HSPB8 over-expression prevents disruption of blood-brain barrier by promoting autophagic flux after cerebral ischemia/reperfusion injury

Abstract

Heat-shock protein B8 (HSPB8) has been recently reported to confer neuroprotection against ischemia/reperfusion (I/R)-induced cerebral injury in vivo and in vitro. However, the molecular mechanism is still elusive. This study focused on the effect of intracerebroventricular (i.c.v) delivery of lenti-HSPB8 virus against neurological injury in a rat model of cerebral I/R and explored the underlying mechanism. We found that lentivirus i.c.v injection-induced HSPB8 over-expression strongly alleviated infarct volume, improved neurobehavioral outcomes, and reduced brain edema in rat middle cerebral artery occlusion/reperfusion (MCAO/R) model. Concomitantly, HSPB8 over-expression noticeably prevented blood-brain barrier (BBB) disruption after cerebral I/R injury as indicated by the reduction in Evans blue leakage and IgG detection in the ipsilateral hemisphere compared with the vehicle group. Moreover, immunoblotting and immunofluorescence staining of tight junction proteins claudin-5 and occludin showed that HSPB8 over-expression prevented the degradation of these proteins induced by MCAO/R, which indicated the protective effect of HSPB8 on BBB. Western blotting and immunostaining techniques were also utilized to analyze the expression of the markers of autophagy. We found that HSPB8 over-expression promoted autophagic flux, evidenced by increased ratio of LC3 I/II, accumulation of Beclin-1 expression and enhanced p62 degradation. i.c.v injection of 15 μg autophagy inhibitor 3-methyladenine (3-MA) was applied at the onset of reperfusion. The results showed that 3-MA elicited a significant loss of the protective effect of HSPB8 against MCAO/R-induced neurological defect, Evans blue extravasation, and the loss tight junction proteins, suggesting that the BBB protective role of HSPB8 was, at least in part, mediated through autophagy. Collectively, HSPB8 may represent a potential therapeutic agent for preserving BBB integrity following cerebral I/R injury. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/. Cover Image for this issue: doi: 10.1111/jnc.14488.

Keywords: autophagy; blood brain barrier; cerebral ischemia/reperfusion; heat shock protein B8.