Discussion
Chicken colibacillosis, characterized by multiple organ lesions with airsacculitis and associated pericarditis, perihepatitis and peritonitis, initially develops in the respiratory tract and air sacs and then takes the form of sepsis, causing considerable mortality in poultry(Wernicki, Nowaczek, & Urban-Chmiel, 2017). Previous studies have reported that the air-exchange regions of the lung and the airsacs are important sites of entry of E. coli into the bloodstream of birds during the initial stages of infection and that resistance to phagocytosis may be an important mechanism in the development of the disease (Dho-Moulin & Fairbrother, 1999). Therefore, controlling the bacterial infection in lung and airsacs may be a pivotal method to protect against colibacillosis. Therefore, we established the model of colibacillosis by intratracheal inoculation with 2×109 CFU APEC-O78 to mimic the natural infection process.
The gut microbiome, which consists of trillions of bacterial, viral, and fungal microorganisms, is a complex ecosystem that can mediate the interaction of the host and their environment(Blum, 2017). Growing evidences have shown that gut microbiota is closely related to health and various diseases including neuropsychiatric disorders (Cenit, Sanz, & Codoner-Franch, 2017), inflammatory bowel disease (Frank et al., 2007), Type 1 Diabetes(Dedrick et al., 2020), atherosclerosis (D. Y. Li & Tang, 2017) and acute respiratory distress syndrome(Mukherjee & Hanidziar, 2018). Especially, the beneficial role of the gut microbiota through “gut-lung axis” during pulmonary bacterial infections has been explored in some researches (Dumas, Bernard, et al., 2018). Therefore, we conjectured that gut microbiota also played an important role in chicken colibacillosis, an avian respiratory disease. To explore the effects of gut microbiota during colibacillosis, we pretreated chicken with Abx for two weeks to deplete the microbiota(Dumas, Corral, et al., 2018). Two days later, the colibacillosis was induced and relevant indicators were examined. The experimental results indicated that infected with APEC induced significantly pathological damages of heart, spleen, liver, kidney, and lung especially, increased the inflammatory cytokines secretion and gene expression in lung tissues. Importantly, these changes were more severity in gut microbiota-depleted chickens when compared to the control chickens. The air-blood barrier is essential to maintain the homeostasis of the lung microenvironment (L. Y. Zhang et al., 2016). In the present study, the air-blood barrier integrity was markedly impaired showing by increased protein in BALF and decreased expression of tight junction induced by APEC when gut microbiota was depleted. Collectively, these results demonstrated that gut microbiota may contribute to protect against the chicken colibacillosis.
Baicalin, a main component of scutellaria baicalensisGeorgi, , possesses extensive pharmacological activities, such as antioxidant, anti-apoptotic, anti-excitotoxicity and anti-inflammatory(Liang, Huang, & Chen, 2017). In previous study, we have proved that protective effects of baicalin against APEC-induced lung inflammation in chicken colibacillosis. However, the underlying mechanisms remains to be explored. Recently, there is accumulating evidence have reported the potential interactions between the gut microbiota and herbal medicines. These interactions mainly includes that gut microbiota can “digest” herbal medicines into absorbale active small molecules by enzymatically transforming, vice versa, herbal medicines can influence the composition and metabolite of gut microbiota(An et al., 2019; Weersma, Zhernakova, & Fu, 2020). As previously reported, codonopsis pilosula polysaccharide can be digested by gut microbiota to exert its pharmacological effect. Meanwhile, codonopsis pilosula polysaccharide can promote abundance of the beneficial gut microbiota and inhibit the colonization by pathogens(Jing et al., 2018). Whether baicalin has a same mechanism in the protection of chicken colibacillosis was still elusive.
Based on this, we firstly compared the pharmacological effect of baicalin in healthy chickens and microbiota-depleted chicken after infected by APEC to decipher the influence of gut microbiota on baicalin. The results showed that the effect of baicalin on ameliorating the air-blood permeability, inflammatory responses, lung bacterial loads and tissues damages were significantly diminished when gut microbiota was depleted. As we can see, the bacterial loads of lung tissue, BALF protein, production and gene expression of pro-inflammatory cytokines and histological lesion were distinctly elevated, and the expression of tight junction were markedly reduced incomparision with BAI+APEC group and BAI+Abx+APEC group. This phenomenon may imply that gut microbiota plays a pivotal role during the process of baicalin exerting pharmacological effects in colibacillosis. Refer to the previous literature(Jung, Jang, Hong, Hana, & Kim, 2012b), we supposed that baicalin may need to be metabolized by gut microbiota to play its therapeutic effect during chicken colibacillosis. However, the specific mechanism still needs to be deeply explored. Furthermore, we explored the influence of baicalin on gut microbiota in APEC-induced colibacillosis. The results showed that infection with APEC significantly increased the gut bacterial diversity and richness, and increased the relative abundance of Ruminococcaceae_UCG-014 ,Ruminococaceae_unclassified , andClostridiales_vadinBB60_group_unclassified , while these changes were reversed by pretreatment with baicalin.Ruminococcaceae family contains a large number of gut-associated butyrate producing bacteria, and they were positive association with total milk intake in piglets(Morissette, Talbot, Beaulieu, & Lessard, 2018). However, other studies suggestedRuminococcaceae was associated with the development of children with chronic pancreatitis(W. Wang et al., 2020). In addition, studies revelated that Clostridiales was associates with the development of inflammatory bowel diseases (IBD), and was negatively associated with the levels of SCFAs(Saitoh et al., 2015). Furthermore, we also found that treatment with baicalin increased the relative abundance of intestinimonas , which was associated with the production of SCFAs (Shomorony et al., 2020). These results suggested that gut microbiota and its metabolites may be play an important role in the pharmacological effects of baicalin on chicken colibacillosis.
In order to investigate the whether the changes of gut metabolites could provide further insight into specific gut microbiota-related changes and aimed to explore a new perspective targeting gut metabolism to find biomarkers of chickens colibacillosis and reveal the mechanism of the protective role of baicalin on chicken colibacillosis. We analyzed the gut metabolites in chickens from different treatment groups. The results showed that there are significant differences in metabolic composition among CON, APEC, BAI, and BAI+APEC group chickens. Combined analysis of gut microbiota and metabolites indicated that increased of concentration of Xanthine, Deoxyinosine, 21-Hydroxypregnenolone, Serotonin, 3,4-Dihydroxyhydrocinnamic acid, 8,9-DiHETrE, Dihydrofolic acid, and Corticosterone may be associated with the development of colibacillosis induced by APEC in chicken. Xanthine is a plant alkaloid which acts as an intermediate product on the pathway of purine degradation. It also serves as scaffold for various natural and synthetically derived bioactive molecules (Singh, Patra, & Patra, 2018). Deoxyinosine is an abnormal nucleoside and has hypoxanthine as its base moiety (Yoneshima et al., 2016). Studies indicated that exogenous deoxyinosine can be utilized to meet the carbon and energy requirements of growing cells (Carta et al., 2001). 21-Hydroxypregnenolone play an important role in steroid hormone biosynthesis, which does well in helping with memorization, anti-fatigue, and refreshing the neural system (C. Shao et al., 2018). However, other studies showed that 21-Hydroxypregnenolonemay be key biomarkers of hypothalamic-pituitary-adrenal (HPA) axis abnormalities in patients with chronic fatigue syndrome (CFS)(C. Z. Shao et al., 2017). Serotonin, synthesized mainly by enterochromaffin cells (ECs), is an important gut neuroendocrine factor which have ability to regulate gut motility (Ge et al., 2017). Previous studies demonstrated that fecal pellets facilitate serotonin release from enterochromaffin cells by regulating the activation of local mucosal reflexes and stretch reflexes of colonic migrating motor complex to facilitate propulsion (Ge et al., 2017; Heredia, Dickson, Bayguinov, Hennig, & Smith, 2009). Others studies also showed that treatment with prebiotics and probiotics have antidepressive effects by regulating the Serotonin metabolism(H. Li, Wang, Huang, Li, & Zhang, 2019). Oxylipins, namely 8,9-DiHETrE, has been showed that it was associated with development of Alzhemer’s disease risk, type 2 diabetes cardiovascular disease, cerebrovascular events, and acute coronary syndrome (ACS) (Morris et al., 2019; Solati & Ravandi, 2019). Dihydrofolic acid can be converted into tetrahydrofolic acid, and tetrahydrofolic acid is activated to 5,10-methylenetetrahydrofolic acid by the action of serine hydroxymethylase (B. X. Zhang, Qi, & Cai, 2020). Corticosterone, a stress hormone, the main glucocorticoids released from hypothalamus-pitutary-adrenal (HPA) axis. Recently studies indicated that Corticosterone was elevated in mice that received mild stress at a neurobiological level(Fuentes-Verdugo et al., 2020), and others showed that Corticosterone impairs the recall of strong contextual fear memory after reactivation in the ovariectomized rat model of menopause (Kashefi et al., 2020). Importantly, BAI not only significantly reduced the levels of 21-Hydroxypregnenolone, Serotonin, 8,9-DiHETrE, Dihydrofolic acid, and Dihydrocortisol in the gut, but also increased the abundance ofBlautia and Fournierella and the associated metabolites 2-oxoarginine in the chickens suffered colibacillosis. Studies showed that arginine/proline metabolic pathway compounds 2-Oxoarginine, a guanidino metabolite of arginine, were negatively associated with the glioma risk (Huang et al., 2017). Others also suggested that 2-Oxoarginine may play an important role to chelate Mo and reduce its toxicity(Xu et al., 2018). In addition, Intestinimonas and its positively associates metabolites Gentamicin C1a, Selenodiglutathione, L-Arabinose, Serotonin, Norfloxacin, 2-Oxoarginine, Epinephrine, and Xanthine were increased in the gut of chicken that treated with BAI compared to the CON chicken. Gentamicin C1a is considered as a precursor to the synthesis of etimicin (Wei et al., 2019). Selenodiglutathione is a primary Se metabolite conjugated to two glutathione moieties, and it increase intracellular Se accumulation and is more toxic than selenous acid (Fisher et al., 2016; Tobe et al., 2017). L-arabinose, a kind of plant-specific five-carbon aldose, is a naturally occurring constituent of plant polysaccharides, and often extracted from vegetable gum, corn straw or beet(Feher, Gal, Feher, Barta, & Reczey, 2015; Kotake, Yamanashi, Imaizumi, & Tsumuraya, 2016; Moremi, Jansen Van Rensburg, & La Grange, 2020). Recently, L-Arabinose has been used as a food additive, nutritional supplement, and have many pharmacological activities, including inhibited intestinal sucrose activity and triglyceride in blood serum and fat accumulation, increased insulin resistance, promoted the proliferation of probiotics, increased the synthesis and production of organic acids and SCFAs, as well as ameliorates colitis and metabolic syndrome by regulating gut microbiota in mice(Kaats et al., 2011; Y. Li et al., 2019; Osaki, Kimura, Sugimoto, Hizukuri, & Iritani, 2001; Seri et al., 1996; Zhao et al., 2019). Norfloxacin, a fluoroquinolone antibiotic, has an activity and enhances a regulatory T cell-mediated inflammatory response properties(Juanola et al., 2016). These results suggested the protective effect of baicalin on chickens colibacillosis induced by APEC was not only associated with regulating gut metabolites, such as 21-Hydroxypregnenolone, Serotonin, 8,9-DiHETrE, Dihydrofolic acid, and Dihydrocortisol, induced by APEC, but may also associated the production of new gut metabolites by the gut microbiota metabolizing baicalin.
In conclusion, the present study showed that gut microbiota plays an important role in the development of colibacillosis and the protective effect of baicalin on chickens colibacillosis. Moreover, increases the abundance of Clostridiales_vadinBB60_group andRuminococcaceae_UCG_014 , as well as the levels of 21-Hydroxypregnenolone, Serotonin, 8,9-DiHETrE, Dihydrofolic acid, and Dihydrocortisol may be serve as the biomarkers of chicken colibacillosis. In addition, the pharmacological effects of baicalin on chicken colibacillosis may be not only by inhibiting the increase the specific gut microbiota and metabolites induced by APEC, but also by regulating the new metabolites produced by fermentation of baicalin by gut microbiota. However, these results need to be further verified by more experiments.