Mechanism Of Hypertonic Saline Inhibiting Ferroptosis In Cerebral Microvascular Endothelial Cells And Decreasing Permeability Of The Blood-brain Barrier After Cerebral Infarction

Objective:To explore the molecular mechanism of hypertonic saline reducing the BBB permeability through inhibiting ferroptosis of cerebral microvascular endothelial cells.Methods:The rat model of ischemia-reperfusion injury was established by the focal middle cerebral artery occlusion(t MCAO)method,and cerebral microvascular endothelial cells were treated by oxygen and glucose deprivation method(OGD/R).Ferroptosis inhibitor(Ferrostatin-1)and the common antioxidant(N-acetylcysteine)were respectively administrated to intervene rats or cells.Ferroptosis related indicators including malondialdehyde(MDA)and lipid reactive oxygen species(L-ROS)in cells were tested to verify whether ischemiareperfusion injury could induce ferroptosis in cerebral microvascular endothelial cells.The content of Evans blue in rats brain tissue was tested to confirm the damaging effect of ferroptosis in the BBB permeability after cerebral infarction.Subsequently,hypertonic saline was applied to treat rats or cells with ischemia-reperfusion injury.Iron content such as iron staining and indicators related to ferroptosis including changes in the microstructure of mitochondria and expression of glutathione peroxidase 4(GPX4)were detected to determine whether the hypertonic saline could alleviate iron overload and inhibit ferroptosis.Moreover,the optimal therapy of hypertonic saline,including intervening time and concentration,to inhibit ferroptosis of cerebral microvascular endothelial cells were discussed.Finally,the molecular mechanism of hypertonic saline alleviating iron overload and inhibiting ferroptosis was further explored through RNA-seq method,and the results of gene sequencing and the upstream molecular mechanism were confirmed by cellular western blot experiments.Results:Compared with the control group,the content of MDA and L-ROS in the cerebral microvascular endothelial cells treated with glucose and oxygen deprivation(OGD/R)were significantly increased,while the content of MDA and L-ROS were remarkably decreased after the treatment with ferroptosis inhibitors,suggesting that ferroptosis occurred in cerebral microvascular endothelial cells was induced by ischemia-reperfusion injury.Compared with the rats of ischemia-reperfusion group and N-acetylcysteine(one of common antioxidants)group,the BBB permeability was significantly decreased in rats of ferroptosis inhibitor group.Besides,it was found that iron deposition was reduced,mitochondrial atrophy was reduced,and GPX4 expression was up-regulated after treatment with hypertonic saline in rats with cerebral infarction.In addition,further cell experiments suggested that hypertonic saline could downregulate TFRC(transferrin receptor)expression and up-regulate FPN(ferroportin)expression,inhibit PI3K(phosphatidylinositol-3 kinase)phosphorylation and HIF-1α(hypoxia inducible factor-1α)nuclear transduction.Conclusions : Hyperosmotic saline could reduce BBB permeability after cerebral infarction through inhibiting ferroptosis of cerebral microvascular endothelial cells,the potential mechanism could be inhibiting activation of PI3K/HIF-1α pathway,down-regulating TFRC and upregulating FPN expression,and thereby alleviating intracellular iron overload.