Role of hepcidin and its downstream proteins in early brain injury after experimental subarachnoid hemorrhage in rats

Abstract

Early brain injury (EBI) is a major cause of mortality from subarachnoid hemorrhage (SAH). We aimed to study the pathophysiology of EBI and explore the role of hepcidin, a protein involved in iron homeostatic regulation, and its downstream proteins. One hundred and thirty-two male Sprague-Dawley rats were assigned into groups (n = 24/group): sham, SAH, SAH + hepcidin, SAH + hepcidin-targeting small interfering ribonucleic acid (siRNA), and SAH + scramble siRNA. Three hepcidin-targeting siRNAs and one scramble siRNA for hepcidin were injected 24 h before hemorrhage induction, and hepcidin protein was injected 30 min before induction. The rats were neurologically evaluated at 24 h and euthanized at 72 h. Hepcidin, ferroportin-1, and ceruloplasmin protein expression were measured by immunohistochemistry and Western blotting. Brain water content, blood-brain barrier (BBB) leakage, non-heme tissue iron and Garcia scale were evaluated. Hepcidin expression increased in the cerebral cortex and hippocampus after experimental SAH (P < 0.05 compared to sham), while ferroportin-1 and ceruloplasmin decreased (P < 0.05). Hepcidin injection lowered the expression of ferroportin-1 and ceruloplasmin further but siRNA reduced the levels of hepcidin (P < 0.05 compared to SAH) resulting in recovery of ferroportin-1 and ceruloplasmin levels. Apoptosis was increased in SAH rats compared to sham (P < 0.05) and increased slightly more by hepcidin, but decreased by siRNA (P < 0.05 compared to SAH). SAH rats had lower neurological scores, high brain water content, BBB permeability, and non-heme tissue iron (P < 0.05). In conclusion, downregulation of ferroportin-1 and ceruloplasmin caused by hepcidin enhanced iron-dependent oxidative damage and may be the potential mechanism of SAH.

The Dual Role of Hepcidin in Brain Iron Load and Inflammation

“…In physiological conditions hepcidin expression in brain structures and cells (neurons, glial cells, endothelial cells, epithelial cells of choroid plexus) has been consistently observed by in vivo studies in humans and rodents, albeit in low levels ( Krause et al, 2000 ; Pigeon et al, 2001 ; Zechel et al, 2006 ; Wang et al, 2008 , 2010 ; Hänninen et al, 2009 ; Raha et al, 2013 ; Wei et al, 2014 ; Farajdokht et al, 2015 ; Raha-Chowdhury et al, 2015 ; Graf et al, 2016 ; Li Y. et al, 2016 ; Pan et al, 2016 ; Tan et al, 2016 ; Lu et al, 2017 ; You et al, 2017 ; Zhang et al, 2017 ). Data from human and animal studies suggest that local hepcidin is more robustly expressed in pathophysiological states ( Sun et al, 2012 ; Urrutia et al, 2013 ; Tan et al, 2016 ; Xiong et al, 2016 ; You et al, 2017 ; Zhang et al, 2017 ). Similar to other cells, hepcidin main target protein in brain cells is FPN, but also iron import proteins ( Sun et al, 2012 ; Urrutia et al, 2013 ; Tan et al, 2016 ; Xiong et al, 2016 ; You et al, 2017 ; Zhang et al, 2017 ).…”

“…During brain hemorrhage/ischemia liver hepcidin is upregulated, which is accompanied with increased global brain iron content after peak levels of hepcidin ( Xiong et al, 2016 ). Importantly, hepcidin knockout decreases brain iron content in models with brain hemorrhage, but not in physiological conditions ( Tan et al, 2016 ; Xiong et al, 2016 ). Xiong et al (2016) study found a significantly lower brain iron content in hepcidin knockout rodents compared to normal rodents during intracerebral hemorrhage (ICH).…”

“…Data from human and animal studies suggest that local hepcidin is more robustly expressed in pathophysiological states ( Sun et al, 2012 ; Urrutia et al, 2013 ; Tan et al, 2016 ; Xiong et al, 2016 ; You et al, 2017 ; Zhang et al, 2017 ). Similar to other cells, hepcidin main target protein in brain cells is FPN, but also iron import proteins ( Sun et al, 2012 ; Urrutia et al, 2013 ; Tan et al, 2016 ; Xiong et al, 2016 ; You et al, 2017 ; Zhang et al, 2017 ).…”

Brain iron overload following intracranial haemorrhage

“…28 Therefore, it can cause iron accumulation within the cell when overexpressed 29. Hepcidin expression is increased in response to cellular iron overload and inflammation 30.…”

“…A pilot study in human patients suggested a causal relationship between free iron in the CSF following SAH and brain injury 150. Indeed, suppression of hepcidin (and thus inhibition of FP1 internalisation) attenuated brain damage following experimental SAH, suggesting the importance of intracellular iron accumulation 29. This suggests that the initial inflammation caused by iron accumulation stimulates hepcidin expression, which in turn leads to a decreased efflux of iron from these cells.…”