Resveratrol protects barrier function of mouse brain microvascular endothelial cell monolayers with oxygen/glucose deprivation and PM2.5 exposure by maintaining mitochondrial dynamics balance

doi:10.12122/j.issn.1673-4254.2025.12.16

Abstract

Abstract:

Objective To evaluate the effect of resveratrol (RES) on barrier function of mouse brain microvascular endothelial cell monolayers exposed to oxygen/glucose deprivation/reoxygenation (OGD/R) and PM_2.5 and explore the role of mitochondrial fission and fusion in protecting endothelial barrier function. Methods Cultured mouse brain microvascular endothelial cells were exposed to OGD/R, treated with PM_2.5 (100 μg/mL) before OGD/R, or pretreated with RES (40 mg/mL) prior to OGD/R+PM_2.5 exposures. The changes in cell viability were examined with CCK-8 assay, and cell permeability was assessed by measuring transendothelial electrical resistance (TEER) and FITC-dextran permeation. Malondialdehyde (MDA) content and superoxide dismutase (SOD) activity were measured, and intracellular and mitochondrial ROS levels were detected using fluorescent probes. Mitochondrial morphology in the treated cells was observed using Mito-Tracker Red CMXRos. Western blotting was performed to detect the changes in cellular expressions of the tight junction proteins (ZO-1, occludin, and claudin-5) and mitochondrial dynamics-associated proteins (Drp1, Fis1, Mfn2, and OPA1). Results Compared with the normal control cells, the cells exposed to OGD/R or both OGD/R and PM_2.5 showed significantly decreased TEER levels, increased permeability, elevated oxidative stress, and increased ROS fluorescence intensities. Obvious mitochondrial fragmentation and morphological changes in the mitochondria were observed in the exposed cells, which also showed decreased expressions of tight junction proteins and mitochondrial fusion proteins with increased expressions of mitochondrial fission proteins. RES pretreatment of the endothelial cells before the exposures significantly reduced membrane permeability, lowered ROS levels, improved mitochondrial morphology, increased expressions of tight junction and fusion proteins, and decreased fission protein expressions. Conclusion RES can protect barrier function of mouse brain microvascular endothelial cell monolayers exposed to OGD/R and PM_2.5 by modulating mitochondrial dynamics, potentially through promoting mitochondrial fusion and inhibiting mitochondrial fission.

Key words: PM_2.5, resveratrol, cerebral ischemia-reperfusion injury, mitochondrial fission and fusion, Blood-brain barrier

Meng QIN, Siyu SUN, Jiaqi LIU, Yujiao GAO, Hao WANG, Youkun WANG, Ao SUN, Jiachun YAN, Jinbao WANG, Ying YU. Resveratrol protects barrier function of mouse brain microvascular endothelial cell monolayers with oxygen/glucose deprivation and PM_2.5 exposure by maintaining mitochondrial dynamics balance[J]. Journal of Southern Medical University, 2025, 45(12): 2690-2698.

Figures/Tables 9

Fig.1 Viability of brain microvascular endothelial cells exposed to oxygen/glucose deprivation/reoxygenation (OGD/R) and different concentrations of PM2.5 (A), pretreated with RES (B), and both (C) (Mean±SD, n=5). Data normalized to control (set as 1) and statistical comparisons are conducted on normalized data. *P<0.05, **P<0.01, ***P<0.001 vs CON group; #P<0.05 vs OGD/R group; ^^P<0.01, ^^^P<0.001 vs OGD/R+PM2.5 group.

Fig.2 Effect of PM2.5 on TEER (A) and FITC-dextran permeability (B) of mouse brain microvascular endothelial cells with OGD/R (Mean±SD, n=5). Data normalized to control (set as 1) and statistical comparisons are conducted on normalized data. *P<0.05, **P<0.01 vs CON group; #P<0.05, ##P<0.01 vs OGD/R group; ^P<0.05, ^^P<0.01 vs OGD/R+PM2.5 group.

Fig.3 Comparison of MDA (A) and SOD (B) levels in the cells in different groups (Mean±SD, n=5). Data normalized to control (set as 1) and statistical comparisons are conducted on normalized data. *P<0.05, ***P<0.001 vs CON group; ##P<0.01 vs OGD/R group; ^^P<0.01 vs OGD/R+PM2.5 group.

Fig.4 Immunofluorescence staining of ROS in the cells in different groups. A: Immunofluorescence staining of ROS and DAPI (Scale bar=100 μm). B: Quantitative analysis of ROS fluorescence intensity (Mean±SD, n=5). ***P<0.001 vs CON group; ##P<0.01 vs OGD/R group; ^^P<0.01 vs OGD/R+PM2.5 group.

Fig.5 Immunofluorescence staining for detecting mitochondrial ROS in different groups. A: Immunofluorescence staining of ROS and DAPI (Scale bar=100 μm). B: Quantitative analysis of ROS fluorescence intensity (Mean±SD, n=5). ***P<0.001 vs CON group; ##P<0.01 vs OGD/R group; ^^P<0.01 vs OGD/R+PM2.5 group.

Fig.6 Immunofluorescence staining for evaluating mitochondrial membrane potential in different groups. A: Immunofluorescence staining of aggregates (red) and monomers (green) (scale bar=20 μm). B, C: Quantitative analysis of membrane potential fluorescence intensity (Mean±SD, n=5). **P<0.01 vs CON group; #P<0.05 vs OGD/R group; ^P<0.05, ^^P<0.01 vs OGD/R+PM2.5 group.

Fig.7 Immunofluorescence staining for observing mitochondrial morphology in different groups. A: Immunofluorescence staining with Mito-Tracker Red and DAPI (scale bar=20 μm). B: Comparison of mitochondrial fragmentation in each group (Mean±SD, n=5). *P<0.05 vs CON group; #P<0.05 vs OGD/R group; ^P<0.05 vs OGD/R+PM2.5 group.

Fig.8 Expression of ZO-1, occludin, claudin-5 proteins in each group detected by Western blotting. A: Western blots of ZO-1, occludin, claudin-5 and GAPDH. B-D: Expression levels of ZO-1 (B), occludin (C), and claudin-5 (D) proteins normalized by GAPDH levels (Mean±SD, n=5). Data normalized to control (set as 1) and statistical comparisons are conducted on normalized data.**P<0.01 vs CON group; #P<0.05, ###P<0.001 vs OGD/R group; ^P<0.05 vs OGD/R+PM2.5 group.

Fig.9 Expressions of OPA1, Mfn2, Drp1and Fis1 proteins in each group detected by Western blotting. A: Western blots of OPA1, Mfn2, Drp1, Fis1 and GAPDH. B-E: Expression levels of OPA1, Mfn2, Drp1 and Fis1 proteins normalized by GAPDH levels (Mean±SD, n=5). Data normalized to control (set as 1) and statistical comparisons are conducted on normalized data. *P<0.05, **P<0.01 vs CON group; #P<0.05, ##P<0.01 vs OGD/R group; ^P<0.05, ^^P<0.01 vs OGD/R+PM2.5 group.

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