We studied the application of PSE in weaned pig diets, and found that PSE supplementation can improve the intestinal health by reducing the gut inflammation in piglet [9]. However, the potential anti-inflammatory effects of PSE on porcine intestinal cells remain to be elucidated. Therefore, the current study employed the IPEC-J2 cell line to investigate the protective effect and mechanism of PSE on LPS-induced inflammatory response in epithelial cells.
To clarify the anti-inflammatory active ingredients in the PSE, we investigated the anti-inflammatory activity of three PSEs by measuring the concentration of pro-inflammatory cytokines in IPEC-J2 cells induced by LPS. Among three PSEs, PSE-NB could significantly decrease the levels of IL-1, IL-6 and TNF-α in culture supernatants of IPEC-J2 cells induced by LPS (Table 2). Therefore, anti-inflammatory active ingredients of PSE were mainly presented in PSE-NB. Our previous study found that PSE-NB contained higher contents of alkaloids and total phenolic than PSE-PE and PSE-EA [14], respectively. A number of studies suggested that alkaloids and phenolic phytochemicals have significant anti-inflammatory activity [15, 16]. So, the better anti-inflammatory properties of PSE-NB may be due to a higher content of alkaloids and total phenolic among the three PSEs. Similarly, Zakaria et al. found that the methanolic extracts of Piper sarmentosum leaves have exhibited anti-inflammatory activity [8].
We further evaluated the protective effect of PSE-NB on inflammatory response in IPEC-J2 cells induced by LPS. Cytokines, like IL-1, IL-6 and TNF-α, play key role in activating immune cells and amplifying inflammatory responses [17]. Previous studies have indicated that intestinal epithelial inflammation could damage tight junction by down-regulating the expression of tight junction proteins through inflammatory signaling transduction [18, 19]. The overproduction of pro-inflammatory cytokines, such as TNF-α, IL-1, and IL-6, has a negative impact on gut integrity and epithelial function [20]. Our results showed that LPS significantly increased mRNA expression of IL-1, IL-6 and TNF-α (Fig. 1), and reduced mRNA abundance of ZO-1, Claudin-1, Occludin and NHE3 (Fig. 2) in cells when compared with control, which are consistent with previous studies [21]. The PSE-NB treatments was able to attenuate the inflammatory responses and preserve barrier function of epithelial cells induced by LPS evidenced by modulating the mRNA expression of pro-inflammatory cytokines and tight junctions in cells (Fig. 1 and Fig. 2). The current results are consistent with previous animal experiment, which indicated that PSE supplementation down-regulated the mRNA expression of TNF-α, IL-1β, and IL-6 in the ileal mucosal layer of piglets [9]. Therefore, supplementation with PSE-NB might be a potential approach to reduce inflammatory responses in weaned piglets.
Generally, LPS triggers inflammatory and immune responses mainly through the TLR4 receptor, and the activation of TLR4 by LPS induces the activation of NF-κB pathway and ultimately results in the release of pro-inflammatory cytokines [22]. The NF-κB signaling pathway plays a critical role in regulating inflammation and immune which are involved in diverse biological responses [23]. Main family members of signaling pathway including p65 and I-κBα participate in the activation and production of pro-inflammatory mediators and cytokines [24, 25]. In LPS-stimulated inflammation, the activation and phosphorylation of NF-κB are crucial for up-regulated mRNA transcription and production of inflammatory mediators. In this study, our results are consistent with previous findings that LPS induced the activation of NF-κB and increased the phosphorylation level of p65 significantly (Fig. 3A and Fig. 3B). The pre-treatment by PSE-NB reduced protein expressions of p65, and p-p65 significantly. The specific kinase inhibitor, PDTC was used in our study to confirm the role of NF-κB signaling pathway in the inhibition of LPS-induced inflammatory response by PSB-NB in IPEC-J2 cells. When inflammatory signaling pathway was blocked, mRNA expressions of pro-inflammatory cytokines were down-regulated (Fig. 4). Previous studies suggested that the anti-inflammatory mechanism of alkaloids and polyphenolics are related to the mediation of NF-κB pathways in LPS-induced cells [26]. PSE-NB contained higher contents of alkaloids and total phenolic than PSE-PE and PSE-EA [14], and these results together suggested that PSE-NB exerted anti-inflammatory effect by the inhibition of NF-κB pathway in IPEC-J2 cells induced by LPS.
The metabolomics profiles of IPEC-J2 cells were significantly changed by LPS and PSE-NB. PCA and PLS-DA revealed that metabolic perturbations were different among control, LPS treatment and LPS + PSE-NB treatment (Fig. 6 and Fig. 8). Changes in metabolites were related to several metabolic pathways. Based on the data obtained from intracellular metabolites on LPS group vs control group, the enhanced production of pro-inflammatory cytokines and mediators markedly perturbed the levels of secretion of some metabolites and the metabolic pattern of the IPEC-J2 cells. These results suggested that LPS-induced inflammatory response promoted the biosynthesis of phosphoserine and serine through the involvement of gluconeogenesis and glycolysis to activate the innate immune system by controlling T cell proliferation capacity [27, 28], and reduced the levels of betaine and sarcosine to affect the biosynthesis of S-(5'-Adenosyl)-L-homocysteine. Soon afterwards, the lower levels of S-(5'-Adenosyl)-L-homocysteine disturbed the homeostasis of methionine metabolic cycle and intercellular transmethylation reactions [29]. Furthermore, the inhibition of intercellular transmethylation reactions reduced carnitine synthesis and affected fatty acid oxidation process [30]. Additionally, LPS-induced inflammatory response and disturbed nicotinate and nicotinamide anabolism and motivated oxidative stress state of cells due to decreased quinolinic acid production, which is an factor affected tryptophan metabolism [31].
Notably, LPS and PSE-NB both affected tryptophan metabolic pathway. As known that LPS perturbed tryptophan catabolism as well as reduced the production of kynurenine, quinolinic acid, alanine and NADPH and increased the production of tryptamine. We assured that tryptophan can be mostly converted into tryptamine and generate CO2 simultaneously via the action of aromatic amino acid decarboxylase. Decreases in the levels of S-(5'-Adenosyl)-L-homocysteine and NADPH implied tryptamine catabolic disorders, which resulted in tryptamine accumulate. Then tryptamine probably released histamine, a kind of inflammatory mediators, which induced the production of inflammatory mediators and cytokines in different immune cells and affect inflammation of the immune system [32]. However, PSE-NB relatively upregulated the levels of kynurenine, quinolinic acid, alanine and NADPH, resulting in the higher levels of quinolinic acid, 3-hydroxyanthranilic acid, NADP, kynurenic acid, xanthurenic acid and glutamic acid compared to LPS-induced IPEC-J2 cells and showed the recovery of tryptophan catabolism. Meanwhile, PSE-NB relatively decreased the levels of tryptamine and enhanced the levels of 5-hydroxytryptophan, melatonin, serotonin, coenzyme A, 5-hydroxyindoleacetic acid and N-acetylserotonin (Fig. 10, http://smpdb.ca).
Some studies have reported that hydroxylated analogues of kynurenine tryptophan metabolites, such as 3-hydroxyanthranilic acid, xanthurenic acid and 5-hydroxytryptophan, possess antioxidant potential revealing a capability of these metabolites to suppress inflammatory condition [33]. Additionally, kynurenic acid, which is detoxified kynurenine, reduced the release of IL-4 through the specific activation of G protein-coupled receptor 35 (GPR35) [34], and quinolinic acid have a role in protect the body from inflammatory injury via a direct action on neutrophils or vascular permeability [35]. Melatonin, serotonin and N-acetylserotonin also have ability to inhibit production of inflammatory cytokines and superoxide [36, 37, 38]. Meanwhile, increase in the production of glutamic acid (glutamate) and NADP have a favor role involved in the purine metabolism and glutathione metabolism while coenzyme A affect phenylacetate metabolism. Furthermore, we found that PSE-NB affected purine metabolism and heighted the levels of purine nucleotides and nucleobases to modulate energy metabolism which can satisfy the nutritional needs and decrease oxidative stress of LPS-induced IPEC-J2 cells [39, 40, 41].
In the glutathione metabolism, increased NADP production significantly implied that the reaction between oxidized glutathione and NADPH, resulting from the synthesis of NADP and glutathione. The higher levels of NADP proved the fact. Therefore, glutathione probably participates in anti-inflammatory and antioxidative response, resulting in the lower levels of it [42]. Moreover, the higher levels of coenzyme A and phenylacetic acid revealed that the process reduce energy consumption to satisfy the needs of anti-inflammatory of cells. In the same way, depletion of thiamine because of it probably was used for helping convert carbohydrates into energy.
Crosstalk between metabolic and inflammatory pathways is critical for cellular homeostasis. In the present study, we found that the connectivity between these processes in PSE-NB-mediating IPEC-J2 cells induced by LPS. Melatonin, which is the product of the tryptophan catabolic pathway affected by PSE-NB, has anti-inflammatory properties through inhibiting NF-κB activation consistent with previous results regarding to anti-inflammatory activity of PSE-NB on inflammatory signaling pathway [38]. As a result, PSE-NB’ anti-inflammatory mechanisms between protein pathway and metabolic pathway are highly integrated and closely linked.