| Due to the high efficiency and controllability,inhalation anesthetics are widely used in clinical practice.However,over the years,multiple reports have recognized that inhalation anesthetic can also be inherently cytotoxic and are associated with functional neurologic deficits in both experimental animal models and clinical cases.Retrospective analysis of case reports has demonstrated the potential impact of inhalation anesthesia on cognitive trajectories.confirming that isoflurane is closely related to learning and memory damage,cognitive dysfunction,and the development of neurodegenerative diseases,which is a potentially harmful risk to the human central nervous system.Therefore,the mechanism of inhalation anesthetic-induced neurotoxicity is of great significance for safety application of inhalation anesthetics.Isoflurane is an isomer of enflurane.Isoflurane-induced neurotoxicity is complicated,including neuron loss,neuroinflammation and abnormal protein deposition.Previous studies showed multiple forms of cell death result from isoflurane-induced cytotoxicity,but the precise underlying mechanism remains poorly understood.Ferroptosis is an iron-dependent form of regulated cell death which is distinct from apoptosis,classic necrosis,autophagy and other forms of cell death,which is characterized by iron-dependent lipid peroxidation and has attracted tremendous interest owing to its unique relevance for a number of pathological conditions including acute tissue damage,ischemia/reperfusion injuries,cancer,hemochromatosis,and neurodegeneration.In fact,regardless of signaling cross-talk among ferroptosis,apoptosis and autophagy,ferroptosis is more likely to be the potential mechanism associated with cellular oxidative toxicity under certain conditions.In the context of neurodegenerative diseases,reduced glutathione activity,iron overload and lipid peroxidation were observed in neurons.In addition,oxidative stress damage was observed in vivo and in vitro after isoflurane exposure.Therefore,we hypothesized that Ferroptosis may be involved in isoflurane-induced cytotoxicity or even neurotoxicity.However,up to now,relationship between Ferroptosis and the isoflurane induced-cytotoxicity and its potential regulatory mechanisms has not been exactly clarified.Objective:To investigate the degree of isoflurane-induced cytotoxicity,further investigate whether Ferroptosis regulates isoflurane-induced cytotoxicity and its possible regulatory signal pathway,hoping to supplement the regulatory mechanism of isoflurane-induced neurotoxicity.Methods1)Human neuroblastoma cell line SH-SY5Y was used in this study;2)Overexpression and knockdown cell models of Beclinl were established by transiently transfection of cDNA plasmid and siRNA;3)Cells were exposed to indicated concentration of isoflurane for 12h or 24h;4)CCK8(cell counting Kit-8)assay for cell survival detection;5)RT-qPCR was conducted to detect mRNA expression;6)Western blot was conducted to detect protein expression;7)Co-immunoprecipitation(IP)was implemented to detect protein-protein interactions;8)Immunofluorescence(IF)was implemented to detect protein localization;9)Flow cytometry was used to detect apoptosis rate,lipid peroxidation,and cell chelated iron;1 0)The commercial kits were used to detect levels of MDC,GSH,and glutamate.Results:1.Multiple injury mechanisms regulate isoflurane-induced cytotoxic damageMicrotubule-associated protein 2(MAP2)staining was positive in SH-SY5Y cells to confirm the general neuronal characteristics of SH-SY5Y cells.Cell viability was decreased in a concentration-dependent manner(1.2%,2.4%,4.8%of isoflurane)and duration-dependent manner(6h,12h,24h,48h).After exposure to isoflurane(1.2%,2.4%,4.8%)for 24 h,analysis of flow cytometry indicated apoptosis rate increased from 3.63%to 7.75%,8.1%and 10.83%,respectively.Western blot showed that the of MAPLC3-Ⅱ/-Ⅰ ratio increased by 1.2,1.6 and 1.9 folds,the p62 level was down-regulated by 88%,75%,and 56%,respectively.It is indicated that isoflurane induced apoptosis and autophagy in SH-SY5Y cells.Isoflurane-induced cytotoxicity involves multiple forms of regulatory cell death processes.SH-SY5Y cells were co-cultured with Z-VAD-FKM,rapamycin,Necrostatin-1,Ferrostatin-I,Deferoxamine mesylate and isoflurane(1.2%or 2.4%)for 12 or 24 h,respectively.Each of these inhibitors mitigated the decreased cell viability following exposure to 1.2%isoflurane.However,only Ferrostatin-1 and Deferoxamine mesylate significantly prevented the decrease in cell viability after exposure to 2.4%isoflurane.These results indicate that ferroptosis inhibitors are more effective for rescuing cell viability after exposure to high isoflurane concentration.2.Isoflurane induced ferroptosisAfter isoflurane(1.2%and 2.4%)exposure for 24 h,oxidative stress marker malondialdehyde(MDA)increased by 1.3 and 1.5-folds.Isoflurane significantly shifted the fluorescent probe from reduced(r-)to oxidative(o-)C11-BODIPY,flow cytometry analysis showed a concentration-dependent increase in o-C11-BODIPY fluorescence from 3.43%to 23.8%and 27.8%.Isoflurane exposure decreased the intensity of the fluorescence signal after calibrating the metal indicator(Phen Green SK)from 38.67%to 12.33%and 7.67%,respectively.In addition,the level of GSH in SH-SY5Y cells was significantly decreased by 1.4 and 1.72-folds after 24 hours of isoflurane exposure.Above data indicated that isoflurane-induced ferroptosis,which characterized by lipid peroxidation,iron overload,and reduction decreased.3.Effects of isoflurane on ferroptosis-related regulatory pathwaysResults of RT-qPCR showed isoflurane exposure did not alter mRNA and protein levels of TFRC,STEAP3 and DMT1,and there was no significant change in the levels of FPN1 in mRNA and protein level.These results suggested that alternate iron metabolism pathways might be involved in isoflurane-induced ferroptosis.For system Xc-/GSH/GPX4,GPX4 mRNA and protein levels remained unchanged after isoflurane exposure.However,isoflurane-induced cell injury was significantly inhibited after GSH treatment.Although the mRNA level of SLC7A11 was upregulated,there was no significant variation in its protein level after isoflurane exposure.However,the glutamate release assay showed that the extracellular glutamate level decreased by 0.8 and o.55 fold in concentration-dependent manner(1.2%,2.4%).These results indicated that isoflurane-induced ferroptosis might be associated with disorders of system Xc-activity4.Isoflurane induces Beclinl phosphorylation and forms Beclinl-SLC7A11 complexThe level of phosphorylated-Beclinl at Ser93 was significantly upregulated in a concentration-dependent manner of isoflurane.Co-IP analysis confirmed that Beclinl and SLC7A11 form a protein complex after isoflurane exposure.Immunofluorescence staining showed the co-localization between Belinl and SLC7A11 occurred mostly in the cytoplasm in SH-SY5Y cells.These results suggest that isoflurane induces Beclinl phosphorylation and causes protein interaction between Belinl and SLC7A115.Overexpression of Beclinl enhances isoflurane-induced ferroptosis and cytotoxicity.Beclinl-cDNA plasmid was transiently transfected into SH-SY5Y cells.There was no significant changes of SLC7A11 protein levels after Beclinl overexpression.In empty vector control cells,the interaction of Beclinl and SLC7A11 significantly increased by 3.8 times after isoflurane exposure.After overexpression of Beclin1,isoflurane further increased Beclin1/SLC7A11 interaction significantly by 4.85 times.Moreover,In Beclin1-cDNA cells,isoflurane exposure further inhibited glutamate release(0.58 vs 0.42),further inhibited GSH activity(0.63 vs 0.43),further increased MDA levels(1.58 vs 2.02),and further increased oxidized C11-BODIPY fluorescence(24.34%vs 36.67%).It is suggested that isoflurane increase a sensitivity of ferroptosis induction after Beclin1 overexpression.After 24 hours of induction with isoflurane in the empty vector control cells,the cell viability was decreased to 62%.However,After Beclin1 overexpression,isoflurane further decreased cell viability to 45%.It is suggested that expression of Beclin1 enhances isoflurane-induced cytotoxicity.6.Inhibition of Beclinl inhibits isoflurane-induced ferroptosis and cytotoxicity.Beclin1-siRNA was transiently transfected into SH-SY5Y cells.There was no significant changes of SLC7A11 protein levels after silencing Beclinl.In negative-control cells,the interaction of Beclin1 and SLC7A11 significantly increased by 3.02 folds after isoflurane exposure.After silencing Beclin1,isoflurane reduced Beclin1/SLC7A11 interaction significantly by 1.13 times.Moreover,In Beclin1RNAi cells,isoflurane exposure improved the inhibition of glutamate release(0.55 vs 0.85),ameliorated the decrease of GSH activity(0.65 vs 0.87),ameliorated the increase of MDA levels(1.45 vs 1.19),and ameliorated the increase of oxidized C11-BODIPY fluorescence(25.67%vs 4.61%).It is suggested that isoflurane decrease a sensitivity of ferroptosis induction after silencing Beclin1.After 24 hours of induction with isoflurane in the negative control cells,the cell viability was decreased to 64.8%.In Beclin1RNAi cells,isoflurane exposure ameliorated the decrease of cell viability(0.64 vs 0.77).It is suggested that silencing Beclin1 alleviates isoflurane-induced cytotoxicity.Conclusions:1.Multiple forms of cell death result from isoflurane-induced cytotoxicity such as apoptosis and autophagy.However,ferroptosis may play a primary role.2.Isoflurane induces ferroptosis in a concentration-dependent manner.3.Isoflurane induces Beclinl Ser93 phosphorylation and formed Belinl/SLC7A11 complex.4.Beclinl medicated isoflurane-induced ferroptosis may be associated with blocking activity of system Xc-. |
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