Vitamin D3 deficiency induced intestinal inflammatory response of turbot through nuclear factor-κB/inflammasome pathway, accompanied by the mutually exclusive apoptosis and autophagy

Abstract

Vitamin D₃ (VD₃) participated widely in the nuclear factor-κB (NF-κB)-mediated inflammation, apoptosis, and autophagy through the vitamin D receptor (VDR). However, the molecular mechanisms remain not understood in teleost. The present study investigated the functions of VD₃/VDR on intestinal inflammation, autophagy, and apoptosis of turbot in vivo and in vitro. Triple replicates of 30 fish were fed with each of three diets with graded levels of 32.0 (D₀), 1012.6 (D₁), and 3978.2 (D₂) IU/kg VD₃. Obvious intestinal enteritis was observed in the D₀ group and followed with dysfunction of intestinal mucosal barriers. The intestinal inflammatory response induced by VD₃ deficiency was regulated by the NF-κB/inflammasome signalling. The promotion of intestinal apoptosis and suppression of intestinal autophagy were also observed in the D₀ group. Similarly, VD₃ deficiency in vitro induced more intense inflammation regulated by NF-κB/inflammasome signalling. The mutually exclusive apoptosis and autophagy were also observed in the group without 1,25(OH)₂D₃ in vitro, accompanied by similar changes in apoptosis and autophagy increased apoptosis. The gene expression of VDRs was significantly increased with the increasing VD₃ supplementation both in vivo and in vitro. Moreover, VDR knockdown in turbot resulted in intestinal inflammation, and this process relied on the activation of inflammasome mediated by NF-κB signalling. Simultaneously, intestinal apoptosis was promoted, whereas intestinal autophagy was inhibited. In conclusion, VD₃ deficiency could induce intestinal inflammation via activation of the NF-κB/inflammasome pathway, intestinal apoptosis, and autophagy formed a mutually exclusive relation in teleost. And VDR is the critical molecule in those processes.

Keywords: NF-κB; apoptosis; autophagy; inflammasome; inflammation; vitamin D3; vitamin D3 receptor.

Figure 1

Inflammation, apoptosis, and autophagy of intestine with VD₃ treatments in vivo. (A) H&E staining of intestine (Black bars, 200 μm or 50 μm), Black arrows indicate widening of the intestinal lamina propria. (B) Alkaline phosphatase, acid phosphatase activities, and lysozyme activities. (C) The relative mRNA expression level of MUC2, MUC18, CLDN4, TRIC, JAM1, and ZO1. (D) The relative mRNA expression level of VDRA&B. (E) The mRNA expression levels of IL1B, TNF, IL8, IFNG, TGFB1, and IL10. (F) The mRNA expression levels of CASP3, BCL2, BAX, MAP1LC3B, and ATG16L1. (G, H) DNA fragmentation and quantification of the density of the 180 to 200 bp DNA band. (I-K) The level of ASC, intranuclear NF-κB p65, total IκB and phosphorylated IκB, CASP3, BCL2, MAP1LC3B, and ATG16L1 were analyzed and quantitated by western blot. The blots of ASC, IκBα, p-IκBα ^{ser32 ser36}, CASP3, BCL2, MAP1LC3B, and ATG16L1 were used for GAPDH loading control, while the blot of NF-κB p65 was used for Lamin B loading control. Error bars of columns denote SD (n = 3), and columns with different letters above them are significantly different (P < 0.05).

Figure 2

Inflammation, apoptosis, and autophagy of intestinal epithelial cells with VD₃ treatments in vitro. (A) The relative mRNA expression level of CLDN4, TRIC, JAM1, and ZO1. (B) The relative mRNA expression level of VDRA&B. (C) The mRNA expression levels of IL1B, TNF, IL8, IFNG, TGFB1, and IL10. (D) The mRNA expression levels of CASP3, BCL2, BAX, MAP1LC3B, and ATG16L1. (E) Confocal images of TUNEL assay (White bars, 50 μm). (F) Confocal images of MAP1LC3B fluorescence (White bars, 50 μm). (G-I) The level of ASC, intranuclear NF-κB p65, total IκB and phosphorylated IκB, CASP3, BCL2, MAP1LC3B, and ATG16L1 were analyzed and quantitated by western blot. The blots of ASC, IκBα, p-IκBα ^{ser32 ser36}, CASP3, BCL2, MAP1LC3B, and ATG16L1 were used for GAPDH loading control, while the blot of NF-κB p65 was used for Lamin B loading control. Error bars of columns denote SD (n = 3), and columns with different letters above them are significantly different (P < 0.05).

Figure 3

VDR knockdown in vivo on inflammation, apoptosis, and autophagy in the intestine. (A) The relative mRNA expression level of VDRA&B in the intestine treated with VDRA&B siRNAs. (B) The mRNA expression levels of IL1B, IL8, TGFB1, and IL10. (C) The mRNA expression levels of CASP3, BCL2, BAX, MAP1LC3B, and ATG16L1. (D-F) The level of ASC, intranuclear NF-κB p65, total IκB and phosphorylated IκB, CASP3, BCL2, MAP1LC3B, and ATG16L1 were analyzed and quantitated by western blot. The blots of ASC, IκBα, p-IκBα ^{ser32 ser36}, CASP3, BCL2, MAP1LC3B, and ATG16L1 were used for GAPDH loading control, while the blot of NF-κB p65 was used for Lamin B loading control. Error bars of columns denote SD (n = 3), and columns with different letters above them are significantly different (P < 0.05).