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. 2023 Sep 7;28(18):6500.
doi: 10.3390/molecules28186500.

Soybean Antigen Protein-Induced Intestinal Barrier Damage by Trigging Endoplasmic Reticulum Stress and Disordering Gut Microbiota in Weaned Piglets

Lei Wang  1 , Yujia Liu  1 , Daoliang Zhang  1 , Hongyan Ding  2 , Shibin Feng  1 , Chang Zhao  1 , Jinjie Wu  1 , Xichun Wang  1
Affiliations

Soybean Antigen Protein-Induced Intestinal Barrier Damage by Trigging Endoplasmic Reticulum Stress and Disordering Gut Microbiota in Weaned Piglets

Lei Wang et al. Molecules. .

Abstract

Endoplasmic reticulum (ER) stress is a crucial factor in the pathogenesis of intestinal diseases. Soybean antigenic proteins (β-conglycinin and soy glycinin) induce hypersensitivity reactions and intestinal barrier damage. However, whether this damage is associated with ER stress, autophagy, and the gut microbiome is largely unclear. Therefore, in this study, we aimed to investigate the effect of dietary supplementation with soy glycinin (11S glycinin) and β-conglycinin (7S glycinin) on intestinal ER stress, autophagy, and flora in weaned piglets. Thirty healthy 21-day-old weaned "Duroc × Long White × Yorkshire" piglets were randomly divided into three groups and fed a basic, 7S-supplemented, or 11S-supplemented diet for one week. The results indicated that 7S/11S glycinin disrupted growth performance, damaged intestinal barrier integrity, and impaired goblet cell function in piglets (p < 0.05). Moreover, 7S/11S glycinin induced ER stress and blocked autophagic flux in the jejunum (p < 0.05) and increased the relative abundance of pathogenic flora (p < 0.01) and decreased that of beneficial flora (p < 0.05). In conclusion, 7S/11S glycinin induces intestinal ER stress, autophagic flux blockage, microbiota imbalance, and intestinal barrier damage in piglets.

Keywords: autophagy; endoplasmic reticulum stress; gut microbiota; soybean glycinin; β-conglycinin.

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Conflict of interest statement

All authors report no conflict of interest.

Figures

Figure 1
Figure 1
(A) IgE level of weaned piglets in each group. (B) IgG1 level of weaned piglets in each group. (C) 5-HT level of weaned piglets in each group. (D) HIS level of weaned piglets in each group. Data are shown as mean ± standard deviation (SD). * p < 0.05, ** p < 0.01, compared with the control group.
Figure 2
Figure 2
(A) Venn diagram. (B) Core species analysis. (C) Pan species analysis. (D) ANOSIM analysis.
Figure 2
Figure 2
(A) Venn diagram. (B) Core species analysis. (C) Pan species analysis. (D) ANOSIM analysis.
Figure 3
Figure 3
(A) PCoA analysis. (B) NMDS analysis.
Figure 4
Figure 4
(A) The ratio of Firmicutes and Bacteroidetes. (B) LEfSe multi-level species difference discriminant analysis. (C) Evolutionary branch diagram. Data are shown as mean ± standard deviation (SD). ** p < 0.01, compared with the control group.
Figure 4
Figure 4
(A) The ratio of Firmicutes and Bacteroidetes. (B) LEfSe multi-level species difference discriminant analysis. (C) Evolutionary branch diagram. Data are shown as mean ± standard deviation (SD). ** p < 0.01, compared with the control group.
Figure 5
Figure 5
(A) The protein expression of Grp78, CHOP, p-IRE1α, XBP-1s, p-PERK and ATF-6 were measured using Western blot analysis. (B) mRNA levels of Grp78 and CHOP were measured using qRT-PCR analysis. (C) The ultrastructure of intestinal epithelial cells in piglets was observed using a transmission electron microscope; scale bar = 1 μm. The red arrow indicates the damaged endoplasmic reticulum in IPEC-J2 cells. Data are shown as mean ± standard deviation (SD). * p < 0.05, ** p < 0.01, compared with the control group.
Figure 5
Figure 5
(A) The protein expression of Grp78, CHOP, p-IRE1α, XBP-1s, p-PERK and ATF-6 were measured using Western blot analysis. (B) mRNA levels of Grp78 and CHOP were measured using qRT-PCR analysis. (C) The ultrastructure of intestinal epithelial cells in piglets was observed using a transmission electron microscope; scale bar = 1 μm. The red arrow indicates the damaged endoplasmic reticulum in IPEC-J2 cells. Data are shown as mean ± standard deviation (SD). * p < 0.05, ** p < 0.01, compared with the control group.
Figure 6
Figure 6
(A) The protein levels of LC3II and p62 were measured using Western blot analysis. (B) The relative expression levels of p62 mRNA were measured using qRT-PCR analysis. Data are shown as mean ± standard deviation (SD). ** p < 0.01, compared with the control group.
Figure 7
Figure 7
(A) The serum levels of DAO and D-lactate were measured using ELISA kits. (B) HE staining was used to assess intestinal villi and crypts (400×). (C) The GCs were observed using PAS staining (400×). (D) The ratio of villi and crypts. (E) The quantity of GCs. (F) The content of MUC2 was measured using ELISA kits. (G) The positive expressions of tight junction proteins (ZO-1, claudin-1, and occludin) were detected by IHC. Black arrows indicate localization and expression of target proteins. (H) The expression levels of p-ASK1 were measured by Western blot in the jejunum of piglets. (I) The intensity of fluorescence expression of TUNEL staining was statistically analyzed. Green fluorescence indicates apoptotic cells. Blue fluorescence labeled nuclei, green fluorescence labeled apoptotic cells. Data are shown as mean ± standard deviation (SD). * p < 0.05, ** p < 0.01, compared with the control group.
Figure 7
Figure 7
(A) The serum levels of DAO and D-lactate were measured using ELISA kits. (B) HE staining was used to assess intestinal villi and crypts (400×). (C) The GCs were observed using PAS staining (400×). (D) The ratio of villi and crypts. (E) The quantity of GCs. (F) The content of MUC2 was measured using ELISA kits. (G) The positive expressions of tight junction proteins (ZO-1, claudin-1, and occludin) were detected by IHC. Black arrows indicate localization and expression of target proteins. (H) The expression levels of p-ASK1 were measured by Western blot in the jejunum of piglets. (I) The intensity of fluorescence expression of TUNEL staining was statistically analyzed. Green fluorescence indicates apoptotic cells. Blue fluorescence labeled nuclei, green fluorescence labeled apoptotic cells. Data are shown as mean ± standard deviation (SD). * p < 0.05, ** p < 0.01, compared with the control group.
Figure 7
Figure 7
(A) The serum levels of DAO and D-lactate were measured using ELISA kits. (B) HE staining was used to assess intestinal villi and crypts (400×). (C) The GCs were observed using PAS staining (400×). (D) The ratio of villi and crypts. (E) The quantity of GCs. (F) The content of MUC2 was measured using ELISA kits. (G) The positive expressions of tight junction proteins (ZO-1, claudin-1, and occludin) were detected by IHC. Black arrows indicate localization and expression of target proteins. (H) The expression levels of p-ASK1 were measured by Western blot in the jejunum of piglets. (I) The intensity of fluorescence expression of TUNEL staining was statistically analyzed. Green fluorescence indicates apoptotic cells. Blue fluorescence labeled nuclei, green fluorescence labeled apoptotic cells. Data are shown as mean ± standard deviation (SD). * p < 0.05, ** p < 0.01, compared with the control group.
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