Analysis of nitric oxide (NO) production in chicken cells
Chunhui Zhang (1,2), Andrew Bean1*, Kerri Bruce1, Tamara Gough1, Christina Rootes1, Cameron Stewart1, Weixian Zhang2* (1 Australian Animal Health Laboratory, Geelong, Australian) (2 Henan University of Animal Husbandry and Economy, Zhengzhou, China) * Corresponding author
Background:Nitric oxide is a mall gaseous, free-radical molecule. And it is a pluripotent, signaling and effector molecule too. Production of NO has two different Pathways:one is NOS dependent pathway, the other is NOS independent pathway... Free radicals as immune molecules, NO is important as a defence molecule against infectious organisms. Activated macrophages inhibit pathogen replication by releasing effector molecules, including NO. Other immune-system cells produce and respond to NO. NO regulates functional activity, growth and death of many immune and inflammatory cell types. However, the role of NO in nonspecific and specific immunity in vivo and in immunologically mediated diseases and inflammation is poorly understood. NO as an antiviral, its production by phagocytes in response to interferon-γ is associated with antimicrobial activity. In response to dsRNA, NO is rapidly produced in primary fibroblasts. Significant role in antiviral protection. NF-κB and interferon regulatory factor 1 participate in the induction of inducible nitric oxide synthase expression, which subsequently produces NO. NO pathway serves as a strategy to protect the host against viruses. We have previously shown that differences in iNOS expression may be important in the inflammatory response that contributes to pathology. Increased iNOS expression is associated with higher severity of H5N1 (Andrew et al.,2011). Increased NO is associated with a higher severity of H5N1 (Burggraaf et al., 2011).
Methods:TheNitrite/Nitrate colorimetric Assay Kit was used. NO production in DF-1 cells stimulated with LPS and Poly (I:C) were Measured, the production of NO in Blood and Spleen cells from chicken were compared, the production of NO in DF-1 cells infected with different Flu virus were compared.
Results:(i) Both of Nitrate standard curve and Nitrite are very similar, equation of Nitrite standard curve is y=0.02819x, equation of Nitrate standard curve is y=0.02914x. Nitrate curve is most appropriate for our sample analysis. (ii) In cells stimulated with LPS, maximum NO production was observed after 48 h. For poly(IC) stimulation, maximum NO production was at 24 h. Both responses were dose-dependent. (iii) Chicken primary cells were stimulated with LPS and poly(I:C), with TCSN assayed for NO production at 24 h and 48 h. Results indict that for LPS 24h is suitable, but the concentration should be equal or more than 100ug. But Ploy(I:C) is different. The research time should be equal or more than 48h. NO production was not dose-dependent. (iv)The production of NO by DF-1 cells infected with influenza A virus is not dose-dependent.
Discussion and conclusions: Chicken viral diseases have been a challenge for the large lost for many years in China vet clinic. It may be that iNOS expression in chickens becomes dysregulated and this may impact on disease severity through the overproduction of free radicals and the resultant damage to cellular function. reinfection is needed. Broilers always grow fast. Feed additives have play an important role for them. A better understanding of the inflammatory response following Chicken virus infection may help in developing new strategies and approaches for modulating a more positive outcome for these infections in vet clinic. New vaccine needs long research time, and it has a high cost. So, the probiotics such as Lactobacillus should be studied further. Possibly, we can prevent chicken virus disease with probiotics through regulating the expression of iNOS.
Thanks China Scholarship Council for the State Scholarship fund. (NO: 201608410295)