Effect Of P23 On Blood Brain Barrier Injury Induced By Cerebral Ischemia-Reperfusion And Its Mechanism

Background and Objective:Stroke is one of the leading causes of death and disability worldwide,with ischemic stroke accounting for approximately 85% of stroke cases.At present,the primary concern of clinical treatment for ischemic stroke is to restore the blood supply to the ischemic area as soon as possible.However,the subsequent reperfusion after the restoration of blood supply can also aggravate further damage of brain tissues,resulting in more severe neurological damage and brain dysfunction,called cerebral ischemiareperfusion injury(CIRI).One of the pathophysiological features of ischemic stroke is the destruction of the blood brain barrier(BBB),which is characterized by increased permeability due to the degradation of tight junction(TJ)and the enhancement of vesicle transport in brain microvascular endothelial cells.The resulting increased permeability leads to an uncontrolled inflow of blood-derived molecules and fluids,leading to destructive cytotoxicity,vasogenic edema and life-threatening hemorrhagic transformation.Ferroptosis,first reported in 2012,is a form of regulatory cell death which is different from apoptosis and refers to irondependent regulatory and lipid reactive oxygen species(ROS).Many studies have shown that ferroptosis is involved in the damage of BBB caused by CIRI.p23 is a protein with a molecular weight of 23-k Da and is widely expressed in several tissues including heart,brain,kidney,and lung.p23 can bind to a variety of proteins and play a regulatory role in a variety of diseases by regulating the biological function of these target proteins.However,the biological significance of p23 in blood-brain barrier injury and ferroptosis caused by cerebral ischemia-reperfusion has not been reported.This study aims to clarify the changes and regulation of p23 in the process of blood-brain barrier injury and ferroptosis induced by cerebral ischemia-reperfusion and provide new clues for the treatment of cerebral ischemia-reperfusion injury.Materials and methods:(1)Middle cerebral artery occlusion(MCAO)model and brain microvascular endothelial cells(BMECs)oxygen-glucose deprivation/reperfusion(OGD/R)model were used to replicate the cerebral ischemia-reperfusion injury model.To detect the effect of different reperfusion time on BBB permeability: BBB permeability was detected by evans blue.The tight junction protein ZO-1 and occludin between BMECs were detected by Western Blot and q RT-PCR,and the expression of ZO-1was detected by immunofluorescence.To observe the changes of ZO-1expression after intracerebroventricular injection of Fer-1 to inhibit ferroptosis,and to explore the relationship between BBB and ferroptosis.(2)The in vitro BBB model was established by BMECs,and OGD/R model was used to replicate cerebral ischemia-reperfusion injury.The related indexes of ferroptosis and the expression of p23 in different reperfusion time were detected.The contents of glutathione(GSH),malondialdehyde(MDA)and reactive oxygen species(ROS)in BMECs were detected by kits.The changes of COX2 were detected by Western Blot and q RT-PCR.(3)OGD/R model was established after si RNA interfered the expression of p23 in BMECs,and the BMECs permeability and ferroptosis related indicators were detected.The expression of ZO-1,occludin,p23 and COX2 were detected by Western Blot and q RT-PCR.The expression of ZO-1 was detected by immunofluorescence.The contents of GSH,MDA and ROS in BMECs were detected by kits and the changes of other ferroptosis related genes were detected by q RT-PCR(4)OGD/R model was established after transfection with p23 overexpression plasmid in BMECs,and the BMECs permeability and ferroptosis related indicators were detected.The expression of ZO-1,p23 and COX2 were detected by Western Blot and q RT-PCR.The expression of ZO-1 was detected by immunofluorescence The contents of GSH,MDA and ROS in BMECs were detected by kits and the changes of other ferroptosis related genes were detected by q RT-PCR.(5)BMECs were treated with cycloheximide(CHX)with or without p23 overexpression plasmid for various lengths of time and GPX4 protein level was measured by Western blot analysis.The interaction of p23 and GPX4 was detected by immunoprecipitation and immunofluorescence colocalization.Protein docking and immunoprecipitation were used to detect whether the binding of p23 to GPX4 was related to heat shock protein90(HSP90).BMECs were transfected with p23 truncated plasmids and immunoprecipitation assays were performed to detect the binding site of p23 to GPX4.Results:(1)The content of evans blue into the brain increased during cerebral ischemia-reperfusion injury in rats.After OGD/R treatment,the expression of ZO-1 and occludin decreased,and evans blue staining showed that the permeability of BMECs increased,indicating that cerebral ischemiareperfusion leads to BBB injury.After intracerebroventricular injection Fer-1,the expression of ZO-1 in brain tissue was significantly higher than that in cerebral ischemia-reperfusion injury group.The results suggested that inhibition of ferroptosis can protect BBB injury caused by cerebral ischemia-reperfusion.(2)After OGD/R treatment,the content of GSH decreased,the content of MDA and ROS increased,COX2 and p23 expression increased in BMECs.The results showed that ferroptosis is involved in OGD/Rinduced BMECs injury.(3)After si RNA interference with p23 expression by OGD/R treatment,the expression of ZO-1 in BMECs decreased and cell permeability increased,indicating that interfering with the expression of p23 aggravated OGD/R-induced BMECs injury.The content of GSH decreased,the content of MDA increased,the content of ROS increased,and the expression of COX2,ACSL4,SLC7A11 and HO-1 increased,indicating that interfering with the expression of p23 promoted ferroptosis of BMECs induced by OGD/R.(4)After overexpression of p23 expression by OGD/R treatment,the expression of ZO-1 in BMECs increased and cell permeability decreased,indicating that overexpression of p23 could reduce OGD/R-induced BMECs injury.The content of GSH increased,the content of MDA and ROS decreased,and the expression of COX2,ACSL4,SLC7A11 and NOX1 expression decreased,and the expression of FTH1 increased.The results showed that overexpression of p23 inhibited ferroptosis of BMECs induced by OGD/R.(5)After BMECs was treated with OGD/R,the expression of GPX4 was down-regulated;si RNA interfered the expression of p23 significantly decreased GPX4 expression,while overexpression of p23 increased GPX4 expression.The degradation of GPX4 protein decreased significantly after p23 overexpression,indicating that p23 can improve the stability of GPX4 protein.The protein docking and immunoprecipitation results showed that p23,GPX4,and HSP90 interacted with each other,and p23 interacted with HSP90 and GPX4 mainly through its N-terminal domain(1-90aa).The results showed that p23 and HSP90 can form complexes with GPX4 and inhibit the degradation of GPX4,stabilizes the expression of GPX4,and then inhibits ferroptosis and protect BMECs injury induced by OGD/R.Conclusion:(1)Cerebral ischemia-reperfusion can lead to BBB injury and increase the ferroptosis level of BMECs.(2)The expression of p23 was significantly increased in BMECs induced by OGD/R.(3)p23 has protective effect on BMECs injury and ferroptosis induced by OGD/R.(4)p23 mainly bind to HSP90-GPX4 through its N-terminal structure(1-90aa)and inhibits GPX4 degradation,stabilizes the expression of GPX4,and then inhibits ferroptosis and protect BMECs injury induced by OGD/R.Figure: 34,Table: 3,Reference: 114.