Study On The Neurotoxicity Of Realgar Based On MAPK And MAPK Mediated Autophagy Of Nrf2/Keap1/p62 Sinagling Pathway To Promote Apoptosis Of Hippocampal Cells

Objective:It mainly contains arsenic disulfide（As₂S₂）or arsenic tetrasulfide（As₄S₄）.Due to the influence of traditional medication habits,drug-induced arsenic poisoning caused by unscientific and rational use of single realgar or its compound preparation has been reported from time to time.From a clinical point of view,it can damage the nervous system,digestion and skin.Arsenic is the mainly part of realgar.And the brain is the main target organs of arsenic toxicity.More and more attention has been paid for the mechanism of damage caused by realgar for nervous system.Nrf2,as a main transcription factor regulating antioxidant expression in cells,is an important receptor of exogenous toxic substances and oxidative stress.The activity Nrf2 is related to the natural aging process and the pathogenesis of human chronic diseases,such as neurodegenerative diseases and cardiovascular diseases.Autophagy in cell growth,differentiation,death and physiological process,also known as the cell death of typeⅡof programmed.Previous studies of our group have found that realgar continuously activates Nrf2 signaling pathway.The effect of Nrf2-mediated Nrf2-Keap1-p62 axis on autophagy has been rarely reported.Therefore,we will establish the model of p38,ERK1/2,JNK and AKT specific inhibitors and the rat model of Nrf2 sh RNA gene silencing and elucidate the role of MAPK and AKT-mediated Nrf2 in regulating autophagy in realgar-induced neurotoxicity.Studies have shown that autophagy and apoptosis are functionally related,that is,autophagy is required for apoptosis,and for cells that cannot be processed through autophagy,apoptosis may eventually be induced.At present,the role of MAPK and AKT-mediated p62-Keap1-Nrf2 system in autophagy promoting apoptosis in realgar-induced neurotoxicity has not been reported.Thus,We established p38,ERK1/2,JNK and Akt specific inhibitor models and Nrf2 sh RNA gene silencing rat models.It can provide theoretical and practical basis for early effective measures to prevent and control the occurrence of drug-induced chronic arsenic poisoning and to guide clinical rational drug use.Methods:A total of 96 healthy SPF female SD rats,aged 4 weeks and weighing50～60 g,were selected and provided by Experimental Animal Center of China Medical University.They were treated by free drinking water,room temperature maintained at 20～25℃,relative humidity at 40～70%,sunlight 12 h/day,day and night cycle feeding.According to body weight were randomly divided into 16 groups,namely,the control group,the realgar group,p38 inhibitor group,realgar+p38MAPK inhibitor group,ERK1/2 inhibitors,realgar+ERK1/2 inhibitors,JNK inhibitors,realgar+JNK inhibitors,AKT inhibitors group,realgar+AKT inhibitors,3MA inhibitor group,realgar+3MA inhibitor group,the Nrf2 sh RNA group and realgar+Nrf2 sh RNA group.0.5%sodium carboxymethyl cellulose（CMC-Na）aqueous solution was used as suspension medium,realgar dose was 0.9 g/kg,control group was given 0.5%CMC-Na,once a day for 8 weeks.From the 6th week,the inhibitor was intraperitoneally injected,and the control group and realgar group were injected with 1%DMSO.The inhibitor group was given SB203580,PD98059,SP600125 LY249002 and 3MA at a dose of 10 mg/kg,once every 3 days for 3 weeks.The latter 4 groups were injected into the CA1 region of the hippocampus（2.8 mm behind the coordinates of the anterior fontanelle,2.8 mm beside the realgar,and 2.0mm deep）,the sham group and the realgar group were given empty virus,and the remaining 2 groups were given Nrf2-sh RNA,respectively.After three weeks,0.5%sodium carboxymethyl cellulose（CMC-Na）aqueous solution was used as suspension medium,and realgar was given.The treatment was the same as above.After eight weeks of exposure,learning and cognitive ability of rats were detected by open field experiments and novel recognition experiments.The abnormal changes of mitochondria and synapses in hippocampal neurons of rats was observed by tansmission electron microscopy.Nrf2,autophagy related indexes and apoptosis related indexes were detected by Western blot.The interactions between p62 and Keap1,and Nrf2 and p62 were detected by immunoprecipitation method.Results:1.Arsenic accumulation can be detected in the brain tissues of realgar after intragastric administration.2.After realgar treatment,compared with the control group,the total distance of realgar group in the open field test was decreased（P<0.05）,the standing times of touching the wall was decreased（P<0.05）,but the average speed was decreased（P<0.05）;In the experimental stage,the exploration time of the control group was significantly higher than the realgar group（P<0.05）.3.After realgar exposure,the morphology of hippocampal neurons in rats was irregular,with loose and transparent nuclei and serrated surfaces.The synaptic structure is damaged,and the signal transmission between neurons is slowed down or blocked.4.Realgar can activate the ERK1/2,JNK,p38,PI3K/AKT signaling pathways in the brain,and realgar can activate the Keap1/Nrf2 system in the brain of rats through the ERK1/2,JNK,P38,and PI3K/AKT signaling pathways,while the addition of the above signaling pathway inhibitors can significantly reduce the high expression of Nrf2,Keap1,and p62 protein caused by realgar.5.Realgar promoted the binding of Keap1to Nrf2 and p62 to Keap1.Inhibition of Nrf2 weakened the destructive effect of realgar on Nrf2/Keap1 and p62/Keap1 complex.6.Realgar can induce the activation of autophagy-related proteins in rat hippocampus,including autophagy initiation（ULK1,Atg13,Beclin1,etc.）,autophagosome extension and autophagosomal lysosomal formation（Atg4B,LC3II/I,etc.）.When Nrf2 was silenced,the expression levels of these proteins were significantly decreased,suggesting that realgar could regulate autophagy through Nrf2 expression.7.Realgar exposure significantly increased the levels of Caspase8,Caspase3 and BAX/BCL2 in the hippocampus of the brain.After autophagy inhibition,the protein expression levels of Caspase8,Caspase3 and BAX/BCL2 were decreased compared with those in the realgar group.Conclusion:Arsenic in realgar can cross the blood-brain barrier and accumulate in brain tissue,causing central nervous system toxicity.The mechanism of toxicity is related to MAPK and AKT signal molecules mediating the Nrf2/Keap1/p62 singaling pathway to perturb autophagy and promote apoptosis.