Protective Effect Of Melatonin Against Lead-induced Neurotoxicity In PC12 Cells

Objective: Lead(Pb),widely used in industry,is a toxic heavy metal and very stable in the natural environment and can exist for a long time.Lead pollution is one of the important environmental problems in the world.Nearly every organse and system would be affected by Pb exposure,especially the nervous system.However,the mechanism of Pb-induced neurotoxicity is not fully elustrated and oxidative stress is considered to be a key factor of Pb-induced neurotoxicity.Melatonin,secreted by pineal gland,has many physiological functions such as antioxidantive effect,anti-aging,anti-tumor,etc.However,whether it can alleviate the nerve damage caused by Pb is still unclear.In this study,PC12 cells were used to investigate the protective effects of melatonin against Pb-induced neurotoxicity.Methods: After PC12 cells were treated with 25,50,100,200,400 or 800?M Melatonin for 48 hours,cell viability was detected by cell counting kit-8(CCK-8)assay.After pretreated with melatonin(50?M),the cells were exposed to Pb Ac(25?M),then CCK-8 assay was used to detect cell viability and LDH assay were used to evaluate cell damage rate.Hoechst staining was used to detect the apoptosis rate.In addition,the expression of Cleaved-Caspase-3 and the location of Cytochrome C(Cyto C)in cells were detected by immunofluorescence.The cellular and mitochondrial ROS levels were measured by DCFH method and mito SOX,respectively.GSH contents and SOD activities were detected after whole cell protein was extracted.Finnally,Western Blot was used to detect the expression of nuclear factor erythroid 2-related factor(Nrf2)and heme oxygenase-1(HO-1)in PC12 cells treated with MT.Results: MT treatment did not affect the PC12 cell viability indicated by CCK8 assay,but Pb Ac treatment at 25?M for 24 h significantly decreased cell viability(66.16%±8.73%,P<0.01)and increased cell damage rate(25.32%±2.94%,P<0.001)and MT pretreatment could inhibit Pb-induced viability decrease and cell damage.The apoptosis rate increased in the Pb Ac group(30.67%±4.75%,P<0.01)while decreased in the MT pretreatment group(18.15%±1.55%,P<0.05).Further results showed that exposure to Pb Ac could increase the protein level of Cleaved-Caspase-3 and induce Cyto C leaking from mitochondria to cytoplasm,while MT treatment could significantly attenuate these negative effects.Pb Ac treatment could also increase ROS level in cytoplasma and mitochondria and decrease GSH contents(33.63%±2.83%,P<0.01)and SOD activities(26.98%±2.87%,P<0.05),which could be significantly inhibited by MT pretreatment maintaining redox balance.The cell viability,damage rate,ROS level,apoptosis-related proteins and antioxidants were not significantly changed after treatment with melatonin alone.The results of Western Blot showed that MT at 50~400?M induced activation of Nrf2/HO-1 antioxidant pathway.Conclusions: Melatonin could attenuate Pb Ac-induced oxidative stress and cell death,promoting cell survival.Nrf2/HO-1 activation may be involved in the protective effects of Melatonin.