Protective Effect Of Curcumin Against Ethanol-induced Neurotoxicity In PC12 Cells

Objective Alcohol abuse can lead to central nervous system damage through oxidative stress,apoptosis and other mechanisms,affecting attention,memory,cognition and motor function.And in severe cases,it can cause wernicke’s encephalopathy,korsakoff’s syndrome,alzheimer’s disease,dementia,and peripheral neuropathy and other nervous system diseases.Curcumin,a natural polyphenolic compound,has a variety of biological functions such as antioxidant stress,anti-apoptosis.In this study,ethanol-induced PC12 cells neurotoxicity was used as an in vitro model of ethanol-induced neuron injury to investigate the protective mechanism of curcumin on ethanol-induced neuron injury.Methods PC12 cells were cultured and treated with different concentrations of ethanol to establish and screen a successful model of ethanol-induced PC12 cell damage.And PC12 cells were treated with different concentrations of curcumin for24 h to observe the effect of curcumin on cell viability.CCK-8 was used to test the effects of different concentrations of curcumin(1.25,2.5,5,10,20μM)on the survival rate of ethanol-induced PC12 cells,and to determine the optimal concentration of curcumin in protecting injury.The effects of different concentrations of curcumin on lactate dehydrogenase(LDH)release and superoxide dismutase(SOD)content in ethanol-induced PC12 cells were measured by LDH and SOD detection kits.Hoechst 33342 was used for fluorescence staining to observe the effect of curcumin on ethanol-induced PC12 cell apoptosis,and the differentially expressed proteins regulated by curcumin in ethanol-induced PC12 cells were identified by two-dimensional electrophoresis and liquid chromatography tandem-mass spectrometry.Results The results showed that the cells were treated with 400 m M ethanol for24 h can be used as the optimal condition for ethanol-induced PC12 cell injury.And there was no significant difference in cell viability when PC12 cells were treated with different concentrations of curcumin for 24h(P>0.05).Compared with the cells treated with 400 m M ethanol,the PC12 cells treated with a mixed solution containing400 m M ethanol and 5μM curcumin for 24 h significantly increased cell viability(P<0.001),decreased LDH release(P<0.001)and increased the content of SOD in cells(P<0.01).Thus,curcumin can resist ethanol-induced oxidative stress.Hoechst33342 fluorescence staining showed that curcumin inhibited ethanol-induced apoptosis.Besides,curcumin up-regulated the expressions of collapsin response mediator protein-2(CRMP-2),endoplasmin(Hsp90b1),stress-induced phosphoproten1(STIP1),alpha-enolase(ENO1),isocitrate dehydrogenase [NAD]subunit alpha(IDH3),ADP/ATP translocase 1(Slc25a4),ATP synthase subunit alpha,mitochondrial(Atp5f1a)in ethanol-induced PC12 cells.According to bioinformatics analysis,CRMP-2 was mainly involved in neuron development and axonal growth,Hsp90b1 and STIP1,as molecular chaperones,were involved in the processing,transport,folding and degradation of proteins,and the other proteins were related to energy metabolism.Conclusion It suggested that curcumin can against ethanol-induced neurotoxicity by inhibiting oxidative stress and apoptosis in PC12 cells,and its anti-apoptotic mechanism may be related to the PI3K/Akt/GSK3β/CRMP-2 signaling pathway.In addition,Hsp90b1,STIP1,ENO1,IDH3,Slc25a4 and Atp5f1 a may be involved in the protective effect of curcumin on ethanol-induced PC12 cytotoxicity.