| As a pesticide widely used in agriculture and animal husbandry, Avermectin (AVM) has been considered one of the most toxic anti-worm drugs for the environment. Previous studies showed that AVM could cause neurosis, cerebral hemorrhage, edema, apoptosis of neurons and so on, and neuronal apoptosis in vitro and mitochondrial pathway indyced by AVM has been confirmed, but studies on the other mechanism of AVM induced apoptosis of neurons—endocytoplasmic reticulum pathway has not been studied.Cellula nervosa of king pigeon cultivated in vitro as the research target, Neurons in king pigeon were infected with AVM at final concentrations of 2.5μg·L-1, 5μg·L-1 and 10μg·L-1 respectively in vitro at different times which were 6h, 12h and 24h. They were divided into 10 groups. cell survival rate, changes of cell endocytoplasmic reticulum construction, oxidative stress and intracellular Bcl-2, Cyt-c, DADD153, GRP78 and Caspase-12 mRNA expression level were detected to study time and dose-response relationship of AVM and neuronal toxicity, and furthermore explore the endocytoplasmic reticulum mechanism of neuronal toxicity of AVM.The results were as followed.1. Transmission electron microscopy, MTT and Tunel were selected to observe the cytotoxic effect of AVM at different dosages and times against the cellula nervosa, AVM could lead to nerve apoptosis observed from morphology, it was showed colinearity definitely between apoptosis ratio and the density of AVM;2. Detective kit of oxidative stress was selected to some indexes, such as emergence of active oxygen (ROS), activity of reduced glutathione (GSH-Px) and activity of super peroxide dismutase in cells, the result indicated that it could lead to oxidative stress of nerve cells, decrease activities of reduced glutathione (GSH-Px) and super peroxide dismutase (SOD) and increase the level of active oxygen (ROS) in different groups, it showed a determinate dosage and time relationship, it hinted that AVM could lead to stress of endocytoplasmic reticulum by oxidative stress and apoptosis induced by endocytoplasmic reticulum level;3. The Bcl-3 mRNA expression level in cells was detected by semi-quantitative RT-PCR, the result indicated that AVM could lead to decrease of Bcl-2 mRNA expression in pigeon nerve cells, thus the protective function of Bcl-2 was lost, which could cause the apoptosis;4. The GRP78 mRNA expression level in cells was detected by semi-quantitative RT-PCR, the result showed that GRP78 mRNA expression increased as the increasing of the dosage and time, the dosage of AVM was at 5μg/mL could reach peak value at 24h, and decrease soon, it hinted that self protective function of endocytoplasmic reticulum could achieve best level;5. The Cyt-c mRNA expression level in cells was detected by semi-quantitative RT-PCR, the result showed that Cyt-c expression was low in common circumstance, and it increased conspicuously as the increasing of drug action, this illustrated that nonage nerve apoptosis had been carried out;6. The GADD153 mRNA expression level in cells was detected by semi-quantitative RT-PCR, the result showed that AVM could induced the increasing of GADD153 mRNA in pigeon nerve cells, and lead to cascade reaction, cause apoptosis by endocytoplasmic reticulum;7. The Caspase-12 mRNA expression level in cells was detected by semi-quantitative RT-PCR, the result showed that AVM could lead to increasing of aspase-12 mRNA expression level in pigeon nerve cells, it was signature genes of apoptosis by endocytoplasmic reticulum, which identified that AVM could lead to apoptosis by endocytoplasmic reticulum.From the perspectives of cell morphous, apoptosis, oxidative stress and molecular expression of apoptosis by endocytoplasmic reticulum, the endocytoplasmic reticulum mechanism of AVM toxic action was clarified in this paper, it provided valuable clues for further research and enriched the toxicology information of AVM. |
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