Auraptene Enhances Junction Assembly in Cerebrovascular Endothelial Cells by Promoting Resilience to Mitochondrial Stress through Activation of Antioxidant Enzymes and mtUPR

Abstract

Junctional proteins in cerebrovascular endothelial cells are essential for maintaining the barrier function of the blood-brain barrier (BBB), thus protecting the brain from the infiltration of pathogens. The present study showed that the potential therapeutic natural compound auraptene (AUR) enhances junction assembly in cerebrovascular endothelial cells by inducing antioxidant enzymes and the mitochondrial unfolded protein response (mtUPR). Treatment of mouse cerebrovascular endothelial cells with AUR enhanced the expression of junctional proteins, such as occludin, zonula occludens-1 (ZO-1) and vascular endothelial cadherin (VE-cadherin), by increasing the levels of mRNA encoding antioxidant enzymes. AUR treatment also resulted in the depolarization of mitochondrial membrane potential and activation of mtUPR. The ability of AUR to protect against ischemic conditions was further assessed using cells deprived of oxygen and glucose. Pretreatment of these cells with AUR protected against damage to junctional proteins, including occludin, claudin-5, ZO-1 and VE-cadherin, accompanied by a stress resilience response regulated by levels of ATF5, LONP1 and HSP60 mRNAs. Collectively, these results indicate that AUR promotes resilience against oxidative stress and improves junction assembly, suggesting that AUR may help maintain intact barriers in cerebrovascular endothelial cells.

Keywords: antioxidant; auraptene; blood-brain barrier; endothelial cell; mitochondria; mtUPR.

Figure 1

Effects of Auraptene (AUR) on junctional proteins expression in bEnd.3 cells. (A) bEnd.3 cells (5 × 10³ cells per well) seeded in 96-well plates were incubated in media containing 0, 1, 2, 4 µM of AUR for 24 h. Cell viability was measured by SRB assay. (B) bEnd.3 cells with vehicle (Veh) or 1 µM AUR were stained for ZO-1 (green) and DAPI (blue); Scale bar: 20 µm. Arrows indicate an increase of ZO-1 expression. (C) Relative ZO-1 intensity was quantified using ImageJ. (D) ZO-1, VE-cadherin and occludin expression were analyzed by Western blot after treatment of vehicle or 1 µM AUR for 24 h. (E–G) The protein levels of ZO-1, VE-cadherin and occludin were quantified using ImageJ. Data are presented as mean and ± SEM of three independent experiments (* p < 0.05, ** p < 0.01, *** p < 0.001 compared to Veh).

Figure 2

Increase of genes encoding antioxidant enzymes in bEnd.3 cells by AUR treatment. (A,B) mRNA expressions for ROS scavenging antioxidant enzymes and GSH recycling-related genes were analyzed using qPCR with bEnd.3 cells treated with vehicle or 1 µM AUR. (C,D) bEnd.3 cells were incubated with vehicle or 1 µM AUR. The cells were stained with 5 µM CM-H₂DCFDA or 5 µM MitoSOX^TM and analyzed by flow cytometry. Total ROS was determined by DCFDA-stained cells (C) and mitochondrial ROS was determined by MitoSOX^TM—Stained cells (D). Median fluorescence intensity (MFI) values are analyzed by FlowJo program. Data are presented as mean and ± SEM of three independent experiments (* p < 0.05, ** p < 0.01 compared to Veh).

Figure 3

Loss of mitochondrial membrane potential accompanying induction of mtUPR in bEnd.3 cells by AUR treatment. (A) Oxygen consumption rate (OCR) was measured in bEnd.3 cells treated with vehicle or 1 µM AUR for 24 h. (B) bEnd.3 cells were incubated with vehicle or 1 µM AUR. The cells were stained with 100 nM TMRE and analyzed by flow cytometry. (C) Mitochondrial membrane potential was determined by TMRE-stained cells. The Median fluorescence intensity (MFI) values are analyzed by FlowJo program. (D) Expression of mRNA for mitochondrial unfolded protein response (mtUPR) genes was examined after 1 µM AUR treatment using qPCR. (E) Expression of mRNA for mitochondrial unfolded protein response (mtUPR) genes was examined with bEnd.3 cells incubated in oxygen-glucose deprivation (OGD) condition for 3 h after pretreated vehicle or 1 µM AUR 24 h. Data are presented as mean and ± SEM of three independent experiments (* p < 0.05, ** p < 0.01 compared to Veh, # p < 0.05 compared to OGD).

Figure 4

Protective effects of AUR pretreatment on the reduction of junctional protein by OGD in bEnd.3 cells.bEnd.3 cells were incubated in OGD condition for 3 h after treated with vehicle or 1 µM AUR 24 h. bEnd.3 cells were stained with claudin-5 (red) (A), ZO-1 (green) (B) and VE-cadherin (red) (C) with DAPI (blue); Scale bar: 20 µm. Arrows indicate junctional protein disruption. (D) Relative fluorescence intensity of markers was quantified using ImageJ. (E) ZO-1 and VE-cadherin expression were analyzed by Western blot with bEnd.3 cells incubated in OGD condition for 3 h after treatment of vehicle or 1 µM AUR for 24 h. Data are presented as mean and ± SEM of three independent experiments (* p < 0.05, *** p < 0.001 compared to Veh, # p < 0.05, ### p < 0.001 compared to OGD).

Figure 5

Schematic representation of the effect of AUR in normal condition and the protective mechanism in vitro ischemic injury model. AUR treatment can enhance junction assembly and induce resilience to oxidative stress in cerebrovascular endothelial cells by altering levels of mtUPR and antioxidant enzymes, resulting in low mitochondrial membrane potential. This reaction contributes mitochondrial stress resilience and it exhibits by alleviating degradation of junctional proteins when the cerebrovascular endothelial cells were put in OGD condition, in vitro ischemic injury model.