Exp Neurobiol.  2019 Apr;28(2):216-228. 10.5607/en.2019.28.2.216.

C3a Receptor Inhibition Protects Brain Endothelial Cells Against Oxygen-glucose Deprivation/Reperfusion

• Ahmad S
• Kindelin A
• Khan SA
• Ahmed M
• Hoda MN
• Bhatia K
• Ducruet AF

• KMID: 2450214
• DOI: http://doi.org/10.5607/en.2019.28.2.216

Abstract

The complement cascade is a central component of innate immunity which plays a critical role in brain inflammation. Complement C3a receptor (C3aR) is a key mediator of post-ischemic cerebral injury, and pharmacological antagonism of the C3a receptor is neuroprotective in stroke. Cerebral ischemia injures brain endothelial cells, causing blood brain barrier (BBB) disruption which further exacerbates ischemic neuronal injury. In this study, we used an in vitro model of ischemia (oxygen glucose deprivation; OGD) to investigate the protective effect of a C3aR antagonist (C3aRA, SB290157) on brain endothelial cells (bEnd.3). Following 24 hours of reperfusion, OGD-induced cell death was assessed by TUNEL and Caspase-3 staining. Western blot and immunocytochemistry were utilized to demonstrate that OGD upregulates inflammatory, oxidative stress and antioxidant markers (ICAM-1, Cox-2, Nox-2 and MnSOD) in endothelial cells and that C3aRA treatment significantly attenuate these markers. We also found that C3aRA administration restored the expression level of the tight junction protein occludin in endothelial cells following OGD. Interestingly, OGD/reperfusion injury increased the phosphorylation of ERK1/2 and C3aR inhibition significantly reduced the activation of ERK suggesting that endothelial C3aR may act via ERK signaling. Furthermore, exogenous C3a administration stimulates these same inflammatory mechanisms both with and without OGD, and C3aRA suppresses these C3a-mediated responses, supporting an antagonist role for C3aRA. Based on these results, we conclude that C3aRA administration attenuates inflammation, oxidative stress, ERK activation, and protects brain endothelial cells following experimental brain ischemia.

Keyword

bEnd.3 cells; Ischemia; OGD; Inflammation; Oxidative stress

MeSH Terms

Blood-Brain Barrier
Blotting, Western
Brain Ischemia
Brain*
Caspase 3
Cell Death
Complement C3a
Complement System Proteins
Encephalitis
Endothelial Cells*
Glucose
Immunity, Innate
Immunohistochemistry
In Situ Nick-End Labeling
In Vitro Techniques
Inflammation
Ischemia
Neurons
Occludin
Oxidative Stress
Phosphorylation
Reperfusion
Stroke
Tight Junctions
Caspase 3
Complement C3a
Complement System Proteins
Glucose
Occludin