| Selenium (Se) is recognized as an essential nutritional trace element for organisms. It plays a vital part in normal physiology of a wide range of organisms, including birds. There have been considerable evidences suggest that Se plays important roles in many aspects of health. Se defi-ciency could be correlated with the incidence of Keshan desease and Kaschin-Beck disease in human and white muscle disease of livertock. It was also found that Se is essential for the normal function of brain. Neurodegeneration disease, for example, epilepsy, PD and AD were correlated with Se-deficiency, and Se compound was the first choice for therapy. The peculiar pathological changes of brain injury in avians, encephalomalacia is also correlated with Se-deficiency. Se plays the biological function through the selenoproteins. Until now,30seleproteins from17families were identified. Selenoprotein W (SelW) was one of the selenoprotein families. It was detected in many tissues of humanbeing and animals. The level of SelW in brain was higher than other organs. Especially in embryo brain, the expression of SelW was prior to other selenoproteins. SelW played an important role in brain for its antioxidant function, but the mechanism of SelW in brain injury which was induced by Se-deficiency remained unclear.For further research of the function of SelW and the mechanism in brain injury induced by Se-deficiency, the experiments in vivo and vitro were carried out:Two hundred male chickens (1day old; Weiwei Co. Ltd., Harbin, China) were divided into two groups (100chickens per group). The chickens fed either the commercial granulated diet (C group) or the Se-deficient granulated diet (L group, from the Se-deficient region of Heilongjiang Province in China, containing0.033mg/kg) for55days. Food and water were provided ad libitum. Following euthanasia with sodium pentobarbital, the Brain tissues (including Cerebral gray matter, Cerebral white matter, marrowbrain, cerebellum, thalamus and brain stem) were quickly removed at15,25,35,45and55days old, some of the tissues were turned out homogenate for the dectection of antioxidant index; some were fixed in10%formalin in phosphate buffer, for the detection of apoptosis in tissues by TUNEL; others blotted and then rinsed with ice-cold sterile deionized water, frozen immediately in liquid nitrogen, and stored at-80℃until required.The chicken embryo neurons were isolated and cultured. LD50of H2O2for neurons was measured by MTT assay. The conditions of cell transfection were optimized through the transfection of siRNA and PcDNA3.1/PEGFP. The SelW RNAi and overexpression cell models were stimulated by H2O2, which concentrations were setted by LD50(10,20and50μM). The expression levels of target gene wer detected by quantitive real-time PCR (qPCR).Based on the Se-deficeincy model of chicken, the SelW RNAi and overexpression neurons models, the expression levels of SelW, apoptosis factors (P53、caspase-3、Bax、Bak-1、Bcl-2) and inflammation cytokines (cox-2、PTGEs、TNF-α、iNOS、NF-κB) were detected by qPCR. The protein expression of cox-2, PTGEs, NF-κB, caspase-3, Bax, Bcl-2and SelW were detected by Western blot. The apoptosis of brain tissues and neurons were detected by TUNE and AO/EB assay.The results showed that:1. The Se-deficeincy model of chicken was established successfully through the observation of clinical symptoms and pathological changes.2. The results of antioxidant index showed that the content of GSH and the activity of GSH-Px were decreased; the content of H2O2was increased accompany by the activity of CAT were decreased; the content of GSH and the activity of iNOS were increased; the content of MDA and-OH were also increased. It assumed that the loss of antioxidant ability might be the mechanism of chicken brain injury induced by Se-deficeincy.2. The qPCR results showed that the expression level of SelW in overexpression model was3-4times higher than the control group, but it was only50%in RNAi models. It means that the two models were established successfully. For each blank of culture plate (six blanks), the best condition of cell transfection were4μL HP reagent and1μg PcDNA3.1/SelW for overexpression model;30nM siRNA and3μL RNAiMAX for RNAi model3. The expression levels of P53, caspase-3, Bax and Bak-1mRNA in brain tissues of Se-deficeincy chicken were increased, but the level of BCL-2were increased firstly and then decreased, there were significant differences between the control group and the L group (P<0.05). In overexpression model, the levels of P53, caspase-3, Bax and Bak-1were decreased, but the level of BCL-2was increase; in RNAi model, the results were opposite. Statistical datas also showed the significant differences. And the ratio of cell apoptosis was higher in RNAi model. The levels of protein expression of caspase-3, Bax and Bcl-2were accordated with the levels of mRNA.It could be concluded that brain could be protected from oxidative stress through the expression levels of apoptosis factors suppressed by SelW.4. The expression levels of cox-2, PTGEs, TNF-a, iNOS and NF-κB mRNA of brain tissues of Se-deficeincy chicken were increased. There were significant differences between the control group and the L group (P<0.05). The levels of protein expression of cox-2, PTGEs and NF-κB were in accord with the levels of mRNA. In overexpression model, the levels of cox-2, PTGEs, TNF-α, iNOS and NF-κB mRNA were decreased, but in RNAi model, the results were opposite. The levels of protein expression of cox-2, PTGEs and NF-κB were in accord with the levels of mRNA. Statistical datas also showed the significant differences. It could be concluded that brain could be protected from oxidative stress through the expression level of NF-κB suppressed by SelW.In conclusion, the results showed that the chicken brain injury could be induced by Se-deficeincy, and the expression levels of brain tissues were regulated by the content of Se in feed. SelW could protect the inflammation and apoptosis in brain from the expression of inflammation cytokines and apoptosis factors. The expression level of SelW was decreased in chicken brain under Se-deficeincy, which leaded to the antioxidant ability decreased. It might be one of the mechanisms that the chicken brain injury induced by Se-deficeincy,... |
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