Naringenin Inhibits Oxidative Stress Damage Through Nrf2/HO-1 Pathway And The Mechanism Of Neuroprotective Effect In Epilepsy Rat

Objective:Epilepsy is a chronic brain disease caused by multiple causes,and characterized by excessive and repeated hypersynchronous discharges of neurons in the brain.Oxidative stress injury,glutamate excitatory toxicity,calcium overload and other factors may induce neuron to discharge trigger epilepsy aberrantly.The chain reaction of oxygen free radicals produced by oxidative stress is the core pathological link of neuron damage.Previous studies have shown that during epileptic seizures,the amounts of free radicals in the brain significantly increased,and large amounts of ROS were produced and released,which would cause the imbalance in cells,leading to series of phenomena such as DNA damage,destruction of cell membrane structure,damage of mitochondria and other organelles,and finally causing cell apoptosis.Therefore,inhibiting oxidative stress and scavenging oxygen free radicals might be important strategies for epilepsy treatment.The keap1-nrf2-are signaling pathway plays an important role in the cellular antioxidant process.Keap1 provides a scaffold for E3 ubiquitin ligase CUL3,which can mediate the degradation of Nrf2 through ubiquitin-proteasome,keeping Nrf2 at a lower level to ensure daily life activities.During oxidative stress,the related upstream signaling pathway is activated,making the degradation of Nrf2 terminated,leading it to enter into the nucleus,promoting the transcription of the downstream antioxidant-related gene HO-1,and participating in the cellular antioxidant defense mechanism.Naringenin is a flavonoid with strong antioxidant activity.In this study,the rat model of epileptic continuous state and the oxidative stress model of human neuroblastoma cells were used as the research objects.Through in vivo and in vitro experiments,the neuroprotective effect of Naringenin on oxidative stress injury of nerve cells and the possible mechanism of action were investigated,which provided an important research basis for the antioxidation treatment of epilepsy.Specifically,this study investigated whether Naringenin inhibits the neurotoxic damage induced by hydrogen peroxide（H2O2）and the role of Nrf2/HO-1 in this process,and further elucidated whether Naringenin has neuroprotective effects in pirocarpine-induced SE juvenile rats,and whether it participates in neuroprotective mechanism in vivo by regulating Nrf2/HO-1.Methods:1.The status epilepticus（SE）model of rats induced by lithium chloride-pilocarpine was established,and the experiment was divided into control group,SE group and Naringenin group respectively.The cognitive level of rats was observed by water maze experiment.HE,Nissl and TUNEL staining were used to observed the pathological changes of hippocampal tissue,the changes of living neurons and programmed dead cells respevitvely.The localization of Nrf2 and HO-1 proteins in cells was observed by immunofluorescence.Immunohistochemistry（IHC）and Western blot were used to observe the expression levels of Nrf2 and ho-1 proteins.2.Human neuroblastoma cell（SH-SY5Y）was cultured in vitro,and the proliferation activity of each group was detected by MTT;the level of reactive oxygen species（ROS）was detected by DCF-DA fluorescence staining;the mitochondrial membrane potential was detected by jc-1 fluorescence staining;the Annexin V/PI was used to estimate apoptosis rate;the expression changes of bcl-2,Bax,caspase-3 and Cyto C were detected by Western blot respectively.3.The transfection of Nrf2 shRNA plasmid was verified by Western blot.To observe the effect of silencing Nrf2 gene on apoptosis,Western blot was used to detect the expression levels of Nrf2 and HO-1 proteins in each group after Nrf2 gene silencing.The expression levels of Nrf2,HO-1,pERK,ERK,pAKT and AKT were detected by Western blot.Immunofluorescence（IF）was used to observe the nuclear immigration of Nrf2 and the localization of HO-1 protein.Annexin V/PI staining was used to detect the apoptosis rate.MTT was used to detect cell proliferation activity.Results:1.EEG recording results showed that the EEG rhythm of rats in the control group was regular,and no abnormal waveform was observed;the EEG of epileptic rats showed widespread spinous wave rhythm.2.The Morris water maze experiment results showed that compared with the control group,the escape latency of the epileptic model group rats was significantly prolonged;the evasive latency of Naringenin intervention group was significantly shortened.Compared with the control group,The Times of crossing the platform in the epileptic model group were significantly reduced,and The Times of crossing the platform in the Naringenin intervention group were significantly increased.3.HE staining results showed that the hippocampal structure and morphology of rats in the control group were basically normal,the cells were uniformly stained and arranged in a more orderly manner,the nuclei and nucleoli were normal in size,and the cytoplasm and nuclei were clear,presenting light red and light blue.In group 12h after epilepsy,the neuronal cells were swollen,the cytoplasm was transparent,the nuclei and cytoplasm were stained unevenly,and the cell structure and morphology were disordered.In ld group after epilepsy,pyramidal cells were significantly lost,neurons were reduced in size,the nuclei were hyperchromatic,and nucleoli were reduced or even disappeared.The nuclei and cytoplasm were stained dark blue and purplish blue,with uneven coloring,different sizes and disordered morphology.In groups 3d,7d and 14d after epilepsy,the morphology and structure of cells gradually recovered compared with ld after epilepsy.The staining of nuclei and cytoplasm of neurons was more uniform and the morphology was basically regular.4.Nissl staining results showed that neurons in the hippocampal vertebral bodies of rats in the control group were tightly arranged,with complete morphology,clear nucleoli,abundant intracytoplasmic nysch,and no obvious neuron loss.Compared with the control group,hippocampal neurons in the ld group after epilepsy were significantly lost and disorganized,with cell shrinkage,chromatin agglutination,nucleation,and significantly reduced intracytoplasmic nidenite.After epilepsy,the number of pyramidal cells in groups 3,7,and 14d gradually increased,the intercellular space shrank,and the arrangement was more orderly,the distribution of intracytoplasmic nissl was abundant,and the phenomenon of nucleolar hyperchromatism,shrinkage and fragmentation gradually improved.5.Western blot results showed that compared with the control group,Nrf2 expression level increased 12h after epilepsy,peaked 1d after epilepsy,and gradually decreased on3d,7d and 14d.6.Western blot results showed that,compared with the control group,the expression level of HO-1 increased 12h after epilepsy,peaked 1d after epilepsy,and gradually decreased on 3d,7d and 14d.7.HE staining results showed that,compared with the control group,the cell morphology and structure of the epileptic group were obviously disordered,pyramidal cells were significantly lost,neurons were reduced in size,nuclei were hyperchromatic,and nucleoli were reduced or even disappeared.Compared with the epileptic group,the cell morphology and structure of the Naringenin intervention group were significantly restored,pyramidal cell loss was reduced,and nucleus and cytoplasm staining was more uniform than that of the epileptic group.8.To observe the effect of Naringenin on the pathological damage of hippocampal tissue in epileptic rats by Nissl staining.The results showed that,compared with the control group,the hippocampal neurons in the epileptic group lost significantly,arranged disorganized,the cells were observed to shrink,chromatin clumped,and the number of Nissenden decreased significantly.Compared with the epileptic group,the hippocampus neurons in the Naringenin intervention group had clear margins,only a small amount of chromatin agglutination,and the amount of Nissl was significantly increased.9.TUNEL staining results showed that the nuclei of hippocampal neurons in the control group were blue,with orderly cell arrangement and uniform staining,and apoptotic neurons were occasionally observed.Compared with the control group,the number of TUNEL positive cells in hippocampal neurons of the epileptic group was significantly increased.Compared with the epilepsy group,the number of TUNEL positive cells in the Naringenin intervention group was significantly reduced.10.Western Blot results showed that Naringenin could up-regulate the hippocampal Bcl-2/Bax ratio and inhibit the activation of Caspase-3 in epileptic rats.11.Western blot results showed that compared with the control group,the expression of Nrf2 in hippocampal tissues of epileptic rats was increased.Compared with the epilepsy group,Nrf2 expression was significantly increased in the Naringenin intervention group.12.Western blot results showed that compared with the control group,the expression of HO-1 in the hippocampal tissues of the epileptic rats was increased.Compared with the epilepsy group,the expression of HO-1 protein in the Naringenin intervention group was significantly increased.13.MTT results showed that Naringenin inhibited the proliferation of SH-SY5Y cells induced by H2O2 in a dose-dependent manner.14.Intracellular ROS was detected by DCF-DA fluorescence staining,and the results showed that ROS in H₂O₂ group was significantly increased.Naringenin intervention can significantly inhibit the increase of ROS release induced by H₂O₂.15.The results of mitochondrial membrane potential test showed that the mitochondrial membrane potential in H₂O₂ group was significantly reduced.Naringenin intervention can inhibit the decrease of mitochondrial membrane potential induced by H₂O₂.16.Flow cytometry showed that the apoptosis rate of cells in H₂O₂ group was significantly increased,and Naringenin intervention could inhibit the increase of H₂O₂-induced apoptosis.17.Western Blot results showed that Naringenin could up-regulate bcl-2/Bax ratio under oxidative stress,the release of cytochrome c and the activation of caspase-3 respectively.18.Western Blot results showed that Naringenin up-regulated HO-1 expression,and immunofluorescence results showed that up-regulated HO-1 was mainly distributed in cytoplasm.19.Naringenin activated Nrf2,and Western Blot results showed that Naringenin up-regulated Nrf2 expression.Naringenin promoted Nrf2 to enter the nucleus,and immunofluorescence results also showed that Naringenin promoted Nrf2 to enter the nucleus.20.Immunoprecipitation experiment results showed that Nrf2 and KEAP1 proteins in the control group were mutually binding,and the binding of Nrf2 and KEAP1 was significantly reduced after naringin intervention.21.Western Blot results showed that the expression levels of Nrf2 and HO-1 proteins in SH-SY5Y cells decreased after shRNANrf2 transfection.22 Naringenin activates ERK1/2 and PI3K/Akt.Western Blot results showed that ERK1/2 and PI3K/Akt were activated by Naringenin in a time-dependent manner.23.ERK1/2 inhibitor PD98059 and PI3K/Akt inhibitor LY294002 inhibit the up-regulation of HO-1 expression induced by Naringenin,suggesting that ERK1/2 and PI3K/Akt participate in the up-regulation of HO-1 expression induced by Naringenin.24.Western Blot results showed that after silencing Nrf2,Naringenin was inhibited to increase the expression of Nrf2 and HO-1 in SH-SY5Y cells induced by H2O2.25.Flow cytometry showed that Nrf2 silencing partially reversed the inhibition of Naringenin on H2O2-induced SH-SY5Y cell apoptosis.26.After pretreated with HO-1 active inhibitor ZnPP,it was found that ZnPP reversed the inhibition of Naringenin on H₂O₂-induced oxidative damage,indicating that up-regulated HO-1 expression of Naringenin was involved in inhibiting H₂O₂-induced oxidative damage.Conclusion:1.Naringenin improves spatial learning and memory in epileptic rats.2.Naringenin inhibits brain injury in epileptic rats by antiapoptotic pathway.3.Naringenin promots the expression of Nrf2 and HO-1 in hippocampal neurons of epileptic rats.4.Naringenin may inhibit oxidative damage of SH-SY5Y cells by mitochondrial related anti-apoptosis pathway.5.Naringenin up-regulates Nrf2 expression by promoting the dissociation of Keap1 and Nrf2.6.Naringenin up-regulates Nrf2-dependent HO-1 expression by activating ERK1/2 and Akt pathways.In conclusion,Naringenin has a neuroprotective effect on the brain injury induced by pilocarpine in young rats with epilepsy.By activating ERK1/2 and Akt pathways,Naringenin up-regulates Nrf2/HO-1 pathway,thereby inhibiting H₂O₂-induced oxidative damage of SH-SY5Y cells.