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Article

Bacillus coagulant HYI (BC-HYI) Alleviates LPS-Elicited Oxidative Stress by Engaging the Nrf2/HO-1 Signaling Pathway and Regulates Gut Macrobiotics in Laying Chickens

by
Tianhang Lu
†,
Le Wang
†,
Qiong Wu
,
Hua Zhang
,
Defeng Cui
,
Bowen Liu
,
Jinjin Tong
* and
Yonghong Zhang
*
College of Animal Science and Technology, Beijing University of Agriculture, Beijing 100191, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Appl. Microbiol. 2023, 3(4), 1178-1194; https://doi.org/10.3390/applmicrobiol3040081
Submission received: 21 August 2023 / Revised: 19 September 2023 / Accepted: 28 September 2023 / Published: 9 October 2023
(This article belongs to the Special Issue Applied Microbiology of Foods 2.0)
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Abstract

:
In the current study, Bacillus coagulants had a role in combating oxidative stress by inhibiting the growth of intestinal pathogens. However, there are few studies on reducing the mechanisms of oxidative stress. Therefore, this study aimed to explore the effects and underlying mechanisms of B. coagulant HYI (BC-HYI) treatment on growth and intestinal functions in laying chickens under LPS-induced oxidative stress. The in vivo experimental group included five groups of laying chicks: normal control, LPS group, B6 group, B7 group and B8 group. The test consisted of six repetitions in each group, with six animals in each repetition. In the in vitro experiment, an LPS-induced oxidative stress model of chicken fibroblast DF-1 cells was established, and the DF-1 cells were divided into control group, LPS-treated group, B5 group, B6 group and B7 group. On the one hand, we found that BC-HYI can inhibit pathological changes in some intestinal tissues. On the other hand, BC-HYI supplementation has a dual effect on the gut microbiota, promoting the proliferation of beneficial microbes such as Barbarella, Lactobacillus, and Antibacterial while maintaining symbiotic balance. The abundance of Barbarella, Bactericide, and Cloistral was significantly different between the LPS group and the BC-HYI group (p < 0.01). Moreover, compared with the LPS group, BC-HYI significantly decreased reactive oxygen species levels and prevented cell apoptosis (p < 0.01). It used to prevent oxidative stress by activating the Nrf2-ARE/HO-1 signaling pathway, enhancing the scavenging of free radicals, and reducing oxidative damage. BC-HYI alleviated oxidative stress in laying chickens by modulating the gut microbiota and activating the Nrf2-ARE/HO-1 signaling pathway. In summary, laying chickens and cell experiments indicate that BC-HYI supplementation can improve the enzyme function of antioxidants, regulate intestinal barrier function and activate the Nrf2-ARE/HO-1 signaling pathway to regulate intestinal barrier function.

1. Introduction

The overuse of antibiotics has afflicted society with an epidemic of antibiotic-recalcitrant infections and subsequent harmful inflammation, resulting in lowered immunity, cellular damage, secondary infections, and intestinal microbial imbalances [1]. Thus, the search for alternative antibacterial and noninflammatory agents is urgently needed. As an essential protectiveness organ, the intestinal tract is subjected to potential pathogens and consequent inflammation, which can cause diarrhea, slow growth, and even death in commercially raised animals like chickens if the intestinal flora becomes imbalanced [2]. Because of this risk, probiotic preparations have attracted considerable research attention because they could be used to reestablish the intestinal flora balance and restore immunity quickly.
Probiotic preparations are formulations containing live microorganisms that can modify the composition of the intestinal macrobiotic within the body, thus improving the organism’s health, fostering better digestion and absorption and restoring a healthy immune response [3]. Abaya-Loyola et al. [4] found that Bacillus coagulant (B. coagulant) can improve physical fitness by enhancing the body’s digestion and absorption of nutrients. This is because B. coagulant can scavenge free radicals and boost the activity of antioxidant enzymes in the host, resulting in an antioxidant effect. B. coagulant can synthesize anti-inflammatory cytokines as immune regulatory agents by activating the CONFAB signaling pathway through direct interaction between the bacterial cell wall’s outer layer and immune cell receptors [5]. Our previous studies showed that BC-HYI improved heat resistance and chicken feed utilization and reduced LPS-induced inflammatory damage by activating the TLR4/NF-AB-signaling pathway to promote cytokine production [6]. However, the mechanism of BC-HYI’s reduction in oxidative stress (OS) in laying chickens has yet to be elucidated.
Reactive oxygen species (ROS) is a toxic substance produced during the body’s metabolic process. According to [7], accumulating excessive amounts of these substances leads to oxidative stress (OS), which disrupts the balance within cells and results in mitochondria dysfunction. Fao et al. showed that the presence of heavy metals like Cr(VI) could increase the level of cellular ROS, resulting in abnormal mitochondrial membrane potential (MMP) and decreased catalepsy activity [8]. We evaluated cellular oxidative stress levels by detecting reactive oxygen species (ROS) content combined with antioxidant enzyme results in the article.
Wei et al. found that ROS content increased in diabetic rats, and the abundance of Firmicutes and Bacteroides in the gut microbiota was higher [9]. High levels of ROS and increased abundance of Enterococcus faecalis were found during the development of inflammatory bowel disease (IBD) [10]. Bacterial lipopolysaccharide (LPS) increased ROS and damaged chicken embryonic lung cells by activating NF-κB and Nrf2 signaling pathways [11]. The probiotic, B. coagulans, has been reported to have multiple effects in animals, including shifting the intestinal population of microbes towards the beneficial types, accelerating growth, promoting healthy immune responses, and inhibiting pathogen invasion [12]. In a dextran sulfate sodium (DSS)-induced mouse model of IBD, treatment with B. coagulans significantly modulated the cytokines, IL-4, IL-6, IL-8 and IL-10, upregulated the expression of claudin and mucin, and promoted repair of the intestinal barrier [13]. Furthermore, 200 mg/kg of B. coagulans improved growth performance and increased the activity of antioxidant enzymes and the abundance of intestinal bifidobacteria and bacilli in pigs [14]. Laying hens administered with B. coagulans R11 showed increased total antioxidant capacity (T-AOC) and superoxide dismutase (SOD) activity and reduced the MDA concentration [15]. B. coagulans also reduced the prevalence of harmful bacteria in cows with diarrhea and prevented oxidative damage [16]; however, the mechanism by which gut microbiota lower OS has not been worked out in detail. In our experiments, the beneficial effect of BC-HYI on the antioxidant stress of laying chicks was evaluated in vivo by detecting changes in intestinal tissue structure and intestinal microbiota quantity combined with the contents of antioxidant enzymes in blood and tissue. In in vitro experiments, the antioxidant mechanism of BC-HYI was explored by detecting the amount of reactive oxygen species (ROS) combined with the content of antioxidant enzymes in cells and its impact on gene and protein expression.
The normal growth of laying chickens during the rearing period is essential to ensure their economic value for later egg laying. Laying chickens are susceptible to OS during husbandry resulting in decreased immunity, diarrhea, vomiting, weight loss, and even death [17]. If BC-HYI can change the metabolites in the intestine by increasing the content of beneficial bacteria, it can even change the expression of genes and proteins through this pathway to increase antioxidant capacity. First of all, BC-HYI is a newly discovered strain of Bacillus coagulans with powerful functions. We comprehensively illustrated the role of BC-HYI through in vitro and in vivo experiments. We used functional prediction to combine intestinal microbiota functions and signaling pathways, which serves as a basis for subsequent exploration. Therefore, for laying chickens, feeding probiotic BC-HYI in advance can not only improve immunity, but also reduce mortality and increase survival rate when oxidative stress occurs. This provides some data support for the research and development of antibiotic alternatives and ensuring the economic benefits of animal husbandry. Therefore, this study aimed to explore the research goal of this investigation, which was to ascertain the ways in which BC-HYI balanced the gut macrobiotic and reduced LPS-induced oxidative stress. In this paper, we presented data on the functional mechanism underlying the use of BC-HYI in promoting the steady state of gut macrobiotic and reducing OS in laying chickens.

2. Materials and Methods

2.1. Bacteria Strains and Cultivation

B. coagulant HYI (BC-HY1) was obtained from the Chinese General Microorganism Culture Collection Center which produced exopolysaccharides (CGMCC No. 24423). B. coagulant HYI was inoculated into an LBS broth medium (Boombox Biotechnology Co., Ltd., Beijing, China) and grown at 37 °C in a shaker incubator (210 rpm) for 22 h. The pH was kept at 6.2. Lactobacillus rhamnosus GG (LGG) was obtained from Hebei Agricultural University and cultured statically at 37 °C in a D Man-Rosa-Sharpe (MRS) medium (Boombox Biotechnology Co., Ltd., Beijing, China) to stationary phase. The purity and identity of the strains were checked, and bacterial liters were determined by serial dilution, spreading on agar plates, and colony counting. Serial dilutions were performed to obtain the desired inoculate (1.0 × 105, 1.0 × 106, 1.0 × 107 and 1.0 × 108 CFU/L).

2.2. Animals and Sample Collection

A total of 288 one-day-old laying chickens were donated by the Beijing Sadhu Yuk ou Poultry Industry Co., Ltd. (Beijing, China). After a 10-day adaptation, the animals were split into five groups and assigned randomly to different feeding conditions. (1) The basal diet was administered to the control (CON) group; (2) the LPS group, which received basal diet feeding; (3) B6 group, which received basal diet feeding supplemented with 1.0 × 106 CFU/mL of BC-HY1; (4) the B7 group, which received basal diet feeding supplemented with 1.0 × 107 CFU/mL of BC-HY1; (5) the B8 group, which received basal diet feeding supplemented with 1.0 × 108 CFU/mL of BC HY1. On Day 28, Groups 2–5 received 2 mg/kg of LPS administered by gavage. Blood, intestinal and fecal samples were collected within 6 h of LPS gavage, and the samples were collected and stored at −80 °C for future analysis.

2.3. Histological Analysis

Upon collection, intestinal tissue samples from chickens were immediately fixed in 10% neutral buffered formalin to preserve their structural integrity. The paraffin-embedded samples were thinly sliced into sections measuring approximately 5 μm thick to obtain thin sections suitable for microscopic examination. To analyze the cellular morphology and tissue composition, thin sections of the intestinal tissue were prepared and subjected to local staining using the widely employed hematoxylin and eosin (H&E) technique. Furthermore, the stained sections were examined under an IX71 visible light microscope (Olympus, Japan). This microscope provides high-resolution imaging capabilities, allowing for detailed observation and analysis of the stained tissue sections.

2.4. High-Throughput Sequencing Was Performed on the 16S rRNA Genes

To analyze the microbial composition of fecal samples, DNA extraction was performed using the Fast DNA® SPIN for salt kit (MP Biomedicals, Solon, OH, USA) following the recommended protocol. To target bacterial 16S rRNA genes, the V3–V4 region was selected for amplification. To achieve this, specific primers were used, namely 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). These primers were selected based on their ability to amplify the target region, allowing a comprehensive assessment of bacterial communities in fecal samples. After PCR amplification, the resulting product was purified using a preparative DNA gel extraction kit (Oxygen BioSciences, Union City, CA, USA). The libraries were sequenced on the Illumina MiSeq PE300 platform, a widely used NGS system known for its high-throughput sequencing capabilities. In order to analyze the obtained sequencing data, the online service provided by the Majorbio cloud platform (WWW.Majorbio.com) (accessed on 15 March 2023) was used. Lease analysis, a statistical method commonly used in ecological research, was used to further study the relationship between microbial taxa and environmental factors.

2.5. ABTS Free Radical Scavenging Ability

According to the study protocol of Jo et al. [18], we used a similar method to that of Kim et al. (2019) to assess the ABTS radical scavenging activity of the samples. In summary, bacterial cell suspensions, treated with varying BC-HYI or LGG concentrations, were combined with a diluted ABTS solution (1 L). After thoroughly mixing and preparing the mixture, it was incubated in a dark environment at room temperature for 6 min.

2.6. Cell Lines and Culture Conditions

The DF-1 cell line, derived from glass fibers and obtained from ATCC (SCRC-1040), has been widely used in various studies. In this particular experiment, cells were cultured in a DF-1 medium supplemented with 13% fetal bovine serum (FBS), which provides essential nutrients and growth factors for cell proliferation and maintenance. The temperature of the incubator was maintained at 37 °C, which is the ideal temperature for the growth of avian cells. Additionally, the incubator provides a humidity level of 90% to prevent cell dehydration and promote a suitable cellular environment. To maintain proper atmospheric composition, the incubator maintains a 5% CO2 concentration, which is essential to maintaining the pH balance of the culture medium. To prevent microbial contamination and ensure the sterility of cell cultures, 1% (v/v) penicillin DF-1 medium was supplemented with streptomycin (Solarbio Science and Technology Co., Ltd., Beijing, China). The culture medium was refreshed every 48 h to provide fresh nutrients and remove metabolic waste products produced by the cells.

2.7. Cell Treatments

To evaluate the antioxidant properties of BC-HYI, DF-1 cells were categorized into different groups: Control (CON), LPS treatment (LPS), CB5 (1.0 × 105 BC-HYI), CB6 (1.0 × 106 BC-HYI), and CB7 (1.0 × 107 BC-HYI). DF-1 cells were seeded at a density of 1.2 × 106 cells/L (100 µL per well) in a 96-well plate and incubated for 24 h. The control and LPS groups were cultured in DMEM devoid of FBS and antibiotics for 6 h. In the BC-HYI + LPS group, cells were co-cultured with BC-HYI for 6 h, followed by washing with PBS to remove the macrobiotics. Then, DMEM without FBS containing LPS (at the concentration determined by the CCK-8 assay) was added for 6 h to induce oxidative stress, excluding the control group.

2.8. Reactive Oxygen Species (ROS) Assay

The levels of reactive oxygen species (ROS) in DF-1 cells were determined utilizing a fluorescent dye-based assay employing 2′,7′-dichlorofluorescein acetate (DCFH-DA, D2215-25 mg, Landholder, Beijing, China). DF-1 cells were cultivated in a 6-well plate, with each well containing approximately 1.2 × 106 cells, and the culture medium was replenished upon reaching 80% confluence. After the respective treatments, the cell culture medium was aspirated, and afterward, the cells underwent a single phosphate-buffered saline (PBS) wash.
Following the initial steps of the experiment, the DF-1 cells were subjected to further treatment to assess the level of reactive oxygen species (ROS) present within the cellular environment. To accomplish this, the cells were treated with a solution of 2′,7′-dichlorofluorescein diacetate (DCFH-DA), which is a non-fluorescent compound that can be converted to a fluorescent form in the presence of ROS.
The DCFH-DA solution, prepared at 10 µM and diluted in serum-free medium, was added to the cells and incubated at 37 °C for 20 min. During this incubation period, the DCFH-DA molecules entered the cells and became trapped within the cytoplasm.
After the incubation period, the DCFH-DA solution was carefully discarded, and the cells underwent three consecutive washes with a fresh supply of serum-free medium. This washing step was essential to remove any residual DCFH-DA solution and ensure accurate measurement of the ROS-induced fluorescence.
It is important to note that the initially non-fluorescent DCFH-DA molecule can undergo oxidation in the presence of ROS, generating a fluorescent compound known as 2′,7′-dichlorofluorescein (DCF). The formation of DCF fluorescence is directly proportional to the levels of ROS present in the cellular environment.
To visualize and capture images of the fluorescence, a fluorescence microscope manufactured by Olympus Corporation (Olympus, Japan) was utilized. The microscope was equipped with appropriate filters, including an excitation filter with a wavelength of 488 nm and an emission filter with a wavelength of 525 nm. These filters were specifically chosen to maximize the detection and visualization of the DCF fluorescence.
Under the fluorescence microscope, the DF-1 cells were observed, and images were captured using suitable magnification settings. The intensity of the fluorescence observed in the captured images directly corresponded to the levels of ROS within the cells. Higher fluorescence intensity indicated higher levels of ROS production, while lower intensity suggested lower ROS levels.
This fluorescence microscopy-based method allowed for qualitatively assessing ROS levels in the DF-1 cells. By analyzing the captured images, researchers could gain insights into the cellular oxidative stress status and evaluate the effects of different treatments or interventions on ROS generation.
In conclusion, treating DF-1 cells with the DCFH-DA solution followed by fluorescence microscopy enabled the measurement and visualization of ROS levels within the cells. This technique provided valuable information regarding cellular redox status and contributed to a better understanding of the role of ROS in cellular processes and diseases.

2.9. Antioxidant Enzyme Activity and MDA Content in Jejunum Samples, Serum, and Cultured Cells

DF-1 cells were seeded at a density of 1.2 × 106 cells/mL in a 6-well plate and incubated for 24 h. The jejunum samples were added to a tissue lysis solution (Nanjing Jiancheng) and macerated. After the abovementioned treatment, the superoxide dismutase (SOD), catalase (CAT), Glutathione peroxidase (GSH-Px), total antioxidant capacity (T-AOC) and malondialdehyde (MDA) assay kits (Nanjing Jiancheng Biological Engineering Research Institute Co., Ltd., Nanjing, China) were used to measure the content in cells, serum and jejunum samples [19]. The BCA assay kit (Nanjing Jiancheng) was used to determine the protein content of cells and tissues.

2.10. Measurement of Cell Apoptosis

To quantify the population of apoptotic cells, we employed the Annex V apoptosis detection kit I (BD Biosciences, San Diego, CA, USA) labeled with FITC, following the instructions provided by the manufacturer. After rinsing the cells twice with chilled PBS, we resuspended them in a 1X binding buffer at a 1 × 106 cell/L density. Next, 1 × 105 cells in a 100 µL aliquot were transferred to a 5 mL culture tube, and 5 µL of Annexin V and 5 µL of propidium iodide were added. Gentle vortexing ensured thorough mixing, and the mixture was then incubated at room temperature (25 °C), shielded from light, for 15 min. Following the incubation, the cells were analyzed using a flow cytometer (BD LSRII, San Diego, CA, USA) within 1 h. The acquired flow cytometry data were processed using BD Facsimile software version 8.0. The numbers of late apoptotic and early apoptotic cells were recorded in the Q2 and Q3 quadrants, respectively.

2.11. Real-Time Quantitative Polymerase Chain Reaction

Total RNA extraction and cDNA synthesis using the SteadyPure Universal RNA Extraction Kit (Accurate Biology, Changsha, China) and the Pro Taq HS SYBR Green premixed qPCR kit (Accurate Biology, Changsha, China) were performed following the manufacturer’s instructions. PCR primer sequences for the gallus genes were designed and selected by Primer3Plus and NCBI Website (https://www.ncbi.nlm.nih.gov (accessed on 18 September 2023)) as presented in Table S1. GAPDH as housekeeping gene was used to normalize target gene transcript levels. Real-time PCR was performed using Premix Ex TaqTM with SYBR Green (Accurate Biology, Changsha, China) and ABI Stepone Real-Time PCR System 7500 Fast Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA). The thermocycle protocol included 30 s at 95 °C followed by 40 cycles of 5 s denaturation at 95 °C, 30 s annealing/extension at 60 °C, and then a final melting curve analysis to monitor the purity of the PCR product. The 2−ΔΔCt method was used to estimate messenger RNA (mRNA) abundance. Relative gene expression levels were normalized by eukaryotic reference gene GAPDH.

2.12. Western Immunoblot Analysis

To extract total cellular protein from DF-1 cells and jejunum tissue, we utilized RIPA dialysis buffer obtained from Nanjing Kanchenjunga Bioengineering Institute in Nanjing, China. The protein concentration in the supernatant was determined using a BCA protein assay kit provided by the same institute. Equal amounts of protein (30 µg) were taken from each sample and separated by 10% SDS-PAGE. Subsequently, the proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Immobile, Shanghai, China) after blocking the membrane. After that, the membranes were incubated overnight at 4 °C with specific primary antibodies, followed by a 1 h incubation with secondary antibodies at room temperature. Based on the manufacturer’s instructions, bioluminescence was detected using an ECL luminescence reagent. The internal control alpha-tubulin (α-tubulin) was used to normalize the relative protein expression. Quantitative analysis of fluorescence intensity was conducted using Image software.

2.13. Statistical Analyses

Data shown in the study were obtained from at least three independent experiments and all data in different experimental groups were expressed as the mean ± SD. Statistical analyses were performed using GraphPad Prism version 7.0 (San Diego, CA, USA). The flow cytometry data were analyzed by BD FACSDiva Software version 8.0. One-way analysis of variance (ANOVA) followed by a least significant difference multiple comparison test was used. Values of p < 0.05 were considered statistically significant while p < 0.01 was considered a trend.

3. Result

3.1. Effects of BC-HYI on Intestinal Integrity and Antioxidant Enzyme Activity

In the control group, the intestinal histology displayed no indications of edema, inflammatory cell infiltration, or any other irregularities. The jejuna tissue exhibited a normal arrangement with well-organized, uniformly distributed intestinal villi (Figure 1A). Conversely, in the LPS group, the jejuna villi exhibited severe disarray, atrophy, and reduction in length, with a noticeable presence of detached or necrotic villi. Remarkably, both the B7 and B8 groups demonstrated significantly fewer structural abnormalities in the jejunum compared to the LPS group.

3.2. BC-HYI Reduces Oxidative Stress in Laying Chickens through the Nrf2/HO-1 Signaling Pathway

The interaction between BC-HYI and the Nrf2 signaling pathway was investigated to assess its impact on oxidative stress (OS) in chickens (Figure 1B). In the LPS group, the expression levels of Nrf2 and HO-1 were significantly increased (p < 0.05), while SOD exhibited a significant elevation (p < 0.01) compared to the control group. Conversely, Keap-1 expression showed a significant decrease (p < 0.05). Notably, the B8 group demonstrated significant regulation in the gene expression levels of GST, Nrf2, NQO1, HO-1, and SOD compared to the LPS group (p < 0.01), with a significant downregulation of Keap-1 (p < 0.05). Additionally, the B8 group exhibited significantly increased expression levels of SOD (p < 0.05) and significantly elevated expression of Nrf2 and NQO1 (p < 0.01) compared to the control group. The protein levels of Nrf2 and HO-1 in the intestinal tissue were also evaluated (Figure 1C,D). The LPS group displayed a significant reduction in Nrf2 and HO-1 protein levels compared to the control group (p < 0.05), while the B8 group showed a remarkable increase (p < 0.01).
The intestinal redox status was determined to reflect the oxidative stress and antioxidative function of laying chickens supplemented with BC-HYI (Table 1). Compared with the control group, the MDA (p < 0.01) activity in the serum and jejunal of the LPS group increased significantly (Table 1). However, the activities of SOD (p < 0.05), and CAT (p < 0.01) in the serum of the B8 group were significantly increased; the activities of GSH-PX (p < 0.05), CAT (p < 0.05) and T-AOC (p < 0.01) in the jejunum of the B8 group were significantly increased, and the content of MDA was decreased in comparison to the LPS group (p < 0.05).

3.3. BC-HYI Regulated the Gut Microbiota in Laying Chickens

The effect of BC-HYI on intestinal flora of laying chickens was detected by 16S rRNA gene sequencing. PCA of the binary Jaccard distance showed that the samples from the LPS group clearly pooled separately from those in the control group, while the B8 group samples were close to those in the control group (Figure 2A). Compared with the control group, the Chao index and ACE index in the LPS group were significantly decreased, and the Simpson index was significantly increased (Table S1). Moreover, we found that 367 OTUs common among all groups and few specific OTUs in each group (Figure 2B). In contrast, the B8 groups had lower OTUs, which were improved by higher doses of BC-HYI (Figure 2C).
To characterize the bacterial populations in each experimental group, the taxonomic compositions of gut microbial communities were analyzed at the phylum and genus levels. The predominant taxa identified across all groups were Vermiculite, Bactericide, and Arachnophobia at the phylum and family classification. At the genus level, the major types included Barbarella, Bactericide, Lactobacillus, Flautist, Alcestis, Antibacterial, Cloistral, and Heisenberg (Table S2). In the LPS group, there were significant decreases in the abundance of Barbarella, Arachnophobia, Flautist (p < 0.01), and Antibacterial, while Bactericide, Cloistral, and Thrombosis (p < 0.01) showed a significant increase. Comparatively, the B8 group exhibited significant decreases in the abundance of Barbarella, Flautist, Alcestis, Arachnophobia (p < 0.01), and Antibacterial compared to the LPS group, while the abundance of Bactericide (p < 0.01), Cloistral (p < 0.01), and Thrombosis significantly increased.
Distinct bacterial lineages were identified through the application of linear discriminant analysis effect size (Lease) when examining the effects of different treatments. Significant differences in bacterial composition were identified when comparing the control group with other treatment groups. The LPS group was mainly enriched in Antibacterial, Pneumococcus and Bactericidal (Figure 3A,B). Notably, as shown in Figure 3C,D, the bacteria with significant differences in the B8 group were mainly enriched in Barbarella, Alcestis, Cyanobacteria, and Streptococcal compared with the LPS group. Using the OTU abundance data from the Crust database, we annotated the KEGG pathway functions, screened the pathways for statistically significant differences, and presented the results in a chart (Figure 3E,F). Functions related to the Nrf2 signaling pathway and polysemous biosynthesis were more prevalent in the LPS group. In contrast, functions involved in the Linoleum acid metabolism, cell division, penicillin, cephalopod biosynthesis, and lac tam resistance were more frequent in the B8 group.

3.4. Effect of BC-HYI on OS Survival and Antioxidant Enzyme Levels

In assessing the ABTS radical scavenging capacity of BC-HYI, we used Lactobacillus rhamnosus (LGG) as a control. According to the results in Figure 4A, the free radical scavenging capacity of BC-HYI and LGG increased as the number of bacterial cells increased. Notably, BC-HYI exhibited a stronger free radical scavenging capacity than LGG, with significant differences observed, especially in the B6 and B7 groups (p < 0.01).
To assess the potential of BC-HYI in preventing LPS-induced oxidative stress (OS), we initially evaluated its impact on the viability of DF-1 cells at 6 and 12 h. As depicted in Figure 4A, BC-HYI at concentrations ranging from 1 × 103 to 1 × 108 CFU/L did not significantly affect DF-1 cell viability. Therefore, subsequent experiments utilized BC-HYI BP at concentrations ranging from 1 × 105 to 1 × 107 CFU/L for 6 h. To induce OS and reactive oxygen species (ROS) production, DF-1 cells were exposed to 8 Ag/L LPS for 6 h (Figure S1). The viability of cells in the LPS group significantly decreased (p < 0.01) compared to the control group. However, the addition of BC-HYI to the cell culture medium reversed this viability loss and dose-dependently increased the survival of LPS-treated DF-1 cells (Figure 4C).

3.5. Effect of BC-HYI on LPS-Induced ROS Generation and Apoptosis in DF-1 Cells Associated with Nrf2 Signaling

To assess the impact of BC-HYI maltreatment, we measured acellular ROS formation in cells treated with LPS. As shown in Figure 5A, the acellular ROS level in DF-1 cells exposed to 8 μg/L LPS was significantly higher than that in the control group (p < 0.01). However, the addition of BC-HYI at concentrations of 1 × 106 CFU/L and 1 × 107 CFU/L effectively attenuated the increase in reactive oxygen species (ROS) production compared to the LPS group without bacterial treatment (p < 0.01). Treatment with 1 × 105 CFU/L (B5) also demonstrated a significant reduction in ROS production (p < 0.05). These results indicate that BC-HYI maltreatment efficiently suppresses LPS-induced ROS generation, potentially through its direct antioxidant activity by scavenging free radicals. However, further investigations are required to elucidate the underlying mechanism in detail.
We determined the effect of BC-HYI on apoptosis in cultured DF-1 cells. We found that the percentages of late apoptotic cells and early apoptotic cells were significantly increased after the LPS challenge compared to the control (p < 0.01). BC-HYI maltreatment significantly decreased the percentages of late and early apoptotic cells under LPS-induced OS conditions (p < 0.01). All BC-HYI liters showed anti-apoptotic capacity, but the B7 group exhibited the highest activity (Figure 5B).
Here, we further investigated whether B. coagulans affect the regulatory properties of LPS-damaged on these antioxidant genes (Figure 5E). The expression levels of Nrf2, SOD and GST (p < 0.05), HO-1 and NQO1 (p < 0.01) genes were significantly decreased in the LPS group compared with the control group. Compared with the LPS group, the expression levels of NQO1, Nrf2, GST, HO-1, SOD (p < 0.01) and Keap-1 (p < 0.05) genes were significantly increased in the B7 group. The expression levels of the proteins HO-1 (p < 0.05), Nrf2 and NQO1 (p < 0.01) in the DF-1 cells by the LPS-treated group are reduced compared to those in the control group. While compared with the LPS-damaged group, the DF-1 cells in the B7 group exhibit greatly increased levels of NQO1, Nrf2, and NQO1 (p < 0.01).These showed that BC-HYI had a positive regulatory effect on the Nrf2/Keap1 pathway of LPS-induced oxidative damaged cells. In summary, laying chickens and cell experiments indicate that BC-HYI supplementation can improve the enzyme function of antioxidants, regulate intestinal barrier function and activate the Nrf2-ARE/HO-1 signaling pathway to regulate intestinal barrier function (Figure 6).

4. Discussion

Dietary supplementation of probiotic strategies helps control the development of enteritis by altering intestinal epithelial integrity and microbiota structure. Our results clearly indicated that supplementation of BC-HYI reduces LPS damage to maintain the intestinal structural integrity in laying chickens by increasing the activity of antioxidant enzymes Pasteurella and Lactobacillus and abundance of beneficial bacteria in Faecalibacterium, as well as reduced abundance of harmful bacteria in Bacteroides and Clostridium. The results of the article indicate that BC-HYI can change the intestinal environment by increasing the content of beneficial bacteria, and may also affect the expression of genes and proteins in the Nrf2 signaling pathway to increase antioxidant capacity.
With the development of colitis, the accompanying inflammation generates a large amount of ROS, and the oxidative stress causes damage to intestinal epithelial cells. In the present study, LPS-induced oxidative stress is similar to the pathological characteristics of the stress responses in the body to curative colitis, as well as LPS-induced oxidative stress commonly adopted in the process of drug discovery [20]. It is well known that effective scavenging of hydroxide radicals is beneficial for living organisms [21]. BC-HYI showed higher free radical scavenging ability than LGG, especially in the CB6 and CB7 groups. These results are consistent with previous antioxidant studies of B. coagulant on HT-29 cells [22]. In addition, York-Duran et al. [23] reported that the scavenging of ROS includes both enzymatic and non-enzymatic systems.
The activity of antioxidant enzymes was increased by BC-HYI with a concomitant reversal of the apoptotic response induced by LPS.ROS. It is an active signaling molecule which plays a regulatory role in oxidative stress by regulating oxidative indicators, increasing formaldehyde (MDA) content and decreasing the expression of antioxidant enzyme genes [24]. Researchers also found that Se-enriched Bacillus subtitles significantly reduced MDA content and increased SOD, CAT, and GPX activities in oxidative stressed fish [25]. Correspondingly, Bacillus coagulant T242 increased T-SOD and CAT activities by inhibiting oxygen radical production and activating the Nrf2 signaling pathway in HT-29 cells [26], consistent with our findings. In addition, Chaos et al. showed that Bacillus coagulant 12 increased the expression of Nrf2, HO-1, AKT, P-ACCT, and Bcl-2 proteins, thereby reversing antitoxin-induced apoptosis in mouse kidney cells [27]. This result may be due to the activation of antioxidant stress response by B. coagulant, mainly through Nrf2 and MAPK signaling pathways.
Consistent with the findings of Kai et al., the present study found that BC-HYI reduced the number of harmful bacteria while increasing the relative abundance of beneficial bacteria [17] It has been reported that OS was induced in mice by whole-body radiation, the ROS content was significantly increased, the antioxidant enzyme activity was decreased, the numbers of Bactericide and Streptococcus fatalistic were increased, and the abundance of Arachnophobia was decreased in the gut [28]. Similarly, studies by Wu et al. demonstrated that treatment with 2 × 107 CFU/L B. coagulant increased the abundance of lactobacillus and decreased cloistral in porcine colon [29]. In our previous investigations, it was demonstrated that BC-HYI has the capability to generate organic acids, including lactic acid, satyric acid, and propionic acid [30]. These organic acids play a crucial role in connecting the gut macrobiotic with various diseases. For instance, Saks et al. [31] revealed that Bacillus coagulant SANK 70258 could effectively treat colitis by increasing the abundance of Arachnophobia and the levels of satyric acid in the gut. Based on our current findings, we propose that BC-HYI modifies the composition of the gut macrobiotic by producing metabolites such as short-chain fatty acids and interactions. Moreover, BC-HYI regulates the Erna and protein expression levels of TLR4, reducing intestinal permeability and improving LPS-induced colitis and overall gut health. These results are consistent with the findings reported by [32].
In conclusion, this study highlights the efficacy of BC-HYI in reducing LPS-induced oxidative damage and reducing its severity in laying chickens. BC-HYI reduced mortality by enhancing the abundance of beneficial intestinal microbes, maintaining intestinal homeostasis, and relieving oxidative stress caused by environmental challenges. Thus, BC-HYI has the potential to be a safe natural antioxidant in feed and also can be used to develop new storage-stable probiotics aimed at improving intestinal health, providing more possibilities for replacing antibiotics. However, the mechanism of Bacillus coagulant antioxidant activity needs to be further studied to provide a theoretical basis for research leading to improved production, which is of great value to the livestock and poultry breeding industry.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/applmicrobiol3040081/s1, Figure S1: Effects of LPS on Viability and Oxidative Stress of DF-1 Cells; Table S1: Effect of BC-HYI on alpha diversity of intestinal microbiota of laying chickens.; Table S2: Effects of BC-HYI on the abundance of intestinal microbiota.

Author Contributions

T.L.: Conceptualization, Data curation, Formal analysis, Writing—original draft, Writing—review and editing, Validation. L.W.: Methodology, Conceptualization, Validation, Data curation, Writing—original and draft. Q.W.: Formal analysis, Investigation, Validation, Writing—review and editing. H.Z.: Formal analysis, Investigation, Methodology, Supervision, Validation, Writing—review and editing. D.C.: Formal analysis, Data curation, Investigation, Methodology. B.L.: Data curation, Investigation, Methodology, Resources. J.T.: Funding acquisition, Project administration, Resources, Supervision, Writing—review and editing. Y.Z.: Funding acquisition, Resources, Supervision, Writing—review and editing. All authors have read and agreed to the published version of the manuscript.

Funding

This research received financial support from various funding sources. Specifically, the Beijing Livestock Industry Technology Innovation Plan (BAIC05-2022), National Science Foundation of Beijing Province (KM202010020009), and National Natural Science Foundation of China (32202881) provided the necessary funding for this study.

Institutional Review Board Statement

Approval for all animal studies conducted in this research was obtained from the Animal Welfare Committee of the Institute of Animal Science, Chinese Academy of Agricultural Sciences (Ethics Code Permit BUA 2021-003).

Informed Consent Statement

Not applicable.

Data Availability Statement

Restrictions apply to the availability of these data. Data was obtained from Shanghai Meiji Biomedical Technology Co., Ltd. and are available Majorbio with the permission of https://login.majorbio.com/ (accessed on 18 September 2023).

Acknowledgments

The support from the Beijing Livestock Industry Technology Innovation Plan played a crucial role in advancing the research and technological innovations in the livestock industry. This funding initiative promotes the development of novel approaches, techniques, and solutions to address key challenges and enhance the overall efficiency and sustainability of the livestock sector in Beijing. Additionally, the National Science Foundation of Beijing Province provided financial assistance to support the research endeavors in the field of science and technology within the region. This foundation aims to foster scientific discoveries, promote interdisciplinary collaborations, and facilitate the translation of research outcomes into practical applications that benefit the society and economy of Beijing.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. The impact of BC−HYI on mitigating oxidative stress in laying chickens through the Nrf2 signaling pathway is depicted in Figure 1. (A) Representative H&E staining images of jejunum sections. (B) mRNA expression levels of genes associated with the Nrf2 signaling pathway. (C) Immunohistochemical analysis of the jejunum in laying chickens. (D) Protein levels of Nrf2, HO−1, and NQO1 in the jejunum. The data are presented as mean ± SD and represent three independent experiments. Different letters (a–d) indicate significant differences between groups (p < 0.05).
Figure 1. The impact of BC−HYI on mitigating oxidative stress in laying chickens through the Nrf2 signaling pathway is depicted in Figure 1. (A) Representative H&E staining images of jejunum sections. (B) mRNA expression levels of genes associated with the Nrf2 signaling pathway. (C) Immunohistochemical analysis of the jejunum in laying chickens. (D) Protein levels of Nrf2, HO−1, and NQO1 in the jejunum. The data are presented as mean ± SD and represent three independent experiments. Different letters (a–d) indicate significant differences between groups (p < 0.05).
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Figure 2. BC−HYI alters the composition of gut microbiota in the presence of LPS. (A) PCoA analysis of each group. (B) Venn diagram of OTUs from cecum samples. (C) Relative abundance of gut microbes at the phylum level. (D) Relative abundance of gut microbes at the genus level. Different letters (a–c) indicate significant differences between groups at p < 0.05.
Figure 2. BC−HYI alters the composition of gut microbiota in the presence of LPS. (A) PCoA analysis of each group. (B) Venn diagram of OTUs from cecum samples. (C) Relative abundance of gut microbes at the phylum level. (D) Relative abundance of gut microbes at the genus level. Different letters (a–c) indicate significant differences between groups at p < 0.05.
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Figure 3. BC−HYI alters LPS−induced changes to gut microbiota. (A) LEfSe analysis of CON vs. LPS. (B) LDA discriminant result histogram for CON and LPS. (C) LEfSe analysis of B8 vs. LPS. (D) LDA discriminant result histogram for B8 and LPS. (E) Functional prediction of gut microbiota for CON and LPS. (F) Functional prediction of gut microbiota for B8 and LPS.
Figure 3. BC−HYI alters LPS−induced changes to gut microbiota. (A) LEfSe analysis of CON vs. LPS. (B) LDA discriminant result histogram for CON and LPS. (C) LEfSe analysis of B8 vs. LPS. (D) LDA discriminant result histogram for B8 and LPS. (E) Functional prediction of gut microbiota for CON and LPS. (F) Functional prediction of gut microbiota for B8 and LPS.
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Figure 4. BC−HYI regulates LPS−induced antioxidant enzyme activity in DF-1 cells. (A) Effect of BC−HYI and LGG on free-radical scavenging capacity of DF−1 cells. (B) Effect of BC−HYI CFU/mL on DF−1 viability at 6 and 12 h. (C) Effect of increasing titer of BC−HYI on viability of LPS−damaged DF−1 cells. (D–H) Content of MDA, T−SOD, T−AOC, GSH−Px, and CAT. The results are representative of three independent experiments and expressed as means ± SD. Different letters (a−d) indicate significant differences between groups (p < 0.05).
Figure 4. BC−HYI regulates LPS−induced antioxidant enzyme activity in DF-1 cells. (A) Effect of BC−HYI and LGG on free-radical scavenging capacity of DF−1 cells. (B) Effect of BC−HYI CFU/mL on DF−1 viability at 6 and 12 h. (C) Effect of increasing titer of BC−HYI on viability of LPS−damaged DF−1 cells. (D–H) Content of MDA, T−SOD, T−AOC, GSH−Px, and CAT. The results are representative of three independent experiments and expressed as means ± SD. Different letters (a−d) indicate significant differences between groups (p < 0.05).
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Figure 5. The impact of BC−HYI on reducing oxidative damage in DF−1 cells through the Nrf2 signaling pathway. (A) Representative images demonstrate the fluorescence of DCFH−DA in DF−1 cells. (B,C) Relative levels of reactive oxygen species (ROS). (D) Effects of BC−HYI on apoptosis of DF−1 cells induced by LPS. (E) Gene expression levels of the Nrf2 signaling pathway. (F) Immunological analysis of DF−1 cell jejunum. (G) Protein levels of Nrf2, HO−1, and NQO1 in DF−1 cells. The results, presented as means ± SD, are representative of three independent experiments. Different letters (a–e) indicate significant differences between groups (p < 0.05).
Figure 5. The impact of BC−HYI on reducing oxidative damage in DF−1 cells through the Nrf2 signaling pathway. (A) Representative images demonstrate the fluorescence of DCFH−DA in DF−1 cells. (B,C) Relative levels of reactive oxygen species (ROS). (D) Effects of BC−HYI on apoptosis of DF−1 cells induced by LPS. (E) Gene expression levels of the Nrf2 signaling pathway. (F) Immunological analysis of DF−1 cell jejunum. (G) Protein levels of Nrf2, HO−1, and NQO1 in DF−1 cells. The results, presented as means ± SD, are representative of three independent experiments. Different letters (a–e) indicate significant differences between groups (p < 0.05).
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Figure 6. Mechanism of B. coagulans HYI antioxidant effects through activation of the Nrf2 signaling pathway. Nrf2: nuclear factor erythroid 2−related factor 2; Keap−1: kelch−1ike, ECH−associated protein l; HO−1: heme oxygenase 1; CAT: catalase; SOD: superoxide dismutase; NQO1: quinone oxidoreductase.
Figure 6. Mechanism of B. coagulans HYI antioxidant effects through activation of the Nrf2 signaling pathway. Nrf2: nuclear factor erythroid 2−related factor 2; Keap−1: kelch−1ike, ECH−associated protein l; HO−1: heme oxygenase 1; CAT: catalase; SOD: superoxide dismutase; NQO1: quinone oxidoreductase.
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Table 1. Effect of BC-HYI on redox status in laying chickens.
Table 1. Effect of BC-HYI on redox status in laying chickens.
AnalyteControlLPSB6B7B8
Serum
T-SOD (U/mL)189.33 ± 9.97 Aa93.89 ± 5.49 Bb202.42 ± 8.01 Aa221.58 ± 10.08 Ab248.11 ± 6.62 Ab
T-AOC (U/mL)35.88 ± 3.71 Aa11.25 ± 2.83 Bb12.31 ± 4.14 Bb23.24 ± 4.37 Ab27.31 ± 3.54 Aa
MDA (nmol/mL)2.97 ± 0.44 Aa16.24 ± 0.07 Bb10.92 ± 2.25 Ba7.58 ± 0.76 Ab6.40 ± 0.19 Ab
CAT (U/mL)4.19 ± 0.51 Aa1.82 ± 0.41 Ab5.05 ± 0.73 Aa7.35 ± 0.19 Bb7.80 ± 0.79 Bb
GSH-Px (U/mL)519.72 ± 57.98461.97 ± 8.29650.52 ± 19.65653.90 ± 8.78665.16 ± 9.92
Jejunum
T-SOD (U/mg prot)54.98 ± 5.1632.74 ± 3.2742.80 ± 2.543.14 ± 3.1145.82 ± 5.48
T-AOC (U/mg prot)2.59 ± 0.16 Aa0.97 ± 0.39 Ab2.76 ± 0.44 Ab5.08 ± 0.25 Bb5.12 ± 0.02 Bb
MDA (nmol/mg prot)0.69 ± 0.20 Aa9.16 ± 0.85 Bb6.42 ± 0.39 Ba3.16 ± 0.22 Ab1.52 ± 0.28 Ab
CAT (U/mg prot)5.37 ± 0.22 Aa0.81 ± 0.10 Bb2.33 ± 0.13 Ab3.91 ± 0.53 Ab4.09 ± 0.11 Ab
GSH-Px (U/mg prot)20.16 ± 3.03 Aa18.36 ± 1.90 Aa24.66 ± 1.47 Ab32.44 ± 2.05 Ab36.26 ± 0.94 Ab
a, b Values within a row with different letters differ at p < 0.05; A, B Values within a row with different letters differ at p < 0.01.
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MDPI and ACS Style

Lu, T.; Wang, L.; Wu, Q.; Zhang, H.; Cui, D.; Liu, B.; Tong, J.; Zhang, Y. Bacillus coagulant HYI (BC-HYI) Alleviates LPS-Elicited Oxidative Stress by Engaging the Nrf2/HO-1 Signaling Pathway and Regulates Gut Macrobiotics in Laying Chickens. Appl. Microbiol. 2023, 3, 1178-1194. https://doi.org/10.3390/applmicrobiol3040081

AMA Style

Lu T, Wang L, Wu Q, Zhang H, Cui D, Liu B, Tong J, Zhang Y. Bacillus coagulant HYI (BC-HYI) Alleviates LPS-Elicited Oxidative Stress by Engaging the Nrf2/HO-1 Signaling Pathway and Regulates Gut Macrobiotics in Laying Chickens. Applied Microbiology. 2023; 3(4):1178-1194. https://doi.org/10.3390/applmicrobiol3040081

Chicago/Turabian Style

Lu, Tianhang, Le Wang, Qiong Wu, Hua Zhang, Defeng Cui, Bowen Liu, Jinjin Tong, and Yonghong Zhang. 2023. "Bacillus coagulant HYI (BC-HYI) Alleviates LPS-Elicited Oxidative Stress by Engaging the Nrf2/HO-1 Signaling Pathway and Regulates Gut Macrobiotics in Laying Chickens" Applied Microbiology 3, no. 4: 1178-1194. https://doi.org/10.3390/applmicrobiol3040081

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