Study On The Mechanism Of Excess Manganese-induced Apoptosis In Chicken Brain Tissues And Neurocytes

Manganese?Mn?is an essential trace element for humans and animals,as Mn is widely used in industry and agriculture,it caused Mn pollution.Nervous system is one of the objects of excess Mn,Mn pollution can make workers appear mental illness symptoms,and affects mental development of children.The accidental ingestion of excess Mn by livestock and poultry will appear apathetic symptoms,and even deaths,resulting in loss in livestock and poultry production.The purpose of this study was to investigate the effects of excess Mn on apoptosis in chicken brain tissues and chicken embryonic neurocytes,to provide basic data for the neurotoxicity of chicken,and to provide references for the prevention and treatment of animal Mn poisoning in livestock and poultry production.This test included tests in vivo and vitro.In vivo test,240 1-day-old Hyline male chickens were fed on a standard diet for 7 days and were randomly divided into four groups:control group was fed by standard diet?containing 127.88 mg/kg Mn?and three treated groups were fed by three standard diets?containing 600,900,and 1800 mg/kg Mn,respectively?with different levels of manganese chloride.At 30,60,and 90 days after fed,the cerebrum,cerebellum,thalamus,and brainstem tissues were collected for the detections of related indicators.In vitro test,chicken embrynic neurocytes were separated from 6-8 day old chicken embryos and divided into one control group and six Mn treated groups.The control group was cultured in DMEM medium and six treated groups were cultured in DMEM medium supplemented with manganese chloride?MnCl₂ concentrations were 0.5,1.0,1.5,2.0,2.5 and 3.0 mM,respectively?.The cells were harvested at 12,24,36,and 48 hours respectively for detection of related indicators.The clinical symptoms and necropsy of chickens were observed in this experiment,the Mn contents in chicken brain tissues and blood were detected using flame atomic absorption spectrometry and graphite furnace atomic absorption spectrometry,respectively,the microstructure and ultrastructure in chicken brain tissues were observed using hematoxylin-eosin?HE?staining and dual staining of uranyl acetate and lead citrate respectively,viability and apoptosis in chicken embryonic neurocytes were detected using cell counting kit-8?CCK-8?and acridine orange/ethidium bromide?AO/EB?double staining respectively,superoxide dismutase?SOD?activity,total antioxidant capacity?T-AOC?activity,nitric oxide?NO?content,and inducible nitric oxide synthase?iNOS?activity were detected using kits,inflammatory factors nuclear factor-?B?NF-?B?,tumor necrosis factor-??TNF-??,iNOS,cyclooxygenase-2?COX-2?,and prostaglandin E synthase?PTGEs?,cytokines Interleukin-4?IL-4?,IL-7,IL-10,IL-17,Interferon-??IFN-??,and transforming growth factor-?4?TGF-?4?,apoptotic genes p53,B-cell lymphoma 2?Bcl-2?,B-cell lymphoma-extra large?Bcl-x?,B-cell lymphoma 2-associated X protein?Bax?,B-cell lymphoma 2 homologous antagonist/killer?Bak?,fas,and caspase-3,and heat shock proteins HSP27,HSP40,HSP60,HSP70,and HSP90 mRNA expression in chicken brain tissues and chicken embryonic neurocytes were detected using real time quantitative PCR?qPCR?,inflammatory factors NF-?B,iNOS,and PTGEs,apoptotic genes p53,Bcl-2,Bax,and caspase-3,and heat shock proteins HSP60,HSP70,and HSP90 protein expression in chicken brain tissues and chicken embryonic neurocytes were detected using Western blotting,the results showed as follow:?1?The Mn contents in chicken brain tissues and blood increased by excess Mn,there were dose and time effects on Mn contents in chicken brain tissues,and a dose effect on Mn contents in chicken blood,excess Mn led to Mn accumulation in chicken brain tissues.?2?Inflammatory injury were caused by excess Mn in chicken brain tissues,the degree of inflammatory injury increased with the increase of Mn treatment dose and treatment time.Excess Mn induced the mRNA expression of inflammatory factors NF-?B,TNF-?,iNOS,COX-2,and PTGEs mRNA and protein expression of NF-?B,iNOS,and PTGEs,and increased NO content and iNOS activity in chicken brain tissues and chicken embryonic neurocytes,there were dose and time effects on the above indicators in chicken brain tissues and on the protein expression of inflammatory cytokines in chicken embryonic neurocytes,there was a dose effect on the mRNA expression of inflammatory cytokines,NO content,and iNOS activity in chicken embryonic neurocytes.Inflammatory response and inflammatory injury were caused by excess Mn in chicken brain tissues and chicken embryonic neurocytes.?3?Apoptosis in chicken brain tissues were caused by excess Mn in organizational morphological terms,the degree of apoptosis in chicken brain tissues increased with the increase of Mn treatment dose and treatment time.Apoptosis in chicken embryonic neurocytes were caused by excess Mn in cellular morphological terms,the number of apoptosis cells in chicken embryonic neurocytes increased with the increase of Mn treatment dose.The mRNA expression of apoptotic gene p53,Bax,Bak,fas,and caspase-3 mRNA and protein expression of p53,Bax,and caspase-3were induced,and the mRNA and protein expression of Bcl-2 and Bcl-x mRNA were inhibited by excess Mn.There were dose and time effects on apoptotic gene mRNA and protein expression in chicken brain tissues and apoptotic gene protein expression in chicken embryonic neurocytes,a dose effect on apoptosis gene mRNA expression in chicken embryonic neurocytes.Apoptosis in chicken brain tissues and chicken embryonic neurocytes were induced by excess Mn.?4?The SOD and T-AOC activities in chicken brain tissues and chicken embryonic neurocytes decreased by excess Mn,there were dose and time effects on SOD and T-AOC activities in chicken brain tissues and a dose effect in chicken embryonic neurocytes.Oxidative stress in chicken brain tissues and chicken embryonic neurocytes were caused by excess Mn.?5?The mRNA expression of cytokines IL-7 and IFN-?were induced by excess Mn,and mRNA expression of cytokines IL-4,IL-10,IL-17,and TGF-?4 were inhibited by excess Mn in chicken brain tissues and chicken embryonic neurocytes.There were dose and time effects on the mRNA expression of cytokines in chicken brain tissues and a dose effect in chicken embryonic neurocytes.Immune responses were caused by excess Mn in chicken brain tissues and chicken embryonic neurocytes.?6?The mRNA expression of heat shock proteins HSP27,HSP40,HSP60,HSP70,and HSP90,protein expression of HSP60,HSP70,and HSP90 were induced by excess Mn in chicken brain tissues and chicken embryonic neurocytes.There were dose and time effects on the mRNA and protein expression of heat shock proteins in chicken brain tissues and the protein expression of heat shock proteins in chicken embryonic neurocytes,a dose effect on the mRNA expression of heat shock proteins in chicken embryonic neurocytes.Heat shock proteins reacted protective response to Mn-induced apoptosis in chicken brain tissues and chicken embryonic neurocytes.?7?Excess Mn had the most significant effect on mRNA expression of NF-?B and TNF-?in cerebrums,protein expression of NF-?B,iNOS,and PTGEs in the cerebrums,on protein expressions of Bcl-2,HSP60,and HSP90 in thalami,on mRNA expression of NF-?B,IL-4,and IL-17 and protein expression of p53 in the cerebrums and thalami.The toxicity of excess Mn on cerebrums and thalami were more than that on cerebellums and brainstems.Excess Mn led to apoptosis in chicken brain tissues and chicken embryonic neurocytes,oxidative stress,inflammatory injury,immune responses,and heat shock proteins were involved in apoptosis in chicken brain tissues and chicken embryonic neurocytes caused by excess Mn.