Hexavalent Chromium Exposure Induces Intestinal Barrier Damage via Activation of the NF-κB Signaling Pathway and NLRP3 Inflammasome in Ducks

Abstract

Hexavalent chromium [Cr(VI)] is a dangerous heavy metal which can impair the gastrointestinal system in various species; however, the processes behind Cr(VI)-induced intestinal barrier damage are unknown. Forty-eight healthy 1-day-old ducks were stochastically assigned to four groups and fed a basal ration containing various Cr(VI) dosages for 49 days. Results of the study suggested that Cr(VI) exposure could significantly increase the content of Cr(VI) in the jejunum, increase the level of diamine oxidase (DAO) in serum, affect the production performance, cause histological abnormalities (shortening of the intestinal villi, deepening of the crypt depth, reduction and fragmentation of microvilli) and significantly reduced the mRNA levels of intestinal barrier-related genes (ZO-1, occludin, claudin-1, and MUC2) and protein levels of ZO-1, occludin, cand laudin-1, resulting in intestinal barrier damage. Furthermore, Cr(VI) intake could increase the contents of hydrogen peroxide (H₂O₂) and malondialdehyde (MDA), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-18 (IL-18) but decrease the activities of total superoxide dismutase (T-SOD), catalase (CAT), and glutathione reductase (GR), as well as up-regulate the mRNA levels of TLR4, MyD88, NF-κB, TNFα, IL-6, NLRP3, caspase-1, ASC, IL-1β, and IL-18 and protein levels of TLR4, MyD88, NF-κB, NLRP3, caspase-1, ASC, IL-1β, and IL-18 in the jejunum. In conclusion, Cr(VI) could cause intestinal oxidative damage and inflammation in duck jejunum by activating the NF-κB signaling pathway and the NLRP3 inflammasome.

Keywords: NF-κB; NLRP3; duck; hexavalent chromium; intestinal barrier.

Figure 1

Cr(VI) exposure induced intestinal damage. (A) Cr(VI) content in jejunum tissues. (B) Body weight. (C) Jejunum weight. (D) The length of jejunum. (E) Histopathological variation in jejunum tissues (scale bar = 200 μm). (F) Length of intestinal villus. (G) Crypt depth. (H) The ratio of villus height to crypt depth (VH/CD). All data were presented as mean ± SEM; n ≥ 3 for each group. The symbol “*” denotes a statistically significant difference from the control group (*P < 0.05, ***P < 0.005 and ****P < 0.001).

Figure 2

Effects of Cr(VI) exposure on intestinal epithelial barrier function. (A) PAS staining was used to determine the distribution of goblet cells (red arrows) in the intestine (scale bar = 200 μm). (B) Ultrastructure of the jejunum (scale bar = 2 μm). The red and green arrows represent changes in tight connections and microvilli, respectively. TJ stands for tight connection; MV stands for microvilli. (C) mRNA levels of genes related to the intestinal barrier. (D–G) Effects of Cr(VI) exposure on tight-junction protein expression levels in the duck jejunum. (E) ZO-1/GAPDH. (F) Occludin/GAPDH. (G) Claudin-1/GAPDH. (H) ImageJ analysis of MUC2 immunofluorescent staining results. (I) DAO content. (J) Immunofluorescence staining of MUC2 protein expression in jejunum tissue. The symbol “*” denotes a statistically significant difference from the control group (*P < 0.05, **P < 0.01, ***P < 0.005 and ****P < 0.001).

Figure 3

The effects of Cr(VI) on inflammation and oxidative stress in the jejunum. (A) TNF-α concentration. (B) IL-1β concentrations. (C) IL-18 concentrations. (D) H₂O₂ concentrations. (E) MDA concentrations. (F) T-SOD activity. (G) CAT activity. (H) GR activity. The symbol “*” denotes a statistically significant difference from the control group (*P < 0.05, **P < 0.01, ***P < 0.005 and ****P < 0.001).

Figure 4

The influence of Cr(VI) on the NF-κB signaling pathway in jejunum tissues. (A) mRNA levels of genes involved in the NF-κB signaling pathway. (B) Quantitative analysis of NF-κB signaling pathway-related protein expression. (C) Western blot reveals that expression levels of NF-κB signaling pathway-related proteins. (D) A heatmap depicts the relationship between NF-κB signaling pathway-related mRNA and protein levels in jejunum tissues. The symbol “*” denotes a statistically significant difference from the control group (*P < 0.05, **P < 0.01, ***P < 0.005 and ****P < 0.001).

Figure 5

Effects of Cr(VI) exposure on NLRP3 inflammasome activation in jejunum tissues. (A) Pyroptosis-related gene mRNA levels. (B) Quantitative analysis of pyroptosis-related factor protein levels. (C) Western blot demonstrates pyroptosis-related protein expression. (D) A heatmap depicts the relationship between the expression of pyroptosis-related genes and proteins in jejunum tissues. The symbol “*” denotes a statistically significant difference from the control group (*P < 0.05, **P < 0.01, ***P < 0.005 and ****P < 0.001).