Protection And Mechanism Of Ferroptosis Inhibition By Activin A On Cerebral Ischemia/reperfusion Injury

Background:The key for the treatment of acute ischemic stroke is to rebuild the blood flow as soon as possible,and save the ischemic penumbra.However,the existence of ischemia/reperfusion injury limits the benefits of vascular recanalization therapy in the real world,and its molecular mechanism is still unclear,which limits the implementation of related drug prevention strategies.Activin A(Act A)is one of the members of the transforming growth factor-?(TGF-?)superfamily,which has the functions of resisting ischemic injury and promoting the recovery of neurological function.Our previous study has confirmed that Act A can reduce the apoptosis of PC12 cells induced by oxygen and glucose deprivation(ODG),and the mechanism may be related to the activation of Act A/Smads signal pathway,the decrease of inflammatory factors and increase of the activity of antioxidant enzyme SOD.Ferroptosis is an iron-dependent programmed cell death characterized by accumulation of lipid peroxidation,which has been confirmed to be involved in the ischemic stroke and ischemia/reperfusion injury.Studies have shown that Act A I-type receptors are involved in Erastin-induced ferroptosis in HK-2 human renal tubular epithelial cells through the nuclear factor erythroid-2-related factor 2(Nrf2)signaling pathway,but whether Act A and its pathways are involved in neuronal ferroptosis and whether it play a role in cerebral ischemia/reperfusion injury by regulating ferroptosis is unclear.In this study,middle cerebral artery occlusion(MCAO)of mice and oxygen and glucose deprivation/reoxygenation(OGD/R)of HT22 cells was adopted to establish models of cerebral ischemia/reperfusion injury and ferroptosis model of HT22 cells was established by Erastin induction.Cellular iron content and lipid peroxidation levels was detected,Western blot,Real-time quantitative PCR was used to determine the expression of key proteins and genes of ferroptosis,and the intervention of exogenous Act A was used to clarify the effect of Act A on cerebral ischemia/reperfusion injury,on ferroptosis and the molecular mechanism of Act A regulating cerebral ischemia/reperfusion injury through ferroptosis.This study includes the following contents:1.The effect of Act A on cerebral ischemia/reperfusion injury in mice and its effect on ferroptosisObjective:To clarify the effect of Act A on cerebral ischemia/reperfusion injury of mice,and to explore the effect of Act A on key indicators of ferroptosis and possible signaling pathway proteins.Methods:Twenty-four C57BL/6 male mice were divided into vehicle sham group,Act A sham group,vehicle MCAO group,and Act A MCAO group.The MCAO/reperfusion model in mice was established by suture method.The neurological deficit was evaluated by Bederson score,the volume of cerebral infarction area was analyzed by TTC staining,and the content of malondialdehyde(MDA)was determined by ELISA,iron kit was used to detect tissue iron content,and Western-Blot was used to detect the expression of ferroptosis marker proteins GPX4,ACSL4 and possible pathway protein Nrf2.Results:Compared with the vehicle MCAO group,the neurological deficit score and the cerebral infarction volume of the mice in the Act A intervention MCAO group were significantly reduced(P<0.05);compared with the vehicle sham group,the iron content and MDA level in the vehicle MCAO group were significantly increased,the expression of ferroptosis marker protein GPX4 was significantly decreased,and the expression of ACSL4 was significantly increased(P<0.01);compared with the mice in the vehicle MCAO group,the iron content and MDA level of the MCAO mice in the Act A group were significantly decreased,the expression of GPX4 protein was significantly increased,and the protein expression of ACSL4 was significantly decreased(P<0.01);compared with the mice in the vehicle sham group,the expression of Nrf2 protein in the vehicle MCAO group was significantly increased(P<0.01);and the expression of Nrf2 protein in the MCAO mice in the Act A group was further increased compared with the mice in the vehicle MCAO group(P<0.05).Conclusion:Act A has a protective effect on cerebral ischemia/reperfusion injury,and exogenous administration of Act A can reduce the neurological deficit score and reduce the infarct volume.Act A can alleviate the key events of ferroptosis such as brain tissue iron overload,increased lipid peroxidation level,increased ACSL4protein expression and decreased GPX4 expression caused by cerebral ischemia/reperfusion injury.Act A significantly upregulated Nrf2 protein expression while protecting mice from MCAO/reperfusion injury.2.Act A protects neurons from OGD/R injury by inhibiting ferroptosisObjective:To clarify the effect of Act A on OGD/R injury of HT22 neuron and its effect on ferroptosis;to explore the possible mechanism of neuronal ferroptosis inhibition by Act A;and to further confirm that Act A exerts its protective effect on neuronal OGD/R injury by inhibiting ferroptosis.Methods:The mouse hippocampal neuron HT22 cell line was selected,and the OGD/R of HT22 cells was established by sugar-free medium and the tri-gas(94%N₂,5%CO₂,1%O₂)method followed by reoxygenation for 24 h.Cell viability was determined by CCK-8 method,LDH was used to determine cell damage,and the ferroptosis-related indicators were detected.Erastin-induced HT22 cells were used to construct a neuronal ferroptosis model,Act A was administered exogenously.Cellular iron and lipid peroxidation levels was detected,Western-blot was used to detect marker proteins of ferroptosis,and Real-time quantitative PCR was used to detect the expression of key genes of ferroptosis.Detecting the expression of Nrf2,a possible protein for Act A pathway.Results:The results showed that compared with the control group,the survival rate of cells in the OGD/R group was significantly decreased,and the LDH levels was significantly increased(P<0.01),indicating that model of OGD/R was successfully established.Compared with the OGD/R group,the cell viability of the OGD/R-pre+Act A and OGD/R-post+Act A groups were significantly increased(P<0.01);and pretreatment with Act A further improved cell survival compared with the administration of Act A after OGD(P<0.01);compared with the OGD/R group,LDH,iron content,and ACSL4 protein expression were significantly decreased,while GPX4 protein and SLC7A11 expression were significantly increased in the two Act A intervention groups(P<0.01);the levels of MDA decreased significantly in the OGD/R-pre+Act A group(P<0.01).The optimal concentration of Erastin to induce ferroptosis in HT22 cells was 10?M,and the optimal effect time was 8h;compared with the control group,the levels of LDH and iron in the Erastin group were significantly increased(P<0.01),the expression of the ferroptosis marker protein GPX4 was decreased,the expression of ACSL4 was increased,and the m RNA expression of the ferroptosis key gene SLC7A11 was decreased(P<0.01),suggesting the successful establishment of ferroptosis model;compared with the Erastin modeling group,the iron content,MDA level,the ACSL4 protein expression was decreased,the GPX4 expression was increased(P<0.01),and the SLC7A11 m RNA expression was increased in the Act A intervention group(P<0.05);compared with the control group,the expression of Nrf2 protein in the Erastin group was increased(P<0.01);and compared with the Erastin group,the expression of Nrf2 protein in the Act A intervention group was further increased(P<0.01).Pretreatment with the ferroptosis inducer Erastin before OGD/R decreased the cell survival rate(P<0.01);pretreatment with Act A before OGD/R increased the survival rate of cells(P<0.01);intervention of Act A with Erastin at the same time,the cell survival rate decreased(P<0.05).The expression of ACSL4 was increased,and the expression of GPX4 was decreased when Erastin was administered before OGD/R(P<0.01);and the expression of ACSL4 was decreased and the expression of GPX4 was increased when Act A was administered before OGD/R(P<0.01);compared with Act A intervention group,the expression of ACSL4 was increased and the expression of GPX4 was decreased in the group treated with Act A and Erastin simultaneously(P<0.01).Conclusion:Act A has a protective effect on OGD/R injury of HT22 neurons;Act A can inhibit Erastin-induced cellular iron overload,lipid peroxidation,and decrease the expression of ACSL4 and increase the expression of GPX4 and SLC7A11,and can promote expression of Nrf2 during neuronal ferroptosis;Act A protects neurons from OGD/R injury by inhibiting ferroptosis.3.Act A inhibits OGD/R ferroptosis in HT22 cells by up-regulating Nrf2Objective:To further elucidate the molecular mechanism of Act A protecting neurons from OGD/R injury by inhibiting ferroptosis.Methods:The HT22 cell line OGD/R model was established.The experimental groups were divided into control group,OGD/R group,OGD/R+Act A group,OGD/R+Act A+Nrf2 inhibitor(ML385)group.The concentration of Act A was100ng/ml,which was added 12 hours before modeling,and the concentration of ML385 was 5?M,which was added at the same time as Act A.Cell viability was measured by CCK-8 method,cell damage was measured by LDH,iron and lipid peroxidation levels of MDA were measured by ELISA,and the expressions of ferroptosis marker proteins ACSL4 and GPX4 were measured by Western-blot.Results:After 24 hours of hyperglycemia and reoxygenation,the relevant indicators were detected,and the results showed that compared with the OGD/R Act A intervention group,cell viability was significantly decreased when treated with Act A and the Nrf2 inhibitor ML385 simultaneously(P<0.01);the LDH levels and cellular iron contents were significantly increased(P<0.01)and the lipid peroxidation MDA level was increased in Act A and the Nrf2 inhibitor co-treated group(P<0.05);also the expression of ferroptosis marker protein ACSL4 was significantly increased,and the expression of GPX4 was significantly decreased in co-treated group(P<0.01).Conclusion:Act A inhibits OGD/R ferroptosis in HT22 cells by upregulating Nrf2,thus protecting neurons from OGD/R injury.