Hydrogen Sulfide Gas Exposure Induces Necroptosis and Promotes Inflammation through the MAPK/NF- κ B Pathway in Broiler Spleen

Abstract

Hydrogen sulfide (H₂S) is one of the main pollutants in the atmosphere, which is a serious threat to human health. The decomposition of sulfur-containing organics in chicken houses could produce a large amount of H₂S, thereby damaging poultry health. In this study, one-day-old broilers were selected and exposed to 4 or 20 ppm of H₂S gas (0-3 weeks: 4 ± 0.5 ppm, 4-6 weeks: 20 ± 0.5 ppm). The spleen samples were collected immediately after the chickens were euthanized at 2, 4, and 6 weeks. The histopathological and ultrastructural observations showed obvious necrosis characteristics of H₂S-exposed spleens. H₂S exposure suppressed GSH, CAT, T-AOC, and SOD activities; increased NO, H₂O₂, and MDA content and iNOS activity; and induced oxidative stress. ATPase activities and the expressions of energy metabolism-related genes were significantly decreased. Also, the expressions of related necroptosis (RIPK1, RIPK3, MLKL, TAK1, TAB2, and TAB3) were significantly increased, and the MAPK pathway was activated. Besides, H₂S exposure activated the NF-κB classical pathway and induced TNF-α and IL-1β release. Taken together, we conclude that H₂S exposure induces oxidative stress and energy metabolism dysfunction; evokes necroptosis; activates the MAPK pathway, eventually triggering the NF-κB pathway; and promotes inflammatory response in chicken spleens.

Figure 1

Histopathological and ultrastructural changes in chicken spleens. Histopathological changes and ultrastructural changes in chicken spleen tissues after 6 weeks of H₂S exposure. (a) represents histopathological changes of chicken spleen tissues (400x). A: the normal spleen cells including the central artery (yellow box) and trabecula (green box). B: abundant immune cells in the control spleen including lymphocytes (green box) and macrophages (yellow box). C–F: the spleen damage features: white pulp hyperplasia (C), multiplied lymphatic nodules (D), red pulp congestion (E), splenic cord hyperplasia (F). G: reduced lymphocytes (green arrow) and macrophages (yellow arrow). H: spleen cell necrotic features: karyorrhexis (green box), karyolysis (yellow box), and hematocytosis (red box). (b) represents ultrastructural changes of chicken spleen tissues. A: the normal mitochondrial structure (black box). B: spleen cell necrotic features: mitochondria swelling and vacuolation (yellow box), cell membrane breakage, dissolution, and cytosolic content spillover, accompanied with extensive formation of vesicles (red box).

Figure 2

The relative expressions of related necroptosis in chicken spleens. The effect of H₂S on the mRNA (a) and protein (b) expressions of related necroptosis in chicken spleens. The results are from at least three independent experiments. Data are represented as the means ± SD (n = 10). β-Actin and GAPDH were selected as the reference of mRNA and protein expressions, respectively. ∗ shows significant difference from the corresponding control groups (p < 0.05); ∗∗ shows significant difference from the corresponding control groups (p < 0.01).

Figure 3

The antioxidant capacity in chicken spleens. The effect of H₂S on the antioxidant capacity in chicken spleens. They represent GSH, CAT, T-AOC, SOD and iNOS activities, and NO, H₂O₂ and MDA contents. ∗ shows significant difference from the corresponding control groups (p < 0.05); ∗∗ shows significant difference from the corresponding control groups (p < 0.01).

Figure 4

The ATPase activities and energy metabolism-related expressions in chicken spleens. The effect of H₂S on energy metabolism of chicken spleens. (a) represents the activities of Na⁺-K⁺-ATPase, Ca²⁺-Mg²⁺-ATPase, Ca²⁺-ATPase, and Mg²⁺-ATPase of spleen tissues. (b) and (c) represent the mRNA and protein expressions of spleen tissues, respectively. The results are from at least three independent experiments. Data are represented as the means ± SD (n = 10). β-Actin and GAPDH were selected as the reference of mRNA and protein expressions, respectively. ∗ shows significant difference from the corresponding control groups (p < 0.05); ∗∗ shows significant difference from the corresponding control groups (p < 0.01).

Figure 5

The activation of the MAPK pathway. The effect of H₂S on MAPK activation in chicken spleens. (a) and (b) represent the effect of H₂S on the mRNA (a) and phosphorylated protein (b) expressions of JNK, ERK, and p38 in chicken spleens. The results are from at least three independent experiments. Data are represented as the means ± SD (n = 10). β-Actin and total JNK, total ERK, and total p38 were selected as the reference of mRNA and phosphorylated protein expressions, respectively. ∗ shows significant difference from the corresponding control groups (p < 0.05); ∗∗ shows significant difference from the corresponding control groups (p < 0.01).

Figure 6

The activation of the NF-κB pathway and the expressions of inflammatory factors and HSP in chicken spleens. The effect of H₂S on the expressions of the NF-κB pathway (a) and mRNA (b) and protein (c) expressions of TNF-α, IL-1β, HSP70, and HSP90 in chicken spleens. The results are from at least three independent experiments. Data are represented as the means ± SD (n = 10). β-Actin and GAPDH were selected as the reference of mRNA and protein expressions, respectively. ∗ shows significant difference from the corresponding control groups (p < 0.05); ∗∗ shows significant difference from the corresponding control groups (p < 0.01).