Effect Of 15-PGDH In Cerebral Ischemia-reperfusion Injury And Its Mechanism

Background and objective:Stroke is known as an acute cerebrovascular focal lesion.It is the second leading cause of death and one of the leading causes of long-term disability worldwide,imposing a huge burden on human life and health.Stroke includes ischemic stroke,hemorrhagic stroke,and subarachnoid hemorrhage.Ischemic stroke is the most common type of stroke,mainly presenting as focal infarction of the cerebral,spinal cord,or retinal.Early restoration of blood supply to the infarcted area is the first principle of ischemic stroke treatment.However,it is highly likely to induce a more serious injury,known as cerebral ischemia-reperfusion injury（CIRI）.The mechanisms of CIRI involve oxidative stress,inflammation,excitatory toxicity,and various forms of cell death,including necrosis,apoptosis,autophagy,and ferroptosis.Ferroptosis is a non-apoptotic form of programmed cell death caused by iron-dependent lipid peroxidation.It has been confirmed to be involved in a variety of pathophysiological processes such as tumors,inflammation,brain trauma,and neurodegenerative diseases.The role of ferroptosis in CIRI has also been reported that ferroptosis inhibitors can successfully alleviate neurological deficits and cerebral infarction in CIRI,but the specific mechanism needs to be further explored.15-hydroxyprostaglandin dehydrogenase（15-PGDH）is the sole degrading enzyme of prostaglandin E2（PGE2）,which plays an important role in regulating the level of PGE2.By regulating the level of PGE2,via the specific receptor of PGE2,15-PGDH can play a variety of biological functions.The role of 15-PGDH in tumors,acute kidney injury,acute liver injury,tissue repair and reconstruction has been reported many times.Recent reports have revealed that PGE2 significantly regulates ferroptosis induced by cerebral ischemia-reperfusion（CIR）.As the PGE2 degrading enzyme,15-PGDH is also potential to regulate CIRI-induced ferroptosis.Therefore,our study will focus on the effect of 15-PGDH in CIRI,reveal the regulation of 15-PGDH in CIR-induced ferroptosis,and further explore the mechanism,providing more clues for the treatment of CIRI.Methods:（1）Detect the expression level of 15-PGDH in CIRI:After the establishment of the middle cerebral artery occlusion（MCAO）rat model,Western Blot,polymerase chain reaction（PCR）,and immunohistochemical staining were used to detect the expression and location of 15-PGDH in CIRI brain tissue.Oxygen glucose deprivation（OGD）cell model was established,then the expression of 15-PGDH in rat primary cortical neurons was detected.（2）Explore the regulation of 15-PGDH in CIRI:After 15-PGDH overexpressed adeno-associated virus or15-PGDH inhibitor SW033291 was injected into the ventricle,Masao Shmi izu-Sasamata score,triphenyl tetrazolium chloride（TTC）staining,hematoxylin-eosin（HE）staining were adopted to detect neurological function score,cerebral infarction volume,brain water content and pathological structure of the rat brain tissues,to evaluate the effect of 15-PGDH in CIRI at animal level;After transfection with 15-PGDH overexpressed plasmid or 15-PGDH inhibitor SW033291,CCK-8 was adopted to detect cell viability to evaluate the effect of 15-PGDH in CIRI at the cell level.（3）Explore the regulation of 15-PGDH in ferroptosis induced by CIR:Brain tissue samples of patients with brain injury were collected to detect the levels of 15-PGDH,reactive oxygen（ROS）,and malondialdehyde（MDA）,then to analyze the correlation between 15-PGDH and lipid peroxidation.After 15-PGDH overexpressed adeno-associated virus or 15-PGDH inhibitor SW033291 was injected into the ventricle,the levels of Fe²⁺,ROS,MDA,glutathione（GSH）and several ferroptosis marker genes were detected to evaluate the regulation of 15-PGDH in CIR-induced ferroptosis at animal level.Meanwhile,after transfection of 15-PGDH overexpressed plasmid or 15-PGDH inhibitor SW033291,the levels of ROS,MDA,and GSH were detected to evaluate the regulation of 15-PGDH in CIR-induced ferroptosis at the cell level.（4）Explore the regulation mechanism of 15-PGDH in ferroptosis induced by CIR:Glutathione peroxidase 4（GPX4）knockout mice were constructed to verify whether the regulation of CIRI and ferroptosis by 15-PGDH was mediated by GPX4.The regulation of 15-PGDH on PGE2 and PGE2receptors（EP）was detected,and the decisive receptor was determined by the application of the antagonist and agonist.Western Blot and immunofluorescence were used to detect the phosphorylation and nuclear transfer of EP-regulated transcription factors,and luciferase reporter gene and chromatin immunoprecipitation were used to explore the bound and regulation of transcription factors on GPX4.（5）Blood samples of stroke patients were collected,and the level of 15-PGDH in the samples was detected by enzyme-linked immunosorbent assay（ELISA）.According to the occurrence of stroke-related pneumonia,the prediction effect of 15-PGDH on stroke-associated pneumonia and its specific association was analyzed.Results:（1）In the rat MCAO model,the expression of 15-PGDH was decreased;In the cell OGD model,15-PGDH also showed a decreasing trend.（2）At the animal level,15-PGDH overexpression significantly aggravated neurological deficits,brain infarction,brain edema,and damage to brain structure,inhibition of 15-PGDH alleviated cerebral infarction and brain edema.At the cell level,15-PGDH overexpression decreased the cell viability after OGD injury in rat primary neurons,while15-PGDH inhibition increased the cell viability.（3）Correlation analysis of the levels of 15-PGDH and ROS and MDA in brain tissue samples of patients with brain injury showed that 15-PGDH was positively correlated with ROS and MDA.At the animal level and cell level,15-PGDH overexpression significantly increased the levels of ROS,MDA,and Fe²⁺after CIRI,while decreased the level of GSH.On the contrary,15-PGDH inhibition significantly decreased the levels of ROS,MDA,and Fe²⁺,while increased the level of GSH.（4）Overexpression of 15-PGDH significantly decreased GPX4 expression,while inhibition of 15-PGDH increased GPX4 expression.The knocked out of GPX4 eliminated the improvement effect of SW033291 on cerebral infarction and cerebral edema caused by CIRI,as well as ferroptosis induced by CIR.Overexpression of 15-PGDH inhibited the PGE2/EP4 pathway,while inhibition of 15-PGDH promoted the PGE2/EP4 pathway,and reversal of EP4 can eliminate the effect of 15-PGDH in CIRI.SW033291 promoted the phosphorylation and transformation into the nucleus of EP4-regulated transcription factor c AMP-response element-binding protein（CREB）and nuclear factor kappa-B（NF-κB）.The luciferase reporter gene and chromatin immunoprecipitation showed that CREB and NF-κB directly bind to GPX4and promote the expression of GPX4.（5）The level of 15-PGDH decreased in patients with stroke-associated pneumonia（SAP）.Logistic regression analysis showed that reduced 15-PGDH was a risk factor for SAP,and there was an L-shaped relationship between the level of 15-PGDH and the risk of SAP.Conclusions:（1）The expression of 15-PGDH is decreased in CIRI.（2）The 15-PGDH/PGE2/EP4 axis transcriptionally down-regulates GPX4expression through CREB and NF-κB to promote ferroptosis.（3）15-PGDH reduction is a risk factor for SAP.Figure:25,Table:6,Reference:121.