| Objective:Alzheimer's disease has become one of the most serious diseases in the world,and the incidence rate of disease has been increasing year by year.The main pathological features of Alzheimer's disease are:Deposition of Amyloid?-peptide(A?)and formation of senile plaques by cell debris in the brain of patients;Neurofibrillary tangles(NFTs)formed by tau hyperphosphorylation and neuronal death.At present,the pathogenesis of Alzheimer's disease has not been completely clear.In recent years,studies have shown that the increase of iron in the brain and the disorder of iron metabolism may play an important role in the pathogenesis of Alzheimer's disease.With the increase of iron content,a large amount of reactive oxygen species(ROS)can be formed,which will damage the macromolecular substances including protein and DNA.Ferroptosis is a new cell death was discovered in 2012.Brain sections of Alzheimer's disease patients showed that the distribution of iron in the senile plaques formed by a?and the surrounding cells increased significantly,suggesting that iron deposition and the destruction of iron homeostasis occurred in the brain of Alzheimer's disease patients.The level of iron in brain tissue will gradually increase in the course of neurodegenerative diseases,which may mediate cell iron death.Therefore,targeted reduction of brain iron level,regulation of oxidative stress response and reduction of ferroptosis may become a new method for the treatment of Alzheimer's disease.When oxidative stress occurs,the body will produce antioxidant defense mechanisms such as glutathione peroxidase 4(GPX4)to prevent this damage.GPX4 is also regarded as the core component of iron death.Studies have shown that GPX4 deficiency in degenerative diseases can lead to decreased hippocampal neurons and astrocytes.Promoting GPX4 expression can significantly alleviate Parkinson's symptoms in rats.GPX4 also catalyzes the reduction of lipid peroxides in glutathione-dependent reactions,preventing iron from dying.Therefore,GPX4 can be used as an important target for inhibiting cell iron death,which is worthy of further study.Peroxisome proliferators activated receptor?(PPAR?)belongs to the nuclear receptor superfamily and is a ligand activated nuclear transcription factor.Studies have shown that PPAR?is involved in the regulation of synaptic plasticity by controlling calcium influx and encoding the expression of hippocampal related proteins.The down-regulation of PPAR?expression may decrease the ability of anti-oxidation and anti-inflammatory reaction,Which suggests that PPAR?may be an important target for the treatment of Alzheimer's disease.NF-E2-related factor 2(Nrf2)is one of the most important transcription factors in antioxidant stress defense system.When the body has a strong oxidative stress injury,Nrf2will be separated from Keap1 in the cytoplasm and transferred to the nucleus,combined with antioxidant response elements,to activate the expression of antioxidant gene HO-1.Activation of Nrf2/HO-1 signaling pathway can eliminate the cytotoxicity in degenerative neurological diseases by regulating the accumulation of?-synuclein,neuroinflammation and apoptotic cell death.The purpose of this study is to investigate the effect of PPAR?agonist on ferroptosis and its relationship with Nrf2/HO-1 signaling pathway in APP/PS1mice and cells.Methods:1.Effect of PPAR?agonist on brain injury and ferroptosis in APP/PS1 mice:WT mice and APP/PS1 transgenic mice were selected.Experimental groups:WT group,WT+PPAR?agonist group,APP/PS1 group and APP/PS1+PPAR?agonist group.The accumulation of A?in the brain of mice in each group was detected by immunohistochemical staining,the cognitive ability of mice was detected by Morriswatermaze,the expression of Iba-1 and BDNF was detected by immunofluorescence test,the apoptosis level of neurons was detected by TUNEL method,the expression of apoptosis-related markers Caspase3,Bcl-2 and Bax was detected by Western Blot and the expression of inflammatory cytokines such as IL-1?,TNF-?and IL-6 was detected by ELISA.The expression of FPN1,DMT1,Ferritin and other ferriregulatory proteins was detected by Western Blot.2.PPAR?agonists target GPX4 to inhibit oxidative stress in APP/PS1 mice.Experimental groups:WT group,WT+PPAR?agonist group,APP/PS1group and APP/PS1+PPAR?agonist group.Firstly,the expression level of intracellular reactive oxygen species and the contents of endogenous lipid peroxidation products MDA and 4-HNE were detected;secondly,the expression of GPX4 in each group was detected;finally,bioinformatics analysis,luciferase report and chromatin co-immunoprecipitation were used to examine whether PPAR?can be used as an upstream molecule to promote the transcription and expression of GPX4.3.PPAR?agonist ameliorates iron uptake regulated by ferroptosis by activating Nrf2/HO-1 signal pathway through GPX4.Experimental groups1:WT group,WT+PPAR?agonist group,APP/PS1 group and APP/PS1+PPAR?agonist group.,experimental group 2:SH-SY5Y cells transformed with unloaded plasmid neo as control group(control group),SH-SY5Y cells overexpressing APPsw as APP group,APP cells+PPAR?agonist as PPAR?group,APP cells+PPAR?agonists+GPX4inhibitors as GPX4 group.Firstly,the expression levels of HO-1,Nrf2 and Trx-1 in the brains of wild type mice and APP mice were detected by Western Blot.Secondly,the cells of different experimental groups were treated with PPAR?agonists and GPX4 inhibitors to study their effects on cell viability,apoptosis,the expression of A?1-42and ROS,and the levels of HO-1,Nrf2,Trx-1,iron uptake and proteins related to iron metabolism in each group.Results:1.The results of immunohistochemistry showed that senile plaque was not detected in the brain of WT mice,and a large number of senile plaques containing A?were found in the cerebral cortex and hippocampus of APP/PS1 mice,while GW7647 treatment could reduce the size and number of senile plaques in mice.Compared with WT mice,the escape latency of APP/PS1 mice was significantly increased and the number of cross-platform was significantly decreased(P<0.05).GW7647 treatment could significantly reduce the escape latency and increase the number of cross platforms in APP/PS1 mice,but there was no significant change in swimming speed among groups(P<0.05).Compared with WT mice,the levels of IL-1?,TNF-?and IL-6 in APP/PS1 mice were significantly increased,the expression of Iba-1 was significantly increased,the apoptosis of neurons in hippocampus and cerebral cortex was significantly increased,the expression of Caspase3 and Bax was significantly up-regulated,the expression of Bcl-2 was significantly down-regulated,the expression of BDNF was significantly decreased,the expression of FPN1 was significantly decreased,and the expression of DMT1 and ferritin was significantly increased(P<0.05).After GW7647 treatment,the expression of IL-1?,TNF-?and IL-6 in APP/PS1 mice decreased significantly,the expression of Iba-1 decreased significantly,the apoptosis of neurons in hippocampus and cerebral cortex decreased,the expression of Caspase3 and Bax decreased,the expression of Bcl-2 increased,the expression of BDNF increased,the expression of FPN1 increased,and the expression of DMT1 and ferritin decreased(P<0.05).The above results suggest that abnormal iron metabolism occurred in the brain of mice with Alzheimer's disease,which is closely related to the occurrence and development of Alzheimer's disease.2.Compared with WT mice,the expression of ROS,MDA and 4-HNE in APP/PS1 mice was significantly increased,while the expression of GPX4 was down-regulated(P<0.05).However,in the comparison between APP group and PPAR?group,the red fluorescence increased,the expression of MDA and 4-HNE decreased significantly,and the expression of GPX4 increased in PPAR?group(P<0.05).The results of bioinformatics analysis showed that there was a PPAR?binding sequence(PPRE)in intron3 of GPX4.The luciferase reporter gene plasmid containing binding site sequence or binding site mutation was constructed according to this sequence.The results showed that GW7647treatment significantly increased GPX4-WT luciferase activity,while GPX4-MUT had no significant change after GW7647 treatment(P<0.05).GW7647 treatment significantly increased the enrichment level of intron 3 PPAR?in GPX4 of APP/PS1 mice.(P<0.05).These results suggest that the activation of PPAR?-related proteins can significantly inhibit the oxidative stress damage induced by ROS overexpression and lipid peroxidation,and the mechanism is related to the fact that PPAR?can bind to GPX4 intron 3 to regulate GPX4transcription and promote GPX4 expression(P<0.05).3.PPAR?agonist ameliorates iron uptake regulated by ferroptosis by activating Nrf2/HO-1 signal pathway through GPX4.We found that compared with the wild type group,the expression of HO-1,Nrf2 and Trx-1protein in the brain tissue of the AD model group was lower than that of the wild type group(P<0.05).Compared with Neo/SH-SY5Y cells,the activity of APPsw/SH-SY5Y cells was significantly decreased,the apoptosis rate was significantly increased,the expression of A?1-42 and green fluorescence was significantly increased,the expression of ROS was significantly increased,the expression of HO-1,Nrf2,Trx-1 protein was significantly decreased,the ability of iron uptake was significantly increased,the expression of Ferritinand DMT1 was significantly increased,and the expression of FPN1 was significantly decreased(P<0.05).After GW7647 treatment,the activity of APPsw/SH-SY5Y cells increased,the apoptosis rate decreased significantly,the expression of A?1-42 decreased significantly,and the red fluorescence increased significantly,indicating that the expression of ROS decreased significantly,the expression of HO-1,Nrf2,Trx-1 protein increased,the ability of iron uptake decreased significantly,the expression of Ferritinand DMT1 decreased significantly,and the expression of FPN1 increased significantly(P<0.05).After administration of GPX4 inhibitor RSL3,the cell viability decreased,the apoptosis rate increased,the expression of A?1-42 increased,the green fluorescence enhanced,the expression of HO-1,Nrf2,Trx-1 protein decreased,the ability of iron uptake increased,the expression of Ferritinand DMT1 increased,and the expression of FPN1 decreased(P<0.05).Conclusion:PPAR?agonist can inhibit oxidative stress and alleviate Ferroptosis,and its mechanism is related to activation of Nrf2/HO-1 signaling pathway. |
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