According to our results, C. perfringens challenge caused damages on jejunal barrier of broilers and increased the permeability of jejunal mucosa, allowing antigenic substances (LPS, etc.) to enter the blood and internal environment, which in turn triggered jejunal inflammation and oxidative stress, as well as systemic inflammation, reducing the ability of intestinal digestion and absorption, finally impaired the growth performance of broilers. However, dietary EA supplementation exerted anti-inflammatory and antioxidant effects in the jejunal mucosa, which protected and improved the intestinal barrier, preventing the invasion of antigenic substances, and finally improved the growth performance of broilers. Meanwhile, the supplementation of dietary EA also relieved the cecal microbiota imbalance caused by the C. perfringens challenge, protecting the health of broilers (Fig. 6).
Toll-Like Receptors (TLRs) are important members of pattern recognition receptors, TLR4 could recognize LPS, which is unique to Gram-negative bacteria, and TLR2 could recognize peptidoglycans (PGN), which is abundant in Gram-positive bacteria . TLRs can trigger subsequent inflammatory responses through MyD88 dependent or independent signaling pathways that activate NF-κB and finally lead to the release of pro-inflammatory mediators, including TNF-α, IL-1β, IL-6, IL-8, and iNOS . In inflammatory bowel disease, LPS or cytokines (E.g IL-6 and IFN-γ) can activate JAK/STAT signaling pathway to regulate the expression of pro-inflammatory mediators, including Claudin-2 and iNOS . In our results, C. perfringens challenge increased the mRNA expression of TLR4, TLR2, NF-κB, JAK3, and STAT6, while dietary EA supplement relieved these adverse effects. Due to the deficiency of appropriate antibodies available for use in studies of chickens, we did not determine the protein levels and phosphorylation status of components in these signaling pathways. A series of studies [7, 12, 30] have reported the activation process of TLR/NF-κB or JAK/STAT signaling pathways in broilers with C. perfringens challenge. Similar to our results, EA was proved to possess a protective effect on concanavalin A-induced hepatitis in mice via decreasing the expressions of TLR2 and TLR4 and suppressing NF-κB signaling pathway . C. perfringens challenge in this study has no obvious effect on the mRNA expression of MyD88, which may indicate TLRs activate NF-κB through MyD88 independent signaling pathways. Some studies [29, 31] have reported EA inhibited the phosphorylation of JAK1, JAK2, STAT1, and STAT3 to exert anti-inflammatory effects in keratinocytes or rats, but no report has been found on the impact of EA on JAK3/STAT6 in any animals. In human and mice, the activation of JAK3/STAT6 pathway was related to the differentiation of monocytes and the enhancement of Th2 inflammatory response (the release of IL-4, IL-5, and IL-13) . It means that C. perfringens challenge may trigger the Th2 inflammatory response related to the JAK3/STAT6 pathway in jejunal mucosa of broilers, while EA relieves this hazard in this pathway.
During the inflammatory response, the activation of TLR/NF-κB and JAK/STAT signaling pathways can induce the release of a variety of pro-inflammatory cytokines, which will lead to the activation of immune cells and the production of more cytokines . TNF-α and IL-1β are pleiotropic pro-inflammatory cytokines, whose dysregulations are linked with a wide range of pathological conditions, such as infection, metabolic syndrome, and inflammatory bowel disease . IL-8 is a very potent trigger to immune cells’ migration and proliferation, which guides neutrophils to the direction of inflammation . TGF-β and IFN-γ also play an important role in a variety of inflammation-related diseases; C-reactive protein promotes the inflammatory response of atrial fibrillation through the overexpression of TGF-β related to the TLR4/NF-κB/TGF-β pathway in HL-1 cells, which is related to heart arrhythmia , while IFN-γ was reported to contribute to the hepatic inflammation in HFD-induced nonalcoholic steatohepatitis by STAT1β/TLR2 signaling pathway in mice . iNOS is related to immune response via macrophage defence mechanism, its expression and the increase of NO levels can cause various inflammation-related pathophysiological conditions, the cell wall components of bacteria (mainly through LPS) can activate the JAK/STAT signaling pathway and subsequently activate NF-κB to initiate iNOS transcription . In our study, C. perfringens challenge caused up-regulations on the mRNA expressions of pro-inflammatory mediator genes TNF-α, IL-1β, and iNOS in jejunal mucosa, while the EA diets down-regulated the mRNA abundances of TNF-α, IL-1β, IL-8, and iNOS. A series of studies [12, 24, 30] have proved that C. perfringens challenge can cause an up-regulation on pro-inflammatory mediator genes in the intestine of broilers, including TNF-α, IL-1β, IL-8, TGF-β, IFN-γ, and iNOS. Meanwhile, the alleviating effect of EA on inflammatory mediators, including TNF-α, IL-1β, IL-8, and iNOS has been widely reported in mice or rats [15, 16], which are in line with our results and further indicated that EA reduced inflammatory mediators in broilers may be through NF-κB and STAT signaling pathways. However, the C. perfringens challenge or dietary EA levels show no significant effect on TGF-β and IFN-γ in our results, which may be related to the difference on C. perfringens strains and frequency of the challenge.
The activation of inflammatory pathways and the release of inflammatory mediators can affect the antioxidant, barrier, and absorption functions of the jejunum, which are vital to the growth performance and health of broilers.
Oxidative stress plays an important role in NE. SOD can convert O2•− into H2O2, CAT then transforms the generated H2O2 into H2O, thus preventing the harmful effects of oxidative radicals . In our results, SNE induced by C. perfringens decreased the antioxidant capacity of jejunal mucosa by reducing the activities of SOD and CAT, resulting in an increase on MDA concentration, while the dietary EA supplementation relieved these adverse effects. EA itself has good antioxidant capacity , which may jointly explain the antioxidant mechanism of EA in SNE induced by C. perfringens. Moreover, in the oxidized fish oil-induced oxidative stress of mice, the supplementation of EA in diet increased the total antioxidant capacity (T-AOC) and the activities of the glutathione peroxidase (GSH-Px) and SOD, while decreased the MDA concentration in the intestine . Another report demonstrated that EA exerted anti-inflammatory and antioxidant functions against streptozotocin-induced diabetic nephropathy in rats via reducing the activation of NF-κB and increasing the nuclear translocation of Nrf2 to up-regulate GSH, γ-GCL, and SOD activities .
Tight junction proteins are vital structures of the physical barrier in jejunal mucosa, which form a seal between intestinal epithelial cells and prevent the transmission of macromolecules . In the present study, C. perfringens challenge decreased the jejunal mRNA expressions of ZO-1 and occludin in broilers and increased the mRNA expression of claudin-2, the dietary supplementation of EA relieved these adverse effects. ZO-1 and occludin are barrier-forming proteins, whose reduction mean damage of tight junctions; whereas claudin-2 is a pore-forming protein, whose increase can increase the permeability of intestinal barrier . As many studies reported [9, 12], the infection of C. perfringens can reduce the mRNA expression of ZO-1 and occludin in broilers through the activation of NF-κB. While pomegranate and pomegranate leaf, which rich in EA, can relieve the decrease of ZO-1 and occludin caused by alcoholic liver disease or hyperlipidemia in the intestine of mice . The infection of C. perfringens can increase the expression of claudin-2 in intestine of broilers , which may be explained as a result of 'cross-talk' caused by IL-6 between JAK/STAT, SAP/MAPK, and PI3K signaling pathways . Interestingly, the mRNA expression of occludin was increased in the broilers only fed the diet with EA supplementation; in another study , thymol and carvacrol supplementation demonstrated a similar effect on the mRNA expression of occludin in broilers challenged with C. perfringens.
D-xylose crosses the intestinal mucosa via a Na+-dependent mobile-carrier mechanism, in the case of malabsorption syndrome, the entry of D-xylose from the gut lumen to the portal vein is damaged, resulting in reduced concentrations of D-xylose in blood . DAO is an intracellular enzyme in the small intestinal epithelia and released into the peripheral circulation as a result of intestinal villi damage, so the level of serum DAO could reflect the severity of intestinal mucosal injury . In our study, the decrease of plasma D-xylose concentration indicated that C. perfringens challenge had impaired the intestinal absorption function, while the increase of DAO activity in serum may imply the relation to the impaired intestinal epithelium. The supplement of dietary EA alleviated the decrease of plasma D-xylose concentration, but had no effect on DAO activity in serum. Similar to our results, the arginine additive alleviated an increase on plasma D-xylose concentration caused by the C. perfringens challenge . LZM can cleave peptidoglycan of the cell wall in Gram-positive bacteria, resulting in the loss of cellular membrane integrity and cell death . In our results, C. perfringens infection increased the activities of iNOS and LZM in jejunal mucosa, while the supplement of EA in diet relieved these adverse effects. LZM was up-regulated in the gastrointestinal tract of patients affected by chronic inflammation, which was related to the LZM-mediated processing of luminal bacteria in the colon that triggered the pro-inflammatory response . These up-regulations of iNOS and LZM in our result further explained the mechanism of chronic inflammation caused by SNE.
Damage of the intestinal barrier and absorption function was also reflected in the microstructure of jejunum. The results of jejunal morphology, including VH, CD, and V/C ratio by HE staining, were important indexes that intuitively reflected jejunal health and absorption surface. C. perfringens challenge seriously destroyed the villi structure and reduced the absorption surface of nutrient, which is in line with the results reported previously [12, 25]. On the contrary, the dietary EA supplementation alleviated the jejunal lesions, indicated the good condition of enterocytes and efficient ability of nutrient absorption. In the mice model , EA effectively alleviated the intestinal damage caused by oxidized fish oil via significantly increasing the VH and V/C, while improving the mucous epithelium injury. Also, thymol and carvacrol alleviated the ileal lesion and improved V/C ratio in broilers with C. perfringens infection . Furthermore, the antioxidant and anti-inflammatory effects of EA may explain the mechanism that is beneficial to the health of intestine and villus-crypt architecture.
Intestinal NE lesions and mucosal atrophy greatly compromises epithelial permeability and mucosal barrier function, which may result in adverse effects on internal environment homeostasis and production performance of broilers, therefore, these serum inflammation biomarkers were used to evaluate the systemic inflammatory response intensity of broilers. LPS is an endotoxin produced by Gram-negative bacteria, its increase in blood reflected the bacteria translocation to liver, spleen, and blood . IL-6 is an important cytokine of inflammatory bowel diseases, which can activate the JAK/STAT signaling pathway and promote the release of various inflammatory factors . CRP is synthesized in liver, mainly in response to IL-6, and can be combined with the pathogen LPS to activate the classical complement pathway . PCT is a diagnostic marker of bacterial infection, which is produced by LPS, TNF-α, and IL-6 acting on neuroendocrine cells or special cells in the liver and spleen. MPO is a sign of neutrophil aggregation and inflammation, its activity is a marker of neutrophil infiltration into the intestine. C. perfringens infection increased the concentrations or activities of LPS, IL-6, CRP, PCT, and MPO, causing a higher stress of systemic inflammatory response in broilers, while the supplement of EA in diet relieved these adverse effects. In line with our results, Lactobacillus acidophilus supplementation significantly decreased the serum LPS content in broilers with C. perfringens challenge , while EA treatment can decrease the mRNA expressions of TNF-α and IL-6 in the liver and intestine of oxidative stress mice . In NE model caused by C. perfringens, probiotic powder containing Lactobacillus plantarum decreased the MPO activity in the ileum mucosa of broilers . Overall, our study reflected that EA alleviated the systemic inflammatory response caused by C. perfringens challenge, possibly by protecting the integrity of intestinal mucosa and reducing the expression of inflammatory factors.
On the other hand, intestinal microbiota is involved in intestinal nutrition, defense, and immunity. The high diversity of intestinal microbiota is beneficial to maintain the stability of the intestinal microenvironment and defend against the invasion of pathogenic microorganisms . In our study, only the dietary EA supplement increased the alpha diversity, including observed species and Shannon index, which may mean an improvement in intestinal health; but the beta diversity analysis showed no difference of the microbial community structure among groups, which may be related to the microbiota from different parts of the intestine and the time of sample collection. In broilers challenged by C. perfringens and Eimeria , the effects of dietary lauric acid supplement or the challenge on microbiota in the jejunum were distinct from those in the cecum, as well as the change of microbiota was more significant in jejunum; however, these treatments did not promote significant difference of taxa abundance and diversity in cecum, which may explain our results. In terms of microbial abundance, C. perfringens challenge increased the relative abundance of Firmicutes and decreased the relative abundance of Desulfobacterota. Similarly, EA increased Firmicutes relative abundance and showed a trend of lowering Desulfobacterota and Campilobacterota relative abundances. It has been reported that Firmicutes improved the utilization of energy in the diet and the ratio of Firmicutes to Bacteroides was often positively associated with weight gain . However, both EA and C. perfringens challenge resulted in an increase in Firmicutes abundance and a decrease in Desulfobacterota abundance, which may be related to the longer time interval between challenge and sample collection, as well as the immune regulation of broilers, especially in the EAXCP group, which may mean that the challenge has played an immune-stimulating effect with the presence of EA. In rats with stress-induced depressive-like behavior , fecal microbiota transplantation ameliorates gut microbiota imbalance and intestinal barrier damage through increasing Firmicutes and decreasing Desulfobacterota and Bacteroidetes at phylum levels; this treatment also reduced the loss of villi and epithelial cells, suppressed the inflammatory cell infiltration, and increased the expression of ZO-1 and occludin in the ileum. These results were amazingly similar to ours, which may indicate that the microbiota displayed a similar mechanism in the intestinal protection of broilers. Campylobacter was believed to be closely related to the zoonotic campylobacter disease , the EA-induced decrease of Campilobacterota relative abundance may have a protective effect on the health of broilers. At the genus level, only the main effect of C. perfringens challenge showed a trend of heightening Ruminococcus]_ torques_group abundance. The increase of [Ruminococcus]_ torques_group abundance was reported in irritable bowel syndrome, which phylotype was associated with severity of bowel symptoms . Another study showed that the Ruminococcus]_ torques_group seemed to be especially involved in controlling paracellular permeability , which may be another factor that SNE affects intestinal permeability of broilers in our result.
LEfSe analysis revealed the different phylotypes of cecal microbiota between groups. Compared with cecal microbiota in Control group, the increased abundance of Oscillospiraceae in CP group was thought to be linked to intestinal inflammation . The effect of Butyricicoccaceae abundance on inflammation was lacking in reports, but it was thought to be an important butyrate producer , which may be beneficial to the recovery of the intestines. Gordonibacter_pamelaeae has been reported to have the function of transforming EA into urolithin , its high abundance was also observed in the EAXCP group, which may mean the transfer of the microbiota between the CP and EAXCP groups, because they were kept in the same pheasantry room. Compared with cecal microbiota in Control group, a main increase on the abundances of Turicibacter_sp_H121 and Romboutsia_ilealis in EA group was found in our results. The increase on Turicibacter_sp_H121 was also observed in cecal microbiota of EAXCP group compared with CP group, which may indicate that EA is beneficial for Turicibacter_sp_H121, but the effect of the increase is unclear. Romboutsia ilealis is a beneficial intestine bacterium, whose decrease in the response to Streptococcus agalactiae infection of zebrafish was considered harmful . The cecal microbiota between EAXCP and other three groups were quite different. In EAXCP group, the increased abundance of Sellimonas has been reported as a potential biomarker of homeostasis gut recovery after dysbiosis events , Bacteroidales was thought to be involved in the synthesis of fatty acids and are beneficial to the health of the host , Erysipelotrichaceae was highly abundant in good FCR broilers , and mice fed with normal diet possessed more abundant than those fed with high fat diet on Monoglobaceae and RF39 . Rhodobacteraceae is widely reported in aquatic animals or marine environments and has no adverse effects on host health. In Control group, the abundance of Synergistes was reported to be negatively correlated with the levels of IL-1β, IL-6, and TNF-α in serum samples from piglets , but Phascolarctobacterium predominated among the Clostridia in low FCR birds . Dietary supplementation with medium-chain a-monoglycerides can decrease the abundances of Cerasicoccus, and improve productive performance and egg quality in aged hens . Comparing with birds in CP group, those in EAXCP group had higher abundance of Faecalibacterium, which was enriched in chickens with the higher BW . It was speculated that Clostridiales_vadinBB60_group might also be beneficial bacteria in intestinal tract . Bifidobacterium_breve was probiotic which has been verified . Comparing with birds in EA group, those in EAXCP group had a higher abundance of Subdoligranulum, which was negatively correlated with CRP and IL-6 in human . Fusobacterium mortiferum was often reported in clinical infections of human, but its strains, which were isolated from poultry caeca, also can produce bacteriocin-like substances inhibiting Salmonella enteritidis . For another, birds in EA group had higher abundance of Muribaculaceae, which negatively correlated with inflammatory markers in high fat-high sucrose diet-induced insulin resistant mice . Eubacterium_xylanophilum_group was thought to be lactic acid- and SCFA-producing bacteria, which enhanced intestinal homeostasis and ameliorated weaning stress in piglets . Escherichia coli showed higher levels in broilers with smaller BW . In rats, Elusimicrobium was thought to be beneficial bacteria, whose increase can protect the intestinal barrier . Overall, C. perfringens challenge caused an adverse effect on the cecal microbiota of broiler chickens, dietary EA supplementation led to a small beneficial effect, while the simultaneous effect of dietary EA and challenge seems to stimulate the immune function of broilers and made them possess a better cecal microbiota. Furthermore, the cecal microbiota of the EAXCP group seems very different from other groups, which may explain the significant interaction between dietary EA level and C. perfringens challenge in our results.
Finally, growth performance is the most comprehensive indicator of commercial broiler quality. SNE induced by C. perfringens usually reduces the performance of broilers without serious clinical symptoms and high mortality [4, 6]. In this study, the main effect of C. perfringens infection lowered BW and ADG of flocks, as well as enhanced FCR. However, the addition of EA in diet increased ADG and decreased FCR in broilers. Previous studies [26, 42] reported that SNE challenge reduced ADG, body weight gain, and feed intake, while heighten FCR in broilers, which were similar to our results. The supplementation of plant extracts, including tannin and polyphenol compounds, have been proved to be effective against NE [12, 46], but the effect of EA on the growth performance of broilers has not been reported. In laying quails, EA improved the feed conversion and egg quality . In addition, pomegranate extract was reported to have a positive effect on the growth and slaughter performances of broilers . These improvements provided by EA may be explained by improving the intestinal barrier function and microbiota structure, thereby indirectly increasing the performance of broilers raised without anti-resistant diets .