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.