The Neuroprotection Of Carvacrol On Iron-overload Injury In SH-SY5Y Cells And Study Of Its Mechanism

BackgroundIntracerebral hemorrhage(ICH)is a stroke subtype that is associated with high mortality and morbidity.After ICH,the hematoma could induce a series of reactions,which might lead to the secondary brain injury resulting in the severe neurological dysfunction.Previous studies have confirmed that iron-mediated oxidative damage and apoptosis might play an important role in the mechanism of secondary brain injury in ICH.An excess of ferrous iron can catalyze lipid peroxidation and produce lots of free radicals,causing oxidative stress injury.These process often are correlated to the edema of peripheral tissue,neuroinflammation and neuronal apoptosis.Some studies have reported that carvacrol is neuroprotective in central nervous system diseases.Our previous study also found that carvacrol has a neuroprotective effect on ICH in mice,but the specific mechanism is not clear,lacking scientific research.In this study,we established an iron overload injury model in vitro and to study the mechanism of neurotoxicity ferrous iron on SH-SY5Y cells and the protection of carvacrol.And that clarify the neuroprotective mechanisms of the carvacrol on iron overload injury in experimental ICH from the SH-SY5Y cell pointThis study includes three parts.Part one:Cytotoxic damage of ferrous iron on SH-SY5Y cells and the cytoprotection of carvacrol;Part two:The effect of ferrous iron on oxidative stress and NFκB expression in SH-SY5Y cells;Part three:The mechanism of carvacrol on NFκB expression in SH-SY5Y cells after iron overload injuryPart Ⅰ Cytotoxic injury of ferrous iron on SH-SY5Y cells and the cytoprotection of carvacrolObjective To establish iron overload injury model in vitro and study the cytotoxic damage of ferrous iron on SH-SY5Y cells and the cytoprotection of carvacrol.Methods The SH-SY5Y cells were treated with different dose of ferrous iron to induce neurotoxic damage.Some cells were pretreatment with carvacrol before Fe2+exposure,to study the effect of carvacrol on Fe2+induced cytotoxicity.We used CCK-8 assay to detect cell viability and to assess Fe2+cytotoxicity;we used TUNEL staining and flow cytometer analysis to assess cell apoptosis;we also performed Western Blot to analyze the expression of apoptosis-related proteins.Results Fe2+exposure decreased cell viability in a dose dependent manner.And the decrease in viability of cells caused by Fe2+exposure was significantly prevented by pretreatment of carvacrol.The result of TUNEL positive staining in situ detection and Flow cytometric analysis also exhibited that Fe2+significantly increased the number of apoptosis positive cells,compared to untreated cells.Pretreatment of carvacrol,however,could partially attenuated the proportion of apoptotic cells.Exposure to Fe2+significantly increased Bax,Caspase3 and decreased Bcl-2 expression,while pretreatment with carvacrol inhibited up-regulation of Bax,Caspase 3 and down-regulation of Bcl-2.Conclusions Fe2+exposure decreased cell viability and significantly increased expression of apoptosis-related proteins,resulting the cell apoptosis.Carvacrol treatment,however,could partially attenuated cell apoptosis and contributed the neuroprotective effect against iron toxicity.Part Ⅱ The effect of ferrous iron on oxidative stress and NFκB expression in SH-SY5Y cellsObjective To explore the effect of Fe2+exposure on oxidative stress and NFκB expression in SH-SY5Y cellsMethods We assayed ROS with the non-fluorescent probe DCFH-DA.Mitochondrial Membrane Potential in SH-SY5Y cells was detected by JC-1 assay kit.Real time-PCR and Western blot analysis were used to determine the expression of NFκB p65 mRNA and protein.The expression and localization of intracellular NFκB p65 was examined by the immunofluorescence staining.The signaling protein expression of mitogen-activated protein kinases(MAPKs)pathway was also analyzed by Western Blot.Results Incubation of SH-SY5Y cell with Fe2+significantly increased ROS production and decreased mitochondrial membrane potential.Both of which,however,could be attenuated by Carvacrol.Both NFκB p65 mRNA and protein levels increased in a concentration-and time-dependent manner after Fe2+exposure.The exposure to Fe2+significantly increased the nuclear translocation with a phenomenon of NFκB p65 protein mainly located in the nuclear.Meanwhile,the exposure to Fe2+increased the expression of phospho-p38,phospho-ERK and phospho-JNK.Conclusion Carvacrol attenuated Fe2+-mediated oxidative stress and increased mitochondrial membrane potential.Treatment with Fe2+also significantly increased the expression of NF-κB P65 and the phosphorylation of p38,ERK,JNK in SH-SY5Y cellsPart Ⅲ The mechanism of carvacrol on NFκB expression in SH-SY5Y cells after iron overload injuryObjective To explore the effect of carvacrol on NFκB expression in SH-SY5Y cells after iron overload injury and study of its mechanism.Methods Cells were pretreated with carvacrol and then stimulated with Fe2+.Western blot analysis and immunofluorescence staining was conducted to examine the effects of carvacrol on Fe2+-induced upregulation of NF-κB p65 proteins and nuclear translocation in SH-SY5Y cells.Western blot analysis was also conducted to examine the effects of carvacrol on the activation of IKK and MAPKs in SH-SY5Y cells.The mRNA levels of pro-inflammatory cytokines were determined using RT-PCR.Cells were pre-incubated with different MAPKs inhibitors and then treated with Fe2+.NFκB p65 levels were measured by western blot analysis.Furthermore,some cells were pre-incubated with MAPK/JNK inhibitors or NFκB inhibitor an then treated with Fe2+.Caspase 3 levels were measured by western blot analysis.Results Carvacrol significantly inhibited Fe2+-induced activation of NF-κB and expression of the pro-inflammatory cytokines.Pretreatment with carvacrol inhibited Fe2+-induced JNK and IKK phosphorylation,while carvacrol had no apparent effect on ERK and p38 phosphorylation.Concomitantly,both MAPK/JNK inhibitors and NFκB inhibitor markedly inhibited the increase of caspase-3 protein expressions in SH-SY5Y cells.Conclusion Carvacrol could protect SH-SY5Y cells against apoptotic cell death,and these protective effects might be related to the inhibitory of JNK/MAPK and IKK activation,and subsequently decreased the production of NF-KB-mediated prion-flammatory factors.