Fluoride stimulates the MAPK pathway to regulate endoplasmic reticulum stress and heat shock proteins to induce duodenal toxicity in chickens

Abstract

Fluoride is one of the essential trace elements for body. However, excessive fluoride poses a major threat to human and animal health. Fluorosis may cause pathological damage of the duodenum, but the underlying mechanism needs to be further studied. This study was to investigate the effects of long-term exposure to sodium fluoride (0, 500, 1,000, 2,000 mg/kg) on the duodenum of chickens. The results showed that after NaF exposure, intestinal epithelial cells were disarranged, necrotic or even exfoliated, goblet cells and mucus secretion were increased, and inflammatory response was induced in duodenal tissue. Oxidative stress, endoplasmic reticulum stress (ERs), and heat shock proteins (HSPs) are an adaptive response, however long-term, excessive changes are detrimental. Fluorosis activates ERs through IRE1, PERK and ATF6 pathways, increases the expression of HSP60, HSP70 and HSP90, and causes apoptosis and oxidative damage in duodenal tissue. In addition, fluorosis can activate the MAPK signaling pathway. This article can provide a reference for exploring the potential duodenal toxicity of sodium fluoride.

Keywords: MAPK; duodenal toxicity; endoplasmic reticulum stress; fluoride; heat shock protein.

Figure 1

Effects of different NaF doses on the chicken duodenum's histopathology (A) and ultrastructure (B). (A) The histopathological changes of the duodenum were observed by H&E staining. In the control group, the tissue structure was standard, and the duodenum damage was more evident with the increased NaF exposure. Intestinal epithelial cells are not arranged neatly, some cells are necrotic and shed (red arrow), goblet cells and mucus secretion are increased (blue arrow). (B) The ultrastructural changes of the duodenum were observed by TEM (scale bar 2.0 µm, 2000 ×). Mitochondrial swelling with vacuolization, cristae fragmentation or even disappearance (red letter M), and disordered ER arrangement (blue letter ER). Yellow letter N, nucleus.

Figure 2

Effects of NaF on Inflammatory factors in chicken duodenum. The mRNA levels of IL-1β, IL-2, IL-6 and IL-8 were detected by RT-PCR. *p < 0.05, **p < 0.01 and ***p < 0.001 were compared to the CON group.

Figure 3

Effects of NaF on OS in chicken duodenum. The levels of CAT, GSH, MDA and SOD were detected by commercial kits. *p < 0.05, **p < 0.01 and ***p < 0.001 were compared to the CON group.

Figure 4

Effects of NaF on apoptosis in chicken duodenum. The protein levels of BAX, BCL-2, Fas, Cyt-C, Cleaved Caspase3, Caspase8 and Caspase9 were detected by western blot. *p < 0.05, **p < 0.01 and ***p < 0.001 were compared to the CON group.

Figure 5

Effects of NaF on ERs in chicken duodenum. The protein levels of ATF4, ATF6, GRP78, IRE1, p-PERK, PERK, CHOP and Caspase12 were detected by western blot. *p < 0.05, **p < 0.01 and ***p < 0.001 were compared to the CON group.

Figure 6

Effects of NaF on HSPs in chicken duodenum. The protein levels of HSP60, HSP70 and HSP90 were detected by western blot. *p < 0.05, **p < 0.01 and ***p < 0.001 were compared to the CON group.

Figure 7

Effects of NaF on MAPK pathway in chicken duodenum. The protein levels of p-JNK, JNK, p-p38, p38, p-ERK and ERK were detected by western blot. *p < 0.05, **p < 0.01 and ***p < 0.001 were compared to the CON group.