Steap4 Promotes Neuronal Ferroptosis In Cerebral Ischemiareperfusion Injury Via Activating P38/JNKk Signaling Pathway

Objective:Cerebrovascular disease is one of the common diseases that threaten human health and affect human life.Ischemic stroke(IS)is the most common type of cerebrovascular disease,the number of patients is about 80% of the total number of patients with cerebrovascular disease.Its pathological mechanism may be due to the blockage of blood supply in the brain,leading to cerebral ischemia,hypoxia,brain necrosis,edema,and affecting the normal neurological function.The mortality rate of ischemic stroke is high,the disability rate is higher,the quality of life of patients with ischemic stroke may be seriously affected.So far,the only internationally recognized treatment for ischemic stroke is revascularization.However,because patients and their families do not understand the early clinical manifestations of ischemic stroke,they do not seek medical treatment in time,and miss the best opportunity of vascular recanalization treatment,the prognosis of the disease may be very poor.In addition,cerebral ischemiareperfusion injury may occur after vascular recanalization treatment or during conservative treatment without vascular recanalization treatment,and the condition will be further aggravated.Therefore,it is urgent to explore the early diagnosis and effective treatment of ischemic stroke at this stage.Neuronal death induced by cerebral ischemia-reperfusion injury is the most direct factor to damage neural function.During cerebral ischemia-reperfusion injury,due to the destruction of blood-brain barrier,neuroexcitotoxicity,neuroinflammation and other reasons,neurons will accumulate excessive reactive oxygen species(ROS),reactive nitrogen species(RNS)and other free radicals,damage organelles,DNA,lipid and so on in neurons,and then lead to neuronal death.On the other hand,the morphological features of neurons were cell shrinkage,nuclear pyknosis,DNA fragmentation,cell swelling,rupture and so on,which indicated that a variety of cell death forms existed simultaneously in the process of cerebral ischemia-reperfusion injury.In recent years,it has been found that a new form of cell death,called ferroptosis,can induce cerebral ischemia-reperfusion injury.Ferroptosis is related to the accumulation of reactive oxygen species in lipids mediated by iron overload.As the initiating factor of ferroptosis,the accumulation of iron ions in cells is very important to trigger iron death.Excessive free iron ions in cells can promote lipid peroxidation.When glutathione peroxidase 4(GPX4)is inactivated,polyunsaturated fatty acids(PUFAs)in cell membrane are increased,The imbalance of PUFA oxidation initiates ferroptosis.At present,it is believed that lipid oxidation disorder and the formation of lipid reactive oxygen species will eventually lead to cell membrane damage and perforation,but the exact molecular mechanism of this damage is still unclear.Six transmembrane prostatic epithelial antigen 4(Steap4),located at 7q21.12,expresses a protein that functions as a metal reductase that transfers electrons from intracellular NADPH to extracellular iron or copper ions.In the study of osteoclast differentiation,it was found that STEAP4 can promote the increase of intracellular iron ions and promote osteoclast differentiation.In the mouse model of colitis,the high expression of STEAP4 leads to iron overload in mitochondria,which plays an important role in promoting the development of colitis.However,the expression of STEAP4 and its biological role in acute ischemic stroke have not been reported all over the world.In this study,we preliminarily investigated the changes in the expression of STEAP4 after cerebral ischemia reperfusion injury and the potential regulatory mechanism of STEAP4 on ferroptosis of neurons induced by cerebral ischemia reperfusion injury.Methods:1.Animal and cell models of cerebral ischemia reperfusion injury were established to analyze the expression level of STEAP4 in the disease model.(1)A transient middle cerebral artery occlusion(tMCAO)model was established in mice,and the volume of cerebral infarction was detected by TTC staining.The expression of STEAP4 in serum of the sham operation group and the disease group was detected by ELISA assay.The mRNA and protein expression levels of STEAP4 in brain tissues of mice were detected by qRT-PCR and Western Blot.The neuronal markers NEUN and STEAP4 in the brain tissues of mice in each group were double-stained by immunofluorescence method to observe the distribution of STEAP4 expression in tissues and cells.(2)A model of oxygen and glucose deprivation reoxygenation(OGD/R)was established in N2 a cells.The mRNA and protein expression levels of STEAP4 were detected by qRT-PCR and Western Blot.2.To study the effect of STEAP4 on ferroptosis of neurons after cerebral ischemia reperfusion injury.(1)In the OGD/R model of N2 a cells,STEAP4 was knocked down by siRNA transfection and overexpressed by overexpressed plasmid transfection.,(2)Change of cell viability was detected by CCK8 method.The damage of cells was detected by LDH assay.(3)The expression differences of ferroptosis related proteins GPX4,SLC7A11,DMT1 and FPN1 in each group were detected by Western Blot.Iron detection kit was used to detect the difference of iron content in cells.The changes of reactive oxygen species(ROS)in cells were detected by 2',7'-Dichlorodihydrofluorescein diacetate(DCFH-DA)probe.The contents of GSH and MDA were detected by GSH and MDA kit.3.The mechanism of STEAP4 aggravating ferroptosis of neurons after cerebral ischemia reperfusion injury.(1)Steap4 was knocked down and overexpressed in the OGD/R model of N2 a cells,and the protein expression changes of p-p38,p-JNK,p38 and JNK were detected by Western Blot.(2)In the OGD/R model of N2 a cells,the p38 inhibitor SB202190 and the JNK inhibitor SP600125 were added at the same time as the overexpression of STEAP4,and the cell viability was detected by CCK8 assay.The damage of cells was detected by LDH assay.The expression differences of ferroptosis related proteins GPX4,SLC7A11,DMT1,and FPN1 in each group were detected by Western Blot experiment to determine whether the promotion of ferroptosis by STEAP4 was partly dependent on the activation of p38/JNK signaling pathway.Results:1.The expression of STEAP4 protein in serum of tMCAO mice was significantly higher than that of sham operation group.The mRNA and protein expression levels of STEAP4 in brain tissue of tMCAO mice were higher than those in sham operation group.The results of immunofluorescence double staining showed that the expression level of STEAP4 protein in the ischemic penumbra brain tissue neurons of tMCAO mice was higher than that of sham operation group.2.After OGD/R injury in N2 a cells,the mRNA and protein expression levels of STEAP4 were higher than those in the normal control group.3.Transfection of Steap4 siRNA-1 and Steap4 siRNA-2 can reduce the protein expression levels of Steap4 in OGD/R cells.Steap4 knockdown can reduce OGD/Rinduced neuronal death and enhance cell viability.Steap4 knockdown can reduce the iron content in OGD/R neurons,increase the protein expressions of GPX4,SLC7A11 and FPN1,decrease the protein expression of DMT1,decrease the content of ROS in cells,increase the content of intracellular GSH and decrease the expression of MDA,suggesting that inhibition of Steap4 can inhibit the ferroptosis of neurons caused by cerebral ischemia reperfusion injury.4.Transfection of STEAP4 plasmid can significantly up-regulate the protein expression levels of STEAP4.Overexpression of STEAP4 can enhance OGD/ R-induced neuronal death and decrease cell viability.Overexpression of STEAP4 can increase the iron content in OGD/R neurons,decrease the protein expressions of GPX4,SLC7A11 and FPN1,increase the protein expression of DMT1,increase the content of ROS in cells,decrease the content of intracellular GSH and increase the expression of MDA,suggesting that STEAP4 can promote the ferroptosis of neurons caused by ischemia reperfusion injury.5.In the case of OGD/R injury,Steap4 knockdown can inhibit the phosphorylation of p38 and JNK proteins,while overexpression of Steap4 can promote the phosphorylation of p38 and JNK proteins.6.In the case of OGD/R injury,the expression of ferroptosis related proteins GPX4,SLC7A11 and FPN1 were increased,and the expression of DMT1 were decreased after the addition of p38 inhibitor SB202190 and JNK inhibitor SP600125 at the same time of overexpression of STEAP4,compared with the only overexpression of STEAP4 group.It is suggested that STEAP4 can promote ferroptosis of neurons through the activation of p38/JNK signaling pathway.Conclusions:1.Steap4 was highly expressed in serum and brain tissue neurons of mouse tMCAO model and in OGD/R model of N2 a cells.2.During OGD/R in N2 a cells,STEAP4 knockdown can alleviate ferroptosis of neurons and enhance cell viability.Overexpression of STEAP4 can promote ferroptosis of neurons and inhibit cell viability.3.Steap4 can aggravate iron death of neurons by promoting the p38/JNK signaling pathway.Inhibitors of p38 and JNK partially reverse STEAP4 induced ferroptosis of neurons.