| Backgroud:Hypoxic-ischemic encephalopathy(HIE)is an essential disease causing neonatal brain damage and death,involving different pathophysiology and various neurological dysfunctions.There are limited treatment options for HIE and currently the only method to improve neurological damage in HIE is hypothermia,but the survivors still suffer from severe sequelae such as cerebral palsy,cognitive impairment and anxiety.Therefore,there is an urgent need for clinicians to find effective alternative therapies for HIE.GSS is a compound obtained by sulphonated modification of the phytoestrogen genistein,and has a strong water solubility.Previous studies have shown that GSS exerts excellent neuroprotective effects in brain injury in both t MCAO rats and neonatal rats,but the mechanism of effect in HIE model is unknown.Ferroptosis,recently identified as an iron-dependent form of cell death,is closely associated with HIE,and HIE neurological damage can be alleviated by inhibiting ferroptosis.Therefore,this experiment constructed a neonatal rat HIE model,which aimed to clarify whether GSS plays a vital role in inhibiting ferroptosis during the recovery period of cerebral ischemia and hypoxia,and to explore its molecular mechanism of effect,so as to provide sufficient experimental basis for further revealing the neuroprotective effect and mechanisms of GSS.Objective:To observe the changes of ferroptosis during ischemia and hypoxia in neonatal rat brain tissue.To clarify the cerebral protective effect of GSS on HIE neonatal rats.To elucidate the mechanism by which GSS modulates STAT3-Hepcidin signaling pathway through GPR30 to alleviate ferroptosis in HIE neonatal rats,exerting the cerebral protective function.Methods:Seven-day-old healthy SD rats,both male and female,weighing 12-18 g were selected to construct a neonatal rat HIE model by applying the Rice-Vannucci method.To determine the changes of ferroprosis during ischemia and hypoxia in neonatal rat brain tissue,we collected brain tissue samples at 1 d,3 d,7 d,14 d,21 d,21 d and 28 d post ischemia and hypoxia in neonatal rats.DAB enhanced Prussian blue iron staining test was applied to examine brain iron deposition and brain damage in neonatal rats.WB experiments were designed to detect the expression of GPX4,x CT,ACSL4 proteins in the injured lateral cortex.To further elucidate whether GSS exerts a cerebral protective effect by regulating the STAT3-Hepcidin signaling pathway through GPR30 to alleviate ferroptosis in HIE neonatal rats.The treatment was continued for 21 days by intraperitoneal injection of1 mg/kg GSS,while the agonist of ferroptosis,Erastin,and the inhibitor of GPR30,G15,were administered for intervention.Right brain/left brain weight ratio for detecting cerebral infarction after ischemia and hypoxia in neonatal rats.Y-maze test was used to assess the cognitive performance after ischemia and hypoxia in neonatal rats.Elevated cross-maze and open-field tests were used to evaluate anxiety-like behavior after ischemia and hypoxia in neonatal rats.WB experiments were designed to detect iron metabolism related indicators TFR,FHC,FPN1,ferroptosis related indicators GPX4,x CT,ACSL4,estrogen receptor GPR30,ERα,Erβ,STAT3-Hepcidin signaling pathway related indicators STAT3,p-STAT3,Hepcidin protein expression in the injured lateral cortex.Transmission electron microscopy experiments were performed to observe mitochondrial damage characteristic of neuronal ferroptosis in cortical neurons of the side of brain injury.DAB enhanced Prussian blue iron staining test was applied to examine brain iron deposition and brain damage in neonatal rats.Immunofluorescence staining was performed to determine the expression of Neu N and GPX4 proteins in the cortex of the brain injury side.Immunohistochemical staining was employed to examine the expression of 4-HNE,Hepcidin proteins in the cortex of the brain injury side.Molecular docking techniques were used to analyse the binding ability of GSS with the estrogen receptors GPR30,ERα and Erβ.Results:1.Changes of ferroptosis during ischemia and hypoxia in neonatal rat brain tissue.Iron staining showed no obvious iron aggregation in the Sham group at any time point,while iron aggregation on the ischemic side of the brain gradually increased in the HI group compared to the Sham group,with the greatest iron deposition and brain loss at day 21 post-modelling,and then gradually recovered.Anatomical images of the whole brain showed a progressive increase in brain damage from edema to infarction,with the greatest brain damage at day 21 post-modelling,and then gradually recovered.WB results showed that compared with the Sham group,the expression of GPX4 protein in the HIE group gradually decreased over time,with the most significant decrease at the 21 st day and then stabilized;the expression of x CT protein in the HI group remained unchanged at the early stage and began to decrease at the 14 st day;the expression of ACSL4 protein in the HI group increased at the 21 st day and decreased at the rest of the time.In addition,as neonatal rats developed,expression of GPX4,x CT,and ACSL4 protein changed in Sham group,in which GPX4 protein expression tended to increase,x CT protein expression tended to increase and then decrease,and ACSL4 protein expression tended to decrease and then increase.2.GSS has neuroprotective function in HIE neonatal rats.NeuN immunofluorescence and right brain/left brain weight ratio results showed that the fluorescence intensity of Neu N was significantly reduced in the HI group,with massive neuronal loss,showing significant brain atrophy,while GSS treatment significantly reduced neuronal loss and promoted recovery of brain injury.Y-maze results showed that compared to the Sham group,the spontaneous alternating and exploratory behaviours were reduced in the HI group and significantly improved by GSS treatment.The elevated cross-maze results showed that compared to the Sham group,the frequency and duration of entering the open arm and the probe were greatly reduced in the HI group,while GSS treatment significantly increased the frequency and duration of entering the open arm and the probe.Open field experiments showed that compared to the Sham group rats,the frequency of entering and time spent in the central area were significantly decreased in the HI group,while the frequency of entering and time spent in the central area improved with GSS treatment.3.GSS alleviates ferroptosis in HIE neonatal rats by regulating the STAT3-Hepcidin signaling pathway through GPR30,exerting cerebral protection.(1)GSS alleviates brain injury in HIE neonatal rats by inhibiting ferroptosis.Iron staining and transmission electron microscopy showed that,compared to the Sham group,the HI group showed a remarkable increase in iron deposition,mitochondria atrophy and vacuolization,while GSS treatment reduced iron deposition significantly and mitochondria returned to normal.Immunohistochemical staining and WB results indicated that compared with the Sham group,the expression of GPX4,x CT,TFR and FHC protein significantly decreased and that of ACSL4 and 4-HNE protein significantly increased in the HIE group,while the expression of GPX4,x CT,TFR and FHC protein significantly up-regulated and that of ACSL4 and 4-HNE protein significantly down-regulated after GSS treatment.After the administration of the ferroptosis inducer erastin,we found that erastin intervention significantly abolished the effect of GSS in inhibiting cognitive impairment,anxiety behaviour and brain damage in HIE neonatal rats.Furthermore,transmission electron microscopy results showed that compared with the HI+GSS group,mitochondria were significantly smaller and even vacuolated after erastin intervention.WB experiments showed that compared with the HI+GSS group,the expression of GPX4 and x CT proteins were downregulated and that of ACSL4 protein was upregulated after erastin intervention.(2)GSS regulates STAT3-Hepcidin signaling pathway through GPR30 to improve ferroptosis in HIE neonatal rats.Molecular docking and WB results indicated that GSS regulates the expression of the three estrogen receptors: GPR30 > ERβ > ERα.Immunohistochemical staining and WB results revealed that compared with the Sham group,the expression of GPR30 protein was reduced,the expression of p-STAT3 and Hepcidin was increased and the expression of FPN1 protein was reduced in the HI group,while the expression of GPR30 was increased,the expression of p-STAT3 and Hepcidin was reduced and the expression of FPN1 protein was increased after GSS treatment.After the intervention with G15,a GPR30 inhibitor,we found that G15 significantly abolished the effects of GSS in improving cognitive impairment,anxiety and brain damage in HIE neonatal rats.In addition,transmission electron microscopy results revealed that compared with the HI+GSS group,the mitochondrial volume was significantly smaller and even vacuolated after G15 intervention;WB results showed that the expression of GPX4 and x CT proteins were down-regulated and that of ACSL4,p-STAT3 and Hepcidin proteins were up-regulated after G15 intervention compared with the HI+GSS group.Conclusion:1.ferroptosis is involved in the pathological process of HIE,and persistently enhances over time.2.Long-term treatment with GSS alleviates HI-induced cognitive impairment and anxiety in neonatal rats.3.Long-term treatment with GSS inhibits HI-induced ferroptosis in neonatal rat brain tissue.4.GSS inhibits HI-induced ferroptosis in neonatal rats by regulating STAT3-Hepcidin signaling pathway through GPR30 to improve neurological impairment. |
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