DHA Attenuates Hypoxia/Reoxygenation Injury by Activating SSeCKS in Human Cerebrovascular Pericytes

Yanli Yu,Haibin Fang,Zhen Qiu,Zhongyuan Xia,Bin Zhou

doi:10.1007/s11064-019-02915-0

Yanli Yu, Haibin Fang + Show 3 more

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https://doi.org/10.1007/s11064-019-02915-0

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Abstract

Docosahexaenoic acid (DHA) can alleviate cerebral ischemia/reperfusion injury by reducing blood–brain barrier permeability and maintaining its integrity, accompanied by an increased Ang-1/Ang-2 ratio; however, the underlying mechanisms of these effects remain unclear. Src-suppressed C kinase substrates (SSeCKS), a substrate of protein kinase C, plays an important role in maintaining cell junctions and cell morphology and regulating cell permeability. However, whether DHA can increase SSeCKS expression and then mediate the Ang-1/Ang-2 ratio still needs to be studied. Human cerebrovascular pericytes (HBVPs) cultured in vitro were divided into groups, treated with or without DHA along with SSeCKS siRNA to knockdown SSeCKS expression, and then subjected to 24 h of hypoxia followed by 6 h of reoxygenation. Cell viability; lactate dehydrogenase (LDH) release; and Ang-1, Ang-2 and VEGF activity were detected by using ELISA kits. The apoptosis rate was assessed by TUNEL flow cytometry. Expression of the SSeCKS, Ang-1, Ang-2 and VEGF proteins was evaluated by western blotting. Pretreatment with 10 μM or 40 μM DHA efficiently attenuated hypoxia/reoxygenation (H/R) injury by activating SSeCKS to increase the Ang-1/Ang-2 ratio and downregulate VEGF expression in HBVPs, as evidenced by decreased LDH release and apoptotic rates and increased HBVPs viability. Meanwhile, after we used SSeCKS siRNA to knock down SSeCKS protein expression, the protective effect of DHA on HBVPs following H/R injury was reversed. In conclusion, DHA can activate SSeCKS to increase the Ang-1/Ang-2 ratio and downregulate VEGF expression in HBVPs, thus reducing H/R injury.

Highlights

Secondary injuries caused by traumatic brain injury and subarachnoid hemorrhage, such as cerebral ischemia, vasospasm and cerebral edema, are important factors in determining the recovery of neurological function and survival outcome [1]
Apoptosis ratios of Human cerebrovascular pericytes (HBVPs) under various treatments were measured by using Annexin V-fluorescein (AV) and propidium iodide (PI) apoptosis detection kit (Invitrogen, Carlsbad, CA, USA) by flow cytometry
The same differences were observed between the HD + H/R and SS + HD + H/R groups. These results suggested that the protective effect of Docosahexaenoic acid (DHA) on HBVPs is achieved by upregulating suppressed C kinase substrates (SSeCKS)

Summary

Introduction

Secondary injuries caused by traumatic brain injury and subarachnoid hemorrhage, such as cerebral ischemia, vasospasm and cerebral edema, are important factors in determining the recovery of neurological function and survival outcome [1]. Ischemic stroke is the sudden onset of cerebral blood circulation disorder and a pathological condition characterized by an initial restriction of blood to the brain [2]. Recanalization therapy provokes cerebral ischemia/reperfusion injury and impairs brain homeostasiss, which increases vascular permeability, The vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1)/Tie-2 signaling pathways are closely related to continuous changes in microcirculation and barrier function after secondary injury [5]. VEGF is primarily responsible for the early promotion of vascular network formation, and plays an important role in protecting cerebral endothelial cells from H/R-induced injury. Ang-2 is an antagonist of Ang-1, and the early and direct administration of Ang-1 or induction of Ang-1 expression was found to reverse imbalance in the Ang-1/ Ang-2 ratio in a middle cerebral artery ligation mouse

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