Based On The Nrf2/ARE Pathway To Explore The Mechanism Of Iron Metabolism After The Regulation Of I/R By The Membrane Iron Transporter Protein And The Intervention Of Naotaifang

Objective:To investigate the changes of Hephaestin(Heph)expression in hippocampus of rats at different time points after cerebral ischemia-reperfusion,and to clarify the significance of Heph involved in cerebral iron metabolism in cerebral ischemia-reperfusion injury.Study on Heph regulate iron metabolism based on Nrf2 / ARE Pathway,we investigated the mechanism of Naotai Formula extract(NTE)Anti-cerebral ischemia-reperfusion injury.Methods:The model of focal cerebral ischemia and reperfusion was reproduced by MCAO method.In the first part of the experiment,8-week-old male SD rats were randomly divided into sham operation group,cerebral ischemia-reperfusion group(2h group,12 h group,24 h group,48 h group and 72 h group),and the Zea Longa neurological score Immunohistochemistry was used to detect the expression of Heph m RNA in hippocampal CA2 region at different time points.Real-time PCR was used to detect the expression of Heph m RNA in hippocampus of rats at different time points.In the second part,8-week-old male SD rats were randomly divided into sham operation group,model group,low dose group(4.5g / kg),brain dose group(9g / kg),brain High dose group(18g / kg)and deferiprone group(125mg / kg),deferiprone group 1h preoperative treatment of intragastric administration(ig),the other groups of preoperative treatment ig administration 3days,all groups were treated with continuous ig for 2 days.The neuronal seizures of hippocampal CA2 area were measured by HE staining and DAB-intensified Perl’s histochemical staining to detect the iron accumulation in hippocampal CA2 area.Real-time PCR was used to detect the hippocampus Nrf2 m RNA,HO-1 m RNA and Heph m RNA.The expression of Nrf2,HO-1 and Heph protein was detected by Western blot.Results:1.The sham group showed a low level of Heph in hippocampal CA2 region.The Heph expression increased gradually at 2h-24 h and decreased gradually at 48h-72 h after cerebral ischemic reperfusion.Compared with the sham group,the Heph expression maintain a high level at 24 h after cerebral ischemic reperfusion(P <0.01).2.The sham group showed a low level of Heph m RNA in hippocampal CA2 region The Heph m RNA expression increased at 2h-24 h after cerebral ischemia and reperfusion,which was the most obvious at 12 h.3.The neurological score of the model group was significantly higher.Compared with the model group,the neurobehavioral scores of NTE and DFP groups were significantly decreased(P <0.01).4.The model group showed a large range of infarction.Compared with the model group,the brain infarct volume was significantly decreased in NTE and DFP groups(P <0.01).5.The cells in the hippocampal CA2 region of the model group were necrotic and the number of iron-containing granules increased.Compared with the model group,the number of cell necrosis and the number of iron-containing granules were decreased in NTE and DFP groups.6.The expression of Nrf2 m RNA in the hippocampus group was significantly higher than that in the sham operation group(P> 0.05).Compared with the model group,the expression of Nrf2 m RNA was increased in NTE and the DFP groups(P>0.05).Compared with the model group,the expression of HO-1 m RNA and Heph m RNA were significantly increased in NTE and the DFP groups(P<0.01,P<0.05).7.The expression of Nrf2 protein in model group increased,and the expression of HO-1 and Heph protein decreased,but there was no significant difference between the model group and the sham operation group(P> 0.05).Compared with the model group,the expression of Nrf2,HO-1 and Heph were increased in NTE and the DFP groups(P <0.01,P <0.05).Conclusion:1.The expression of Heph protein in the hippocampal CA2 region of the sham operation group showed a slight increase in the expression of Heph protein in the hippocampal CA2 region.The expression of Heph protein increased gradually after 2 h-24 h and decreased gradually at 48 h-72 h.Compared with the sham-operated group,The expression of Heph in hippocampus after cerebral ischemia and reperfusion showed a certain regularity,Heph was closely related to brain iron metabolism.2.Intracellular iron overload is involved in cerebral ischemia reperfusion injury.3.Naotai formula can relieve cerebral ischemia-reperfusion injury.The mechanism might be associated with activing Keap1-Nrf2/ARE signaling pathways,promoting HO-1 generation,advancing the expression of Heph,and then reducing brain iron deposition,inhibit oxidation of iron mediated Stress injury,play a role in the protection of neurons after cerebral ischemia and reperfusion.