Molybdenum And Cadmium Co-Induce Autophagy Via Calcium Homeostasis Disorder And Unfolded Protein Response In Duck Brain

To investigate whether molybdenum（Mo） and cadmium（Cd）combined-induce the brain toxicity of duck under the involvement of calciumhomeostasis disorder and unfolded protein response（UPR）mediated autophagy,ammoniummolybdate(（NH₄）₆Mo₇O₂₄·4H₂O)and cadmiumsulfate（3Cd SO₄·8H₂O）were selected as the experimental Mo and Cd sources,respectively.160 healthy 8-day-old ducks（Anas platyrhyncha）were divided into four groups in random（40 ducks for each group）and fed a basal diet containing different doses of Mo（100 mg/kg）or/and Cd（4 mg/kg）for 16 weeks.On the 16^thweek,ducks were selected fromeach group to collect the brain tissues and then they were stored as different detection.Macroelement contents,ATPases activities,tissue lesions and autophagosomes,LC3II fluorescent puncta,and calciumhomeostasis,UPR and autophagy related gene and protein levels were detected by inductively coupled plasma mass spectrometer（ICP-MS）,microplate reader,transmission electron microscope（TEM）,confocal microscopy,RT-q PCR and western blotting,respectively.The results were as follows:1.In comparison with control group,in all treated groups,Ca²⁺ATPase,Mg²⁺ATPase,Na⁺-K⁺ATPase and Ca²⁺-Mg²⁺ATPase activities and K and Na contents were greatly decreased,Mg and Cacontents were greatly increased,calciumhomeostasis related genes mRNA and proteins（CaMKIIα,CaN,IP₃R and CRT）levels were obviously upregulated,and the above element contents were extremely changed in Mo+Cd group.Thus,Mo or/and Cd were able to cause disturbance of calciumhomeostasis in duck brain.2.In comparison with control group,in all treated groups,UPR associated genes（PERK,GRP78,e IF2α,ATF4,IRE1 and ATF6）mRNA expression levels and PERK,IRE1and ATF6 proteins expression levels were obviously upregulated,and the above genes and proteins expression levels were highly significantly upregulated in Mo+Cd group.Thus,Mo or/and Cd can activate UPR in duck brain.3.In comparison with control group,in all treated groups,autophagosomes and LC3II immunofluorescence puncta,upregulated the mRNA expression levels of autophagy related genes including Beclin-1,Atg5,LC3A,LC3B,and the proteins expression levels of Beclin-1 and LC3II/LC3I were markedly elevated,the mRNA expression levels of dynein and p62 and p62 protein expression level were markedly declined,and the above genes and proteins expression levels changed more significantly in the Mo+Cd group.Thus,Mo or/and Cd were able to induce autophagy in duck brain.In summary,Mo or/and Cd can result in calciumhomeostasis dysregulation,initiation of UPR and induction of autophagy.Therefore,autophagy activated by Mo or/and Cd in duck brain could be associated with crosstalk between calciumhomeostasis dysregulation and UPR,and combined Mo and Cd could aggravate brain toxicity and injury in ducks.