Study Of The Role And Mechanism Of Ferroptosis In Astrocytes Induced By Hyperhomocysteine

Objectives:Stroke is currently the second leading cause of death worldwide,of which ischemic stroke is the most common.The long-term disability caused by stroke not only affects the functional status of the patient,but also has a profound psychological and social impact on stroke survivors and their families.The mechanisms of ischemic stroke development are complex,and it is generally accepted that hyperhomocysteine（HHcy）is one of the major risk factors for the development of ischemic stroke.Currently known clinical options for the treatment of ischemic stroke are mainly aimed at thrombolytic therapy,such as recombinant tissue-type fibrinogen activator（t PA）,which is known to be commonly used in the treatment of acute ischemic stroke,but this treatment has a strict time window and most patients cannot receive treatment in a timely manner,so it is important to explore the mechanisms of ischemic stroke development to find more effective preventive and therapeutic measures.Ferroptosis is an important form of cell death that is strongly associated with the development of stroke,but the mechanism is not fully understood.Astrocytes are components of the blood-brain barrier（BBB）and neurovascular units（NVU）,which function to maintain the stability of the intracerebral environment and protect neurons,and play a role in the development of stroke and other diseases.We thus hypothesize that BBB destruction due to ischemic stroke causes risk factors in the blood,including homocysteine（Hcy）,to enter the brain and directly affect astrocyte function,thereby damaging brain structures.The aim of this study was to investigate the role and mechanism of HHcy on the induction of ferroptosis in astrocytes,and the results will provide experimental support for further exploration of the mechanisms of ischemic stroke development.Methods:1.OGD/R induction in vitro:When the mouse astrocytes density attained 80%,the medium was replaced with sugar-free and serum-free medium.Then 2ml sterile liquid paraffin was uniformly covered on the surface of cell culture medium to form a film of about 0.5mm and were incubated at 37℃with 5%CO₂for 1 hour.After that,the cells were cultured under normal culture conditions（37℃,5%CO₂）for 24 h to terminate oxygen and glucose deprivation.2.Experimental grouping:a.Control group;b.Positive control group:Erastin group;c.HHcy group:30μM,100μM,150μM;d.Inhibitor group:HHcy+Lip-1 group.3.MTT was used to determine the cell survival rate of different concentrations of HHcy（0μM,30μM,100μM,150μM）incubated for 24h.4.The intracellular iron concentration was determined by colorimetry.5.The content of GSH in cells was determined by microplate reader.6.The ROS expression was detected by flow cytometry.7.The expression of LPO was detected by two-photon confocal laser scanning microscopy.8.Mitochondrial morphology of Control group and HHcy group was observed by transmission electron microscopy.9.The expression of Glutathione Peroxidase 4（GPX4）gene was detected by q PCR.10.Western blot was used to detect GPX4 protein expression.Results:1.The results of MTT assay showed a trend of HHcy concentration-dependent decrease in astrocyte survival.2.The results of Fe^3+/2+content assay showed that the intracellular Fe^3+/2+content of astrocytes increased after HHcy-induced ischemia-reperfusion.3.The results of GSH content assay showed that HHcy caused GSH depletion in astrocytes,and the depletion of intracellular GSH was correlated with Fe^3+/2+accumulation.4.The results of flow cytometry showed that HHcy caused altered levels of oxidative stress in mouse astrocytes;correlation analysis showed that the oxidative stress of cells was positively correlated with Fe^3+/2+content.5.The confocal assay results showed that HHcy induced changes in the level of lipid peroxidation in mouse astrocytes;the correlation analysis showed that the lipid peroxidation of cells was positively correlated with Fe^3+/2+content.6.The results of transmission electron microscopy showed that the mitochondrial structure in astrocytes with high concentration of HHcy showed obvious ferroptosis changes.These results suggest that HHcy can cause ferroptosis in astrocytes after ischemia-reperfusion.7.Lip-1 was able to inhibit the intracellular Fe^3+/2+increase,GSH depletion and lipid peroxidation induced by HHcy.8.q PCR and Western blot results showed that HHcy was able to down-regulate GPX4expression in astrocytes;correlation analysis showed that GPX4 levels correlated with HHcy-induced changes in ROS and LPO levels in astrocytes;Inhibitor Lip-1 could inhibit HHcy-induced down-regulation of GPX4 expression in astrocytes.The above results suggest that HHcy may cause cells to undergo ferroptosis by affecting the level of GPX4in astrocytes.Conclusions:1.HHcy can cause ferrptosis of astrocytes.2.Ferroptosis inhibitor can block ferroptosis-like changes in astrocytes induced by HHcy.3.HHcy is able to induce a ferroptosis response in cells by down-regulating the level of GPX4 in astrocytes.