Growth performance and diarrhea incidence
Due to the post-weaning piglets are usually exposed to environmental, nutritional, and psychological stress responses, the combined and additive effects of these factors directly or indirectly contributed to the results of increased diarrhea which can deteriorate gut health of weaned piglets. In the current study, compared with the NC group, piglets fed the diet with supplementation of 50–200 mg kg− 1 GE had lower diarrhea incidences. In a previous study, Ezekwesili et al. investigated the underlying mechanism of antidiarrheal effect of GE, and they suggested that phenolics and alkaloids in GE present antimicrobial activity as well as reduce gastrointestinal motility of piglets [25]. Similarly, Morales et al. and Jaisinghani also demonstrated that quercetin, as a main active phenolics of GE, not only have a good antibacterial activity, but also could reduce intracellular calcium release of the sarcoplasmic reticulum to relaxes smooth muscles and inhibit bowel contraction [26, 27]. Collectively, due to GE was rich in bioactive composition, such as phenolics, alkaloids, etc., which display a remarkable array of antimicrobial and antioxidant activities [28], dietary addition of GE might be advantageous in preventing from ETEC infection, and improving gut health in weaned piglets.
Intestinal mucosal barrier
In general, diarrhea disease cause by enterotoxigenic ETEC infections is a major risk factor for impaired intestinal structure and barrier function of piglets. It has been reported that claudins and occludins are considered to the tight junction protein components of that primarily regulated permeability of uncharged and charged molecules, respectively. Further, ZO-1 is the adaptor protein of that modulated the actin cytoskeleton [29, 30], and NHE3 is a primary mediator of the absorptive route for Na+ entering the intestinal epithelium from the lumen [31]. Thus, all of them play important roles in mediating functional integrity of the junction in epithelial and endothelial cells of intestine [29–31]. Our results showed that ETEC decreased the expression of epithelial tight junctions such as claudin-1, occludin, ZO-1 and NHE3, thereby in turn increased cellular permeability and disturbed intestinal mucosal barrier. Subsequently, luminal antigens rather than bacteria may enter the lamina propria, resulting in inflammation [32]. However, weaned piglets fed a diet supplementation with GE (50–200 mg kg− 1 in the diet) increased the proteins expression of claudin-1, occludin, ZO-1 and NHE3, which are crucial for the formation of a semi-permeable mucosal barrier and the recovery of the barrier function of intestinal tight junctions compared with the NC group. Furthermore, previous studies also suggested that GE was rich in phenolics [33], and phenolics has a positive effect on gut health [34]. Specifically, with respect to quercetin and myricetin, as the main phenolic constituents in GE, which have been demonstrated to enhance the intestinal barrier function [35]. Consequently, it is assumed the abundant phenolics in GE exerted anti-inflammatory effect [36] to improve the intestinal barrier function and increase gut mucosal integrity of piglets.
Feces metabolomics
In mammals, L-pipecolic acid has long been recognized as a metabolite of lysine degradation pathway [37]. In this pathway, peroxisomal sarcosine oxidase (PSO) can catalyze L-pipecolic acid and oxygen to give (S)-2,3,4,5-tetrahydropiperidine-2-carboxylate and hydrogen peroxide (H2O2). As seen in Table 3 and Fig. 6, the findings of our study indicated that L-pipecolic acid was significantly lower in NC compared with BC group and suggested that ETEC might activated the reactions mentioned above, which resulting in the consumption of L-pipecolic acid and the production of H2O2. Whereas the H2O2 accumulated can induced disruption of intestinal epithelial barrier function by a mechanism involving phosphatidylinositol 3-kinase and c-Src kinase [38, 39]. Here, consisted with the results of immunohistochemistry in the present study, and it suggested that H2O2-induced oxidative stress in gut might has been considered to be one of the important pathogenic mechanisms in NC compared with BC group, which disrupted intestinal epithelial tight junctions and barrier functions.
As was well known that the catecholamines are generally associated with stressful events that result in high levels of gram-negative pathogens, such as Escherichia coli [40]. Based on S50 vs NC group, the results suggested that dietary addition with 50 mg kg− 1 GE can decrease the production of stress hormones, such as catecholamines (epinephrine and normetanephrine), and increase the production of 3-methoxytyramine (an in-active metabolite of dopamine) through tyrosine metabolism and catecholamine biosynthesis pathways and finally to inhibit the growth of Escherichia coli and resist oxidative stress in gut. In addition, it has been reported that caffeine can increase the intracellular calcium levels through directly effects on metabolic phosphorylase-like enzymes (PHOS) regulation and calcium mobilization from the sarcoplasmic reticulum [41]. Our results revealed the caffeine in feces was downregulated in S50 compared with NC group and suggested it may be beneficial to a decrease in cytosolic calcium to inhibit pathophysiologic processes which resulting in diarrhea [42]. Moreover, previous studies have suggested that nuclear translocation of nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) can induce melatonin synthesis in macrophages [43]. Whereas GE in S50 group can attenuate inflammatory response, achieve its antioxidant and antimicrobial actions and protect the intestinal epithelial barrier, resulting in decrease of intestinal melatonin synthesis in macrophages compared with the NC group. Meanwhile, small doses of melatonin in S50 group may be propitious to increase intestinal motility [44] to improve digestion and absorption of nutrients in gut. Thus, it suggested that the downregulation of melatonin of piglets in S50 group probably play an important role in increase of ADG.
Based on the fecal metabolomic data of S100 vs NC group, it showed that 100 mg kg− 1 GE upregulated the level of biliverdin and downregulated the level of 5-aminolevulinic acid in gut via porphyrin metabolism pathway. This process started as the condensation of glycine and succinyl-CoA by 5-aminolevulinate synthase (ALAS), and generated 5-aminolevulinic acid. Presumably, the resulting 5-aminolevulinic acid has two fates. On one hand, it may be finally converted into biliverdin via a series of steps, leading to the accumulation of biliverdin in gut (Fig. 6). The biliverdin generated in this process can protect intestine from oxidant and inflammatory injury [45, 46]. On the other hand, 5-aminolevulinic acid was also potentially absorbed into the blood, resulting in the high level of 5-aminolevulinic acid in serum via glycine and serine metabolism pathway (Fig. 7). Here, 5-aminolevulinic acid reduced intracellular carbon monoxide concentration and inhibited oxidative stress and inflammation response [47]. Moreover, phosphorylcholine was downregulated in S100 compared with NC group, which associated with the phosphatidylcholine and phospholipid biosynthesis pathways. It means that most of choline might not be break down in gut but was absorbed into the blood in S100 group, and then choline went to the betaine metabolism pathway and was probably used in betaine biosynthesis (Fig. 7). Notably, it was showed that L-pipecolic acid in feces was significantly higher in S100 group than NC group. Conversely, L-pipecolic acid in feces was significantly lower in NC group than BC group. Our findings suggested that 100 mg kg− 1 GE might inhibit the activity of PSO and reversed the lower levels of L-pipecolic acid caused by ETEC, which in turn prevented the production of H2O2 and decreased oxidative stress level.
Based on S200 vs NC group, L-phenylalanine was downregulated, which is a double-edged sword. On one hand, due to L-phenylalanine is not only an essential amino acid and the precursor of the amino acid tyrosine as well as also a precursor for catecholamines including tyramine, dopamine, epinephrine, and norepinephrine, the lower level of L-phenylalanine might decrease oxidative stress in gut [40]. On the other hand, the lower level of L-phenylalanine also might lead to a decrease of gut hormone secretion, including glucose-dependent insulinotropic peptide (GIP) and cholecystokinin (CCK) [48]. While GIP and CCK are important hormonal regulators of the ingestion, digestion, and absorption of intestinal nutrients [49, 50]. Hence, it suggested that the lower ADG in S200 group probably as a result of the downregulation of L-phenylalanine in gut. Additionally, our results indicated that UMP and dCMP were downregulated in S200 group compared with NC group, which were involved in the pyrimidine metabolism and lactose synthesis pathways and suggested that UMP and dCMP finally may be degraded to β-alanine through pyrimidine metabolism pathway and then β-alanine synthesized probably went to the alanine metabolism pathway. In this pathway, alanine and glyoxylic acid can be converted into glycine and pyruvic acid via serine-pyruvate aminotransferase. Meanwhile, D-glucose probably participated in the biosynthesis of pyruvic acid, leading to the lower level of UMP in lactose synthesis pathway. Then the pyruvic acid generated via two pathways may be absorbed into the blood and was involved in the transfer of acetyl groups into mitochondria pathway (Fig. 7). Furthermore, our present data discovered the lower level of deoxyguanosine in feces was associated with the higher level of inosine-5'-monophosphate (IMP) in serum in S200 group compared with NC group (Table 3S). While the higher level of IMP, as a nucleotide, may be propitious to the growth and maturation of intestinal epithelial cells and plays an important role in intestinal immunity and health [51].
Serum metabolomics
As seen in Table 4 and Fig. 7, based on NC vs BC group, it indicated that ETEC exposure decreases glucosamine 6-phosphate (G6P) and oxidized glutathione (GSSG) levels and increases NADP levels in serum by affecting glutamate and glutathione metabolism pathways, which resulted in accumulation of H2O2 in serum. On one hand, the producing H2O2 is not reduced to water (H2O) which resulted in peroxide interference and cell damage through oxidation of lipids, proteins, and nucleic acids [52]. On the other hand, H2O2 is not a radical but is considered a reactive oxygen species and it can induce a cascade of radical reactions and inactivate pyruvate dehydrogenase (PDH) [53, 54], leading to accumulation of TPP in serum and meaning that pyruvic acid cannot be create acetyl-CoA. While the latter was closely associated with fatty acid biosynthesis. In addition, the lower levels of cytidine monophosphate (CMP) and deoxyuridine triphosphate (dUTP) in NC group compared with BC group revealed that ETEC perturbed pyrimidine metabolism, then might inhibit the process of pyrimidine-related nucleotide biosynthesis.
Interestingly, based on S50 vs NC group, it was found that caffeine was significantly downregulated in feces (Table 3), while it was significantly upregulated in serum (Table 3S) and suggested most caffeine allowed for absorption into blood through intestinal mucosa. Furthermore, the high level of caffeine in serum could lead to an increase of lipolysis and it is usually accompanied by accumulation of glycerol in serum [55]. Then the high level of glycerol could raise blood osmolality, and it in turn probably played a favorable role in an increase of intestinal water absorption and a decrease of sodium efflux into the intestinal lumen, and finally resulted in attenuation of secretory diarrhea caused by ETEC [56]. It is of note that, based on S50 vs NC group, indoleamine 2, 3-dioxygenase 1 (IDO1) or tryptophan 2, 3-dioxygenase 2 (TDO2) drives tryptophan down the kynurenine pathways that produce tryptophan catabolites, such as the high level of kynurenic acid in serum (Table 3S). In this process, it is usually accompanied by the production of folic acid and L-glu, meaning that the generated folic acid and L-glutamate can be synthesized to THF through folate metabolism pathway. Meanwhile, THF also was biosynthesized in serum via two pathways, including pterine biosynthesis pathway and histidine metabolism.
Based on S100 vs NC group, the higher level of L-glu, as a note molecule, was known to be relevant in three pathways, including amino sugar metabolism, nicotinate and nicotinamide metabolism, and glycine and serine metabolism. It revealed that after glutamine synthetase or glutaminase liver isoform (GLS2) converting L-glu into Gln, and glutamine-fructose-6-phosphate aminotransferase (GFPT1) subsequently converted Gln and fructose 6-phosphate (F6P) into L-glu and G6P, which suggested that 100 mg kg− 1GE reversed the ETEC-induced downregulation of G6P. The higher level of G6P in S100 group, in turn, can be converted into N-acetyl-D-glucosamine-6-phosphate (N-AG6P) (via glucosamine 6-phosphate N-acetyltransferase) compared with NC group. Here, downregulation of uridine diphosphate-N-acetylglucosamine and upregulation of ATP in serum indicated that most N-AG6P generated likely can be converted into N-acetyl-D-glucosamine (N-AG) (a polysacchatide) and ATP, via N-acetyl-D-glucosamine kinase (NAGK). The resulting N-AG has been confirmed its anti-inflammatory efficacy for inflammatory bowel disease [57]. It is worth mentioning that betaine, which might be synthesized from choline, can be degraded via two pathways. The first pathway involves the betaine metabolism pathway. Compared with NC group, the higher levels of S-adenosyl-L-homocysteine (SAH), L-methionine, THF and ATP in S100 group indicated that THF cofactors was probably used to carry and activate one-carbon units via the folate-mediated one-carbon transfer pathway, resulting in the remethylation of homocysteine to L-methionine and the synthesis of purine nucleotides and thymidylate [58]. In the second pathway, betaine can be synthesized to dimethylglycine in methionine cycle, then the generated dimethylglycine can be converted into sarcosine and went to the glycine and serine metabolism. Here, the sarcosine synthesized via two routes to create 5-aminolevulinic acid and serine, respectively. Of them, the formation pathway of serine was accompanied by the production of THF. Whereas the producing L-methionine, purine nucleotides and 5-aminolevulinic acid may participate in the processes of attenuated inflammatory responses and inhibited oxidative stress [59–61].
Furthermore, it showed that 200 mg kg− 1 of GE reversed the ETEC-induced upregulation of NADP and TPP in serum via the transfer of acetyl groups into mitochondria and phytanic acid peroxisomal oxidation pathways and thereby participated in the production of acetyl-CoA, and the latter was related to the synthesis of fatty acids and sterols and the metabolism of many amino acids [62]. Meanwhile, similar to the S50 or S100 groups, S200 group also upregulated the levels of THF and L-methionine via the betaine metabolism and pterine biosynthesis pathway compared with the NC group.
It is worth noting that dietary addition of 50, 100 and 200 mg kg− 1 GE all can upregulate the level of THF and reversed the high level of NADP caused by ETEC compared with the NC group (Fig. 5). It suggested that THF is probably a main antioxidative force for GE indirectly [63, 64]. Meanwhile, GE downregulated level of NADP also meaning that the NADP pool is probably maintained in a highly reduced state, which improved antioxidant ability in response to oxidative damage [65].