Hypertonic Saline Alleviates Ischaemic Blood-brain Barrier Permeability By Downregulating Expression Of VEGF Mediated By The NLRP3/IL-1β Signalling Axis In Microglia

Background:Neurological dysfunction and deterioration of brain edema secondary to ischemic stroke plays an important role in endangering the patients’lives.In recent years,the neuroinflammatory response mediated by the inflammatory activation of microglia(NLRP3)plays an important role in the development of ischemic brain injury.And the increase of the blood-brain barrier(BBB)permeability induced by NLRP3 not only induces vasogenic brain edema,but also aggravates the damage of nerve function.It was well documented that hypertonic saline(HS)has obvious anti-inflammatory and organ protective effects.In addition to osmotic dehydration mechanism,current studies have found that HS may exert anti-brain edema through non-osmotic molecular mechanisms.Our previous studies had showed that HS may exert anti-brain edema by decreasing the expression of vascular endothelial growth factor(VEGF)and alleviating BBB permeability induced by cerebral ischemia.However,it is not clear whether the mechanism of HS down regulating VEGF expression is related to the inhibition of NLRP3 inflammasome activation and the corresponding downstream effect pathway of VEGF.Hence,if the above molecular mechanisms are elucidated,it will provide the new molecular mechanisms and new theory basis for HS to treat ischemic brain edema in clinic.Methods:Transient focal brain ischaemia of right middle cerebral artery occlusion(MCAO)was induced by the intraluminal suture method to simulate ischemia-reperfusion brain injury in rats,and the hypoxia-reoxygenation models of microglia and brain microvascular endothelial cells was used to simulate ischemia-reperfusion injury in vivo.The methods including western-blotting,immunofluorescence and signal pathway technique were used to clarify the molecular mechanism for HS to treat ischemic brain edema by down-regulating VEGF protein expression.Firstly,BBB permeability induced by transient focal brain ischaemia were detected by Evans blue,at the same time,the proteins expression of VEGF and tight junctions was detected by western-blotting and immunofluorescence.Secondly,the expression of(NOD-like receptor protein 3,NLRP3)and IL-lbeta(IL-1β)proteins in microglia was detected after HS treatment in vivo and in vitro,and the expression of VEGF protein in astrocytes was detected by incubation medium of HS treating microglia,recombinant IL-1 β,IL-1Ra and NF-KB inhibitors.Finally,permeability of cerebral microvascular endothelial cells in vitro was detected by HRP leakage test after HS treatment and the expression of tight junction proteins in cerebral microvascular endothelial cells was detected by western-blotting and immunofluorescence.The expression of tight junction proteins in endothelial cells was detected after intervention with VEGF receptor2(VEGFR2)blocker,PLCyl and eNOS inhibitors,repectively.Results:The Evans blue exudation in the ischemic cerebral hemisphere induced by focal cerebral ischemia-reperfusion in rats and HRP leakage of cerebral microvascular endothelial cells induced by oxygen-glucose deprivation/reoxygenation(OGD/R)in vitro reduced after HS treatment when compared with other treatments.The protein expression of NLRP3 inflammasome and IL-1β in microglia peri-ischaemic brain tissue in vivo and BV2 microglia treated by OGD/R in vitro decreased after HS treatment.Moreover,the protein expression of IL-1R1,pNF-KB65 and VEGF increased in the astrocytes peri-ischaemic brain tissue and those in the TNC1 astrocytes after OGD/R treatment.However,the expression of VEGF protein decreased significantly after incubated with HS,IL-1Ra and PDTC in vitro,respectively.When compared with OGD/R treatment in vitro or brain ischemia-reperfusion in vivvo,the expression of VEGF proteins decreased significantly in bEnd.3 endothelial cells or cerebral microvascular endothelial cells in the peri-ischaemic brain tissue after HS treatment,however the expression of ZO1 and occludin proteins was up-regulated after HS treatment.At the same time,the expression of VEGF,VEGFR2,p PLCyl and eNOS proteins increased significantly in the bEnd.3 endothelial cells or cerebral microvascular endothelial cells in the peri-ischaemic brain tissue after OGD/R treatment in vitro or brain ischemia-reperfusion in vivo,but the expression of ZO1 and occludin proteins was down-regulated.The expression of ZO1 and occludin proteins was up-regulated after incubated with HS,SU5416,pPLC γ1 inhibitor and eNOS inhibitor in the bEnd.3 endothelial cells.Conclusions:HS can alleviate the BBB permeability induced by cerebral ischemia-reperfusion injury through down-regulating the expression of VEGF in astrocytes and cerebral microvascular endothelial cells,the mechanism of which may be related to reduce the IL-1β release by inhibiting the activation of NLRP3 inflammasome in microglia,and decrease the expression of VEGF in astrocytes through the IL-1β/IL1R1/NF-KB pathway.Then the expression of tight junction proteins such as ZO1 and occludin was up-regulated by activation of endothelial cell VEGFR2/PLCyl/eNOS signaling pathway.Thus we further clarify the specific mechanism for HS to alleviate BBB permeability in cerebral ischemia-reperfusion injury,which is associated with down-regulating the expression of VEGF.At the same time it aslo may provide a new theoretical mechanism and molecular therapeutic target for HS to treat ischemic brain edema in this study.