Effects Of Oxidative Stress On MeHg-induced MTOR-dependent Autophagy

Objective: Methylmercury(Methylmercury,MeHg)is a lipophilic environmental poison that can cause damage to the central nervous system in humans.MeHg has a strong affinity with the brain.When MeHg enters the body,it crosses the blood-brain barrier and accumulates in the brain,which is an important basis for the neurotoxicity of MeHg.MeHg can reduce GSH content through the high affinity of-SH group,inhibit the transcription of antioxidant enzymes,and reduce the scavenging ability of Reactive Oxygen Species(Reactive Oxygen Species,ROS),thus causing oxidative stress.Meanwhile,PI3K/AKT can activate mTOR to inhibit neuronal autophagy,and AMPK/TSC2 can inhibit the activation of mTOR to activate neuronal autophagy.Nacetyl-l-cysteine(N-acetyl-l-cysteine,NAC)is an antioxidant that can remove excess ROS in cells and is widely used in the prevention and treatment of clinical diseases.In this study,in vivo animal experiments were conducted to investigate whether MeHg induces autophagy and oxidative stress in the MeHg-induced neurotoxicity.At the same time,NAC pretreatment was applied to further investigate the effects of oxidative stress on the autophagy signaling pathways of PI3K/AKT/mTOR and AMPK/TSC2/mTOR,providing a theoretical basis for the study of the neurotoxic mechanism of MeHg.Methods: A total of 72 clean grade Wistar rats were provided by Liaoning Changsheng Biotechnology Co.,Ltd.,weighing 160-180 g.Male and female were equally divided.Thirty-two rats were randomly divided into the control group,low-mercury group,medium-mercury group and high-mercury group,with 4 male and 4 female rats in each group.The control group was intragastric with 0.9 % sodium chloride,while the lowmercury group,medium-mercury group and high-mercury group were intragastric with4 μmol/kg,8 μmol/kg and 12 μmol/kg MeHg,respectively.They were poisoned once a day for 28 consecutive days.During the course of exposure,we weighed and recorded changes in body weight on alternate days.The changes of behavioral indexes were detected by behavioral experiments.HE staining was used to observe the pathological damage of rat cerebral cortex.The formation of autophagosomes was observed by transmission electron microscopy.The other forty rats were randomly divided into the control group,the NAC group,the MeHg group,and the NAC+MeHg group,with 5males and 5 females in each group.The control group and the MeHg group were treated with 0.9 % sodium chloride,by subcutaneous injection,while the NAC group and the NAC+MeHg group were treated with 1 mmol/kg NAC by subcutaneous injection.After two hours,the rats were poisoned by gavage.The control group and NAC control group were treated with 0.9 % sodium chloride,while the MeHg group and NAC+MeHg group were treated with 12 μmol/kg MeHg.Treatments were given once a day,for up to 28 days.After the poisoning,the autophagy fluorescence intensity was detected by MDC method,the formation of ROS was detected by flow cytometry,and the changes of GSH,MDA,GSH-Px and total-SH were detected by kits.mRNA and protein expression levels of PI3K/AKT/mTOR and AMPK/TSC2/mTOR pathway related indexes were determined by RT-q PCR and Western blotting.Results: 1.MeHg affected learning and memory,motor function,and spontaneous activity in rats.In the Morris water maze experiment,with the increase of the mercury dose,the movement distance and the escape latency of the rats were increased.With the platform removed,the accumulative duration of movement of the rats in the mercury-exposed group in the platform quadrant was reduced,and the number of crossing the platform quadrant was also significantly reduced.The total moving distance and average active time of the mercury-exposed group in the center of the open field decreased obviously.In the test of claw grip,the claw grip of the rats was obviously weakened.The results of HE staining showed that the pathological damage of cerebral cortex of rats exposed to MeHg was significantly higher than that of control group.The formation of autophagosomes was increased in the mercury-treated group by Transmission electron microscopy.Finally,Western blotting analysis showed that the expression of autophagy-related proteins LC3II/I and Beclin1 increased in a dosedependent manner after MeHg exposure.2.Compared with the control group,the content of ROS and MDA in the MeHg group increased,the content of GSH and-SH decreased,and the activity of GSH-Px decreased.Compared with the MeHg group,the content of ROS and MDA in the cerebral cortex of rats decreased,and the content of GSH and-SH,and the activity of GSH-Px increased significantly in the NAC+MeHg group,with significant differences.3.Compared with the control group,the mRNA transcriptions of PI3 K,AKT,and mTOR in the cerebral cortex of the MeHg group were reduced,and the relative protein expressions were also significantly reduced.The mRNA transcription of AMPK and Beclin1 was increased,and the corresponding protein expression increased.The intensity of autophagy fluorescence was also increased significantly.Compared with the MeHg group,the mRNA and protein expression levels of PI3 K,AKT and mTOR in cerebral cortex of rats in the NAC+MeHg group were significantly increased,the mRNA and protein expression levels of AMPK and Beclin1 were significantly decreased,and the intensity of autophagy fluorescence was significantly decreased,with significant differences.Conclusion: 1.MeHg exposure led to neurotoxic effects in the cerebral cortex and induced autophagy in rats.2.MeHg can induce oxidative stress of cerebral cortex in rats,and NAC has a certain protective effect on MeHg-induced cerebral cortical oxidative stress.3.NAC regulates autophagy mediated by PI3K/AKT/mTOR and AMPK/TSC2/mTOR signaling pathways by antagonizing oxidative stress.