The Regulatory Mechanism Of Sodium-dependent Vitamin C Transporter 2 In Ethanol-induced Neurotoxicity In Prefrontal Cortex Of Rats

Ethanol-induced neurotoxic injury is one of the most costly health problems in the world.The mechanism through which ethanol exerts its neurotoxicity is not well understood.Ascorbic acid(AA)is important as an intracellular antioxidant and participates in numerous cellular functions.Among mammalian tissues,neurons of the CNS contain the highest AA levels,which are thought to be generated and maintained by the sodium vitamin C transporter 2(SVCT2),a specific transporter for AA,which play vital roles in the growth of neurons,the mature of the function antioxidant process.Our previous studies also showed that AA was increased in prefrontal cortex(PFC),nucleus accumbens and striatum dialysates of rats after acute ethanol exposure,and AA content increase and negatively correlated with hydroxyl free radicals,these results provide first evidence that release of endogenous ascorbic acid in the striatum plays an important role in preventing oxidative stress by trapping hydroxyl radical in the central nervous system,which might be a self-defense mechanism to protect the body.But its special mechanism and the role of SVCT2 in the process has not yet been elucidated.Based on above,the experimental techniques including confocal microscopy,HPLC,qRT-PCR and Western-blot were used to investigate the relationship between ethanol neurotoxicity and oxidative stress,the changing of intracephalic AA levels of 4-day binge drinking rats,and to research the role of SVCT2 and its regulatory mechanism during ethanol-induced neurotoxicity further.First,4-day binge ethanol model was established.The results showed that significant objects recognition deficits in objects discrimination experiments,besides,a significant neuronal death in PFC of binge drinking ethanol rats using Fluoro-Jade B(FJB+)staining and the level of 4-HNE-adducted protein and phosphorylated MAPKs proteins were significantly increased by binge drinking.In addition,CSF AA levels increased,while intracellular AA levels decreased,and both recovered to normal after 7 days of withdrawal.This indicated that EtOH-induced neurodegeneration in the PFC was accompanied by oxidative stress,and CSF AA levels increased might be helpful to recover the intracellular AA levels in PFC,and might play an important role in self-defense mechanism during EtOH-induced neurotoxicity.Furtherly,the role of SVCT2 in the process of EtOH-induced neuronal injury weas investigated.The results showed that the cell viability,intracellular AA levels,superoxide dismutase(SOD)and reduced glutathione/oxidized glutathione(GSH/GSSG)ratio of primary cultured cortical neuron were significantly reduced,while the levels of intracellular reactive oxygen species(ROS)and 4-hydroxynonenal(4-HNE)-adducted proteins were significantly increased,meanwhile the expression of SVCT2 was also significantly increased after EtOH treatment.Moreover,neurons treated with sulphinpyrazone(a non selective ascorbic acid transporter inhibitor,Sulf)or transfected with SVCT2 siRNA were more susceptible than controls to certain aspects of EtOH-induced injury,including cell death,dendrite damage and increased oxidative stress.the effect of ethanol induced changes in neuronal injury.These data suggested that SVCT2 plays an important role in neuroprotective effect during EtOH-induced neuronal injury.Many reports showed that SVCT2 is regulated by multiple signal transduction pathways,including PKA,PKC and NO-cGMP-PKG-NF-κB signaling pathways ect.Whether the MAPK and NF-κB pathways participate in the regulation mechanism of SVCT2 is not clear.Our resluts showed that EtOH up-regulated the levels of p-p38,p-JNK and p-ERK,and markedly induced the phosphorylation of IκB-α,but reduced the level of IκB-α.The levels of NF-κB p65 and p50 subunits were significantly increased by EtOH treatment.Moreover,inhibitors of JNK,p38 and NF-κB blocked EtOH-induced up-regulation of SVCT2.In contrast,inhibition of ERK did not have a marked effect on the expression of SVCT2.These results suggest that phosphorylation of JNK/p38 MAPKs and activation of NF-κB may increase the cellular uptake of AA into cells by enhancing SVCT2 expression.MiRNAs are important components of the epigenetic landscape that regulates both the transcription and the translation of protein-coding gene networks.Our miRNA microarray results showed that a number of differentially expressed miRNAs are associated with oxidative stress or MAPK signal pathways in PFC of 4-day binge drinking rats.Among these miRNAs,miR-125a-5p was down-regulated significantly after 4 days of binge drinking,while p38 MAPK phosphorylation and the level of SVCT2 protein were increased significantly.Furthermore,over-expression of miR-125a-5p decreased p38 MAPK phosphorylation and the intracellular AA level in primary neurons that were not exposed to EtOH.Neither EtOH treatment(100 mM for 6 h)nor over-expression of miR-125a-5p had a significant effect on the survival rate of primary cortical neurons,but EtOH treatment(100 mM for 6 h)led to neuron death when miR-125a-5p was over-expressed,which suggests that miR-125a-5p might have a crucial effect on neuronal fate.Furthermore,over-expression of miRNA-125a-5p partly inhibited the EtOH-enhanced expression of phosphorylated p38 MAPK and SVCT2 protein,and the EtOH-induced up-regulation of SVCT2 was blocked by a specific inhibitor of p38.Thus,we propose that miRNA125a-5p might affect the expression of SVCT2 by negative regulation of p38 MAPK.Down-regulation of miR-125a-5p might therefore decrease EtOH-induced oxidative stress.In conclusion,our findings clearly revealed that SVCT2 is one target of self-defense mechanisms that protects against oxidative stress during EtOH-induced neurotoxicity.Expression of SVCT2 was enhanced significantly during 4 days of binge drinking,accompanied by oxidative stress and changes in intracephalic AA levels.CSF AA levels increased,while intracellular AA levels decreased,and both recovered to normal after 7 days of withdrawal.The data showed that the up-regulation of SVCT2 contributed to transport of extracellular AA into neurons during and after binge drinking.Furthermore,we ascertained that SVCT2 exerted a neuroprotective effect against EtOH-induced oxidative stress,as evidenced by the greatly increased susceptibility to EtOH-induced cell death and dendrite damage,and the increased oxidative stress in neurons transfected with SVCT2 siRNA.We also found that the level of SVCT2 protein was regulated by JNK and p38 MAPKs,the NF-κB signaling pathway and miRNA-125a-5p.These results provide evidence supporting the idea that SVCT2 represents a potential target of antioxidative therapies for EtOH-induced neurodegeneration.