4.1. Effect of G. elegans on Intake and Average Gain
Wu et al. [
15] fed piglets with a basal diet containing 0.3% and 0.5%
G. elegans powder for 30 days. The results showed that the weight gains of the experimental groups fed with 0.3% and 0.5%
G. elegans powder were higher than those of the control group, and the feed conversion ratio was significantly reduced by 3.81% and 10.59%, respectively.
In this study,
G. elegans could significantly reduce the material to weight ratio and promote the digestion and absorption of animals which is consistent with the previous report. However, contrary to the discovery of Wang et al. [
16], the additional dose of 20 g/kg
G. elegans whole grass did not significantly improve the growth of piglets, which might be due to the bitter taste of the
G. elegans whole grass dosage form and poor palatability, which may affect the appetite of piglets. The whole grass dosage form should be avoided as much as possible, and the
G. elegans extract form should be used for feeding in actual production and application. According to reports, at the dose of 50 mg/kg,
G. elegans showed remarkable growth-promoting effects but had no significant effect on the average daily feed intake of piglets [
17]. However,
G. elegans has some kind of toxicity [
18], so the residues in blood and muscle are a concern of people. It is reported that
Gelsemium alkaloids are absorbed rapidly in pigs, and the T1/2 values of most
Gelsemium alkaloids ranged from 8 h to 12 h, suggesting that the elimination was slow and there may still be residual levels in pigs [
19]. Our team will further study the residue depletion of
G. elegans in pigs for food safety.
4.2. Effect of G. elegans on Physiological Function of Weaned Piglets
Routine blood tests can effectively reflect the body’s resistance to diseases. The reduction of white blood cells and hemoglobin in the blood can be regarded as the signs of decreased antibody resistance and reactivity. T lymphocytes can not only mediate the cellular immune response of the animal body, but also, the cytokines secreted in the immune response have important regulatory effects on the immune response of the body, including the proliferation, differentiation, and function of immune cells.
Many studies reported that
G. elegans alkaloids triggered the immune response by promoting the expression of pro-inflammatory factors [
20] and affect the activation and proliferation of T lymphocytes [
10]. The results of the present study showed that compared with the control group, the counts of neutrophils and leukocytes in the blood of the
G. elegans-treated group increased, which indicates that
G. elegans improved the cellular immune function and disease resistance of the weaned piglets. In addition, the mean red blood cell volumes in the
G. elegans-treated pigs, although significantly reduced, remained within the normal reference range without adverse effects.
4.3. Evidence That G. elegans Regulates Amino Acid Metabolism and Decreases p38 Activation
In the study, the results of which are presented here, the metabolomics results emphasize a series of metabolites related to amino acids, including glycine and its derivatives and NAC, which suggests that the regulation of amino acid metabolism plays an important role in the immune stimulation of
G. elegans. Glycine has recently been classified as a nutritionally essential amino acid for maximal growth in young pigs. If glycine is insufficient in the body, amino acid metabolism will be affected, resulting in intestinal dysfunction. Studies have shown that glycine can protect a variety of organs, such as the liver, skeletal muscle, and small intestine, from certain harmful substances [
17]. Wang et al. [
21] investigated the cellular protective effects of glycine and indicated that glycine stimulates protein synthesis in IPEC-1 cells and inhibits oxidative stress by increasing intracellular glutathione concentrations. Moreover, various studies have shown that glycine can produce anti-inflammatory effects by decreasing reactive oxygen species and inflammatory mediator levels [
19], inhibiting inflammatory cell aggregation, and reducing lipid peroxidation [
22]. NAC can protect intestinal health and has a therapeutic effect in the treatment of colitis [
23] and hepatitis [
24]. The effects of NAC have been reported to be associated with decreases in the proinflammatory cytokines TNF-α, IL1β, and IL-6 [
23,
25]. As
G. elegans contains many active ingredients, it is difficult to directly attribute the specific metabolic effects of these active ingredients. However, drug effects and other exogenous stimulation always lead to variations in the metabolic network of endogenous metabolites, mainly reflected in the types and quantities of metabolites present. Metabolomics can be used to investigate the overall effect of stimulation on the body through comprehensive and systematic detection and analysis of endogenous small molecule metabolites in biological samples [
26]. Therefore, there is no doubt that the discovery of amino acid-related metabolites may provide new insights into the mechanisms of the immune stimulation of
G. elegans.
Results of the transcriptome analyses identified 199 DEGs in the liver, and through GO enrichment analysis, it was found that these transcripts were mainly enriched in the biological processes of proteins and amino acid metabolism and immune responses. Peptidyl-cysteinenitrosylation and peptidyl-cysteine modification were directly related to cysteine. Four of the down-regulated genes, including NOS2, LTF, S100A9, and S100A8, are well-known to be involved in multiple processes of cysteine regulation. NOS2 encodes a type of oxide synthase that is expressed in the liver and is inducible by a combination of lipopolysaccharide and certain cytokines. It mediates the nitrogenation of cysteine, participates in the inflammatory response, and enhances the production of NO and proinflammatory mediators such as IL-6 and IL-8 [
27]. Thus,
G. elegans can reduce the synthesis of proinflammatory mediators by regulating the expression of nitric oxide synthase. According to previous reports, koumine can inhibit the secretion of NO, ROS, TNF-α, IL-6, and IL-1β and significantly reduce the mRNA and protein levels of iNOS [
1], which is consistent with our observations. S100A9 and S100A8 are both members of the S100 family of proteins. This family of proteins has a wide range of intracellular and extracellular functions and is considered an important regulator of macrophage inflammation, tissue damage, and regulatory stress [
28]. Similarly, the decreased expression of LTF supports the anti-inflammatory effect, as the gene encoding lactoferrin is up-regulated during inflammation, activating the innate immune system through surface receptors, producing immune complexes containing LTF, and triggering the infiltration of monocytes and macrophages [
29]. Consequently, results of the gene expression profile indicate that
G. elegans can significantly regulate the expression of genes related to cysteine metabolism and achieve anti-inflammatory effects.
It is worth mentioning that our previous study reported the same batch of experimental ileum transcriptional spectrum data [
12], and the results suggest that the inflammation-related genes (such as C3, C5, S100A8, IL-8/CXCL-8, CSF2, IL-1/IL1A) were generally down-regulated, and the intestinal inflammatory response was inhibited in the
G. elegans-treated group, compared to the controls, which was similar to the results of the liver transcriptome results in the present study.
With respect to oxidative stress, there is increasing evidence that glycine enhances the intestinal mucosal barrier, reduces intestinal inflammation, and inhibits oxidative stress by inhibiting NF-κβ and TNF-α activation and IL-1 and IL-6 production [
30]. In addition, both glycine and NAC protect the lipopolysaccharide (LPS)-induced intestinal barrier in piglets through the mTOR and MAPK signaling pathways [
17,
31,
32]. MAPK signaling in mammals mainly includes MAPKs, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38, which play important roles in cell growth, proliferation, differentiation, migration, inflammation, and survival [
33,
34,
35,
36]. P38 mitogen-activated protein kinases are activated primarily in response to inflammatory cytokines and cellular stress. In the ileum transcriptomes, the p38 gene was down-regulated in the
G. elegans-treated group compared to the controls. It was speculated that
G. elegans could reduce the activation of p38 in the MAPK signaling pathway by regulating glycine and NAC-related metabolism and could play a role in protecting intestinal cells from oxidative stress. In some pharmacological mechanism studies of
G. elegans, koumine has been observed to significantly reduce phosphorylation of p38 and ERK in the LPS-mediated MAPK signaling pathway in mouse cells, all of which are consistent with the reduction in p38 activation [
1].
4.4. G. elegans Regulates Lipid Metabolism
To explore the influence of
G. elegans on other metabolic pathways, this study conducted a comprehensive network analysis based on the transcriptomics and metabolomics analyses results. In the MetScape analysis [
37], we identified seven metabolic pathways directly related to lipids. These pathways include unsaturated fatty acid metabolism pathways: linoleic acid metabolism and arachidonic acid metabolism, saturated fatty acid metabolism, glycerolipid metabolism, sphingolipid metabolism, steroid hormone biosynthesis, and metabolic pathways. The liver is the main site of fatty acid synthesis in animals. It first synthesizes palmitate and then produces other saturated fatty acids and unsaturated fatty acids. Unsaturated fatty acids exist mainly in the form of phospholipids in cell membranes. Under the action of phospholipase, unsaturated fatty acids produce free arachidonic acid, which is then converted into leukotrienes or prostaglandins by lipoxygenase or cyclooxygenase. The balance of these two substances plays an important role in regulating lipid metabolism [
38].
Additionally, glycosphingolipids are membrane components that can affect numerous cellular events, including homeostasis, adhesion growth, motility, apoptosis, proliferation, stress, and inflammatory responses [
39]. Interestingly, the alteration of lipid metabolites was also reported to be associated with type 2 diabetes risk in metabolomics studies [
40,
41]. In the transcriptome studies, we found that PPAR signaling pathways are involved in the lipid metabolism regulation of
G. elegans in pigs. PPAR ligands activate nuclear hormone receptor family receptors, control many cell metabolism pathways, and play an important role in regulating cell differentiation, growth, and metabolism in higher organisms [
42]. Three subtypes, namely PPAR alpha, PPAR beta, and PPAR-gamma, have been reported. PPAR alpha is expressed in the liver, kidney, heart, muscle, fat, and other organs [
43] and is involved in the fatty acid metabolism and lipid transport in the liver, in lipoprotein oxidation and combination, and the uptake of fatty acids and assembly [
44], and plays an important role in regulate hepatic fat metabolism. DEGs, including CPT-1, PCK1, and PATP, were enriched in the PPAR signaling pathway, andCPT-1 and PCK1 are downstream genes regulated by PPAR.
CPT-1 expression was increased by
G. elegans supplementation. CPT-1 is a mitochondrial enzyme that plays an important role in regulating fatty acid metabolism. Lipid consumption is mainly oxidized by FAO through transport to the mitochondrial matrix, and the transport process is mediated by the CPT system, which is composed of CPT-1, acylcarnitine translocation enzyme, and CPT2 [
45]. The activation of CPT-1 in an obesity (DIO) model has been reported to increase energy utilization and fatty acid oxidation [
46]. Additionally, according to the RNA-seq results, the expression of CPT-1 was increased, and it was speculated that
G. elegans could regulate the lipid metabolism process through CPT-1, thus improving the hepatic function. PCK1 catalyzes gluconeogenesis, that is, the synthesis of glucose, and plays an important role in maintaining glucose homeostasis. By regulating the expression of this gene, blood glucose levels can be maintained within a well-defined range. According to the report, the excessive expression of PCK1 can result in type II diabetes symptoms [
47], and excessive sugar dysplasia may also lead to the occurrence of metabolic diseases such as insulin resistance and hyperglycemia, indicating that PCK1 is important in glucose homeostasis. In the present study, the PPAR pathway inhibited the expression of PCK1, suggesting that
G. elegans could be used to inhibit the excessive production of glycogen via PCK1-mediated regulation of sugar dysplasia, thus improving metabolic diseases such as type II diabetes.