There are trillions of microorganisms in gastrointestinal tract, in which reside many immune cells and cytokines molecules involved in innate and adaptive immune systems to defend against bacterial invasion and maintain the intestinal health. The innate immune system is the first line of defense and consists of physical intestine barrier, chemical and cellular defenses. The adaptive immune response is the second gut defense and is pathogen-specific. Innate immunity occurs immediately while adaptive immunity develops upon pathogen exposure to produce antibody, and is long-lasting (Stambas et al., 2020). Both of them play synergistically effects in intestine defense processes against various harmful external agents and pathogenic invasion from the broken gut epithelial barrier (Zheng et al., 2020). It is clear that the prominent receptors of innate immunity are NOD-like receptors (NLRs) and Toll-like receptors (TLRs) in human gut. TLRs commonly combine with extracellular pathogens substances in gut, and most NLR members recognize the intracellular dangerous debris from pathogenic microbes (Zheng et al., 2020). Once these receptors of immune cells are activated, the intracellular signaling cascades involved in the inflammatory and other immune responses are rapidly induced, such as NOD-like receptor signaling, and Toll and lmd signaling pathway. Inflammasome fulfills a central role in the inflammatory process of innate immunity, which is a supramolecular complex in the cytosol. A canonical inflammasome comprises three parts (Fu et al., 2024): sensor, adaptor and effector proteins. The sensor protein, such as NLRP3, is triggered by intracellular danger signals of PAMPs (pathogen-associated molecular patterns) resulting in its oligomerization. Most inflammasome sensors are members of nucleotide-binding oligomerization domain-like receptor (NLR) family (Fu et al., 2024). And the adaptor protein, apoptosis-associated speck-like protein, is recruited by the sensor, and bridges the effector caspase-1. Caspase-1 then cleaved the proinflammatory cytokines to their matured forms of IL-1β and IL-18 (Fu et al., 2024).

Beside innate immunity, some stimuli from microbes in intestinal lumen can be recognized by adaptive immune system. FcγR is the core receptor in adaptive immune responses, which binds with extracellular pathogenic particles, activating phagosomes along with FcγR-mediated phagocytosis signaling. The engulfed materials in phagosomes are digested under the help of enzymes from the fused lysosomes. Then, the antigen-presenting cells, including dendritic cells (DCs) and phagocytes, are activated to present antigens via antigen processing and presentation pathway (especially by MHC-I and MHC-II molecules) and to produce IL-12 (Trinchieri, 2003). IL-12 participates in the immune cell mediated adaptive immunity via stimulating the differentiation of Th1 cells from naive CD4⁺ T cells. The primary effector of gut inflammatory response is CD4⁺ T cells. Th1 cells then induce strong cytotoxicity effects of macrophages and cytotoxic T cells to phagocytize those IgG-coated targets (Bournazos and Ravetch, 2015). In addition, the naive CD4⁺ T cells also differentiate into regulatory T (Treg) and Th17 cells. The differentiation of Th17 cells mainly dependent on IL-1β and aggravates inflammation by production of IL-17. Treg cells secret IL-10 and function as an inhibitor to Th1 and Th17 cells activities, decreasing the secretion of IL-1β and IL-17, thereby limiting inflammation and maintaining the immune homeostasis (Rasquinha et al., 2021).

Unavoidably, some conditional pathogenic bacteria residing in intestine might invade intestine especially those evading innate immune system by using a variety of strategies. After invasion, bacteria release macromolecules to further target components of NF-κB signaling pathway of intestinal epithelial cells, resulting in intestinal inflammation and diarrhea (Bruno et al., 2009). Thus, inhibiting inflammation and protecting intestinal epithelial integrity is considered to be an efficacious treatment for preventing pathogens invasion especially in young rabbits, which is more sensitive to seasonal changes due to their specific physiological characteristics, such as few sweat glands, thick coats of fur, high metabolic and growth rates (Ye et al., 2023a). The conditioned infections mostly encountered in intensive breeding farms caused by opportunistic organisms that act singly or jointly including bacteria, viruses, protozoa and mycetes. Furthermore, chronic, progressive and persistent infections might present in rabbits, which are slow, latent, tolerated, hidden infections or virus-associated tumors (Galassi et al., 1986). In commercial rabbits, diarrhea is an important factor influencing profitable production. During the late 2001 and early Spring of 2002 in Mexico, a sharp outbreaks of diarrhea characterized by a high morbidity and mortality occurred in different commercial rabbit farms. The most affected rabbits are aged 5–7 weeks with death rates varied from 16.7 to 35.2% (Rodríguez-De Lara et al., 2008). Similarly, the colibacillosis of rabbits caused by conditional pathogen E. coli occupy 42.8% from 2003 to 2011 in Guiyang, China (Wang et al., 2012). The traditional Chinese medicines can provide a strategy to deal with those disease caused by conditional pathogen via their anti-inflammation and immunity-modulation capability. Some herbal medicines are characterized by properties of against inflammation, oxidative stress, viral infection, and cancer (National Pharmacopoeia Committee, 2015), such as Acanthopanax senticosus (Rupr & Maxim.) Harms (Ciwujia in Chinese), Astragalus membranceus (Fisch.) Bge (Huangqi in Chinese), Indigo naturalis (Qingdai in Chinese), Houttuynia cordata Thunb (Yuxingcao in Chinese), and Glycyrrhizae radix et Rhizoma (Gancao in Chinese). And those herbs are widely used in Chinese medicine and are efficacious in treating various inflammatory diseases including ulcerative colitis and conditioned chronic inflammation caused by pathogens (Lau et al., 2019; Li et al., 2020; Liu X. et al., 2021; Kakdiya et al., 2024; Shu et al., 2024). However, interrelationship of these combined herbs with the intestinal flora remained largely unclear. This study designed an ACS decoction, which was combination of Ciwujia, Huangqi, Qingdai, Yuxingcao, and Gancao at different dosages, and investigated the effects of dietary ACS supplementation on the growth performance, intestinal immunity and intestinal flora structure in young rabbits.

The designed ACS decoction improved the growth performances such as the ADG, the decreased rates of death and diarrhea in ACS group, and increased the concentration of IL-10 in high ACS addition while inhibited contents of IL-1β and IL-12 in low ACS addition in intestine tissues. Furthermore, the data from 16S rRNA gene sequencing produced 311 genera from more than 600 genera, due to large amounts of genera presented at low or very low richness. The Firmicutes was predominant phylum in rabbit intestine and many new bacteria were needed to be revealed in future.

We further calculated the correlation coefficients of the relative abundance of each bacterium with the cytokines concentrations, and the predictive abundances in KEGG immune signaling pathways inferred by PICRUSt2 (Douglas et al., 2020), respectively. It obtained 24 differential genera between ACS and control groups, which presented clear collaborative effects on immunity responses on rabbit intestine (Supplementary Table S4; Figure 9B). The related factors and signaling pathways interplay formed complex regulatory networks of innate and adaptive immunity systems in rabbit (Figure 10), in which bacteria in intestine activate NF-κB or NLRP3 inflammasome through Toll and lmd signaling pathway and NOD-like receptor signaling to produce IL-1β in innate immunity, and also stimulate T cells differentiation to produce interleukin by APC via FcγR-mediated phagocytosis and IL-17 signaling pathways in adaptive immunity.

Of those 24 differential genera, nine differential genera between ACS groups and control presented promotion effects on gut inflammation, consisted of Alistipes, CAG_352, Candidatus_saccharimonas, Christensenellaceae_R_7_group, Clostridia_UCG_014, Monoglobus, Muribaculaceae, RF39, V9D2013_group in digesta of small and large intestines of rabbits (Supplementary Table S4; Figure 10B). Among them, abundances of genus Alistipes, CAG_352 and Monoglobus were inhibited by ACS supplement but negatively related with immunity pathways such as NOD-like receptor signaling and Toll and lmd signaling, resulting in promotion on gut inflammation. In ulcerative colitis mice model, the abundance of Alistipes is increased and the expressions of NLRP3 and caspase-1 are elevated (Li C. et al., 2024). The inflammation status in rats is related with the enhanced CAG-352 (Ma et al., 2022). Oral opioids cause the patient fecal richness of Monoglobus, and promoting systemic inflammation by activating TLR2/4 (Wang et al., 2022). Another six genura were boosted by ACS and positively related with cytokines concentration and immune signaling pathways. The abundance of Candidatus saccharimonas was positively related with IL-1β concentration and five signals including NOD-like receptor signaling, IL-17 signaling, antigen processing and presentation, Th17 cell differentiation, FcγR-mediated phagocytosis pathways. It does not know how the genus affect those signals while it is reported that the abundance of C. saccharimonas is increased in the colitis-associated carcinogenesis mice model harbored severe inflammatory in colon (Cruz et al., 2020). Two genera, Christensenellaceae_R_7_group and V9D2013_group, were also detected to relate with IL-1β and IL-10 and more than one signals in young rabbits. The coprophagy prevention augments cecum inflammation and apoptosis with higher content of IL-1β and abundance of Christensenellaceae_R-7_group and V9D2013_group in cecum, and many genes enriched in IL-17 signaling, Th17 cell differentiation, NOD-like receptor signaling, antigen processing and presentation, Toll-like receptor signaling pathways based on transcriptome sequencing of cecum from rabbits (Li, et al., 2024), rats and chicken (He et al., 2022; Lin et al., 2023; Ye et al., 2023a). In the present study, the increase of RF39 in ACS group was positively related with NOD-like receptor signaling together with concentrations of IL-1β and IL10. Broiler chickens with necrotic enteritis show higher mortality and intestinal lesion together with increases of RF39, IL-1β, and IL-17/IL-10 ratios in jejunum (Song et al., 2023), and cecum of mice (Zhang et al., 2020b). The elevated richness with 2–3-folds of Muribaculaceae and Clostridia_UCG_014 in ACS groups was significantly related with IL-1β level in young rabbits. It is reported that the abundances of Muribaculaceae and Clostridia UCG 014 are positively correlated with the content of IL-1β in rabbit (Li et al., 2022; Ye et al., 2023a) and in mice (Xie et al., 2024). All of these works indicated that the effects of the nine differential genera had a role to stimulate immunity and reinforce the inflammation response in rabbit intestine.

After ACS supplement, 17 differential genera showed inhibition effects on gut inflammation with strong relationship, such as Bacteroides and Corynebacterium, etc., in which two genera, Clostridia_UCG_014 and RF39 were multifunctional in ileum and rectum. About seven genera were negatively related with interleukin concentration of IL-1β, IL-12, or IL-10 in different gut fragments, including Corynebacterium, Enterorhabdus, Eubacterium_ruminantium_group, Papillibacter, RF39, Subdoligranulum, and UCG_005. Taking a protein of Corynebacterium as antigen can stimulate IL-12 and Th1 response in immunized sheep and mice (Vale et al., 2016; Barbosa et al., 2024). The abundance of Enterorhabdus is negatively associated with colonic IL-1β in mice (Guo et al., 2022). Similarly, phenolic phytonutrients increases Eubacterium_ruminantium_group, reduces inflammation and the concentration of IL-12 in rumen of lambs (Wang et al., 2023). Oral leaf powder from Moringa oleifera reduces contents of IL-1β, and increase Papillibacter in cecum (Yasoob et al., 2021). RF39 presents the anti-inflammation effects in chicken and sows (He et al., 2022b; Liu et al., 2022; Chen et al., 2024). N-carbamylglutamate or glutamate increases Subdoligranulum and UCG_005 in colonic digesta, increases IL-10 and IL-1β in piglets (Liu et al., 2023a, 2023b). Furthermore, both of Subdoligranulum and Eubacterium are butyrate producers. Butyrate is utilized to protect intestinal barrier integrity, restricted pro-inflammatory cytokines to hinder the entry of opportunistic pathogens, and to help a healthy gut environment (Singh et al., 2023).

Those six differential genera were detected to be related with NOD-like receptor signaling or Toll and lmd signaling pathways, consisting of Bacteroides, Clostridia_UCG_014, Clostridia_vadinBB60_group, dgA_11_gut_group, Parabacteroides, and Phascolarctobacterium. Oral supplementation with cranberry polyphenols increases the abundance of Bacteroides and TLR2 expression in obese mice (Medina-Larqué et al., 2022). The protection by Bifidobacterium is accompanied by an increase of Clostridia_UCG-014, reduce the ileum inflammation in mice (Yue Y. et al., 2023). Polysaccharides increases the proliferation of Clostridia_vadinBB60_group, the bacteria is contributed to alleviate gut inflammation in mice (Tian et al., 2024). The mulberry 1-deoxynijirimycin increases the abundance of dgA-11_gut_group, decreases the inflammation of rabbits (Li S. et al., 2024). The richness of Parabacteroides is positively correlated with intestinal integrity while negatively correlated with TLR4 and NF-κB in chicken (Kong et al., 2021). Treatment of electroacupuncture increases fecal richness of Phascolarctobacterium and inhibits NLRP3 in rats (Zhou et al., 2022). Other types of three differential genera were related with multiple signalings, including Eubacterium siraeum group, E. xylanophilum group, and UCG_010. The Chinese medicine, Qianshan Huoxue Gao, is reported to improve the inflammation and positively correlated with abundance of E. xylanophilum group and E. siraeum group (Zhao et al., 2023; Ye et al., 2023b). The family UCG-010 has a role in anti-inflammatory effects after malic acid supplied to sows (Chen et al., 2024).

The decreased NK4A214_group in ACS groups was the only genus to be positively related with FcγR-mediated phagocytosis pathway. It is not clear how the bacteria affect FcγR-mediated phagocytosis, but it is detected that the genus is negatively correlate with IL-1β and showing anti-inflammation in Hyplus rabbit cecum (Ye et al., 2023a; Li Z. et al., 2024). In the present paper, the coefficient of NK4A214_group abundance with IL-1β concentration was 0.48 although the p-value was not significant (p = 0.12), which indicated the strain might have a role in the immune regulation of ACS in rabbits. Taken together, all mentioned differential 17 genera bacteria tended to have an anti-inflammation effect in supplement of ACS in intestine of young rabbits.

Numerous researches indicate that the relationship between Chinese herbal medicines and intestinal flora is much complicated. Some components in Chinese herbal medicines affect the structure and abundance of intestinal flora, and improve the intestinal environment for the growth of bacteria. In a similar vein, the vibrant intestinal bacteria can also change the catabolism, absorption, chemical modification, potency of the herbal medicines (Zou et al., 2024). And both of herbal medicines and intestine flora might synergistically regulate the immunity of rabbits. In the aqueous extract from Ciwujia (the monarch herb in ACS decoction), the most potent efficacy of phytochemicals contain polysaccharide, isofraxidin, and eleutheroside E (Lau et al., 2019). Dietary Ciwujia polysaccharide decreases the expressions of IL-1β, IL-6, and TNF-α in spleen of piglets (Han et al., 2014; Fan et al., 2019). The component isofraxidin inhibits IL-1β-induced joint inflammation via the regulation of NF-κB signaling (Lau et al., 2019). And Eleutheroside E suppresses the release of inflammatory cytokine TNF-α and IL-6 in cultured macrophages (Lau et al., 2019). Moreover, Ciwujia reduces the counts of E. coli, staphylococci, Pseudomonads and Clostridia in rabbits (Lauková et al., 2016). And Astragalus polysaccharides supplementation significantly decreased the abundance of Bacteroides in cecum and closely associated with the alteration of ADG, FCR, TNF-α, IL-1β in chicken and rat (Qiao et al., 2022; Liu Y. et al., 2021; Zhou et al., 2023). Houttuynin, the component of Yuxingcao, harbors activities of antibacterial, anti-inflammatory, antioxidant, antitumor (Cheng et al., 2023; Lau et al., 2019; Liu X. et al., 2021), and regulation of intestinal flora by repressing the TLR4/NF-кB pathway (Zhang et al., 2021; Zhang et al., 2020a). Indigo Naturalis (Qingdai in Chinese), has been used for treatment on patients with ulcerative colitis in China, and the major active components are indigo and indirubin (Kakdiya et al., 2024). It can reduce Th17 cell differentiation and IL-17, IL-1β, and TNF-α release in mice with ulcerative colitis (Yue L. et al., 2023). Glycyrrhizae Radix et Rhizoma (Gancao in Chinese) is functioned as synergistic effects that enhance the efficacy and reduce the toxicity of other herbs (Li et al., 2019). Huangqi decoction, contained Huangqi and Gancao, increases the diversity and richness in cholestatic mice, alleviated intestinal barrier dysfunction, and decreases the abundance of bacteria such as Alistipes, Bacteroides, and Parabacterorides and hepatic expression of pro-inflammatory factors and NLRP3 (Zou et al., 2021).

In conclusion, dietary supplement with ACS exerted diarrhea and mortality-reducing effects, and improved intestinal immunity by modulating abundance of intestinal flora and immunity responses in young rabbits.