Studies On Apoptosis Mechanism Of King Pigeon Neuron Induced By AVM In Vitro

Avermectin (AVM) was generally used in animal husbandry and agriculture as anantiparasite drug and insecticide because of its excellent helminthicide action and safety. Theresidual AVM in environment has affected ecological system. Though there were manyresearches on toxicity of AVM, the relevcant studies of AVM on cell toxicity in vitro wereseldom.In view of this, the present study established the injury model by adding 10μg·L^-1AVM into King Pigeon neurons in vitro. Using flow cytometry, TUNEL, electrophoreticanalysis of DNA, fluorescent staining, electronmicroscope etc., studied the changes ofapoptotic morphology and biochemistry as well as possible regulative mechanism bydetecting intra-cellular Ca²⁺ concentration([Ca²⁺]i), chromosome DNA damage, mitochondriamembrane potential (△ψm) and Caspase-3 activity. The principal focus of this study was toapproach the mechanism of neuron apoptosis induced by AVM, provide informations forreasonable application of AVMs and preserve the ecological environment. Main results weredescribed as follows:1. 7-8 days Pigeon embryo was selected to dissociate neurons, 2μg·ml^-1 arabinosylcytosinwas added into cultural fluid to removal other cells except neurons. Neuron viability wasdetected by MTT assay and LDH Assay Kit, it indicated that neuron viability was irihibited by10μg·L^-1 AVM and inhibition ratio exhibited time dependent.2. Neuron apoptosis induced by 10μg·L^-1AVM was observed by Giemsa staining, AO/EBfluorescent staining, HE staining, transmission electron microscope.3. The changes of [Ca²⁺]i was detected by Fura-2/AM fluorometric method, applyingagarose gel electrophoresis, alkality SCGE, TUNEL detected neuron DNA damage, it showedthat AVM induced neuron apoptosis by increasing [Ca²⁺]i, activiting endogenousendonuclease, breaking DNA in nulceosome.4.△ψm and Caspase-3 activity were detected by flow cytometry and Colorimetric AssayKit, respectively. The research showed that both neuron△ψm and Caspase-3 activity weredecreased, it revealed that AVM induced neuron apoptosis by activating Caspase access.5. Phosphatidylserine (PS) was detected by flow cytometry. The result showed that PSexternalization. This observation suggested that apoptotic neurons losed membranephospholipid asymmetry and exposed PS on the outer leaflet of the plasmamembrane. Macrophages then phagocytose apoptotic neurons after specific recognition of theexposed PS.