Under the management of modern intensive farming modules, broilers are affected by various stress factors, such as environmental pressure, LPS, and pathogenic microorganisms, that can cause intestinal inflammation (1–3). The intestine serves as an immune barrier and the first line of defense against intestinal microbial infections, consisting of a mucus layer, tight intercellular junctions, antimicrobial peptides, and immunoglobulin A (4). It can prevent the invasion of exogenous pathogens or harmful substances, while also playing a crucial role in maintaining intestinal homeostasis and physical health (3, 5). When the mucus layer and intercellular tight junctions in the small intestine are disrupted, endotoxin produced by the metabolism of metabolizing bacteria enters and leaves the bloodstream, causing systemic and intestinal inflammation, disrupting the intestinal barrier, increasing intestinal permeability, inhibiting the absorption and transportation of nutrients in the small intestine, and leading to various chronic diseases (6, 7).

Intraperitoneal injection of LPS can lead to intestinal inflammation, increased intestinal permeability, and intestinal barrier damage. It has been reported that LPS challenge decreased mRNA abundances of β-defensin 2 (pBD-2), mucin (MUC-4), zona occludens 1 (ZO-1), and occludin in jejunal mucosa of piglets (8–10). LPS can recognize the cell surface transmembrane protein Toll-like receptor 4 (TLR4) and activates signaling pathways via myeloid differentiation factor 88(MyD88), which activates the nuclear factor-kappa B (NF-κB) signaling cascade. NF-κB can promote the production and release of inflammatory factors, thereby participating in inflammatory reactions and causing oxidative reactions in the body (11).

Research has shown that many traditional Chinese medicines can improve LPS-induced intestinal mucosal damage in broiler chickens and inhibit intestinal inflammation and oxidative stress, such as by inhibiting the expression of intestinal inflammation genes and intervening with Nrf2 and NF-κB pathways (12, 13). To address the problem of intestinal inflammation and barrier damage in broilers in poultry farming, we wove the formula of WZS. WZS is composed of Schisandra, Anemarrhenae, Lonicerae, and Glycyrrhizae, and has anti-inflammatory, antioxidant, and immunomodulatory effects. Schisandra has immunomodulatory effects, such as invigorating qi, invigorating fluid, and tonifying the kidney (14). Schisandra extract can ameliorate DSS-induced colitis, and its effect may be associated with suppression of the TLR4/NF-κB/NLRP3 inflammasome pathway and GM regulation (13). Research has shown that the active component of Anemarrhena asphodeloides B can block the production of NF-κB, and antipyretic effects are achieved by inhibiting the p38 mitogen-activated protein kinase pathway (15). In addition, Mangiferin in Anemarrhena has strong antioxidant effects (16). Lonicerin can target EZH2 to alleviate ulcerative colitis by autophagy-mediated NLRP3 inflammasome inactivation (17). Glycyrrhiza uralensis could regulate apoptosis of intestinal mucosal cells, through regulating the expression of apoptosis-related proteins and protective proteins of intestinal mucosa (18).

Therefore, this study investigated whether the addition of WZS to the diet could alleviate LPS-induced intestinal inflammation and barrier damage in broilers by inhibiting the inflammatory pathway and enhancing intestinal tight junction protein gene expression.

LPS activates macrophages in the animal body, leading to the production of large amounts of reactive oxygen species, which causes an imbalance in the production and elimination of excess reactive oxygen species in the body, resulting in lipid peroxidation in the body (21). As one of the products of lipid peroxidation, SOD activity can serve as a marker for evaluating the body’s antioxidant status. Its production is positively correlated with the content of oxygen free radicals, which can prevent the oxidation process initiated by superoxide anions and convert superoxide into hydrogen peroxide (22). MPO is a specific enzyme for neutrophils that reduces hydrogen peroxide and decreases the production of free radicals, and its level can be measured to directly reflect the number and activity of neutrophils (23). The more severe the inflammation, the higher the number of neutrophils and the higher the level of MPO (24, 25). The results showed that LPS could decrease SOD levels and increase MPO content in broilers, and all WZS groups could increase SOD levels in broiler serum to varying degrees, and effectively alleviate MPO content increase, among which the WZS-M group had the best effect. These results indicated that WZS had antioxidant and anti-inflammatory effects, and could significantly reduce the aggregation degree of neutrophils in chickens, therefore, reducing the inflammatory damage degree and oxidative stress damage degree of neutrophils to the body.

It has been shown that the levels of DAO and D-LA can reflect the damage of intestinal stress and are sensitive indicators that can evaluate intestinal permeability (26). LPS can damage the intestinal mucosal barrier in broilers, resulting in increased intestinal epithelial permeability (27). This is similar to the results of the present study. In this experiment, immune stress was able to damage the intestinal tract, leading to increased intestinal epithelial permeability and elevated serum DAO activity and D-LA levels. In both stress phases, all WZS groups were able to significantly reduce the serum levels of DAO and D-LA in broilers, indicating that WZS alleviated the intestinal epithelial damage caused by LPS by reducing the serum levels of DAO and D-LA in broilers.

The digestive and absorptive capacity of the intestine is usually reflected by VH, CD, and VH/CD indicators (28, 29). Mucosal integrity, digestive enzymes, transporter proteins, and intestinal microorganisms play an important role in the digestion and absorption of nutrients. Nutrient absorption occurs mainly in the small intestine, and to improve nutrient absorption in the small intestine, VH needs to be increased to expand the contact area between nutrients and the small intestine (30). It also requires a decrease in CD to promote the proliferation of intestinal epithelial cells, which further improves intestinal absorption. In the present study, under LPS stimulation treatment, broilers in the 21-day-old LPS group had significantly increased jejunal CD and significantly reduced VH/CD than the CON group, while the drug administration group was able to significantly reduce jejunal CD and increase VH/CD, among which the WZS-H group had the best effect. Broilers in the 35-day-old LPS group had significantly increased jejunal CD and significantly reduced VH and VH/CD when compared to the other groups. However, the WZS groups were able to alleviate this result to different degrees, and the effects were significant, among which the WZS-L group had the best effect. A study using fermented plant material added to broiler diets found that it significantly increased the villi length and villi length to crypt depth ratio of broiler jejunum under LPS stress, and significantly reduced the crypt depth of broiler jejunum (31). This is consistent with the results of the present study.

LPS binding to TLR4 activates the MyD88 pathway, which in turn induces nuclear displacement of NF-κB and induces gene expression of inflammatory factors, thereby activating lymphocytes to secrete anti-inflammatory cytokines to participate in the body’s immunity. If this signaling pathway is overactivated, the mRNA expression of NF-κB increases, which in turn causes an elevated expression of inflammatory factors leading to an inflammatory response (32). The results of the present study revealed that the mRNA expression of TLR4 in the jejunum of broilers in the LPS group was elevated to different degrees compared with the CON group in both stress phases, indicating that it was immune stress that led to the overexpression of TLR4 mRNA in the jejunum of broilers. Furthermore, the mRNA expression of MyD88 and NF-κB and the downstream signaling molecules of TLR4 were also significantly elevated and led to the protein amount of NF-κB. The expression of these genes, as well as NF-κB protein, was significantly increased in all administration groups, indicating that WZS can reduce the receptors necessary for LPS to attack cells, cut off the MyD88 pathway for LPS to invade cells, and reduce the overexpression of NF-κB genes and protein by decreasing the expression of TLR4 in chick jejunum. The expression in the WZS-L group showed the most obvious effect.

Overexpression of signaling molecules on the TLR4 signaling pathway leads to increased transcription of inflammatory factor genes, which in turn causes massive production of inflammatory factors in the organism. The test results showed that the mRNA expression of IL-6, IL-1β, and TNF-α in the jejunum of broilers in the LPS group increased significantly during the stress phase, indicating that immune stress leads to the overexpression of genes of inflammatory cytokines in the chick organism, resulting in an increase of inflammatory cells in the organism and causing damage to the organism. The mRNA expressions of IL-6, IL-1β, and TNF-ɑ in the jejunum of broilers in each administration group of this experiment were reduced to different degrees, indicating that the herbal compound can inhibit the gene expression of these inflammatory factors. Among them, the inhibition effect observed in the WZS-H group was better. This may be due to the fact that WZS can reduce the overexpression of related signaling molecules on the TLR4/MyD88/NF-κB signaling pathway, thus reducing the gene expression of inflammatory factors. It has also been shown that the increase of IL-1β disrupts the intestinal tight junctions and disrupts intestinal permeability (10). IL-10 could limit the activation of innate and adaptive immune cells to maintain homeostasis and protect the host from immune pathological damage, autoimmune damage, and allergic reactions induced by infection. During the stress period, IL-1β mRNA expression in the jejunum of broilers in the LPS group showed a significant increase, the expression of IL-10 mRNA in the jejunum of broilers in the LPS group was significantly reduced, and the mRNA expression of IL-10 in the jejunum of broilers in each WZS group was increased to different degrees, with the expression level of the WZS-H group being the highest and the effect being the best.

Tight junctions (TJs) are key components of the intestinal mucosal barrier and play an important role in regulating intestinal permeability by maintaining the integrity of the intestinal barrier and ensuring normal barrier function (33). ZO-1, Oclaudin-1, and white Claudin-1 are important components of TJs (34). In the present study, immune stress resulted in impaired jejunal tight junction structures characterized by a significant decrease in the mRNA expression of ZO-1, Occludin-1, and Claudin-1 in the jejunum of the LPS group, consistent with the results of previous studies (34, 35). The mRNA expression of Claudin-1, ZO-1, and Oclaudin-1 in the jejunum of broilers in each WZS group was elevated to varying degrees. This was due to the elevated intestinal permeability, which reduced the protective effect of the intestinal barrier on the intestine (36). Supplementing WZS in feed can alleviate the damage of LPS stress to the intestinal tight junction structure of broilers, and reduce the damage of inflammation to the body. However, the optimal dosage of WZS in feed needs to be further studied in subsequent experiments.

Dietary supplementation of WZS can reduce intestinal inflammation, reduce intestinal barrier damage, and intervene in LPS-induced stress damage by inhibiting the NF-κB/TLR4/MyD88 signaling pathway.

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author.

The animal study was approved by the Animal Ethics Committee of the South China Agricultural University (Guangzhou, China). The study was conducted in accordance with the local legislation and institutional requirements.

HS and CL conceived and designed the experiments. HS, BY, and SY completed the animal test. HS, XZ, and MY completed the laboratory test. HS and HW analyzed the data. HS and XZ wrote the original manuscript. HS, CL, XZ, HW, DS, and SG reviewed and edited the manuscript. CL supervised this study and provided funding. All authors contributed to the article and approved the submitted version.