The problem of residues and resistance caused by antibiotics abusing has caused widespread concerns. Probiotics, as a green feed additive with the potential to replace antibiotics [14], are widely promoted due to their ability to significantly improve production efficiency and welfare of the animal husbandry industry [15]. Our previous study has found that, instead of aureomycin, BaSC06 increased the growth performance of piglets by improving average daily gain [16]. In the current study, the similar results were obtained, which are consistent with other results. Supplementation of 0.2% probiotics Bacillus (including Bacillus subtilis, Bacillus coagulans, and Lactobacillus acidophilus) in the diet could significantly increase the average daily gain of fattening pigs, but has no significant effect on the average daily feed intake and F/G [17]. Balasubramanian et al. (2016) found that the Bacillus compound not only significantly increased the average daily weight gain, but also improved F/G [18]. It indicated that Probiotic BaSC06 can replace antibiotics both in piglets and fattening pigs. Many studies have been verified that the beneficial effects of probiotics on animal growth is related to improve intestinal health, including digestion absorption function, antioxidative capacity, immune function, and microbiota composition [19-22].
The activity of digestive enzymes reflects the digestion and absorption capacity of feed nutrients in monogastric animals. Our research found that BaSC06 could increase trypsin and amylase activity in the intestine of fattening pigs, thereby promoting the digestion and utilization of protein and carbohydrates in the diet. Other study also found that Brevibacillus brevis FJAT-1501-BPA, Bacillus subtilis supplementary significantly improved the feed conversion rate and growth performance of weaning piglets through regulating trypsin, amylase, and cellulase detected in the feces [23, 24]. Besides, our results indicated that the intestinal chymotrypsin and lipase activities in the group supplemented with BaSC06 are lower, which may be related to the consumption of a large amount of lipase and chymotrypsin after feeding BaSC06 to promote the hydrolysis of fat and protein in the body. Similar results were found in broilers which were feed with compound probiotics (Bacillus subtilis, Lactobacillus acidophilus, and Bacillus licheniformis) [25]. In addition, BaSC06 could increase the sucrase and lactase activity without affecting maltase activity, then further improve the absorption of nutrients. Goyal et al. (2013) have found that feeding rats with Lactobacillus La1 can increase sucrase activity in the intestine; what’s more, pre-oral administration of LGG increases sucrase and lactase activity and helps mice effectively resist oxidative stress induced by flagellate infection [26]. We speculate that the changes in digestive enzyme activity detected in this study may be due to the secretion of probiotics and the stimulation of endogenous enzyme synthesis in intestines [27].
When the protein is hydrolyzed in the intestine to produce free amino acids, a large number of di- and tripeptides are also produced, which are the main absorption forms of protein [28]. γ- Glutamytransferase can regulate the glutamine nutrition metabolism of intestinal epithelial cells, improve the uptake, transport, and utilization of amino acids by cells, and thus maintain intestinal mucosal integrity, improve intestinal health, and create favorable conditions for digestion and absorption [29, 30]. Transporters are involved in the transmembrane transport of di- and tripeptides. Current studies have found that there are at least five small peptide transporters, of which the most widely studied are PEPT1 and PEPT2. PEPT1 is highly expressed in the brush border of the jejunum epithelium of pigs and mediates intestinal absorption of di- and tripeptides [31, 32]. Studies have shown that when continuous feeding of Bacillus subtilis, the expression level of small peptide transporter PEPT1 mRNA in the ileum mucosa of broiler chickens is significantly higher than that in the basic diet group and the antibiotics group. The digestion and absorption mainly by upregulating the expression of the small peptide transporter PEPT1 in the ileum improves the absorption and transport of small peptides by the intestine [33]. However, we found that mixed BaSC06 and Kitasamycin did not affect the activity of γ-Glutamytransferase in intestinal mucosal cells of fattening pigs, but BaSC06 alone decreased the activity of γ-Glutamytransferase significantly. BaSC06 may enhance the cell’s absorption capacity, resulting in reduced demand for γ-Glutamytransferase. Interestingly, we also found that adding BaSC06 alone could increase the expression of s PEPT1 mRNA by 6 to 7 times, which may promote the transport and absorption of small peptides in vivo. Furthermore, Na+/K+-ATPase can hydrolyze ATP to help the body obtain energy, transport Na+ against chemical gradients, and regulate the electrochemical balance of Na+ and K+ inside and outside the intestinal cell membrane. It is particularly important to maintain Na+-dependent transporters such as SGLT1 (Sodium-dependent glucose transporter 1) and GLUT2 (Glucose transporter 2), distributing in the small intestine epithelium of animals. They both mediate the transport of glucose molecules across the brush border of the intestinal epithelium [34, 35]. Therefore, AKPase and Na+/K+-ATPase activity and the expression of glucose transporter on intestinal mucosa can reflect the absorption function of small intestinal epithelial cells well. We observed that instead of antibiotic, BaSC06 increased the activity of Na+/K+-ATPase in jejuna mucosa and the expression of SGLT1, which indicated that as an alternative of antibiotic, BaSC06 could influence the carbohydrates metabolism and improve the absorption ability of intestinal epithelial cells, while the certain further research was needed to clarify the certain effects of BaSC06.
With the generation of radicals during the process of energy metabolism, the intestine, as an organ that connects with the outside, is extremely easy to attack by excessive oxygen free radicals, which can trigger cell membrane lipid peroxidation, protein denaturation, and DNA lysis. Oxidative stress causes damage to intestinal mucosal cells and intestinal tissues of animals and metabolic disorders of the body, and then induces a series of intestinal diseases, leading to abnormal intestinal functions [36-38]. Normally, the levels of oxidation and reduction in the body maintain a dynamic balance, which can be broken by a variety of endogenous and exogenous harmful stimuli. If the levels of free radicals and ROS exceed the reduction level of the antioxidant system, the antioxidant capacity of the body decreases, resulting in oxidative damage to biological macromolecules [39]. A large number of studies have shown that free radicals can cause peroxidative damage to cell membrane lipids and produce MDA which involved in attacking polyunsaturated fatty acids in biological cell membranes, further triggering lipid peroxidation, leading to more severe oxidative stress damage [40, 41]. However, many studies have shown that probiotics have excellent antioxidant functions. For instance, Zhao et al. (2017) found that Lactobacillus casei significantly reduced MDA content in the serum of pigs and relieved Lipopolysaccharide (LPS)-induced oxidative stress [42]. In the present study, BaSC06 can reduce the MDA content in the intestinal mucosa of fattening pigs by increasing CAT and GSH-Px, indicating that the strain has good antioxidant activity and prevent intestinal mucosa from oxidative stress damage. This is consistent with previous BaSC06 results in piglets and rats in our group [16, 43].
In addition to antioxidant enzymes, some important phase II detoxifying enzymes such as NQO1, GST, HO-1, and γ-GCS can also play an antioxidant role in the antioxidant process. NQO1 is a type of flavin protease that is ubiquitous in eukaryotic cells. It can catalyze the reduction of quinone compounds and avoid the formation of unstable semiquinones. Therefore, NQO1 can protect cells from oxidative damage caused by biological heterologous substances such as quinones [44]. HO-1 is an inducible phase II detoxifying enzyme [45], the expression of which is regulated by Nrf2 [46], and its increase in expression can also be used as an adaptive mechanism for cell self-protection under oxidative stress [47, 48]. Wang et al. (2017) found that HO-1 is a sensitive index for oxidative stress [9]. In this study, the expression of NQO1 mRNA in the BaSC06 group was significantly up-regulated, while the HO-1 gene showed the opposite trend. The up-regulation of NQO1 gene expression indicates that BaSC06 enhances the self-protection ability of intestinal mucosal cells. The decrease of HO-1 indicates a reduction in oxidative stress, which was resulted from higher antioxidant enzymes activity and NQO1 gene expression induced by BaSC06 treatment. The above results suggest that BaSC06 can regulate the jejuna mucosa phase II detoxifying enzyme-related genes at the transcription level, improve the intestinal antioxidant function, and effectively resist oxidative damage induced by biological heterologous substances and other stress factors.
The Nrf2/Keap1 signaling pathway is important for the body to resist oxidation. In the normal state, the binding of Nrf2 and its inhibitor Keap1 is in resting, while stimulated by oxidative stress factors, Nrf2 and Keap1 rapidly dissociate and move into the nucleus, recognize and bind to antioxidant response elements (AREs). Besides, the transcription and translation of cytoprotective genes such as downstream antioxidase and phase II detoxifying enzymes are initiated, thereby improving the cell's antioxidant capacity [49, 50]. Recent studies have shown that the mechanism by which microorganisms activate the Nrf2 signaling pathway protects the intestinal epithelium from oxidative stress and other endogenous stimuli, and maintains intestinal epithelial cell homeostasis and cell self-protection [51-53]. Chowdhury et al. (2014) found that Bacillus megaterium RB-05 by secreting low fucose to up-regulate the translation and accumulation of Nrf2 and Keap1 in nuclear and then stimulate the expression of downstream transcription factors like HMOX1, NQO1, GSTA2, SOD1 and GPX1 [54]. However, no significant change in the expression of Nrf2 and Keap1 was observed, but we found that BaSC06 significantly up-regulated the expression of phosphorylated Nrf2 protein in the jejuna mucosa of fattening pigs. We also observed similar results on intestinal porcine epithelial cells-1 (IPEC-1) cell experiments [9]. Therefore, we speculate that the activation of the Nrf2/Keap1 signaling pathway indicated by higher p-Nrf2 protein expression in the jejunal mucosa of fattening pigs induced by BaSC06 could promote Nrf2 nuclear translocation, then start transcription and translation of downstream genes (such as the aforementioned antioxidase GSH-Px and CAT, phase II detoxifying enzymes, etc.), resulting in improving the total intestinal antioxidant capacity, protecting the intestine from oxidative damage, maintaining the normal physiological function of the intestine, and ultimately promoting digestion and absorption. These results are similar to that of previous studies on antioxidants of other probiotics [53, 55].
In addition to increasing antioxidant enzymes, probiotics can also reduce oxidative stress by reducing ROS production. Reactive nicotinamide adenine dinucleotide phosphate oxidase (NOX) is the main source of reactive oxygen species in vitro. NOX is a complex composed of the catalytic subunit gp91phox and the regulatory subunits P22phox, P47phox, P67phox, P40phox, and Rac [56]. Among these subunits, P47phox is considered to play a key role in regulating NOX activity [57]. When stimulated by stress factors such as cytokines, hormones, and inflammatory factors, P47phox undergoes phosphorylation and configuration changes. Subsequently, P47phox binds to subunits such as P67phox in the cytoplasm and is transferred to the cell membrane. The catalytic subunit gp91phox and the regulatory subunit P22phox bind and interact with each other, eventually activating NOX and generating a large amount of ROS [58-60]. Probiotics alter NOX activity by regulating P47phox expression. Rashid et al. (2014) showed that oral administration of probiotics could protect P47phox protein expression in rats from vascular endothelial dysfunction induced by common bile duct infarction [61], while Tapia-Paniagua et al. (2015) also suggested that probiotic SpPdp11 down-regulating the transcription of NOX in Solea senegalensis [62]. In our former study, we didn’t found that BaSC06 replacement affects NOX activity and the expression of P47phox in piglets [16]. However, we found that BaSC06 alone could inhibit the production of excess ROS by down-regulating the expression of NOX active subunit P47phox protein in IPEC-1 [9], which is consistent to our present result on fattening pigs.
There are abundant lymphoid tissues in the intestinal mucosa, lymphocytes, plasma cells and macrophages responses to harmful antigens from the gut, such as food toxins, bacteria and viruses. Therefore, intestines are the first immune defense system of the digestive system which is known as intestinal mucosa immunity [63]. The interaction between microorganisms and intestinal epithelial cells is the beginning of the host immune response, which can eliminate potential pathogenic microorganisms [64, 65]. The level of IL-6 and IL-8 in the intestinal mucosa of piglets decreased significantly when Enterococcus faecium was added to the diet [66]. Drouaultholowacz et al. (2006) found that a probiotic with anti-inflammatory effect can effectively prevent intestinal inflammation by increasing the level of anti-inflammatory factors and reducing the level of pro-inflammatory factors [67]. It has been reported that LGG can effectively reduce the level of pro-inflammatory factors in the intestinal mucosa of mice, increase the level of anti-inflammatory factors, and enhance the immune response of mice infected with flagellates [68]. In vitro, it was found that co-culture of Lactobacillus reuteri and IPEC-1 could significantly inhibit the overexpression of TNF-α and IL-6 caused by Escherichia coli K88 or lipopolysaccharide, and significantly promote the expression of IL-10 [69]. The results in our study also showed that BaSC06 could inhibit the development of intestinal inflammation in fattening pigs by regulating the expression of pro-inflammatory cytokines such as IL-6, IL-8 and MCP1 in the intestinal mucosa. However, other studies have also reported that probiotics themselves can stimulate the immune system of the intestinal mucosa [70, 71]. For example, Anderson et al. (2016) showed that Lactobacillus fermentum AGR1487 can up-regulate the expression of proinflammatory factors by activating the TLR signaling pathway and induce the inflammatory response in sterile rats. The different effects of microorganisms on the intestinal mucosal immune system may be due to the differences between test conditions and strains [72].
As an innate immune cells in the gut, macrophages play a key role in maintaining intestinal homeostasis, regulating cytokine secretion and generating immune response [73-75]. Macrophages can sense the stimulation from microorganisms and activate polarization, while the diversity of microorganisms will lead to the polarization difference of M1 or M2 types of macrophages [76-78]. Classical activation of M1 macrophages in response to INF-γ is characterized by high ability to express antigens, high expression of iNOS, IL-6, TNF-α and IL-1β mRNA and nitric oxide (NO), which has the ability to kill cells endogenous pathogens and tumor cells [79]. In contrast, activation of M2 macrophages is promoted by various factors (such as IL-4), which is consistent with the high mRNA expression of Arg1, Fizz1, Ym1, and MR, which plays an important role in in the inflammatory response, removing debris, blood vessels generation as well as tissue repairing and reconstruction [80]. In this study, BaSC06 could up-regulate the expression of M1(iNOS) and M2 (Arg) macrophage marker protein simultaneously, which could promote the phagocytosis and sterilization of cells, and enhance the anti-inflammatory and anti-infection ability of cells. Also, Fu et al. (2019) reported that BaSC06 induced M1 phenotype polarization directly and M2 phenotype polarization via modifying microbiota [81]. The polarization imbalance of macrophages, such as the over enhancement of M1 polarization, will lead to immune damage, while the over enhancement of M2 polarization will promote chronic infection, and then induce various diseases [82]. Therefore, we speculated that BaSC06 could maintain the intestinal immune balance of fattening pigs. E. coli Nissle 1917 can also induce the expression or secretion of pro-inflammatory and anti-inflammatory cytokines in macrophages [78, 83]. Our previous results of intestinal mucosal cytokines showed that BaSC06 enhanced the immune tolerance of intestinal mucosa in fattening pigs and inhibited the intestinal mucosal inflammation. Therefore, the expression of macrophage polarization marker protein may adapt to this change in the microenvironment, avoid the extreme situation of M2 type immune tolerance, maintain the balance of M1 and M2 polarization, and then promote pro- and anti-inflammatory balance to maintain intestinal homeostasis. Bacillus amyloliquefaciens enhanced phagocytic bactericidal function by inducing M1 polarization of mouse bone marrow-derived macrophages [84]. Bacillus amyloliquefaciens reduced the intestinal inflammation and diarrhea rate by inhibiting the Mitogen-activated protein kinase (MAPK) inflammation signal pathway in piglets [85]. There were no significant changes in the total protein and phosphorylated protein of proteins related to the M1 polarization signaling pathway detected in this study, such as STAT1 and IRF3, which indicates that BaSC06 affect other pathways such as NF-κB [86], IRF5 [87, 88] or JAK to activate macrophage M1 polarization. And STAT6 is the major signaling protein of M2 macrophage polarization signaling pathway mediated by IL-4 [86]. Studies have shown that the inhibition of STAT3 leads macrophages polarizes toward the M1 phenotype [89]. In the current study, the expression of STAT3 protein was significantly up-regulated in BaSC06 supplement groups, which indicates that BaSC06 could promote the polarization of macrophages to the M2 phenotype by activating the STAT3 signaling pathway. In vitro, Shiraishi et al. (2012) have found that glucagon-like peptides-1 can up-regulate the expression of phosphorylated STAT3 protein and activate STAT3 to mediate the polarization of macrophages toward the M2 phenotype [90], which is consistent with our results.
Maintaining intestinal microorganisms balance is essential for intestinal development and homeostasis, which further effectively resist pathogenic bacteria. Intestinal microorganisms are mainly classified into about 50 phyla. In the human intestine, Firmicutes and Bacteroidetes are dominant, while Proteobacteria, Actinobacteria, Clostridia etc. only account for a few [91]. We found that the dominant bacteria in the cecum contents of fattening pigs were Firmicutes and Bacteroidetes. The Firmicutes spherical or rod-shaped mainly includes gram-positive bacteria with low G+C content. The most important class is Bacillus, including Bacillus, Enterococcus, Lactobacillus, Lactococcus and other beneficial bacteria, which play an important role in maintaining the gut health of animals [92]. Bacteroidetes are Gram-negative bacteria and the second-largest bacterium, live in the intestines of healthy people or animals. Proteobacteria are also Gram-negative bacteria, which is the largest of the bacteria bacterium, including many pathogenic bacteria, such as E. coli, Salmonella, Vibrio, Helicobacter and so on. The beneficial effects of probiotics may come from inhibiting the growth of pathogenic bacteria and promoting the growth of beneficial flora in the gastrointestinal tract. Probiotic colonization and its effect on members of the intestinal microbiota are highly species-specific. Enterococcus faecium and Lactobacillus can reduce the abundance of E. coli and increase the abundance of anaerobic bacteria in piglets' ileum and cecum [93]. Bacillus can occupy the surface of intestinal mucosa together with other anaerobic microorganisms to form a biological barrier to protect the intestinal mucosa and prevent the invasion of pathogenic bacteria. They can also inhibit the growth and reproduction of harmful microorganisms such as Clostridium, improve the disease resistance of piglets, and then improve its growth performance [11, 94]. Bacillus increases the abundance of Bacillus, Bifidobacterium, and Lactobacillus in the gut of pigs, while the abundance of E. coli is relatively reduced [95-98]. This study found that compared with Anti group, BaSC06 did not affect the diversity of the intestinal microorganisms of the fattening pigs, but it could improve the structure of it, especially significantly increase the proportion of Gram-positive bacteria (Firmicutes), and reduce the proportion of gram-negative bacteria (Bacteroidetes, Proteobacteria). Therefore, the probiotic BaSC06 can promote the balance of the intestinal microorganisms, improve the microbial barrier function, strengthen the dominant position of the Firmicutes, and significantly reduce gram-negative pathogen infection, thereby promote the intestinal health of fattening pigs.