NF-κB pathway took part in the development of apoptosis mediated by miR-15a and oxidative stress via mitochondrial pathway in ammonia- treated chicken splenic lymphocytes

摘要

Ammonia, a kind of gas with pungent smell, is harmful to livestock and people, and has bad influence on the atmosphere. However, the mechanism of splenic toxicity caused by ammonia is still poorly understood. The aim of present study was to investigate the effect of ammonia on chicken splenic lymphocytes from the perspective of apoptosis. Chicken splenic lymphocytes were divided into the control group and the two ammonium treatment groups (1 mmol/L and 5 mmol/L ammonia), and were cultured for 24 h. CCK-8, flow cytometry (FC), fluorescence microscope, quantitative real-time PCR (qRT-PCR), and Western blot were used to study the differences between different groups. The results showed that ammonia exposure increased the release of calcium (Ca)~(2+) and reactive oxygen species (ROS) from mitochondrion. Besides, we found an increase in mRNA levels of glutathione perox-idase (GPx), inflammation-related genes (nuclear factor-KB (NF-κB), cyclooxygenase-2 (COX-2), inducible nitric (iNOS), tumor necrosis factor-α (TNF-α), and transforming growth factor-β (TGF-β)), apoptosis-related genes (B-cell lymphoma-2 (BCL-2), Bcl-2 associated X protein (BAX), Cytochrome c (Cytc), apoptotic protease activating factor 1 (APAF1), Caspase-9, and Caspase-3), and an increase in protein levels of NF-kB, iNOS, BAX, Cytc, Caspase-9, and Caspase-3. At the same time, we found a decrease level of GPx protein expression, and a decrease level of glutathione S-transferase (GST) mRNA expression, and a decrease level of heme oxygenase-1 (HO-1) and BCL-2 mRNA and protein expression in splenic lymphocytes exposed to ammonia. Meanwhile, miR-15a expression increased under ammonia exposure. In summary, these results indicated that ammonia induced oxidative stress, promoted the release of Ca~(2+), Cytc, and ROS from mitochondria, and then induced mitochondria-mediated inflammatory response, finally triggered apoptosis in chicken splenic lymphocytes.