Formulation of Huangqin Decoction Inuence Immune Function and Fecal Microbiome of Chicks Suffered Escherichia Coli O78 Challenge

Ethnopharmacological relevance: Huangqin Decoction (HQD), a traditional Chinese medicine formula from the Shang Han Lun written by Zhang Zhongjing, has been used in China for nearly two thousand years. According to traditional Chinese medicine and a previous literature, HQD has the effect of clearing heat and removing toxin, antidiarrhea, and relieving pain. Therefore, HQD were used to prevent or cure many diseases, such as inammation, diarrhea, malaria, and other acute or chronic gastrointestinal diseases. Materials and methods: HQD consist of four components: Scutellariae Radix (huangqin, HQ), Paeoniae Radix Alba (shaoyao, SY), Jujubae Fructus (dazao, DZ), Licorice (gancao, GC). A total of 80 1-day-old male Esa brown chicks were divided into eight groups (n=10): Control group (CG), model group (MG), Enrooxacin group (ENR, 10 mg/kg·BW), HQD group (HQD, 500 mg/kg·BW), HQD-GC group (GC absent HQD, 500 mg/kg·BW), HQD-HQ group (HQ absent HQD, 500 mg/kg·BW), HQD-DZ group (DZ absent HQD, 500 mg/kg·BW) and HQD-SY group (SY absent HQD, 500 mg/kg·BW). The chicks, which were given HQD, herb absent HQD, or enrooxacin orally at 19 days of age for 7 days, and then were intraperitoneally injected with inoculum of E. coli O78(cid:0)fed continuously for 5 days as before. Results: It showed that E. coli O78 challenge decreased the average daily gain (ADG) and increased the mortality rate of chicks, increased the heart index and the liver index, decreased the bursal index, and had no effect on the spleen index. E. coli O78 challenge increased the serum lysozyme (LZM) and IL-1β, TNF-α, About herb absent groups, the results shown that SY and DZ play a key role in reducing the level of inammatory factors and keeping fecal microbiome balance respectively. what’s more, we highlighted that HQ is indispensable in HQD, HQ not only play a key role in reducing the level of inammatory factors, but also keep the balance of fecal microora.


Introduction
Colibacillosis refers to any localized or systemic infection caused by avian pathogenic Escherichia coli, caused by speci c serotypes or opportunistically pathogenic Escherichia coli, is one of the crucial bacterial diseases of poultry (Chen et al., 2016). Young birds, in which the protective immune system is not fully developed, are more vulnerable. E. coli serotypes O1, O2, and O78 are the most found in domestic breeds with colibacillosis (Carli et al., 2015). Although various antibiotics are typically used to prevent and control colibacillosis, cumulative reports have demonstrated that drug resistance of E.coli O78 has increased owing to the spreading of resistance genes such as extended spectrum betalactamases (ESBL) and/or plasmid-mediated Amp-C beta-lactamases (Amp-C) (Obeng et al., 2012).
Therefore, potential antibiotic alternatives to reduce antimicrobial drug usage in poultry production are urgently needed.
HQD, a traditional Chinese medicine formula from the Shang Han Lun written by Zhang Zhongjing, has been used in China for nearly two thousand years (Ying et al., 2015). The classical formula consists of four components: Scutellariae Radix, Paeoniae Radix Alba, Jujubae Fructus, Licorice. Clinical studies have shown that HQD is safe and effective for treatment of complex gastrointestinal symptoms, like ulcerative colitis and associated cancer (Chen et al., 2016). The researchers suggest that this is because HQD could regulate the structure of intestinal ora and inhibit the intestinal in ammatory signaling pathway (Yao et al., 2019). Basic studies have shown that the main effectors of HQD are baicalin, paeoni orin, polysaccharides and avonoids, which can regulate the number of mast cells and downregulate the expression of in ammatory factors through the activation of TLR4/MyD88/NF-κB, IL-6/JAK/STAT3 and STAT3/NF-κB/IL-6 signaling pathways (Wu et al., 2021), thus play immune regulation and anti-in ammatory effect (Wang et al., 2020). HQD had no inhibitory effect on Escherichia coli O78 in vitro, but the antibacterial effect was enhanced after intestinal ora metabolism (Yang et al., 2017), indicating that the bacteriostatic effect of HQD is mostly by regulating the metabolites and balance of intestinal bacteria rather than directly. Therefore, this study hypothesized that the HQD is effective for treatment of chicken colibacillosis by regulating the immune function and intestinal micro ora structure.
To investigate the molecular mechanisms of HQD in chick model of arti cially infected chicken colibacillosis, the expression levels of lysozyme, in ammatory cytokines in serum, and Toll-like receptors in spleen and bursal were measured. Furthermore, the gut microbiota was pro led using Illumina HiSeq 2500 sequencing of the bacterial 16S rDNA gene V3-V4 region.

E. coli O78 preparation and infection
The E. coli O78 strain (preservation number: CVCC1490; China Veterinary Microorganism Strains Preservation Management Center, Beijing, China) was aerobically cultured in Luria-bacterial liquid medium at 37 °C for 24 h. The bacterium solution was injected into the chicks for strain rejuvenation. E. coli O78 was isolated by coating the liver on MacConkey agar plate at 37 °C for 24 h. The colonies were suspended in 50 mL of sterile sodium chloride at pH 7.0. The number of bacteria was determined by plating dilutions of the suspension onto agar plate. Chicks were intraperitoneally injected with 0.5mL of inoculum with a concentration 0.6×10 9 cfu/mL at 27 days of age.

Preparation of HQD granules for chicks
According to the prescription of HQD, HQ, SY, DZ and GC were soaked in a ratio of 3:2:2:2 for 60 minutes and boiled for 40 minutes. After ltration, the residue of the HQ was dried in a drying oven, then the grinded residue was mixed with the concentrated ltrate in the pelletizer, and then cut to wet granules following desiccation. SY, DZ, GC, HQ were not used in the preparation of the granules of HQD-SY, HQD-DZ, HQD-GC, HQD-HQ by the way like the HQD granules. The residue other than HQ was used to prepare HQD-HQ.

Chicks and treatments
A total of 80 1-day-old male Esa brown chicks were randomly assigned into one of 8 treatment groups with 10 chicks per group. Chicks in control group (CG) and model group (MG) were fed a basal diet formulated (Shiyang Agricultural Group Co., LTD, Jinzhong, China) to meet nutritional requirements of chicks during the whole experimental period. Birds in Enro oxacin Group (ENR) were fed a basal diet supplemented with Enro oxacin 10 mg/kg·BW; birds in HQD group (HQD) were fed a basal diet supplemented with HQD granules 500 mg/kg·BW; birds in group of HQD-HQ were fed a basal diet supplemented with HQD-HQ granules 500 mg/kg·BW; birds in group of HQD-SY were fed a basal diet supplemented with HQD-SY granules 500 mg/kg·BW; birds in group of HQD-DZ were fed a basal diet supplemented with HQD-DZ granules 500 mg/kg·BW; birds in group of HQD-GC were fed a basal diet supplemented with HQD-GC granules 500 mg/kg·BW. All supplement drugs were administered after intramuscular injection of E. coli O78 at 27 days of age, and they were added up to 32 days of age. All the chicks had free access to food and drinking water during the whole experimental period. The experimental animal protocol for this study was approved by the Animal Care and Use Committee of China Agricultural University.

Visceral index
At 32 days of age, to calculate the chicks' visceral index, the living weight of chicks were weighed and liver, heart, spleen and bursal were collected. The formula is: Visceral index (g/kg) = visceral weight (g)/live weight (kg).

Primer
Primer sequences ( To evaluate the effects of drugs on innate immune substances and cytokines in chicks with colibacillosis, the blood was collected, and the serum was separated. The level of LZM (lysozyme) in serum was quanti ed using ELISA kits for chicken following manufacturer's instructions. The levels of TNF-α, IL-1β, IL-6, and IL-10 in serum were quanti ed using ELISA kits (Biosciences, China) following manufacturer's instructions. Take three samples from each group and repeat each sample three times. The results were expressed as content of LZM or cytokines in per mL in serum of chicks.
2.6. Quantitative real-time PCR (RT-qPCR) To evaluate the effect of drugs on Toll-like receptor expression in immune organs of chicks with colibacillosis, the bursal and spleen was collected. Total RNA was extracted from spleen and bursal using Trizol reagent (Takara, Japan). According to the Bio-Rad kit, 1μL DNA was taken for polymerase chain reaction. GADPH was used to normalize based on CT value. All primers were synthesized by Wuhan ServiceBio Technology Co., Ltd. Take three samples from each group and repeat each sample three times. Primer sequences are shown in the Table 1. for 20 s, with a final elongation step at 72 °C for 5 min. Ampli ed products were separated on 2% agarose gels, the puri ed product was used as a second PCR template for ampli cation. The above steps were repeated for puri cation, and then Qubit dsDNA Assay Kit (Life Technologies, USA) was used for quantitative detection. According to the concentration of PCR products, equal amounts of samples were mixed, Barcoded V3+V4 amplicons were sequenced by the paired-end method with Illumina MiSeq at Shanghai OE Biotech. Co., Ltd.

Bioinformatics analysis
Raw sequences were denoised using Trimmomatic and FLASH software and ltered according to their barcodes and primer sequences with QIIME v.1.5.0. Chimeras were identi ed and excluded using the UCHIME algorithm v.4.2.40. Optimized, high-quality sequences were clustered into operational taxonomic units (OTUs) at 97% sequence identity against a subset of the Silva 16S sequence database (Release 119 1). Taxon-dependent analysis was carried out using the Ribosomal Database Project (RDP) naive Bayesian classi er, with an 80% bootstrap cutoff. Alpha diversity (Shannon and Simpson indices), abundance (Chao1 and ACE indices), and Goods coverage and rarefaction were analyzed with mothur v.1.31.2. Principle coordinates analysis (PCoA) was conducted to visualize differences in nasal mucosa community composition. PCoA plots were generated based on Bray-Curtis indices. The linear discriminant analysis effect size (LEfSe) algorithm was used to identify the taxa responsible for the differences between the treatment and control groups. The biomarkers used in the present study had an effect-size threshold of two.

Statistical analysis
Statistical analysis of data from ELISA and RT-qPCR assays was carried out using a one-way analysis of variance (ANOVA) was performed to detect differences between mean values, which were then analyzed further for signi cance with LSD test. The chi-square test was used to analyze the differences in mortality. In all cases, p < 0.05 were considered signi cant.

HQD reduced the mortality of colibacillosis and increased the average daily gain in chicks
Compared with the CG, chicks challenged with E. coli O78 at 27 days of age showed ru ed feathers, closed eyes, reluctance to move, defecated white or green stools and soiled vent. Post-mortem examination showed that E. coli O78 infection led to severe perihepatitis, pericarditis, granuloma, and bleeding spots in the small intestine (Fig. 1). E. coli O78 infection reduced average daily gain (ADG) between 1 and 32 days of age (p <0.05), which was reversed by HQD ( Fig. 2A). There was no signi cant difference between HQD-HQ, HQD-DZ, HQD-GC, HQD-SY and ENR supplementation compared with HQD. E. coli O78 infection increased mortality at day 5 post E. coli O78 challenge, which was reversed by HQD, 500 mg/kg HQD-GC and 500 mg/kg HQD-SY (Fig. 2B). Moreover, there was no effect on mortality in chicks challenged with E. coli O78 at the dose of 250 mg/kg except for the HQD group. Therefore, the dose of 500 mg/kg groups were more worthy of further study.

HQD changed the visceral index in chicks with colibacillosis
In contrast, the heart index and the liver index were increased in chicks challenged with E. coli O78, as compared with control group, which was reversed by HQD and ENR. Chicks with supplementation challenged with E. coli O78 led to reduced visceral index in the bursal, which was reversed by HQD and ENR. Chicks challenged with E. coli O78 were no effect on spleen index, which was increased by HQD. Compared with HQD, HQD-DZ and HQD-SY have the same effect as HQD, HQD-GC resulted in decreased spleen index, while HQD-HQ resulted in increased liver index in chicks challenged with E. coli O78. The results showed that HQD exerted signi cant protective effects against the organ index of arti cial induced colibacillosis, in which HQ played the main role (Table 2). Table 2 Visceral  E. coli O78 infection increased the protein levels of lysozyme in serum, as compared with that of control group, which was signi cantly reversed by HQD and ENR administration. HQD-GC and HQD-SY have the same effect as HQD, but HQD-DZ and HQD-HQ couldn't reverse the increase in lysozyme protein level. The results showed that HQ and DZ in HQD played an important role in eliminating the effect of E. coli O78 on lysozyme (Fig. 3).
3.4. Effect of HQD on E. coli O78-induced in ammatory cytokines in serum ELISA assay was conducted to determine protein level of in ammatory cytokines in serum. E. coli O78 infection increased the protein levels of IL-1β, TNF-α and IL-10 in serum, as compared with control group, indicating activation of in ammatory response following E. coli O78 infection. These alterations were signi cantly reversed by HQD and ENR administration. The protein level of IL-6 was increased by E. coli O78 challenged in serum of chicks, as compared with control group, however, HQD reduced its protein levels to even below control level. The results showed that HQD could reduce the in ammatory reaction in chicks challenged by E. coli O78 for 5 days. Compared with the HQD group, the protein levels of IL-1β and IL-10 were not different, while the levels of IL-6 protein were signi cantly increased in all herbs absent HQD. And the level of TNF-α protein in HQD-SY and HQD-HQ groups were signi cantly higher. The results indicated that the herbs in HQD have the effect of down-regulating pro-in ammatory factors IL-6.
Meanwhile, HQ and SY play a key role in down-regulating TNF-α (Table 3). Table 3 The

Effect of HQD on mRNA level of Toll-like receptors in spleen and bursal of chicks
Further to explore the effect of HQD on the immune function of chicks challenged with E. coli O78, the mRNA levels of TLR4, TLR5 and TLR15 in spleen and bursal were detected. Activation of Toll-like receptor signaling pathway plays an important role in defense against invading pathogens. E. coli O78 challenge enhanced mRNA level of TLR4, TLR5 and TLR15 in spleen, as compared with control group, indicating activation of the innate immune system in the spleen following E. coli O78 infection. And this alteration was signi cantly reversed by all administration. Compared with CG, E. coli O78 challenge had no effect on mRNA level of TLR4, TLR5 and TLR15 in bursal, but the mRNA levels were signi cantly increased after administration. The results indicated that the administration groups could regulate the anti-infection ability of chicks suffered E. coli O78 by increasing the mRNA levels of TLR4, TLR5 and TLR15 in the bursal. There was no signi cant difference between the results of other herb absent formulae and those of HQD group, indicating that HQD acted indirectly on immune organs (Table 4). Table 4 The  Alpha diversity analysis showed a clear decline in bacterial diversity in response to E. coli O78 treatment, and a clear increase in diversity in response to HQD treatment. Compared with HQD group, the Shannon index of HQD-DZ group was signi cantly decreased, indicating that DZ had an important role in maintaining the intestinal micro ora structure of chicks challenged with E. coli O78 (Table 5).
A plateaued rarefaction curve of OTUs indicated that the sequencing depth covered all the species in the samples. E. coli O78 infection increased the OTUs, as compared with control group, which was reversed by HQD and ENR administration. Principal coordinate analysis (PCoA) showed similarity among samples, with similarity indicated by distance in the diagrams. Treatment with E. coli O78 altered the composition and structure of the gut microbiota according to PCoA. Treatment with HQD partially inhibited E. coli O78induced changes in the gut microbiota (Fig. 4). Table 5 The  3.6.2 HQD regulated structure of the gut microbiota The gut microbiota community structure was reported using histograms at the phylum and genus levels. All samples contained abundant Firmicutes, Bacteroidetes and Proteobacteria. Compared with the control group, MG decreased the relative abundance of Bacteroidetes and Firmicutes and increased the proportion of Proteobacteria. These alterations were signi cantly reversed by HQD administration. Compared with HQD group, the HQD-DZ signi cantly increased in the abundance of Proteobacteria, which is a marker of intestinal microbial dysregulation, and other herb absent formulae had varying degrees of in uence on the three phylum levels. These results indicate that each herb in HQD in uences structural segregation of the gut microbiota, in which DZ plays an important role. The ENR group can't recover the changes in the abundance of dominant bacteria caused by E. coli O78. In contrast, the abundance of Proteobacteria was further increased. The results suggest that HQD is more bene cial than ENR in the treatment of E. coli O78 (Fig. 5).
More than 30 genera were identi ed in all samples. Bacteroides, Faecalibacterium, Lactobacillus and Prevotella were dominant communities in the control group but were reduced by E. coli O78 treatment. Compared with the control group, Escherichia-Shigella becomes the dominant genus, but were reduced by HQD treatment. The results showed that the proportions of most bacteria returned to control levels following HQD treatment.
The gut microbiota community structure of HQD, HQD-GC and HQD-SY were like that of CG group, indicating that HQD, HQD-GC and HQD-SY had protective effect on the structural changes caused by E. coli O78 Challenge. Compared with HQD group, the proportion of Escherichia-Shigella in HQD-DZ and HQD-HQ groups were increased, indicating that DZ and HQ in HQD had a certain regulating effect on the increase Escherichia-Shigella (Fig. 6).

Discussion
Avian pathogenic E. coli O78 is the primary cause of colibacillosis, which is associated with remarkable economic losses in the poultry industry worldwide (Xing et al., 2020). Antibiotics have been widely used to prevent or combat E. coli O78 infection in chicks, but it also has been a surge in antibiotic-resistant bacteria. So, considering the incidence of multi-drug-resistant of E. coli O78 and its potential risk for both human and animal health (Subedi et al., 2018), it is imperative to develop alternative strategies to reduce E. coli O78-infection-induced deleterious effect (Johar et al., 2021). Previous studies have shown that HQD has no inhibitory effect on E. coli O78 in vitro, but this effect has been found in vivo studies (Ma et al., 2018). Therefore, it is speculated that the treatment effect of HQD on colibacillosis is independent of its direct bactericidal effect, but dependent on the intestinal metabolism, regulation of intestinal micro ora structure and immune function.
In the present study, we observed that HQD, by down-regulating the mRNA expression of TLR4, TLR5 and TLR15 in the spleen, further decreasing the serum LZM and IL-1β, TNF-α, IL-10, IL-6 levels, improves the immune function. Meanwhile, HQD could keep the balance of fecal microbiome of chicks challenged with E. coli O78, which may be function in Toll-like receptors signaling pathway. About herb absent groups, the results shown that SY and DZ play a key role in reducing the level of in ammatory factors and keeping fecal microbiome balance respectively, What's more, we highlighted that HQ is indispensable in HQD, HQ not only play a key role in reducing the level of in ammatory factors, but also keep the balance of fecal micro ora.
In agreement with previous study, E. coli O78 challenge led to increasing mortality, decrease average daily gain, and raise organ indexes of heart and liver in chicks (Huang et al., 2018), In addition, it also led to severe perihepatitis and pericarditis (Wernicki et al., 2017). In this study, E. coli O78 led to increasing mortality, decreasing average daily gain, and raising organ indexes of heart and liver in chicks, it was attenuated by HQD administration. However, HQD-DZ and HQD-HQ lost this attenuated effect indicating that HQ and DZ in HQD were indispensable in decreasing mortality and increasing ADG.
In previous study, the method of intramuscular injection was used to challenge the E. coli O78, it allows E. coli O78 to crossing the rst immune barrier and entering the bloodstream, and then initiates the secretion of in ammatory factors which are critical factors produced by epithelial cells and macrophages following bacterial infections (Zhong et al., 2014). In the present study, E. coli O78 challenged resulted in increasing LZM, as well as enhancing protein level of in ammatory factors, including IL-1β, IL-6, TNF-α and IL-10 in serum of chicks, which was attenuated by HQD administration, indicating that HQD could reduce the in ammatory factors level of chicks with colibacillosis. What's more, HQ and DZ in HQD play a key role in reducing protein level of LZM, and HQ and SY play a key role in down-regulating TNF-α.
It's worth noting, in the present study, that E. coli O78 challenge led to increasing the level of IL-10 that is an anti-in ammatory factor, has the effect of anti-in ammatory and immunosuppressive. Some researchers believe that anti-in ammatory cytokine IL-10 can reduce the intensity of in ammatory response in severe infection, but on the other hand, the inhibition of the body's defense ability leads to the persistence of microbial infection (Houra et al., 2020). Therefore, it is believed that the increase of IL-10 in infection may be a manifestation of severe injury of the body (Goodman et al., 2011). In present study, E. coli O78 increased protein level of IL-10, it may indicate that E. coliO78 led to severe injury of body.
TLR could identify the microbe related molecular patterns, activate MyD88-dependent signaling pathway, activate transcription factor NF-κB (Luo et al., 2012), and produce IL-1β, IL-6, TNF-α and other cytokines (Wullaert 2010), enhance the immune function of the body (Song and Lee 2012). We observed that E. coli O78 infection led to enhancing the mRNA level of Toll-like receptors in spleen, including TLR4, TLR5 and TLR15, but has no effect in bursal. These up- Enro oxacin has a good therapeutic effect, but it leads to dysregulation of intestinal ora, which is related to its extensive bactericidal action. It leads to the dissolution of a large number of bacteria in the gut releases more LPS, triggering an in ammatory response, which also explains the increased levels of in ammatory cytokines (Temmerman et al., 2021). Therefore, it is of great signi cance to maintain the structural stability and diversity of intestinal ora (Yang et al., 2017). In the present study, the abundance and diversity of intestinal micro ora in chicks infected with E. coli O78 were signi cantly decreased (p<0.05), indicating that E. coli O78 broke the immune barrier in vivo following the blood circulation, or E. coliO78 challenged the immune function of the body, and increased large number of opportunistic pathogens in the intestine. Finally, E. coliO78 caused the decrease of intestinal micro ora diversity and disrupted the balance of intestinal micro ora. Fortunately, HQD can effectively attenuate this phenomenon. What's more, compared with HQD, the Shannon index in HQD-DZ group was signi cantly decreased, indicating that DZ had an important role in maintaining the intestinal micro ora structure of chicks challenged with E. coli O78.
The analysis of intestinal micro ora structure in each group showed that the phylum level micro ora structure of the model group was changed, the proportion of Proteobacteria increased, and the proportion of Firmicutes decreased. Proteobacteria is considered to be a marker of intestinal microbiota imbalance, and a large number of Proteobacteria in the gut re ects stunted growth or an unstable intestinal microbiota structure. But HQD attenuates this change and keeps the intestinal micro ora in balance. Compared with HQD, HQD-DZ increased the harmful bacteria Proteobacteria levels, which indicated that DZ in HQD play a key role in keeping fecal microbiome balance in the phylum levels.
Compared with the control group, the genus structure of the micro ora in model group was changed, the proportion of ProteobacteriaFaecalibacterium, Lactobacillus and Prevotella decreased, and Escherichia-Shigella became the dominant genus, as was reversed by HQD treatment similar to control group, indicating that HQD had protective effect on the micro ora structural suffering E. coli O78 Challenge.
What is more, HQD-DZ and HQD-HQ increased the harmful bacteria Escherichia-Shigella, indicating that HQ and DZ play a key role in keeping fecal microbiome balance in the genus levels in HQD.

Conclusion
In conclusion, we con rmed HQD by down-regulating the mRNA expression of TLR4, TLR5 and TLR15 in the spleen, further decreasing the serum LZM and IL-1β, TNF-α, IL-10, IL-6 levels, improves the immune function, and reverse the change of fecal microbiome in chicks challenged with E. coli O78. About herb absent groups, the results show that SY and DZ play a key role in reducing the level of in ammatory factors and keeping fecal microbiome balance respectively, what's more, we highlighted that HQ is indispensable in HQD, HQ not only play a key role in reducing the level of in ammatory factors, but also keep the balance of fecal micro ora.

Declarations
Ethics approval and consent to participate The experimental animal protocol for this study was approved by the Animal Care and Use Committee of China Agricultural University.

Consent for publication
Not applicable.

Availability of data and materials
All data generated or analysed during this study are included in this published article.

Competing interests
The authors declare that they have no competing interests.