The involvement of miR-6615-5p/Smad7 axis and immune imbalance in ammonia-caused inflammatory injury via NF-κB pathway in broiler kidneys

Abstract

Ammonia (NH₃), a toxic gas, has deleterious effects on chicken health in intensive poultry houses. MicroRNA can mediate inflammation. The complex molecular mechanisms underlying NH₃ inhalation-caused inflammation in animal kidneys are still unknown. To explore the mechanisms, a broiler model of NH₃ exposure was established. Kidney samples were collected on day 14, 28, and 42, and meat yield was evaluated on day 42. We performed histopathological examination, detected miR-6615-5p and mothers against decapentaplegic homolog 7 (Smad7), and determined inflammatory factors and cytokines in kidneys. The results showed that excess NH₃ reduced breast weight and thigh weight, which indicated that excess NH₃ impaired meat yield of broilers. Besides, kidney tissues displayed histopathological changes after NH₃ exposure. Meanwhile, the increases of inducible nitric oxide synthase (iNOS) activity and nitric oxide content were obtained. The mRNA and protein expression of inflammatory factors, including nuclear factor-κB (NF-κB), cyclooxygenase-2, prostaglandin E synthases, and iNOS increased, indicating that NF-κB pathway was activated. T-helper (Th) 1 and regulatory T (Treg) cytokines were downregulated, whereas Th2 and Th17 cytokines were upregulated, suggesting the occurrence of Th1/Th2 and Treg/Th17 imbalances. In addition, we found that Smad7 was a target gene of miR-6615-5p in chickens. After NH₃ exposure, miR-6615-5p expression was elevated, and Smad7 mRNA and protein expression were reduced. In summary, our results suggest that NH₃ exposure negatively affected meat yield; and miR-6615/Smad7 axis and immune imbalance participated in NH₃-induced inflammatory injury via the NF-κB pathway in broiler kidneys. This study is helpful to understand the mechanism of NH₃-induced kidney injury and is meaningful to poultry health and breed aquatics.

Keywords: ammonia; broiler kidney; immune imbalance; inflammatory injury; miR-6615/Smad7 axis.

Figure 1

Effects of NH₃ on histopathology in chicken kidneys on D 42 (H&E staining, ×400). (A1 and A2) Histopathological observation in the control group. (B1 and B2) Histopathological observation in NH₃ group 1. (C1 and C2) Histopathological observation in NH₃ group 2. Abbreviations: C, cast; MC, mononuclear cell; rbc, red blood cell.

Figure 2

Effects of NH₃ on miR-6615-5p and Smad7 in chicken kidneys. (A) Complementary sites between miR-6615-5p and Smad7. (B) Relative expression of miR-6615-5p. (C) Relative mRNA expression of Smad7. (D) Relative protein expression of Smad7.

Figure 3

Effects of NH₃ on iNOS and NO in chicken kidneys. (A) iNOS activity. (B) Relative mRNA expression of iNOS. (C) Relative protein expression of iNOS. (D) NO content.

Figure 4

Effects of NH₃ on IκBα, NF-κB, COX-2, and PGEs in chicken kidneys. (A) Relative mRNA expression of IκBα. (B) Relative mRNA expression of NF-κB. (C) Relative mRNA expression of COX-2. (D) Relative mRNA expression of prostaglandin E synthases (PGEs). (E-F) Relative protein expression of IκBα, NF-κB, and COX-2.

Figure 5

Effects of NH₃ on cytokines in chicken kidneys. (A) Relative mRNA expression of IL-1β. (B) Relative mRNA expression of IL-2. (C) Relative mRNA expression of IL-4. (D) Relative mRNA expression of IL-6. (E) Relative mRNA expression of IL-12β. (F) Relative mRNA expression of IL-17. (G) Relative mRNA expression of IFN-γ. (H) Relative mRNA expression of TGF-β₁.

Figure 6

miR-6615/Smad7 and immune imbalance participated in NH₃-caused inflammatory injury through NF-κB pathway in broiler kidneys.