In the present study, the survival rate of fish in each dietary treatment group did not differ significantly, indicating that a high dose of soy antigen protein did not kill carp. However, the growth performance (SGR, WG, FCR, and PER) of fish was significantly impaired by soybean meal. Interestingly, the WG decreased by 40% in soybean meal dietary treatment, while the 11 + 7S dietary treatment only decreased by 8.9%compared to dietary CON. In addition, the palatability of the feed containing soybean meal as a protein source is significantly lower than that of the two test diets containing purified soybean proteins. In the experiment, it was observed that the appetite of the 11 + 7S dietary treatment was much higher than that of the soybean meal dietary treatment. Secondly, soybean meal contains other anti-nutritional factors, such as proteinase inhibitors, lectin, saponin, and oligosaccharide that inhibits the growth performance (Table 3). Proteinase inhibitors can complex with the corresponding protease and inhibit its activity in the intestine of fish (Santigosa et al., 2010). Lectin can cause histological changes in the intestine of fish, which leads to a decrease in the growth performance of fish (Iwashita et al., 2009), and saponin is a crucial factor causing inflammation of the intestine (Kortner et al., 2012). The disparity in growth performance between the 11 + 7S and SBM dietary treatments suggested that the effect of other anti-nutritional factors in soybean meal on the growth performance of carp may be more pronounced. Moreover, this difference may be due to the body's adaptation to antigens. Carp developed a tolerance to β-conglycinin, glycinin, and other anti-nutrient factors of soybean meal so carp could tolerate high levels of anti-nutrient factors in the diet. The previous study by Uran (2009) showed that the gene expression levels of IL-1β and TNF-1β significantly increased when fed with soybean meal to carps in the early stage of intestinal inflammation, which began to recover after five weeks (Urán et al., 2009). The tolerance effect of the fish immune system to anti-nutritional factors in soybean meal requires further investigation.
The intestinal barrier can prevent the invasion of pathogenic bacteria and protect the health of the fish, while a tight junction is an integral part of the intestinal barrier. Several studies indicate that occludin, claudin-3c, and claudin-11 play a barrier-forming role in fish, whereas claudin-7 appears to have pore-forming characteristic (Chasiotis et al., 2011; Chasiotis et al., 2012; Krause et al., 2010). Previous studies on mammals found that β-conglycinin reduces the mRNA expression of tight junction: occludin and ZO-1, and increases the permeability of intestinal epithelial cells (Zhao et al., 2014), which shows that β-conglycinin directly damages intestinal epithelial cells and destroy it’s integrity. Furthermore, previous studies reported that addition of glycinin in the fish diet significantly reduced the expression of tight junction proteins like occludin, claudin3c, and claudin7 in the intestine (Jiang et al., 2015; Zhao et al., 2017). In the present study, both soybean meal and the mixture of β-conglycinin and glycinin significantly reduced the gene expression level of occludin and claudin-3c in the MI and DI of carps and increased the mRNA levels of claudin-11 in PI and DI. These findings suggest that soy meal and soy antigen protein may damage tight junction components.
Pre-inflammatory cytokines such as IL-1β, TNF-α and anti-inflammatory cytokines such as TGF-β1 play important roles in regulating intestinal inflammation (Fast et al., 2007). Although the anti-nutritional factors in soybean protein cause inflammation in the intestine of fish, some results showed that glycinin and β-conglycinin were the cause of intestinal inflammation, which increased the gene expression of tumor necrosis factor (TNF-α) and interleukin-1 β (IL-1β) (Li et al., 2017; Jiang et al., 2015). In our experiment, both soybean meal and the mixture of β-conglycinin and glycinin increased the expression of TNF-α and IL-1β in PI and DI. Furthermore, both soybean meal and the mixture of β-conglycinin and glycinin decreased the expression of TGF-β1 in PI and MI. These results indicated that whether it was soybean meal or these two antigen proteins to replace fish meal induced intestinal inflammation and the most severe place was at DI. Interestingly, although soybean meal has more anti-nutritional factors, the gene expression of a pro-inflammatory factor in the soybean meal dietary treatment is less than that of the 11 + 7S dietary treatment, indicating that soy antigen protein is the main factor causing intestinal inflammation.
The intestinal mucosal system is in a dynamic equilibrium between cell proliferation and apoptosis and both inflammation and oxidative stress can induce cell apoptosis and ultimately affect health (Fuchs et al., 2011; Buttke et al., 1994). Although the three main apoptotic pathways are the mitochondrial pathway, death-ligand pathway, and endoplasmic reticulum pathway, caspase plays a vital role in the activation and apoptotic cascade reaction, which eventually leads to apoptosis, caspase-3 is considered to be the essential enzyme in the process of apoptosis. Previous experiments on Atlantic salmon Salmo salar have shown that dietary soybean meal increased the mRNA levels of apoptosis in the intestine (Sperstad et al., 2007; Sahlmann et al., 2013), both the purified glycinin and β-Conglycinin also induced apoptosis in the intestine of fish (Jiang et al., 2015; Duan et al., 2019). In the present study, although the gene expression of caspase-3 in the PI and MI did not change significantly, soybean meal and the mixture of β-conglycinin and glycinin increased the gene expression of caspase in the DI. In addition, as the key promoter in the death ligand apoptosis pathway, the expression levels of caspase-8 increased significantly in both the SBM and 11 + 7S dietary treatment, whereas it had not changed in PI and MI. This data suggests that the apoptosis triggered by soybean meal and the combination of β-conglycinin and glycinin may have occurred predominantly in DI cells. Furthermore, soybean meal did not raise the gene expression of caspase-9 in DI of carp, although the mixture of β-conglycinin and glycinin increased the gene expression of caspase-9 in DI. The previous studies showed that caspase-9 is an important initiating factor in mitochondrion dependent apoptosis pathway (Sharifi et al., 2009). This suggests that the soy antigen protein is the primary agent responsible for triggering apoptosis in intestinal cells; nevertheless, this hypothesis has to be investigated further. Such studies show that anti-nutritional factors in soybean protein can destroy the tight junction of the intestine, induce intestinal apoptosis and cause oxidative damage to the intestine, which eventually results in poor growth performance.
AMPK is a highly conserved protein kinase that widely exists in eukaryotes. It can regulate the metabolism of fatty acids, cholesterol, and glucose and affect nutrients’ metabolism through downstream proteins (Hayashi et al., 2000; Barnes et al., 2004). The expression of AMPK increased in the body under a low energy state and phosphorylated to provide energy. In the present study, the expression of AMPK in the intestine shows different results of soybean meal and the mixture of β-conglycinin and glycinin. Soybean meal significantly increased the expression level of AMPK in MI, whereas the combination of β-conglycinin and glycinin significantly decreased it in PI and DI; however, the mechanisms underlying these results require further investigation. ACC is one of the target proteins regulated by AMPK and is the rate-limiting enzyme of fatty acid synthesis (Abu-Elheiga et al., 1995). ACC is phosphorylated and its activity declines when fatty acids are delivered into mitochondria to create ATP (Hardie et al., 2006). Based on the findings of the current study, which show that both soybean meal and the combination of β-conglycinin and glycinin decreased the expression level of ACC in PI, MI, and DI, and taking into account the results of the metabolic changes, it is possible that both soybean and the purified soy antigen protein significantly reduce the restriction of ACC on the synthesis of fatty acids, allowing for their consumption. mTOR is considered a direct receptor of ATP level, usually inhibited under low nutritional conditions, such as low amino acid and cellular ATP content. 4E-BP is the downstream effector of TOR, and the improvement of its expression level would reduce the expression of TOR (Azar et al., 2009; Tain et al., 2009). In the present study, soybean meal decreased the expression level of TOR in PI and MI and increased the level of 4E-BP in PI, while the mixture of β-conglycinin and glycinin decreased in PI, MI, and DI and increased the level of 4E-BP in PI, MI, and DI. The differential expression of four energy control genes indicates that soybean meal and the purified soy antigen protein may influence the energy metabolism of fish intestines, particularly DI.
Metabolomics is commonly used to study the end products of cell metabolism (Wang et al., 2011). Taurine and cysteine were decreased in the muscle of turbot fed a plant-based diet, whereas glucose and tyrosine were increased, and dimethylamine and threonine were decreased in the liver metabolome (Wei et al., 2017). Based on the pathway analytic result in our study, glycerophospholipid metabolism and sphingolipid metabolism in intestines had been changed by the mixture of β-conglycinin and glycinin. Furthermore, lipids and lipid-like molecules in the intestines significantly increased. The changes in lipid substances and lipid metabolism pathway in the intestine indicated that soybean meal significantly increased the consumption of lipids in the intestine of fish to supplement the intestine with energy consumed by oxidative damage and cell regeneration, which was similar to the decrease of the expression level of ACC. Moreover, the fold change of diosmetin and genistein between the 11 + 7S and Control dietary treatments was 8.6 and 0.6, respectively, which means that the mixture of β-conglycinin and glycinin increased the content of diosmetin 8.6 and 0.6 times. Diosmetin, one flavonoid complex distributed widely in each plantation, has a strong antioxidant effect and anti-bacterial functions (Boutin et al., 1993). The previous studies showed that glycinin and β-conglycinin could cause oxidative damage to the fish intestine and reduce the activity of intestinal antioxidant enzymes (Zhang et al., 2014; Jiang et al., 2015; Luo et al., 2023). Therefore, carp seems to absorb and utilize genistein and diosmetin from the diet to compensate for decreased antioxidant capacity in the intestine. The above data analysis suggests that the combination of β-conglycinin and glycinin can alter the intestinal energy consumption.
Although AKG did not influence feed utilization rate and weight gain in the present study, it effectively prevented the negative influences, such as reducing the expression of inflammatory cytokines, including IL-1 and TNF, in the DI. Furthermore, AKG improved the expression of anti-inflammatory cytokines in the DI and reduced the injury caused by β-conglycinin and glycinin on the apoptosis and tight junction of intestinal epithelial cells. Additionally, according to the metabolic pathway analysis, AKG can influence alanine, aspartate, glutamate metabolism, sphingolipid metabolism, pyrimidine metabolism, arginine biosynthesis, pantothenate and COA biosynthesis, and glycophorophospholipid metabolism. As a result, the availability of AKG has the potential to prevent the damage that soybean protein would otherwise cause to the intestinal health of fish.