The Effect Of Alpha-synuclein Gene On Apoptosis Of Neural Cells Induced By Avermectin In Pigeon

Because of its superior efficacy Avermctin are widely used for pesticide in agriculture and veterinary medicine in the world. A large number of studies showed the residues of avermectin drugs or their metabolites in livestock feces have adverse effects on non-target organisms. The studies which indicate many non-target animals present poisoning reactions of central nervous system after exposure to AVMs are abundant. However, most studies have addressed the effects of AVMs on soil-dwelling invertebrates, freshwater organisms and livestock. To date, the researches about the effects of AVMs on birds are scarce. Our previous research found that pigeons are sensitive to AVM and sub-chronic AVM poisoning can cause pigeons showing obvious symptoms of central nervous system. Further studies showed that AVM can cause apoptotic morphological and biochemical changes in neurons in vivo and in vitro. Using the conventional method we prepared antibodies against pigeon a-Syn gene to detect the gene expression of a-Syn in pigeon brain tissues after acute AVM intoxication and found that the expression of a-Syn gene in treatment groups was significantly higher than the control. Many studies showed that a-Syn gene is involved in human neural cell apoptosis and further lead to neurodegenerative diseases. In order to investigate the role of a-Syn gene in the apoptosis induced by AVM, we prepared two poisoning models. In the subchronic model in vivo, eighty60-day old American king pigeons (Columba livid) were randomly allocated into four equal group:a control group, a low-dose group, a middle-dose group and a high-dose group fed the basal diet spiked with0,20,40and60mg AVM/kg diet respectively and the birds were treated for30d,60d and90d respectively. In the vitro model, neurons separated form pigeon embryos were cultured and were treated with0μg·L-1,2.5μg·L-1,5μg·L-1,10μg·L-1,15μg·L-1and20μg·L-1AVM for24h. The change of apoptosis related indicators, the expression of cytokine related genes and the expression of heat shock protein genes in pigeon brain tissues and neurons before and after knockdown a-Syn gene were detected. The main results are as following:(1) Using the7-8days’ pigeon embryos as experimental material the standard methods was used to separate and extract the brain neurons. The Neurobasal medium was used to optimized culture brain cells. NSE immunocytochemistry in nerve cells showed strong positive reaction, suggesting that the cultured neurons were high purity, good activity and could be used to detect subsequent targets. (2) A AVM sub-chronic toxicity model was copied successfully, and the poisoning animals showed significant neurological symptoms such as staggering, depression and ataxia et al.(3) The primary cultured pigeon embryo brain cells were transfected successfully by Stealth RNA with a best combination of1μL RNAi MAX+0.9μL siRNA for each well in a24-well plate, which transfection efficiency was greater than80%. The effective interfering sequence-a-Syn-Stealth (142) was screened, which can reduced the expression of mRNA level of a-Syn to44%comparing to the control group.(4) The LC50of AVM to the neurons was between15-20μg/L in vitro.(5) Apoptosis-inducing situations of AVM to neurons in vitro were detected using TUNNEL and HOECHST staining. The effect of AVM inducing neuronal apoptosis was proved.(6) The up-regulation of Caspase-3, Caspase-8, Caspase-9, cytochrome c and Bax and the down-regulation of Bcl-2was found in cerebrum, the cerebellum and the optic lobe in vivo and in neurons in vitro suggesting that AVM could induce nerve cell apoptosis. After knocking down a-Syn gene the expression of pro-apoototic gene Caspase-3, Caspase-8, Caspase-9, cytochrome c, and Bax was significantly lower than before the interference and the expression of anti-apoototic gene Bcl-2was significantly higher than before the interference, indicating that a-Syn gene plays an important role in neuronal apoptosis indeced by AVM.(7) The up-regulation of a-Syn gene was found in cerebrum, the cerebellum and the optic lobe in vivo and in neurons in vitro after treated by AVM and the reverse effect was observed after knocking down a-Syn gene.(8) The activities of chymotrypsin, peptidyl glutamyl hydrolase and trypsin which are the three representative hydrolases in UPP pathway decreased significantly in cerebrum, the cerebellum and the optic lobe in vivo and in neurons in vitro after treated by AVM, indicating that AVM could affect the function of UPP pathway.(9) The up-regulation of IL-1β, IL-4, IL-6, IL-12β, IL-26, TNF-α and IFN-a and the down-regulation of IFN-y in cerebrum, the cerebellum and the optic lobe in vivo and the up-regulation of IL-1β,IL-4, IL-6and TNF-α and the down-regulation of IFN-γ in in neurons in vitro were found. The expression of IL-1β, IL-4, IL-6, TNF-α and IFN-y decreased after knocking down a-Syn gene. The results indicated that a-Syn gene could be involved in neuroinflammation.(10) The up-regulation of Hsp60, Hsp70and Hsp90in cerebrum, the cerebellum and the optic lobe in vivo and in neurons in vitro was found. The expression of Hsp60, Hsp70and Hsp90decreased after knocking down a-Syn gene. The results showed that AVM induced the stress response in brain tissues and in neurons of pigeon.In present study, it indicated that a-Syn gene play an important role in the nerve cell apoptosis induced by AVM. UPP pathway was blocked by AVM and then the expression of a-Syn gene increased abnormally. The increase expression of a-Syn gene could induce apoptosis not only directly but also indirectly by pass of neuroinflammation. Furthermore, AVM could induced the stress response in cerebrum, the cerebellum and the optic lobe in vivo and in neurons in vitro. This study supply theory and experimental data for AVM toxicology of birds.