Role Of Arginase 1 In Early Vascular Endothelial Dysfunction After Subarachnoid Hemorrhage And The Relevant Mechanism

Background:The damage of vascular endothelial cells is closely related to the damage of blood brain barrier,microthrombus,leukocyte adhesion and imflammation in the early stage of brain injury(EBI)after SAH.Arginase 1(ARG1)is a key regulator of vascular endothelial cell function and has been identified to play an important role in a variety of vascular endothelial dysfunctional diseases.However,the effect of ARG1 on vascular endothelial function during EBI is still unclear.The purpose of this paper is to explore the role of ARGI in vascular endothelial injury during the EBI phase after SAH and the related mechanisms.Methods:First part:Weighted gene co-expression network analysis(WGCNA)of the gene chip GSE36791 was performed to explore key genes associated with SAH,and the results were validated by clinical samples.Subsequently,clinical data,blood ARG1 gene expression and ARG1 activity of 115 aneurysmal SAH patients were prospectively collected to explore the relationship between ARG1 and EBI after SAH.Second part:In vivo and Vitro SAH models,the expression of endothelial ARG1 in cerebral arteries after SAH was examined by immunohistochemistry,immunofluorescence,Western Blot,RT-qPCR,etc.The mechanism of ARG1-mediated endothelial apoptosis was explored by arginase inhibitor and siRNA intervention,detection of NO,nitric oxide synthase(NOS),reactive oxygen species(ROS),endothelin 1(ET-1)and so on.Third part:By using the arginase inhibitor nor-NOHA in both in vivo and vitro models of SAH,we observed whether it could protect arterial endothelial cell function,reduce early brain edema and decrease endothelial cell apoptosis and ultimately play a neuroprotective role during the EBI phase.Results:First part:One down-regulated gene(CD27)and six up-regulated genes(ANXA3,ACSL1,PGLYRP1,ALPL,ARG1 and TPST1)were found to be associated with aneurysmal SAH by WGCNA and clinical sample validation,among which ARG1 gene expression and activity were significantly increased in EBI patients.Second part:In SAH mouse model,ARG1 expression in cerebral artery endothelial cells was significantly high after SAH,and gradually increased within 72 h.RT-qPCR analysis of isolated arterial endothelial cells showed that ARG1 expression was significantly up-regulated in arterial endothelial cells compared with ARG1 expression in whole vascular tissues,while ARG2 expression was not significantly upregulated.In vitro experiments also found that ARG1 was highly expressed in vascular endothelial cells after SAH,and was positively correlated with endothelial cell apoptosis.ARG1-mediated competition with NOS substrates may be the mechanism of endothelial cell apoptosis.In other words,the high expression of ARG1 at the EBI stage after SAH caused a decrease of L-arginine in endothelial cell,which in turn led to NOS uncoupling and mediated both a decrease in NO and an increase in ROS,ultimately leading to apoptosis of endothelial cells.Third part:We intervened SAH models in vivo and vitro with the arginase inhibitor nor-NOHA to determine whether it had neuroprotective effects and attenuated EBI.The results showed that treatment with nor-NOHA at 24 h after SAH effectively alleviated early brain edema and improved neurological deficits in mice.Low-dose of nor-NOHA could effectively inhibit the apoptosis of endothelial cells in vitro.Conclusions:The ARG1 gene played an important role in early vascular endothelial dysfunction after SAH by mediating apoptosis of endothelial cells via the NOS uncoupling pathway.The arginase inhibitor nor-NOHA could reduce the early cerebral edema after SAH,decrease the apoptosis of endothelial cells,and thus improve the neurological dysfunction.