Melatonin Improves Nickel-induced Brain Autophagy In Mice Through PI3K/AKT/mTOR Pathway

Nickel(Ni)is ubiquitous in our lives,and it can exist around us through soil pollution,industrial pollution and other ways.Nickel is a neurotoxic environmental pollutant,and nickel and nickel alloy were classified in the 2B carcinogens list by the International Agency for Research on Cancer of the World Health Organization in 2017.Oxidative stress is considered to be the main mechanism of neurotoxicity caused by nickel.Oxidative stress is a state in which the balance between oxidation and antioxidant action in the body is broken,which may lead to the production of oxidative intermediates and produce a variety of negative effects on the body.Secreted by the pineal gland,melatonin(Mt)has a strong neuroendocrine immune regulation activity and the ability to remove free radicals.As an antioxidant,it has a significant antioxidant effect.Nickel is a metal that can cause neurotoxicity through oxidative stress,while melatonin is mainly attributed to its antioxidant effect.However,whether nickel causes autophagy through PI3K/AKT/m TOR pathway in mice brain has not been clearly studied,and whether the toxicity caused by nickel in mice brain can be alleviated by melatonin is still insufficient.By establishing in vivo and in vitro models of nickel-induced neurotoxicity in mice,we studied the effect of nickel on autophagy of the nervous system and verified whether melatonin can alleviate nickel-induced neurotoxicity.Eighty 8-week-old male wild-type C57BL/6 N mice were randomly divided into four groups,including C group,Ni group,Mt group and Mt+Ni group.Ni was fed by tube at 10 mg/kg,while Mt was given by gavage at 2 mg/kg.All drugs were administered at 0.1 ml /10 g for 21 days.During this period,the normal supply of fresh water was ensured and the mice model was established in vivo.NS20 Y cells(mice neuroblastoma cells)were given normal medium,medium containing nickel chloride and medium containing melatonin to establish the in vitro experimental model.Experimental group related to PI3 K inhibitor LY294002 was added in the cell experiment.LY294002 is the first synthesized small molecule known to inhibit PI3Kα/δ/β,bind class I PI3 Ks and other Pi3K-related kinases,and block autophagy formation.LY294002 was used to observe whether nickelinduced autophagy and melatine-alleviated autophagy are mediated through the PI3 K pathway.Through observation of ultrastructure and H&E staining of mice brain cells,combined with fluorescence staining analysis of ROS and MDC of NS20 Y cells,Western Blot and q RT-PCR were used to detect the protein and m RNA levels of autophagy and autophagy pathway PI3K/AKT/MTOR pathway.To explore the neurotoxicity of nickel on mice and the relieving effect of melatonin on nickel,the specific research results are as follows:(1)Nickel induced brain histopathological injury was observed by H&E staining,and nerve cell atrophy and deformation were observed in the nickel group.A reduction in atrophy and deformable nerve cells was observed in the combined melatonin and nickel group compared to the nickel group.The ultrastructure of mice brain was observed by nickel,and many autophagic lysosomes were produced in mice brain.Compared with nickel treatment group,the autophagic lysosome in melatonin and nickel treatment group was decreased.(2)The results of ROS fluorescence staining on NS20Y cells and the determination of T-AOC,GSH-Px,MDA and SOD in mice brain and NS20 Y cells showed that nickel caused oxidative stress in NS20 Y cells(P< 0.05).However,the degree of oxidative stress decreased after the addition of melatonin(P < 0.05),and there was no significant difference between the melatonin group and the control group(P > 0.05).(3)QRT-PCR and Western Blot showed that the expressions of autophagy related indicators ATG5,ATG7,Beclin1,LC3 genes and proteins in the brain tissue and NS20 Y cells of mice after nickel treatment were increased(P < 0.05).These results suggest that nickel can induce autophagy in mice brains.After the addition of melatonin,the expressions of autophagy related genes and proteins were inhibited(P < 0.05),and there was no significant difference between the melatonin group and the control group(P > 0.05),suggesting that melatonin has a alleviating effect on the brain autophagy induced by nickel in mice.MDC fluorescence staining of NS20 Y cells showed that the degree of autophagy was increased after the addition of nickel(P < 0.05),while the degree of autophagy was relieved after the addition of melatonin(P < 0.05).The degree of autophagy of NS20 Y cells in the nickel + inhibitor group was higher than that in the nickel group(P < 0.05).There was no significant difference between melatonin group and control group(P > 0.05).The results of q RT-PCR and Western Blot showed that the expressions of autophagy pathway genes PI3 K,AKT and MTOR were decreased in the brain tissue and NS20 Y cells after nickel treatment(p < 0.05).In addition,the expressions of autophagy pathway genes PI3 K,AKT and MTOR were also decreased in the nickel + inhibitor group(P < 0.05),indicating that nickel induced autophagy through PI3K/AKT/MTOR pathway.The expression of autophagy pathway genes and proteins was inhibited after the addition of melatonin(P < 0.05),and there was no significant difference between the melatonin group and the control group(P > 0.05).In summary,these data indicate that nickel can cause oxidative stress injury and induce autophagy in the brain of mice by inhibiting PI3K/AKT/m TOR pathway,Mt can effectively reduce the oxidative stress caused by nickel and reduce the autophagy induced by nickel through PI3K/AKT/m TOR pathway in the brain of mice.Our results reveal the mechanism of nickel induced brain autophagy in mice and the effect of melatonin on nickel induced neurotoxic damage,providing new insights for the antioxidant application of melatonin.