PTEN/AKT/mTOR pathway involvement in autophagy, mediated by miR-99a-3p and energy metabolism in ammonia-exposed chicken bursal lymphocytes

Abstract

Emission of atmospheric ammonia (NH₃) is an environmental challenge because of its harmful effects on humans and animals including birds. Among all organisms, NH₃ is highly sensitive to birds. Autophagy plays a critical role in Bursa of fabricius (BF)-mediated immune responses against various hazardous substances. Therefore, we designed our work to demonstrate whether NH₃ can induce autophagy in broiler chicken BF. In this study, the downregulated levels of mammalian target of rapamycin and light chain-3 (LC-Ⅰ), as well as the upregulated levels of phosphate and tensin homology (PTEN), protein kinase B (AKT), autophagy related-5, light chain-3 (LC3-Ⅱ), Becline-1, and Dynein, were found. Our results of transmission electron microscopy displayed signs of autophagosomes/autophagic lysosomes, and immunofluorescence assay displayed that NH₃ exposure reduced the relative amount of CD8⁺ B-lymphocyte in chicken BF. Exposure of NH₃ led to energy metabolism disturbance by decreasing mRNA levels of glucose metabolism factors aconitase-2, hexokinase-1, hexokinase-2, lactate dehydrogenase-A, lactate dehydrogenase-B, pyruvate kinase, phosphofructokinase and succinate dehydrogenase complex unit-B, and adenosine triphosphates (ATPase) activities (Na⁺/K⁺ ATPase, Ca²⁺ ATPase, Mg²⁺ ATPase, and Ca/Mg²⁺ ATPase). Moreover, phosphate and tensin homology was found as target gene of microRNA-99a-3p which confirmed that high concentration of NH₃ caused autophagy in chicken BF. In summary, these findings suggested that ammonia induced autophagy via miR-99a-3p, the reduction of ATPase activity, and the alteration of autophagy-related factors, and energy metabolism mediation in BF. Our findings provide information to assess the harmful effects of NH₃ on chicken and clues for human health pathophysiology.

Keywords: ATPase; NH(3); PTEN/AKT/mTOR signaling pathway; autophagy; miR-99a-3p.

Figure 1

Detection of autophagosome formation by TEM. Experimental groups displayed as low, middle, and high NH₃-treated groups. The black arrows indicate autophagosomes/autophagic lysosomes in cytoplasm.

Figure 2

The estimation of CD8⁺ B-lymphocyte by immunofluorescent assay in broilers BF. Experimental groups displayed as low, middle, and high (A–I) NH₃-treated groups.

Figure 3

ATPase activities were measured at 14th, 28th, and 42nd d in all experimental groups. Experimental groups displayed as low, middle, and high NH₃-treated groups. (A) Na⁺/K⁺ ATPase, (B) Ca⁺⁺/Mg⁺⁺ ATPase, (C) Ca⁺⁺ ATPase, and (D) Mg⁺⁺ ATPase. Bar graphs represent data with mean ± SD. Uppercase alphabets displayed significant difference (P < 0.05) of same group at different conducting time, and lowercase alphabets displayed significant difference (P < 0.05) of different groups at the same time.

Figure 4

Relative mRNA ACO-2 (A), HK-1 (B), HK-2 (C), LDHA (D), LDHB (E), PFK (F), PK (G), and SDHB (H) and protein levels (I) of glucose metabolism factors in chicken BF. Experimental groups were displayed as low, middle, and high NH3-treated groups. Bar graphs represent data with mean ± SD. Uppercase alphabets displayed significant difference (P < 0.05) of same group at different conducting time, and lowercase alphabets displayed significant difference (P < 0.05) of different groups at same conducting time.

Figure 5

Relative miR-99a-3p and PTEN levels in chicken BF. Experimental groups displayed as low, middle, and high NH₃-treated groups. (A) alignment of miR-99a-3p with the predicted target region PTEN 3ʹ UTR, (B) levels of miR-99a-3p, and (C) levels of PTEN. Bar graphs represent data with mean ± SD. Uppercase alphabets displayed significant difference (P < 0.05) of same group at different conducting time, and lowercase alphabets displayed significant difference (P < 0.05) of different groups at same conducting time.

Figure 6

Relative mRNA and protein levels of autophagy-related genes in chicken BF. Experimental groups were displayed as low, middle, and high NH₃-treated groups. Bar graphs represent data with mean ± SD. Uppercase alphabets displayed significant difference (P < 0.05) of same group at different conducting time, and lowercase alphabets displayed significant difference (P < 0.05) of different groups at same conducting time.

Figure 7

Heat map showing relative mRNA levels of different genes involving in multiple signaling pathways including glucose metabolism and autophagy. Different colors representing the expression levels of different autophagy and glucose metabolism genes in broilers BF. Red color displaying the highest expression level, white color displaying medium expression level, and blue color displaying relative lowest expression level of autophagy and glucose metabolism genes. The experimental groups including low, middle, and high NH₃-treated groups.