| BackgroundIschemic stroke,also known as ischemic cerebrovascular disease or cerebral infarction,is a disease of brain tissue necrosis caused by cerebral blood supply artery stenosis or occlusion.Ischemic stroke is one of the leading causes of death and severe long-term disability and has become the most common life-threatening neurological disease.About 15 million patients worldwide suffer from ischemic stroke evrey year,thereby resulting in more than half a million deaths and 5 million permanent disabilities.In addition,the incidence of recurrent ischemic stroke after 5 years is 11.3%.Although restoration of blood supply through reperfusion can improve clinical outcomes,rapid reperfusion may also aggravate brain damage.Cerebral ischemia-reperfusion(I/R)injury has become an increasingly serious challenge for stroke patients.Cell death is the predominant common I/R injury feature in all tissues,so it is a stable pathological indicator of I/R injury.Ferroptosis is a non-apoptotic form of regulatory cell death caused by the failure of glutathione(GSH)-dependent lipid peroxide scavenging network.It depends on iron and reactive oxygen species(ROS)and is considered to be the cause of reperfusion injury.Ferroptosis is often accompanied by lipid repair enzyme(glutathione peroxidase 4,GPX4)dysfunction,massive iron accumulation,and polyunsaturated fatty acid lipid peroxidation.It is driven by loss of GPX4 activity and subsequent accumulation of lipid-based ROS species.Due to its high metabolic rate and relatively low antioxidant defense ability,the brain is susceptible to ROS.Moreover,due to the high concentration of polyunsaturated fatty acids in the membrane structure rich in the brain,oxidative damage in the brain is mainly manifested as lipid peroxidation.In addition,the brain is an organ that gradually accumulates iron with age.The accumulation of iron in the brain easily leads to the production of a large number of free radicals and peroxides,which causes the disorder of lipid peroxidation and protein function,and eventually results in the degeneration and death of neurons.Dihydromyricetin(DHM)is extracted from woody vines,and the main active ingredients are flavonoids.Flavonoids have many special effects such as scavenging oxygen free radicals,antioxidant,antithrombotic,anti-inflammatory and anti-tumor.In addition to the general properties of flavonoids,it has been shown that DHM has a strong protective effect against myocardial and liver I/R injury.Additionally,DHM can improve the cerebral damage in ischemic stroke.DHM protects mouse hippocampal neuron HT22 cells from oxygen-glucose deprivation/reperfusion(OGD/R)-induced oxidative stress and apoptosis via activating Nrf2/HO-1 signal pathway.This suggested that DHM may play a protective role in cerebral I/R injury.Nevertheless,whether DHM could weaken cerebral I/R injury in animal model have not been fully explained.Through bioinformatics analysis(http://119.3.41.228:8000/tcmid/ingredient/32748/)of the potential targets of DHM,it was found that sphingosine kinase 1(SPHK1)and mammalian target of rapamycin(mTOR)may be the targets of DHM.It was speculated that DHM may play a protective role in cerebral I/R injure process by regulating SPHK1 and mTOR.Part Ⅰ DHM improved cerebral I/R injury in ratsObjectiveThis work was attempted to explore the therapeutic effect of DHM on cerebral I/R injury in vivo,and analyze whether DHM can affect cerebral I/R injury by regulating ferroptosis and SPHK1/mTOR signaling pathway.Methods1.Middle cerebral artery occlusion/reperfusion(MCAO/R)surgery was used to construct a rat model of cerebral I/R injury2.Zea Longa behavioral scoring was conducted to assess the neurological deficit of rats.3.Wet-dry weighted method was used to measure the water content in brain tissues.4.2,3,5-Triphenyltetrazolium chloride(TTC)staining was performed to detect the volume of cerebral infarcts.5.TUNEL staining was performed to examine the levels of apoptosis in cerebral tissues.6.Western blot was carried out to detect the expression levels of SPHK1,mTOR,p-mTOR and ferroptosis-related proteins ACSL4,PEBP1 and GPX4 in cerebral tissues.Results1.DHM improved neurological deficits in cerebral I/R rats at doage-dependent manner.2.DHM reduced cerebral edema and cerebral infarction volume in cerebral I/R rats at doage-dependent manner.3.DHM inhibited apoptosis in cerebral tissue of cerebral I/R rats at doage-dependent manner.4.DHM repressed the expression of SPHK1,p-mTOR,ACSL4 and PEBP1,and elevated the expression of GPX4 in cerebral tissue of cerebral I/R rats.ConclusionsDHM improved cerebral I/R injury at doage-dependent manner,and inhibited SPHK1/mTOR signaling pathway and the expression of ferroptosis-related proteins.Part Ⅱ DHM reduced OGD/R-induced neuronal ferroptosisObjectiveThis work aimed to explore whether DHM can affect cell viability and apoptosis of OGD/R-induced mouse hippocampal neuronal cells(HT22)and its effects on SPHK1/mTOR signaling pathway and ferroptosis.Methods1.HT22 cells were induced by OGD/R to establish a cell model of brain I/R injury,and then treated with different concentrations of DHM(1,10,20,30 pM)2.CCK-8 assay was performed to detect cell proliferation activity.3.Flow cytometry was conducted to examine cell apoptosis.4.ROS kit was used to assess the levels of intracellular ROS.5.Iron content detection kit to elevate intracellular iron content.6.Western blot was used to examine the expression levels of SPHK1,mTOR,p-mTOR and ferroptosis-related proteins ACSL4,PEBP1 and GPX4 in cells.Results1.DHM increased the proliferation activity of OGD/R-treated HT22 cells at dosage-dependent manner.2.DHM reduced apoptosis of OGD/R-treated HT22 cells at dosage-dependent manner.3.DHM suppressed ROS and iron content in OGD/R-treated HT22 cells.4.DHM inhibited the protein expression of SPHK1,p-mTOR,ACSL4 and PEBP1,and promoted the expression of GPX4 in OGD/R-treated HT22 cells.ConclusionDHM enhanced cell viability and inhibited apoptosis in OGD/R-treated HT22 cells at dosage-dependent manner.DHM modulated SPHK1/mTOR signaling pathway and reduced ferroptosis in OGD/R-treated HT22 cells.Part Ⅲ DHM reduced OGD/R-induced neuronal ferroptosis by regulating GPX4 expression and inhibiting SPHKl/mTOR signaling pathwayObjectiveThe aim of this study was to investigate the mechanism of DHM in improving cerebral I/R injury in OGD/R-HT22 cells.Methods1.OGD/R-induced HT22 cells were treated with 30 μM DHM,and then sunjected to GPX4 knockdown,SPHK1 overexpression or MHY1485(mTOR activator)treatment.2.CCK-8 assay was performed to detect cell proliferation activity.3.Flow cytometry was conducted to examine cell apoptosis.4.ROS kit was used to assess the levels of intracellular ROS.5.Iron content detection kit to elevate intracellular iron content.6.Western blot was used to examine the expression levels of SPHK1,mTOR,p-mTOR and ferroptosis-related proteins ACSL4,PEBP1 and GPX4 in cells.Results1.DHM treatment increased cell viability and inhibited apoptosis of OGD/R-treated HT22 cells,which was abrogated by GPX4 silencing.2.DHM treatment reduced ROS and iron content in OGD/R-treated HT22 cells.The influence conferred by DHM treatment was rescued by GPX4 knockdown.3.GPX4 knockdown reversed DHM treatment-mediated inhibition of ACSL4 and PEBP1 expression and promotion of GPX4 expression in OGD/R-treated HT22 cells.4.DHM treatment enhanced the cell viability and inhibited apoptosis of OGD/R-treated HT22 cells,which was rescued by SPHK1 overexpression or MHY1485 treatment.5.DHM treatment decreased lipid ROS and intracellular iron levels in OGD/R-treated HT22 cells.The influence conferred by DHM treatment was abolished by SPHK1 overexpression or MHY1485 treatment.6.DHM treatment resulted in decreased expression of SPHK1 and p-mTOR in OGD/R-treated HT22 cells.The effects of DHM treatment on SPHK1 and p-mTOR expression were rescued by SPHK1 overexpression or MHY1485 treatment.ConclusionDHM reduced ferroptosis in OGD/R-treated HT22 cells by regulating GPX4 expression and inhibiting SPHK1/mTOR signaling pathway. |
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