Weaning is a major contributor to intestinal epithelial barrier dysfunction, characterized by increased intestinal permeability, leading to the invasion of intestinal pathogens and then triggering intestinal inflammation [28, 29]. Therefore, effective strategy is urgently needed in this critical phase especially in piglets and infants of human beings. Embelin, one of the naturally-occurring benzoquinone compound, possesses various biological effects and has attracted considerable attention as a functional ingredient to alleviate inflammation in animals and human beings. Recently, numerous studies have focused on improving the gut health of animals through dietary supplementation with antioxidative and antibacterial nutrients, such as resveratrol, oregano oil, and selenium [30–32]. Previous studies in vitro showed that embelin has potential anti-inflammatory effects, furthermore, embelin is regarded as an extremely potent natural inhibitor of p300/CBP - associating factor (PCAF), which has been observed to activate the inflammatory pathway through acetylation of the NF-κB. Therefore, embelin may have a predictable potential ability to attenuate the intestinal inflammation of weaned animals, especially in piglets, which has been proved to be a suitable animal model for human infants [33]. However, the effect and dose effect of dietary embelin on growth performance, diarrhea, intestinal barrier function and inflammation in weaned piglets are poorly understood. In this work, we demonstrated for the first time that as a natural ingredients, embelin efficiently improved growth performance and diarrhea, ameliorated intestinal barrier dysfunction and inflammation of weaning piglets.
First, the effect of dietary embelin on growth performance and diarrhea was measured. The median lethal dose of embelin is reported to be 2000 mg/kg body weight in mice and rats without any mortality or adverse effects indicating its safety [19]. So we chose 200, 400 and 600 mg/kg embelin as the additive dosage. At the beginning of the study, we collected blood samples and verified that there were no differences between piglets and that they were healthy at the beginning of the study. However, after a 28-day period trial, the control group had a higher incidence of diarrhea, which may be related to weaning events. Dietary embelin increased ADG and decreased F/G compared to the control group, indicating improved growth performance. Moreover, dietary embelin significantly reduced the rate of diarrhea in weaned piglets. Previous studies have shown that body weight gain and nutrient absorption as well as diarrhea rates in piglets are related to oxidative stability, intestinal morphology and intestinal inflammation [34], then we tested the intestinal barrier function of weaned piglets after treating with embelin.
The integrity of the intestinal epithelial barrier acts a key role in the digestion and absorption of nutrients and the inhibition of invasion by pathogenic bacteria [35, 36]. However, numerous mammalian studies have shown that weaning stress can severely disrupt small intestinal barrier function and exacerbate intestinal inflammation within a short period of time after weaning [37–39]. It is well known that intestinal permeability is usually assessed by some blood indicators. D-lactic acid is a specific end product of intestinal bacteria and is released into the blood when the intestinal mucosa is damaged. DAO is an intracellular enzyme synthesized mainly by mammalian intestinal epithelial cells and is mainly found in the cytoplasm. Once the intestinal epithelial barrier is damaged, intestinal DAO is released into the circulation [40]. Therefore, blood D-lactic acid concentration and DAO activity can be used as circulating markers to monitor the extent of intestinal barrier damage. In the present study, the addition of embelin to the diet reduced serum D-lactic acid concentration and DAO activity in weaned piglets, suggesting that embelin has a beneficial effect on reducing intestinal permeability in weaned piglets. Furthermore, tight junctions between intestinal epithelial cells maintain and regulate the intestinal epithelial barrier [41]. The formation of tight junctions requires several unique proteins, such as the claudin family, occludin, and intracellular linker proteins, such as ZOs, which anchor directly or indirectly to the actin-based cytoskeleton and then form a selective permeability barrier [42]. Thus, the expression level and distribution of tight junction proteins are closely related to intestinal barrier function. However, weaning stress can disrupt intestinal tight junctions by down-regulating the expression of intestinal barrier-related genes in pigs [43, 44]. The results of this study showed that the addition of embelin to the diet significantly upregulated the expression of claudin-1, occludin and ZO-1 in the small intestine of weaned piglets. Thus, embelin can improve intestinal barrier function by maintaining the expression of tight junction proteins, which may be partly responsible for embelin's ability to reduce intestinal permeability and inflammatory response to weaning stress injury in piglets.
Accumulating evidence has confirmed that inflammation is an important inducing factor in intestinal barrier disruption [45, 46]. Some cytokines are thought to be critical in the predisposition to and exacerbation of some gastrointestinal dysfunctions. NF-κB is a central mediator of intestinal inflammatory diseases [47] and has been shown to play a role in the control of intestinal permeability [48]. Moreover, pro-inflammatory cytokines such as TNF-α, IL-1β, IL-6 have been shown to exert negative roles in maintaining the tight junction and cytoskeleton structure and function. In the present study, embelin feed inhibited NF-κB activity and the pro-inflammatory factors TNF-α, IL-1β, and IL-6, which is consistent with previous studies. Evidence to date demonstrates that the functions of NF-κB are modulated by diverse post-transcriptional modifications. Among these modifications, acetylation has been shown to enhance the nuclear localization of NF-κB and lead to the transcription of NF-κB target genes [49]. NF-κB is mainly acetylated by PCAF. PCAF acetylates multiple lysine residues of NF-κB, including Lys-122, -123, -218, -221 and − 310, activating its transcriptional activity [50]. In the present study, dietary supplementation with embelin at 3 dosages decreased PCAF activity, suggesting that embelin may attenuate NF-κB activity by inhibiting the acetylation activity of PCAF.
To further explore the molecular mechanism of embelin in attenuating intestinal inflammatory response,we investigated the effects of embelin on the activation of PCAF/NF-κB signaling pathway which has been considered as a key inducer of inflammation. It was reported that embelin rapidly inactivates PCAF activity and has a strong apoptosis-inducing effect on leukemia cell lines and on prostate and pancreatic cancer cells through the inhibition of DNA binding [51]. In our study, embelin decreased PCAF activity and NF-κB acetylation suggesting that embelin-induced decreased PCAF activity may reduce inflammatory cytokines in weaned piglets. Furthermore, the upregulation of PCAF and NF-κB in weaned piglets was reversed by embelin treatment, so we propose that the PCAF-NF-κB pathway may be involved in the embelin-mediated alleviation the inflammatory response after weaning, siRNAs inhibits PCAF in cultured IPEC-1 cell, indicating that nonembelin-mediated inhibition of PCAF has similar effects. Activated PCAF can stimulate the activation of NF-κB signal pathway and then increase the expression of various inflammatory cytokines' genes, including IL-6, TNF-α and IL-1β. Therefore, reducing the inflammatory response mediated by the PCAF/NF-κB signaling pathway may play a protective effect in the intestine of weaned pigs.
In conclusion, the present study indicated for the first time that dietary embelin supplementation exerts beneficial effects on ameliorating the intestinal inflammation in weaned pigs. The mechanisms of action might be closely related to improving tight junction protein expression, suppressing intestinal inflammatory mediators released, alleviating intestinal inflammation via downregulation of acetylation of NF-κB by PCAF.
Nevertheless, there were some limitations in this study. In this experiment, due to the imperfect experimental plan, the optimal concentration of embelin in the piglet diet was not further clarified. In addition, there may be other potential mechanisms that have not been explored in this study. For example, embelin itself has a certain antibacterial effect, which may be an important factor in improving the gut mircobiota and intestinal health. Future experiments could focus on evaluating the anti-inflammatory and antibacterial properties of embelin, and further clarification of its role in piglet diet production and human infant health.