, Treg cells, IL-4, IL-10, and TGF-β1 levels and decreased Th1 cells, Th17 cells, IFN-γ, IL-6, and IL- 17A levels in activated BV2 cells and hippocampus in CIH mice. The protein levels of p-ERK1/2, TLR4, p-p38, p-p65, and Bax, apoptosis rate, MDA concentration, Bcl-2 protein level, cell viability, and SOD and GSH-PX concentrations decreased after the activation of BV2 cells. Pte inhibited gliocytes from activating T-cell immune imbalance through p-ERK signaling to alleviate oxidative stress injury in nerve cells.

Fig 1. Open field test.

Fig 2. Role of Pte in CIH-induced nerve injury and glial cell activation in mice.

(A) Nissl staining. (B) Hematoxylin eosin staining. (C, D) TUNEL staining. (E, F) Protein levels of Bax and Bcl-2 were detected using western blot. * P < 0.05 vs normal group. # P < 0.05 vs CIH group. Scale bar, 50 μm.

Fig 3. Protein expression of GFAP in brain tissues examined using immunohistochemistry.

Scale bar, 100 μm.

Fig 4. Role of Pte in glial cell activation in mice.

(A) Protein expression of IBA-1 in brain tissues was examined using immunohistochemistry. Scale bar, 100 μm. (B) Detection of IBA-1 protein expression using western blot.

Fig 5. Role of Pte in CIH-induced oxidative stress in mice.

(A) mRNA levels of CREB and BNDF detected using RT-qPCR. (B, C) Protein levels of p-CREB and BNDF detected using western blot. (D, E) Concentrations of MDA, BNDF, SOD, and GSH-PX detected using ELISA and biochemical analysis. * P < 0.05 vs. normal group. # P < 0.05 vs. CIH group.

Fig 6. Protein expression of occludin and ZO-1 in brain tissues examined using immunohistochemistry.

(A, C) Protein expression of occludin in brain tissues examined using immunohistochemistry. (B, D) Protein expression of ZO-1 in brain tissues examined using immunohistochemistry. *P < 0.05 vs. normal group. # P< 0.05 vs. CIH group. Scale bar, 50 μm.

Fig 7. Role of Pte in CIH-induced immune imbalance in mice.

(A–H) Proportions of Th1, Th2, Th17, and Treg cells in the peripheral blood was analyzed using flow cytometry. (I) Concentrations of IFN-γ, IL-6, IL-17A, IL-4, IL-10, and TGF-β1 were detected using ELISA. *P < 0.05 vs. normal group. # P < 0.05 vs. CIH group.

Fig 8. Role of Pte in BV2-activated cells.

(A, B) Protein levels of Bax, Bcl-2, ERK1/2, p-ERK1/2, p38, p-p38, TLR4, p65, and p-p65 detected using western blot. (C-E) CCK8 to detect cell viability. *P < 0.05 vs. group 0, # P < 0.05 vs. LPS + IFN-γ. (F) Flow cytometric analysis of apoptosis. *P < 0.05 vs. group 0. # P < 0.05 vs. LPS + IFN-γ group.

Fig 9. Role of Pte in BV2-activated induction of immune imbalance in cells.

(A) Concentrations of MDA, SOD, and GSH-PX were detected using biochemical analysis. (B-F) Proportions of Th1, Th2, Th17, and Treg cells in BV2 were analyzed using flow cytometry. (G) Concentrations of IFN-γ, IL-6, IL-17A, IL-4, IL-10, and TGF-β1 were detected using ELISA. *P < 0.05 vs. group 0. # P < 0.05 vs. LPS + IFN-γ group.