Research Of Pomelo Peel Essential Oil On Alleviating Cerebral Ischemia-reperfusion Injury And Mechanism Related To Ferroptosis

Background and objectiveCerebral ischemia reperfusion injury（CIRI）causes damage to the structure or function of cells in the central nervous system,which is the main cause of the high fatality rate of various diseases such as stroke,myocardial infarction,cardiac arrest/resuscitation and organ transplantation.Exploring the pathophysiological mechanism of CIRI and neuroprotective drugs development in this field have always been research hotspot.Among them,antioxidants or other treatments focus on anti-oxidation have shown positive effects.It’s beneficial to improve the adverse cosequences of neuron death caused by ischemia/reperfusion.Ferroptosis is recognized as a new form of regulated cell death characterized by iron-depended lipid peroxidation and implicated in CIRI.Furthermore,antioxidant intervention can inhibit ferroptosis and alleviate brain tissue damage.Pomelo peel essential oil（PPEO）obtained from Rong County,Guangxi,is considered to have potential medical value based on significant antioxidant and anti-inflammatory activities.But it’s not clear whether PPEO can protects against CIRI.In this study,PPEO from the outer peel of Shatian pomelo was extracted by distillation and its main components were identified.Then three experimental models including middle cerebral artery occlusion/reperfusion（MCAO/R）,CA/CPR,oxygen and glucose deprivation/reperfusion（OGD/R）were used to explore the role of PPEO against CIRI.On the basis of that,the underlying mechanism of PPEO were preliminarily explored in three main aspects related to ferroptosis:redox imbalance,lipid and iron metabolism as well as modulation of upstream factor on ferroptosis was preliminarily explored.We revealed the mechanisms related to ferroptosis involed in CIRI and the neuroprotective targets of PPEO,which providing new therapeutic strategies in cerebral functional recovery after recanalization or cardiopulmonary resuscitation.Method1.Extraction of PPEO:Ripe pomelos（Citrus maxima（Burm.）Merr.cv.Shatian Yu）were purchased from Shatian village,Rong Country,Guangxi Zhuang Autonomous Region.The specimen was identified by Guangxi institute of botany,Guangxi Zhuang autonomous region and Chinese academy of sciences.The yellow part of the outer layer of the fruit peel was cut into small pieces.Then PPEO was extracted by distillation after soaking for 4 hours.The components of PPEO was identified by GC/MS analysis.2.Experiments on MCAO/R animal model:total of 70 adult male Sprague-Dawley（SD）rats were used for experiment.Besides sham group,others were built MCAO/R rat models and then randomly divided into the following five groups:model,vehicle,PPEO₁₀,PPEO₃₀ and PPEO₉₀.Rats from different groups were injeced intraperitoneally with saline,10%glycerol solution and various dose of PPEO（10mg/kg,30mg/kg,90mg/kg）respectively every 12 hours and a total of 4 times.Neurological function assessment was performed after 24 hours of reperfusion.At 48-hour timepoint,rats’brains were taken quickly for 2,3,5-triphenyl tetrazolium chloride（TTC）staining to measure the effect of PPEO on cerebral infarct area.The sections stained with hematoxylin-eosin and Nissl solution for the morphological evaluation.The expression of glutathione peroxidase 4（GPX4）was detected by Western blot and immunofluorescence.3.Experiments on CA/CPR animal model:120 adult male SD rats were divided into six groups.After inducing CA/CPR treatment,other groups besides sham group were randomly divided as follow:model group,vehicle group and three PPEO groups.0.9%saline,10%glycerium and different dose of PPEO（12.5 mg/kg,25mg/kg and 50mg/kg）were administered immediately after resuscitation via femoral vein respectively.Neurological dysfunction scores（NDS）were evaluated at 24 hours after resuscitation.Then H&E staining was used to observe the morphological changes of brain tissue,nissl staining was used to observe the effect of PPEO on nissl bodies in neurons.Explore underlying mechanism of PPEO on alleviating CIRI from three aspects related to ferroptosis:redox System Xc^--GSH-GPX4 pathway,iron content and lipid metabolism.Iron,GSH and MDA contents were detected by assay kit respectively.GPX4 and SLC7A11 level were measured by immunocytochemical staining and western blotting.GPX4 and ACSL4 expression were detected by immunofluorescence.4.Experiments on OGD/R model:Cortical neurons of newborn SD rats was primary cultured and the protective effect of PPEO on the morphology and structure of neurons after OGD/R treatment was observed under microscope.We explored the adapt dose of PPEO,erastin and RSL3 on cultured human neuroblastoma cell line（SH-SY5Y）by cell counting kit-8（CCK8）.Experimental group was set depend on the different demand of detection.It includes control group,OGD/R model group,vehicle group,PPEO group and PPEO+inhibitor group.The level of intracellular reactive oxygen species（ROS）was measured by flow cytometric assays.GPX4 and SLC7A11 level were measured by western blotting.Biochemical detection was used to contrast the effect of PPEO on GSH contents of erastin induced ferroptosis and OGD/R induced changes.In additon,immunofluorescence staining was used to observe the expression and translocation of nuclear factor erythroid 2-related factor 2（Nrf2）,a upstream factor of SLC7A11 and GPX4,to explore the role of PPEO on ferroptosis involved in CIRI from upstream factor.Result1.Extraction and identification of PPEO:The extracted PPEO presents light yellow colour and distinctive fragrant smell.The average mass of PPEO is about 0.84g per ml.A total of 35 compounds were identified from 37chromatographic peaks after GC/MS analysis of the PPEO sample.They are terpenes compounds,alcohol,esters,aldehydes and small quantity of ethers.The major constituents were D-limonene which amounts 50.67%and followed byα-pinene,1,6-Octadien-3-ol,3,7-dimethyl-and Naphthalenone.2.Effects of PPEO on neurological function score,morphological changes of brain tissue and expression of GPX4 protein in focal CIRI rat model:Results of NDS demonstrated that scores in the model group and vehicle group were markedly increased compared with sham group（P<0.01）while PPEO treatments reduced them（P<0.05）.Compared with sham group,TTC staining results in model group showed obvious gray-white infarct area and the infarct percentage was 29%±2.205.Though,infarct percentage value in different dose of PPEO groups was significantly declined,perticularly at dose of 30mg/kg（P<0.01）and the value is about 12.2%±1.594.Correspondly,H&E staining showed that normal brain tissue presents intacted structure,well arranged neurons and clearly nucleoli.PPEO improved morphological changes observed in model and vehicle group,such as loosen orgnization,irregulated neurons with pyknosis or necrosis and significantly widened perinuclear space.Nissl staining for sections suggested that nissl’s bodies was significantly reduced and looks lighter in model and vehicle group while that was recovery in PPEO groups,especially in high-dose group.Western blotting and immunofluorescence measurement of GPX4 both declared that GPX4expression level in model or vehicle group was significantly reduced compared to sham group,but that was recovered by PPEO treatment,especially in PPEO₃₀group（P<0.01）.3.Effects of PPEO on neurological function score,morphological changes of brain tissue and indicators related to ferroptosis in global CIRI rat model:NDS at 24 hours after ROSC showed that scores were improved in PPEO treated groups,specially in the CPR+PPEO₅₀ group（P<0.05）.After H&E and Nissl staining,sections were observed under the microscope.All phenomenon included disordered orgnization of brain tusse,irregular neurons or even dead ones and declined number of Nissl bodies observed in model or vehicle group has been improved significantly by PPEO treatment,especially in PPEO₅₀ group（P<0.05）;Typical ferroptotic mitochondria injury with maller volume,condensed membrane densities,disorganized mitochondria cristae and rupture of outer membrane was observed by transmission electron microscopy in model and vehicle group.As expected,PPEO improved the damaged neurons and mitochondria at the ultrastructural level.Results of ferroptosis related indicators:PPEO reverse the increasing of iron contents induced by CIRI.Only at middle and high dose,PPEO reverse the dicline of GSH contents（P<0.05）.PPEO decreased MDA level（P<0.05）only at high dose.Immunohistochemistry staining and western blotting results declared the decreasing of GPX4 and SLC7A11 induced by CIRI was markedly elevated after treatment of the essential oil of pemolo peel（P<0.05）,especially in PPEO₅₀ group.Immunofluorescence double staining of ASCL4 and GPX4 show that CIRI caused decrease of GPX4 while increase of ACSL4 compare to sham group.However,PPEO rescued the expression of GPX4,specially at dose of 50mg/kg（P<0.01）.Simultaneously the expression of ASCL4 decreased,specially at dose of 25mg/kg（P<0.05）.4.Effect of PPEO on morphological changes in OGD/R-treated primary cortical neurons and effect of PPEO on Nrf2-System Xc^--GSH-GPX4 axis in OGD/R-treated SH-SY5Y cells:We cultured primary cortical neurons of newborn Sprague-Dawley rats and observed PPEO attenuated OGD/R damage of neurons at dose of 25μg/ml.In this trial,the dose of Ersatin and RSL3 are determined at 10μM and 2μM respectively.Flow cytometric measurement results discovered that PPEO exerts a trend to decline the increasing level of ROS caused by erastin or OGD/R but the data is not statistically significant,compared to ferroptosis inhibitor Deferoxamine which used as positive control.However,what’s interesting is that level of ROS raised again after SLC7A11was inhibited by erastin.The followed biochemical measurements showed that PPEO reverse the decrease of GSH induced by erastin and OGD/R,the latter effect can be abrogated by erastin again（P<0.01）.WB findings indicated that.the expression of GPX4（P<0.01）and SLC7A11（P<0.01）were decreased after OGD/R,while PPEO increased the expression of these two proteins which can be neutralized by respective inhibitor.In finally,immunofluorescence detect result showed the fluorescence intensity of Nrf2 in the nucleus slightly increased after OGD/R treatment compare to control group,while PPEO greatly enhanced the fluorescence intensity of Nrf2 in the nucleus more obvious.Furthermore,the fluorescence intensity of Nrf2 in nucleus significantly enhanced after PPEO treatment compare to model group.ConclusionEffects of PPEO against CIRI and the underlying mechanism related to ferroptosis was explored in vivo and in vitro based on MCAO/R,CA/CPR rat model and OGD/R cell model,respectively.PPEO improved NDS and damaged brain tissue caused by CIRI and reduced infarct volume,increased the expression of ferroptosis-related inhibited factors such as GPX4,SLC7A11 and GSH through promoting translocation of Nrf2 from cytoplasm to nucleus.Meanwhile,PPEO decreased contents of iron,ROS and MDA as well as decreased ACSL4 level which promoting ferroptosis.Above results declare that PPEO activated Nrf2-System Xc^--GSH-GPX4 axis to positively modulate antioxidative activity of neurons,so exerts positively neuroprotecive role in CIRI through inhibiting ferroptosis.