Soybean glycinin disrupted intestinal structural integrity related to aggravation of apoptosis and downregulated transcription of tight junction proteins in the intestine of juvenile grass carp (Ctenopharyngodon idella)

Abstract

Glycinin is a major anti-nutritional factor of soybean. Glycinin inhibits growth and impairs intestinal health in grass carp and Jian carp, but the underlying mechanisms are largely unknown. This study used grass carp to examine whether the negative influences of dietary glycinin are related to intestinal apoptosis signalling and tight junctions (TJs). The results showed that 8% dietary glycinin caused higher diamine oxidase activities and D-lactate content in the serum than did the control, indicating impairment of intestinal structural integrity. For the apoptosis indices, in the midgut and hindgut, glycinin aggravated the DNA fragmentation phenomenon, promoted caspase-3, −8, and − 9 activities, and increased cytochrome c protein abundance in the cytoplasm (apart from mitochondria) compared to those the control. In the midgut, glycinin increased the mRNA levels of pro-apoptotic molecules, including caspase-3, tumour necrosis factor-α (TNF-α), B-cell lymphoma 2 (Bcl-2)-associated X (Bax), and apoptotic protease activating factor 1 (Apaf-1), and decreased the mRNA levels of the anti-apoptotic molecule Bcl-2 compared to those in the control. In the hindgut, glycinin increased caspase-3, −8, and − 9, TNF-α, Fas-ligand (FasL), Bax, and Apaf-1 mRNA levels and decreased Bcl-2, myeloid cell leukaemia 1 (Mcl-1), and inhibitors of apoptosis proteins (IAP) mRNA levels compared to those in the control. For TJs, compared to that in the control, glycinin reduced the mRNA abundance of barrier-forming TJs—including claudin-c in the foregut; ZO-1, ZO-2b, occludin, and claudin-7a in the midgut; and ZO-1, ZO-2b, occludin, claudin-3c, claudin-7a, claudin-7b, and claudin-11 in the hindgut. In contrast, glycinin increased the mRNA abundance of pore-forming TJs, including claudin-15a and claudin-15b, in the midgut and hindgut. As an important nuclear factor of TJ transcription, hepatic nuclear factor 4 alpha (HNF-4α) protein abundance was reduced by glycinin in the midgut and hindgut. Glutamine alleviated apoptosis in the midgut. Glycinin aggravated apoptosis associated with both extrinsic and intrinsic signalling and disrupted TJ related to HNF-4α signalling in the midgut and hindgut, ultimately disrupting the intestinal structural integrity of grass carp.