Korean J Physiol Pharmacol.  2016 Jul;20(4):325-332. 10.4196/kjpp.2016.20.4.325.

Resveratrol attenuates lipopolysaccharide-induced dysfunction of blood-brain barrier in endothelial cells via AMPK activation

• Hu M
• Liu B

• KMID: 2371049
• DOI: http://doi.org/10.4196/kjpp.2016.20.4.325

Abstract

Resveratrol, a phytoalexin, is reported to activate AMP-activated protein kinase (AMPK) in vascular cells. The blood-brain barrier (BBB), formed by specialized brain endothelial cells that are interconnected by tight junctions, strictly regulates paracellular permeability to maintain an optimal extracellular environment for brain homeostasis. The aim of this study was to elucidate the effects of resveratrol and the role of AMPK in BBB dysfunction induced by lipopolysaccharide (LPS). Exposure of human brain microvascular endothelial cells (HBMECs) to LPS (1 µg/ml) for 4 to 24 hours week dramatically increased the permeability of the BBB in parallel with lowered expression levels of occluding and claudin-5, which are essential to maintain tight junctions in HBMECs. In addition, LPS significantly increased the reactive oxygen species (ROS) productions. All effects induced by LPS in HBVMCs were reversed by adenoviral overexpression of superoxide dismutase, inhibition of NAD(P) H oxidase by apocynin or gain-function of AMPK by adenoviral overexpression of constitutively active mutant (AMPK-CA) or by resveratrol. Finally, upregulation of AMPK by either AMPK-CA or resveratrol abolished the levels of LPS-enhanced NAD(P)H oxidase subunits protein expressions. We conclude that AMPK activation by resveratrol improves the integrity of the BBB disrupted by LPS through suppressing the induction of NAD(P)H oxidase-derived ROS in HBMECs.

Keyword

AMPK; Blood -brain barrier; LPS; NAD(P)H oxidase; Resveratrol

MeSH Terms

AMP-Activated Protein Kinases*
Blood-Brain Barrier*
Brain
Claudin-5
Endothelial Cells*
Homeostasis
Humans
NADPH Oxidase
Oxidoreductases
Permeability
Reactive Oxygen Species
Superoxide Dismutase
Tight Junctions
Up-Regulation
AMP-Activated Protein Kinases
Claudin-5
NADPH Oxidase
Oxidoreductases
Reactive Oxygen Species
Superoxide Dismutase

Figure

• 

  Fig. 1 LPS decreases blood-brain barrier integrity and reduces the expression of tight-junction proteins in human brain microvascular endothelial cells (HBMECs).(A) Confluent monolayer of HBMECs was cultured on Transwell filters and the diffusion of FITC-conjugated dextran (150 kDa; 100 µg/ml) in LPS (1 µg/ml) was measured at various time points. Control was set up as 100%. Data are expressed as the mean±SEM. N is 5 in each group. *p<0.05 vs control. (B) Trans-endothelial electrical resistance was measured in real time in confluent monolayer of HBMECs. Control was set up as 100%. Data are expressed as the mean±SEM. N is 5 in each group. *p<0.05 vs control. (C) Representative Western blot of tight-junction proteins (occludin and claudin-5). The blot is representative of three blots from three independent experiments. *p<0.05 vs control.

• 

  Fig. 2 Reactive oxygen species (ROS) mediates LPS-reduced tight-junction protein expressions in human brain microvascular endothelial cells (HBMECs).(A) Confluent monolayer of HBMECs were cultured in LPS (1 µg/ml) was measured at various time points. ROS productions were assayed by DHE fluorescence. Data are expressed as the mean±SEM. N is 5 in each group. *p<0.05 vs control. (B and C) Cultured HBMECs were infected with adenovirus containing superoxide dismutase (SOD) for 48 hours and then exposure to LPS of 1 µg/ml for 24 hours. (B) Superoxide anion productions and (C) levels of occludin and claudin-5 expression were measured. N is 5 in each group. *p<0.05 vs control.

• 

  Fig. 3 LPS decreases tight-junction protein levels via upregulation of NAD(P)H oxidase in microvascular endothelial cells.(A) Confluent monolayer of HBMECs were cultured in LPS (1 µg/ml) was measured at various time points. NAD(P)H oxidase activity was was assayed by DHE fluorescence. Data are expressed as the mean±SEM. N is 5 in each group. *p<0.05 vs control. (B and C) Cultured HBMECs were pretreated with apocynin (10 µM) for 30 mins and then co-incubated with LPS (1 µg/ml) for 24 hours. (B) ROS productions were detected by DHE. N is 5 in each group. *p<0.05 vs control. NS indicates no significance. (C) Levels of occludin and claudin-5 expression were measured by Western blot. The blot is representative of three blots from three independent experiments. *p<0.05 vs control. #p<0.05 vs LPS alone.

• 

  Fig. 4 AMPK activation by resveratrol reduces LPS-enhanced production of reactive oxygen species in HBMECs.(A) Cultured HBMECs were treated with resveratrol (10 µM) for 24 hours. The levels of pAMPK and pACC were measured by Western blotting. The picture is a representative picture from 3 independent experiments. (B) Cultured HBMECs were pretreated with resveratrol (10 µM) for 30 min and then co-incubated with LPS (1 µg/ml) for 24 hours. ROS productions were measured by DHE/HPLC. Data are expressed as the mean±SEM. N is 5 in each group. *p<0.05 vs control. #p<0.05 vs LPS alone. (C) Cultured HBMECs were infected with adenovirus containing constitutively active AMPK (AMPK-CA) mutant for 48 hours and then incubated with LPS (1 µg/ml) for 24 hours. ROS productions were measured by DHE/HPLC. Data are expressed as the mean±SEM. N is 5 in each group. *p<0.05 vs GFP alone. #p<0.05 vs LPS alone.

• 

  Fig. 5 AMPK activation by resveratrol prevents LPS-induced loss of tight-junction proteins.(A~C) Cultured HBMECs were pretreated with resveratrol (10 µM) for 30 min and then co-incubated with LPS (1 µg/ml) for 24 hours. (A) Expressions of tight junction protein, occludin and claudin-5, were measured by Western blot. Data are expressed as the mean±SEM. N is 3 in each group. *p<0.05 vs control. #p<0.05 vs LPS alone. NS indicates no significance. (B)The morphology of tight-junction was determined by using staining F-actin. (C) Double-labeled immunofluorescence analysis of E-Cadherin (green) and N-Cadherin (red) proteins. Blue, DAPI stained nucleus. (D) Cultured HBMECs were infected with adenovirus containing constitutively active AMPK (AMPK-CA) mutant for 48 hours and then incubated with LPS (1 µg/ml) for 24 hours. Expressions of tight junction protein, occludin and claudin-5, were measured by Western blot. Data are expressed as the mean±SEM. N is 3 in each group. *p<0.05 vs GFP alone. NS indicates no significance.

• 

  Fig. 6 AMPK activation prevents LPS-induced upregulation of NAD(P)H oxidase subunits.(A) Cultured HBMECs were pretreated with resveratrol (10 µM) for 30 min and then co-incubated with LPS (1 µg/ml) for 24 hours. Expressions of NAD(P)H oxidase subunits, p47phox and p67phox, were measured by Western blot. Data are expressed as the mean±SEM. N is 3 in each group. *p<0.05 vs control. #p<0.05 vs LPS alone. NS indicates no significance. (B) Cultured HBMECs were infected with adenovirus containing constitutively active AMPK (AMPK-CA) mutant for 48 hours and then incubated with LPS (1 µg/ml) for 24 hours. Expressions of NAD(P)H oxidase subunits, p47phox and p67phox, were measured by Western blot. Data are expressed as the mean±SEM. N is 3 in each group. *p<0.05 vs GFP alone. NS indicates no significance. (C) Proposed molecular mechanism for protective effect of AMPK activation on BBB integrity.

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