Crosstalk Of Autophagy To Apoptosis In Arsenic Induced Hippocampal Neuron Injury In Mice

Objective: Arsenic is a metallic element that exists widely in the world.In addition to causing cancerous lesions in the body,long-term exposure to arsenic in the environment has also been shown in recent years that it can cause damage to the central nervous system,which has attracted more and more attention.Pregnant feeding is a critical period of neural development,and arsenic is a known developmental poison that easily breaks through the placental barrier,so the two life stages most vulnerable to arsenic exposure are pregnancy and fetal development.Autophagy is a life phenomenon widely found in eukaryotic cells.Abnormal levels of Autophagy(including under-autophagy and over-autophagy)are associated with many cellular pathological processes and the occurrence of diseases.Apoptosis is thought to be an important part of a variety of processes,including normal cell replacement,normal development and function of the immune system.Improper apoptosis(either too little or too much)is a factor in many human diseases.Whether autophagy and apoptosis interact with each other and promote the occurrence and development of diseases is a hot research topic at present.The neurotoxic mechanism of arsenic is not clear.Some studies have shown that the neuronal apoptosis caused by arsenic exposure may be one of the mechanisms of nerve injury.However,there are relatively few reports on the effect of arsenic exposure on autophagy level.Therefore,this study intends to study the effects of arsenic exposure on autophagy and apoptosis levels as well as the crosstalk between autophagy and apoptosis through the animal model of early life sustained arsenic exposure and in vitro cell arsenic exposure model,so as to explore the mechanism of nerve injury caused by arsenic.Methods: In vivo study: modeling early life arsenic exposure.From day 0 of gestation to day 21 of birth,maternal rats were exposed to sodium arsenite solution in free drinking water at doses of 0,20,40,and 80mg/L,respectively.The female mice were removed during initial weaning(PND21),and the young mice continued to be infected with free drinking water until the end point(PND35)after weaning.Hippocampal tissue sections of PND21 and PND35 were embedded,and the damage of nerve cells in the hippocampal tissue was detected by HE staining.The autophagosomes were observed by transmission electron microscopy(TEM)in the hippocampal tissues of young mice.The expression level of autophagy marker protein LC3 B in hippocampal neurons was observed by in situ immunofluorescence technique.CO-IP technique was used to detect the interaction between FAS and LC3 B and Caspase-8 in the hippocampus of PND21 and PND35 offspring.Western Blot was used to detect the expression of p-m TOR,LC3 B,P62 and other autophagy-related proteins as well as the expression of exogenous apoptosis-related proteins.The learning and memory ability of PND35 pups exposed to arsenic in early life was tested by water maze test.In vitro experiment: A mouse hippocampal neuron line HT-22 was used to establish an in vitro arsenic exposure model.The doses of sodium arsenite were 0,4,8,12 ?m,and the exposure time was 48 h.Rapamycin,an autophagy activator,and 3-MA,an autophagy inhibitor,were used to intervene the autophagy level.LC3 B gene expression was knocked down or overexpressed by plasmid transfection with si RNA.Flow cytometry and TUNEL were used to detect the apoptosis level of HT-22 cells after arsenic exposure/autophagy activator or inhibitor intervention /si RNA intervention,respectively.The expression of LC3 B in HT-22 cells and the co-localization of LC3 B with FAS and Caspase-8 with FAS were observed by immunofluorescence.Protein interactions between LC3 B and FAS and Caspase-8 and FAS were detected by co-immunoprecipitation technique(CO-IP).The expression of autophagy related proteins such as p-m TOR,LC3 B and P62 and the expression of exogenous apoptosis-related proteins were detected by Western Blot.Results: 2.Early life arsenic exposure activates autophagy in hippocampal neurons of young mice.Transmission electron microscopy was used to observe the autophagosomes in the hippocampal neurons of PND21 and PND35 young mice exposed to arsenic in early life.With the increase of arsenic dose,the autophagosomes in the hippocampal neurons of mice increased significantly,showing a dose-response relationship.In situ immunofluorescence showed that LC3 B expression was up-regulated in hippocampal neurons of PND35 pups exposed to arsenic in early life.Western blotting confirmed that the expression levels of autophagy-related protein LC3 B were significantly up-regulated in PND21 and PND35 pups,and the expression levels of p-m TOR and P62 were significantly down-regulated(P<0.05).The results showed that arsenic exposure in early life activated autophagy in hippocampal neurons of PND21 and PND35 pups.1.Arsenic exposure in early life activates exogenous apoptosis of hippocampal neurons in infant mice.WB results showed that PND21 and PND35 pups were induced by arsenic exposure in pregnancy and feeding period,and the expression levels of FAS and FADD were significantly increased,Caspase-8 was activated,and Cleaved-Caspase-8 expression was significantly increased,meanwhile,the expression levels of apoptotic related proteins BAX and Cleaved-Caspase-3 were increased,and the expression levels of anti-apoptotic gene BCL-2 were significantly decreased(P<0.05).The results showed that arsenic exposure in early life activated the exogenous apoptosis of PND21 and PND35 hippocampal neurons.3.Arsenic exposure in early life impaired hippocampal neurons and learning and memory ability of young mice.HE staining to observe PND21 and PND35 seed loss of cells in the rat hippocampus pyramidal layer,pyknotic cells arrange disorder and form.The results suggest that arsenic exposure in early life can damage hippocampal neurons in young mice.At the same time,the results of water maze test showed behavioral impairment.Compared with the control group,the total time and distance to find the platform were significantly increased in the arsenic exposure group(P<0.05),suggesting that the learning and memory ability of the young rats was damaged by arsenic exposure in early life.4.Arsenic exposure activates autophagy and exogenous apoptosis in HT-22 cells.Immunofluorescence results showed that the expression of LC3 B in HT-22 cells was significantly increased under the effect of sodium arsenite(P<0.05),and showed a doseresponse relationship with the increase of exposure dose.WB test results showed that the expression levels of autophagy related proteins P62 and p-m TOR in HT-22 were decreased,while the expression levels of LC3 B were increased in a dose-response relationship(P<0.05).The results suggested that sodium arsenite activated autophagy in HT-22 cells.Flow cytometry and TUNEL results showed that arsenic exposure resulted in up-regulation of HT-22 cell apoptosis.WB results showed that arsenic exposure significantly upregulated the expression of FAS and FADD in HT-22 cells,and significantly increased the expression of Cleaved-Caspase-8.Meanwhile,the expression levels of apoptotic protein BAX and Cleaved-Caspase-3 were increased,and the expression levels of antiapoptotic gene BCL-2 were decreased(P<0.05).Arsenic exposure activates exogenous neuronal apoptosis in vitro.5.Autophagy inhibitors inhibit HT-22 exogenous cell apoptosis.The autophagy level of HT-22 cells was significantly inhibited after treated with the autophagy inhibitor 3-MA.Flow cytometry and TUNEL results showed that the number of apoptotic cells decreased when autophagy was inhibited.WB results showed that the expression levels of exogenous apoptotic pathway related proteins FAS,FADD,and Cleaved-caspase-8 in HT-22 cells were significantly decreased under the treatment of 3-MA,while the expression levels of apoptotic related proteins BAX,Cleaved-caspase-3,and anti-apoptotic protein BCL-2 were increased(P<0.05).The results showed that autologous 3-MA inhibited the apoptosis of HT-22 cells.6.Autophagy activator activates HT-22 exogenous cell apoptosis.After Rapamycin treatment,autophagy in HT-22 cells was significantly activated.Flow cytometry and TUNEL activation showed that the number of apoptotic cells increased when autophagy was activated.WB results showed that under Rapamycin treatment,the expression levels of exogenous apoptotic pathway related proteins FAS,Fadd,and Cleaved-caspase-8 in HT-22 cells were significantly increased,the expression levels of apoptotic related proteins BAX,Cleaved-caspase-3 were up-regulated,and the expression levels of antiapoptotic protein BCL-2 were decreased(P<0.05).These results indicated that Rapamycin activated HT-22 exogenous apoptosis.7.Autophagy marker protein LC3 B interacts with exogenous necrosis receptor FAS.Immunofluorescence detection showed that LC3 B and Fas co-localized in HT-22 cells,and arsenic exposure increased the expression of LC3 B and Fas,but the co-localization ratio decreased.Meanwhile,the binding degree of Fas and Caspase-8 was up-regulated(P<0.05).CO-IP results showed that Fas protein interacted with LC3 B protein,and arsenic exposure decreased the interaction between the two proteins,while enhancing the interaction between Fas and Caspase-8(P<0.05).These results suggest that LC3 B may regulate the exogenous apoptosis of HT-22 cells through the interaction between LC3 B and Fas.8.Knocking down LC3 B expression inhibits FAS-dependent exogenous apoptosis in HT-22 cells.Flow cytometry results showed that the apoptosis level of HT-22 cells activated by arsenic exposure was significantly reversed after si RNA interference with LC3 B expression.Immunofluorescence co-localization and CO-IP results showed that the interaction between LC3 B and FAS was enhanced(P<0.05).Meanwhile,the interaction between FAS and Caspase-8 was weakened(P<0.05).WB results showed that the expression of Cleaved-Caspase-8,Cleaved-Caspase-3,and apoptotic marker protein BAX were significantly down-regulated after interference with LC3 B expression,while the expression of antiapoptotic protein BCL-2 was increased(P<0.05).These results indicated that after the interference of LC3 B expression,the exogenous apoptosis pathway of HT-22 cells was inhibited and apoptosis was inhibited.9.Overexpression of LC3 B activates FAS-dependent exogenous apoptosis in HT-22 cells.Flow cytometry results showed that the apoptosis level of HT-22 cells was significantly activated after LC3 B overexpression.Immunofluorescence co-localization and CO-IP results showed that the interaction between LC3 B and FAS was significantly reduced.Meanwhile,the interaction between FAS and Caspase-8 was significantly enhanced(P<0.05).WB results showed that Cleaved-Caspase-8 expression,Cleaved-Caspase-3,BAX expression,and anti-apoptotic BCL-2 expression were increased in HT-22 cells overexpressing LC3B(P<0.05).It was proved that the exogenous apoptosis pathway of HT-22 was activated and apoptosis was activated.Conclusion: 1.Arsenic exposure in early life activated the level of autophagy in hippocampal neurons,enhanced the level of exogenous apoptosis,and impaired the ability of learning and memory.2.Inhibition of autophagy or activation of autophagy can accordingly down-regulate or up-regulate the apoptosis level of HT-22 cells,suggesting that autophagy may have crosstalk effect on apoptosis.3.The cross-talk effect of autophagy on apoptosis is reflected in the interaction between the autophagy marker protein LC3 B and the exogenous necrosis receptor FAS,and autophagy can regulate the necrosis receptor FAS dependent exogenous apoptosis through the key protein LC3 B.