Mechanism Of Activin A On Oxygen-glucose Deprivation/Reoxygenation Of Astrocytes

Cerebral ischemia-reperfusion injury（CI/RI）is a major challenge for clinical treatment and is a key pathological process in ischemic stroke,characterized by initial restriction of blood supply to the brain and subsequent reperfusion reoxygenation,leading to severe damage to brain tissue.Astrocytes,as the most abundant cells in brain tissue,have important regulatory and protective roles for neurons in CI/RI.Activin A（ActA）,a member of the TGF-βsuperfamily,is widely expressed in a variety of tissues and plays important physiological roles in proliferation,differentiation,apoptosis and inflammation.ActA is involved in regulation mainly through the Smads pathway and non-Smad-dependent pathways such as mitogen-activated protein kinase（MAPK）.The MAPK signaling cascade response is important in neurological formation,neurodegenerative diseases,and pain and inflammatory responses in the brain play an important role.Iron death is a newly discovered iron ion-dependent form of cell death.Recent studies have shown that iron death plays an important role in the pathophysiological processes of neurodegenerative diseases,stroke and other diseases.Studies have shown that iron death occurs in neurons during CI/RI and thus exacerbates the damage,and that ActA protects neurons in ischemic stroke,but little is known about its effect on astrocytes.In conclusion,the present study is intended to investigate the effects and mechanisms of ActA on glyoxylate deprivation/reoxygenation（OGD/R）astrocytes,and to provide new ideas for further exploration of new treatments for CI/RI.Purpose:To investigate the effects and molecular mechanisms of ActA on glyoxylate deprivation/reoxygenation（OGD/R）astrocytes.Methods:1.Neonatal C57BL/6 mice were isolated and cultured within 24 h.The primary astrocytes were purified and identified by GFAP immunofluorescence staining.2.CCK8 assay for OGD 2h,4h,6h and OGD/R 6h,12h,24h,48h astrocyte viability.3.Mito-Tracker Red CMXRos staining,ROS kit,lipid oxidation kit,iron ion kit and Western Blot were used to detect changes in mitochondrial activity,iron death-related indicators and iron death-related protein expression levels in the control,OGD/R and OGD/R+ActA groups.4.Western Blot to detect the changes of MAPK signaling pathway-related protein expression levels in control,OGD/R and OGD/R+ActA groups.5.Mito-Tracker Red CMXRos staining,ROS kit,lipid oxidation kit,iron ion kit,and Western Blot were used to detect mitochondrial activity,iron death-related,in the control,OGD/R,OGD/R+ActA,OGD/R+MAPK inhibitor,and OGD/R+MAPK inhibitor+ActA groups.and iron death-related protein expression levels.Results:1.The purity of GFAP immunofluorescence assay is greater than 95%.2.CCK8 results showed that compared with the control group,the cell survival rate decreased to 55.6%in the OGD 6h group（v.s con,P<0.01）,46.8%in the OGD 6h/R 24h group（v.s con,P<0.01）while the cell survival rate in the OGD 6h/R 48h group had decreased to 27.3%（v.s con,P<0.01）.There was a significant difference in the survival rate of cells compared with the control group at all time points when 20 ng/m L of ActA was administered（P<0.01）.Therefore,20 ng/m L ActA given at OGD6h/R 24h was chosen for the follow-up study in this experiment.3.Astrocytes showed a significant decrease in mitochondrial activity and a significant increase in ROS,MDA and Fe²⁺production at OGD6h/R 24h.The addition of ActA showed an improvement in mitochondrial activity and a significant decrease in ROS,MDA and Fe²⁺production.4.Western Blot results showed that the expression levels of FPN1,GPX4 and TF were decreased and the expression of TFR was increased in the OGD 6h/R 24h group,and the expression levels of FPN1 were significantly increased and the expression levels of TF and TFR were significantly decreased after the addition of ActA,while there was no significant difference in the expression levels of GPX4.The expression of related proteins in MAPK signaling pathway was further examined,and the results showed that the expression levels of JNK,P-JNK,P38 and P-P38 were significantly increased,while the expression of ERK and P-ERK was decreased,and all of them were significantly decreased after the addition of ActA.5.Mitochondrial activity was significantly higher and ROS,MDA and Fe²⁺production were significantly reduced in the group with JNK or P38inhibitors compared with the OGD/R group;mitochondrial activity was significantly increased and ROS,MDA and Fe²⁺production were significantly reduced when ActA and JNK/P38 inhibitors were administered simultaneously.6.Western Blot assay showed that GPX4 expression increased in the plus JNK/P38 inhibitor group compared to the OGD/R group,TF and TFR expression were significantly decreased,and FPN1 expression did not change significantly;when ActA and JNK/P38 inhibitors were given simultaneously,GPX4 expression was significantly increased compared to the ActA and JNK/P38 inhibitor groups,and TF and TFR expression was significantly decreased.Conclusion:1.Activin A can inhibit the ferroptosis of astrocytes induced by OGD/R and reduce the damage of astrocytes during OGD/R;2.ActA may exert a protective effect against OGD/R injury in astrocytes by inhibiting the expression of JNK and P38 in the MAPK pathway,thereby promoting the expression of GPX4 and inhibiting the expression of TF and TFR to suppress iron death.