The Pathway Of Realgar Induces Cerebral Cortex Lipid Spectrum Disoder And Disturbs Autophagy Mediated P62-Keap1-Nrf2 Axis To Regulate Neurotoxicity

Objective:Realgar,a traditional Chinese medicine,the main component is As₂S₂or As₄S₄.It is widely used in clinical treatment because of its efficacy in anti-inflammatory,anti-insect and anti-tumor.However,adverse reactions or chronic arsenic poisoning events caused by long-term,excessive and irregular use of realgar alone or in various formulas have often been reported.The arsenic contained in realgar is the real source of its toxicity,and the brain is one of the main target organs of arsenic toxicity.The toxic effect of realgar on the central nervous system has become the focus of attention.Our previous study found that realgar can affect the redox balance in vivo and cause lipid peroxidation in the brain.Lipids are the main component of the bilayer membrane of cells and are the first target for arsenic to attack nerve cells.Lipid imbalance is closely related to the occurrence and development of neurodegenerative diseases（such as Alzheimer’s disease,Parkinson’s disease,etc.）.The cerebral cortex is the key area of the brain involved in learning and memory,but the effect of realgar on the lipid metabolism profile in the cerebral cortex and the underlying mechanisms of neurotoxicity induced by realgar have rarely been reported.Nuclear factor erythroid-derived factor 2-related factor（Nrf2）is an important regulatory element in the cellular antioxidant process.When stimulated by ROS or electrophilic reagents,Nrf2 dissociates from Kelch-like ECH-associated protein 1（Keap1）into the nucleus and promotes the expression of downstream antioxidant enzymes to enhance cellular antioxidant and detoxification capacity.Autophagy plays an important physiological role in maintaining the protein metabolic balance and the homeostasis of cellular.Beclin1 and Phosphatidylinositol 3 kinase submit III（ШPI3K）are considered to be the key factors for the initiation of autophagy.Class II microtubule-associated protein light chain 3（LC3-II）is a sign of autophagosome formation.The degradation of autophagy receptor SQSTM1（p62）is an important mark of autophagic flux.Finally,the autophagosome degrades with the action of Lysosomal associated membrane protein 2a（Lamp2a）.The Keap1 interacting region（KIR）and LC3interaction region（LIR）of p62 recognize Keap1 and LC3,respectively,and mediate the degradation of Keap1 through the autophagic pathway to activate Nrf2.Thus,autophagy can mediate the p62-Keap1-Nrf2 axis to regulate Nrf2 levels.In conclusion,in the first part of this study,an animal model of realgar exposure in vivo was established to investigate the changes of cerebral cortex lipid spectrum and screen potential lipid markers of neurotoxicity after realgar exposure based on lipidomics.On the basis of the first part,the autophagy inhibitor 3-methyladenine（3-MA）was used in the second part to establish the rat model and the pathway of realgar disturbed autophagy and p62-mediated p62-Keap1-Nrf2 axis activating Nrf2 to regulate central nervous system toxicity was studied in detail.Providing research basis and experimental data for further studies of the mechanism by which realgar induces toxicity of central nervous system and offering effective measures to protect human health in the early stages of realgar exposure.Methods:1.ICR mice were randomly divided into 4 groups:control group（Control）,0.15 g/kg realgar group（0.15 g/kg）,0.45 g/kg realgar group（0.45 g/kg）,1.35g/kg realgar group（1.35 g/kg）,13 mice in each group,0.5%sodium carboxymethylcellulose（CMC-Na）（w/v）was used as suspension solvent.The control group was intragastrically administered 0.5%CMC-Na whereas the realgar-treated groups were intragastrically administered 0.15 g/kg,0.45 g/kg or 1.35 g/kg realgar suspension,once a day for consecutive 12 weeks.The level of nerve injury of mice were measured by open field test and novel object recognition task.The structure of neurons,synapses,myelin sheath and the number of autophagosomes were observed under transmission electron microscope.The total arsenic content in the cerebral cortex was determined by atomic fluorescence spectrometry.The contents of malondialdehyde（MDA）,superoxide dismutase（SOD）,catalase（CAT）and glutathione peroxidase（GSH-Px）were determined by kits.The enzyme activities of Lipoprotein-associated Phospholipase A2（Lp-PLA₂）,cytosolic phospholipase A2（c PLA）₂,lysophosphatidylcholine acyltransferase 1（LPCAT1）were determined by Elisa.The distribution and changes of lipid metabolism profiles in the cerebral cortex were determined by non-targeted lipidomics to screen out the lipid markers of realgar-induced neurotoxicity.2.SD rats were randomly divided into 4 groups:control group（Control）,the realgar group（Realgar）,the 3-MA treatment group（3-MA）,and the 3-MA+realgar group（3-MA+R）,8 rats in each group,0.5%CMC-Na（w/v）was used as suspension solvent.The first and third groups were intragastrically administered 0.5%CMC-Na,and the second and fourth groups were intragastrically administered 0.9 g/kg realgar suspension,once a day for consecutive 8 weeks.Starting from the 6th week,the first and second groups were intraperitoneally injected with 5%DMSO solution,and the third and fourth groups were intraperitoneally injected with 20 mg/kg 3-MA,once every 2 days.The exploration ability and spontaneous motor function of the rats were measured by open field test.The structure of neurons,synapses,and the number of autophagosomes were observed under transmission electron microscope.The total arsenic content in the total blood and cerebral cortex were determined by atomic fluorescence spectrometry.The contents of SOD,CAT,total antioxidant capacity（T-AOC）,MDA,oxidized glutathione/total glutathione（GSSG/T-GSH）were determined by kits.The m RNA expression levels of Ulk1,Atg14,Becn1,Atg5,Atg12,Atg16L,Atg4b,Atg7,Atg3,Sqstm1,Nbr1,Map1lc3b,Lamp1,Keap1,Nfe2l2,Ho1,Nqo1,Gclc,Gclm and Gsta1 in the cerebral cortex were determined by RT-q PCR.The protein expression levels of mammalian target of rapamycin（m TOR）,Unc-51-like kinase 1（ULK1）,Atg14,Beclin1,Atg5,Atg12-Atg5,Atg7,Atg4B,p62,LC3Ⅱ/Ⅰ,Nrf2,Keap1,Nrf2,Heme oxygenase（HO-1）,NADPH quinone acceptor oxidoreductase 1（NQO1）,glutamate-cysteine ligase catalytic subunit（GCLC）,Bcl-2 assaciated X protein/B cell lymphoma 2（Bax/Bcl2）,Caspase-8 and Cleaved-Caspase-3 were determined by Western blot.The binding of Keap1-p62,Keap1-p62-LC3,p62-LC3,and p62-Keap1-Nrf2 protein aggregates were determined by Co-immunoprecipitation.Results:1.The arsenic contained in realgar passed through the BBB and accumulated in the brain.Neurons,synapses and myelin showed abnormal changes in the cerebral cortex.The number of autophagosomes were incresed as well as levels of MDA,Lp-PLA₂,and c PLA₂（P<0.05）but the CAT level was significant reduced（P<0.05）.Nontargeted lipidomics detected 34 lipid subclasses including 1603 lipid molecules.The levels of the LPC and LPE were significantly increased（P<0.05）.Under the condition of variable importance for the projection（VIP>1 and P<0.05）,only 28 lipid molecules satisfied the criteria.The 28 lipid molecules were strongly correlated with CAT,SOD,arsenic and GSH-Px.16 lipid molecules were screened out by receiver operating characteristic（ROC）curve（AUC>0.8 or AUC<0.2）,and SM（d36:2）,PE（18:2/22:6）and PE（36:3）were significant changes（P<0.05）in the 0.45 g/kg and 1.35 g/kg groups.The combination of SM（d36:2）,PE（18:2/22:6）and PE（36:3）exhibited the best diagnostic performance（96.4%）,which could be used as sensitive markers to determine realgar-induced neurotoxicity.2.After realgar exposure,the m RNA levels of autophagy initiating genes ULK1,Atg14,Becn1,autophagosome membrane extension genes Atg5,Atg7,Atg16L,Atg4b,and autophagosome forming genes Sqstm1,Nbr1,Map1lc3b were significant incresed（P<0.05）,autophagosome membrane extension gene Atg12 showed a upward trend（P=0.072）,and the autophagosome forming gene Atg3 and lysosomal membrane gene Lamp1 had no significant changes.Autophagy initiating proteins m TOR was significant reduced（P<0.05）,ULK1,Atg13,Beclin1,Atg14,autophagosome membrane extension proteins Atg5,Atg7,Atg12-Atg5,Atg4B,autophagosome forming proteins p62,LC3Ⅱ/Ⅰwere significant increased（P<0.05）.Compared with the Realgar,the m RNA levels of autophagy initiating genes ULK1,Becn1,autophagosome membrane extension genes Atg5,Atg7,Atg16L,Atg4b,Atg12 and autophagosome forming genes Map1lc3b were significant reduced（P<0.05）in 3-MA+R,autophagosome forming genes Sqstm1,Nbr1were significant incresed（P<0.05）,autophagy initiating gene Atg14 showed a downward trend（P=0.073）,and the autophagosome forming gene Atg3 and lysosomal membrane gene Lamp1 had no significant changes.Autophagy initiating proteins m TOR was significant increased（P<0.05）,ULK1,Atg13,Beclin1 were significant decreased（P<0.05）,autophagosome membrane extension proteins Atg5,Atg7,Atg12-Atg5 were significant decreased（P<0.05）,autophagosome forming proteins LC3Ⅱ/Ⅰwas significant decreased（P<0.05）,p62 was significant increased（P<0.05）,Atg14 and Atg4B had no significant changes in 3-MA+R.After realgar exposure,the m RNA contents of Nfe2l2,Keap1,Ho1,Nqo1,Gclc,and Gclm were significant increased（P<0.05）and Gsta1 had no significant change.The protein levels of Keap1,Nrf2,HO-1,Nqo1 and GCLC were significant increased（P<0.05）.Compared with the Realgar,the m RNA contents of Ho1,Nqo1,Gclc,Gsta1were significant increased（P<0.05）in 3-MA+R,Nfe2l2 showed a upward trend（P=0.078）,and Keap1 had no significant change.The protein levels of Keap1,Nrf2,HO-1 were significant increased（P<0.05）in 3-MA+R,GCLC was significant decreased（P<0.05）,and Nqo1 had no significant change.After realgar exposure,the activity of CAT,SOD were significant decreased（P<0.05）,T-AOC was significant decreased（P<0.05）,GSSG/T-GSH and MDA were significant increased（P<0.05）.Compared with the Realgar,the activity of SOD showed a upward trend in 3-MA+R,GSSG/T-GSH and MDA showed downward trends.Co-IP experiments proved that realgar promotes the formation of Keap1-p62,Keap1-p62-LC3,and p62-LC3 protein aggregates,and inhibits the formation of p62-Keap1-Nrf2 protein aggregates.Conclusion:1.The arsenic contained in realgar passed through the BBB and accumulated in the brain,causing central nervous system toxicity.2.Realgar causes the lipid metabolism spectrum disorder in the cerebral cortex.A total of 34 lipid subclasses and 1603 lipid molecules were detected.Finally,the lipid molecules SM（d36:2）,PE（18:2/22:6）and PE（36:3）can be used as potentially sensitive lipid markers to determine realgar-induced neurotoxicity.3.Realgar activates the initiation of autophagy,promotes the formation and extension of autophagosomes,inhibits the degradation of p62,and causes autophagy disorder in the cerebral cortex.4.Realgar causes oxidative damage of the cerebral cortex and activates the Keap1-Nrf2-ARE signaling pathway.5.Realgar-induced p62 accumulation mediates the p62-Keap1-Nrf2 axis to promote the sustained activation of Nrf2 signaling pathway in the cerebral cortex.