| 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 ataxia, cerebral hemorrhage, edema, apoptosis of neurons and so on, but studies on the mechanism of AVM-induced apoptosis of neurons has been less studied.For Pigeon is recommended animal for enviromental monitoring tests , we choose king pigeon as experimental animal. 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 for study subjects. After 24 h's culture, Cell survival rate, changes of cell morphology and biochemical characteristics, LDH leakage rate, NO production, mitochondrial activity, mitochondrial membrane potential, mitochondrial oxidative stress, intracellular free calcium concentration, intracellular CaM mRNA expression, Caspase activity of cells and Caspase-3 mRNA expression level were detected to study dose-response relationship of AVM and neuronal toxicity, and furthermore explore the mitochondrial mechanism of neuronal toxicity of AVM.1.The results of AO-EB dual-fluorescence staining, HE staining, transmission electron microscopy and Tunel showed that AVM can induce neuronal apoptosis from morphology perspective, and the apoptosis index in the Medium and the AVM dose was positively correlated .2.DNA damage situation and phosphatidylserine (PS) of nerve cells were detected by agarose gel electrophoresis, micro-plate reader, and flow cytometry. The results confirmed that AVM can induce apoptosis of neurons and cause membrane symmetry disappear from biochemistry perspective, and this phenomenon was positively correlated with AVM dose.3.By detecting the leakage rate of LDH and the production of NO in neurons, we found that AVM could induce increase membrane permeability of nerve cell and reduce production of NO. 4.Mitochondrial activity was assessed by MTT method, the results indicated AVM could significantly decrease mitochondrial activity in a dose-dependent manner, resulting in mitochondrial injury.5.Mitochondrial membrane potential and cell reactive oxygen species (ROS) were observed by flow cytometry. The results showed that AVM could induce excessive ROS in neurons, reduce mitochondrial membrane potential. Those comfired that AVM could induce neuronal apoptosis by mitochondrial oxidative stress.6.The caspase-3 mRNA expression level and the activity of Caspase-3, 8, 9 were detected by semi-quantitative RT-PCR and colorimetry. With the increase of AVM concentration, the activity of caspase-3 and Caspase-9, and the mRNA expression of Caspase-3 in neurons increased gradually. The activity of Caspase-8 increased in neurons in 2.5μg·L-1 contration, no differences was found in other experimental groups. It showed that AVM could induce neuronal apoptosis through mitochondrial pathway by activate Caspase enzyme system.7.CaM mRNA expression level of intracellular and calcium ion concentration were detected by semi-quantitative RT-PCR and fluorescent probe. The results showed that AVM could decrease CaM mRNA expression level, increase free calcium concentration, disrupt transport of calcium ions, and resulting imbalance of calcium homeostasis. It showed that AVM induce neuronal apoptosis by affect intracellular calcium homeostasis.From the perspectives of cytotoxicity, apoptosis, oxidative stress, DNA damage, mitochondrial membrane potential, activity of mitochondrial enzyme, calcium homeostasis, apoptosis protease, the mitochondrial mechanism of AVM toxicity was elucidated in cell morphology, biochemical characteristics and molecular level. This research can provide new ideas and views for revealing the poisoning mechanism of AVM, replenish AVM ecotoxicological data, provide valuable clues for further study on biological effects of AVM, and provide a scientific basis to protect ecological environment and applyavermectin-type drugs reasonably. |
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