ATP Induces Disruption of Tight Junction Proteins via IL-1 Beta-Dependent MMP-9 Activation of Human Blood-Brain Barrier In Vitro.

Fuxing Yang,Kai Zhao,Jun Zhang,Bainan Xu,Xiufeng Zhang

doi:10.1155/2016/8928530

Abstract

Disruption of blood-brain barrier (BBB) follows brain trauma or central nervous system (CNS) stress. However, the mechanisms leading to this process or the underlying neural plasticity are not clearly known. We hypothesized that ATP/P2X7R signaling regulates the integrity of BBB. Activation of P2X7 receptor (P2X7R) by ATP induces the release of interleukin-1β (IL-1β), which in turn enhances the activity of matrix metalloproteinase-9 (MMP-9). Degradation of tight junction proteins (TJPs) such as ZO-1 and occludin occurs, which finally contributes to disruption of BBB. A contact coculture system using human astrocytes and hCMEC/D3, an immortalized human brain endothelial cell line, was used to mimic BBB in vitro. Permeability was used to evaluate changes in the integrity of TJPs. ELISA, Western blot, and immunofluorescent staining procedures were used. Our data demonstrated that exposure to the photoreactive ATP analog, 3′-O-(4-benzoyl)benzoyl adenosine 5′-triphosphate (BzATP), induced a significant decrease in ZO-1 and occludin expression. Meanwhile, the decrease of ZO-1 and occludin was significantly attenuated by P2X7R inhibitors, as well as IL-1R and MMP antagonists. Further, the induction of IL-1β and MMP-9 was closely linked to ATP/P2X7R-associated BBB leakage. In conclusion, our study explored the mechanism of ATP/P2X7R signaling in the disruption of BBB following brain trauma/stress injury, especially focusing on the relationship with IL-1β and MMP-9.

Highlights

Traumatic brain injury (TBI) often coexists with acute stress disorder (ASD) or post-traumatic stress disorder (PTSD) [1]
Human astrocytes (HAs) were obtained from Jiamay Biolab (Beijing, China); hCMEC/D3 cell lines were purchased from Jiangyin Yuxi Biotechnology (Jiangsu, China); polyclonal anti-ZO-1 and anti-occludin antibodies were acquired from Biorbyt (Cambridge, UK, cat: orb11587, orb11181); Alexa Fluor 594-conjugated AffiniPure donkey anti-rabbit IgG was obtained from Jackson ImmunoResearch Inc. (West Grove, PA, USA, cat: 711-585-152); human interleukin-1β Enzyme-linked immunosorbent assay (ELISA) kit and human matrix metalloproteinase-9 (MMP-9) ELISA kit were purchased from Jiamay (Beijing, China, cat: FHK0016, FHK0144); high glucose Dulbecco’s Modified Eagle’s Medium (DMEM) basal medium was obtained from Hyclone (Logan, UT, United States); and EBM-2 basal medium was purchased from Lonza (Walkersville, MD, USA)
We investigated the effects of benzoyl adenosine 5󸀠-triphosphate (BzATP) on the tight junction proteins (TJPs) of blood-brain barrier and correlated the expression of IL-1β and MMP-9

Summary

Introduction

Traumatic brain injury (TBI) often coexists with acute stress disorder (ASD) or post-traumatic stress disorder (PTSD) [1]. TBI is one of the major health problems that account for high mortality worldwide. According to the World Health Organization, TBI is expected to become the third leading cause of death and disability worldwide by 2020 [2]. TBI/PTSD or TBI/ASD are public health concerns warranting advanced investigation into the underlying mechanisms for appropriate therapeutic intervention [3]. Disruption of blood-brain barrier (BBB) is a common result following brain trauma/stress injury, and little is known about the changes in neural plasticity following brain injury and subsequent cellular responses. There is no effective neuroprotective agent that is available clinically, to prevent the damage to BBB following CNS trauma/stress injury [4, 5]

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