Bacillus coagulans R11 maintained intestinal villus health and decreased intestinal injury in lead-exposed mice by regulating the intestinal microbiota and influenced the function of faecal microRNAs

Environmental Pollution 255 (2019) 113139

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Bacillus coagulans R11 maintained intestinal villus health and decreased intestinal injury in lead-exposed mice by regulating the intestinal microbiota and influenced the function of faecal microRNAs* Si-Cheng Xing a, 1, Chun-Bo Huang a, 1, Jian-Dui Mi a, Yin-Bao Wu a, b, c, Xin-Di Liao a, b, c, * a

College of Animal Science, South China Agricultural University, Guangzhou 510642, China Guangdong Provincial Key Laboratory of Agro-Animal Genomics and Molecular Breeding and Key Laboratory of Chicken Genetics, Breeding and Reproduction, Ministry Agriculture, Guangzhou 510642, Guangdong, China c National-Local Joint Engineering Research Centre for Livestock Breeding, Guangzhou 510642, Guangdong, China b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 9 July 2019 Received in revised form 7 August 2019 Accepted 29 August 2019 Available online 13 September 2019

Lead contamination is an environmental problem, especially in developing countries; due to the nondegradable characteristics of lead, it is easily deposited in human and animal bodies by the food chain. Probiotics are regarded as a good tool to remove lead ions in the intestine and maintain gut health conditions, but previous studies failed to elucidate the relationship among probiotics, the host and the gut microbiota. In the present study, B. coagulans R11 was employed as the “lead removal tool” in leadexposed mouse, and the effects of B. coagulans R11 on intestinal cells, the microbiota and faecal microRNAs were tested. The results indicated that B. coagulans R11 had no negative effects on mouse intestine model cells and helped keep cells in a normal proliferation ratio and reduce the reactive oxygen species and apoptosis ratios under lead exposure conditions. An in vivo mouse experiment also showed that B. coagulans R11 feeding could reduce the intestinal villi damage caused by lead through adjusting the microbiota structure and function, such as increasing the genus abundance of Akkermansia and Alistipes, decreasing the genus abundance of Alloprevotella, Lachnospiraceae, Parabacteroides and Ruminiclostridium, and keeping the protein dltD existing. Host faecal microRNAs may be influenced by lead and B. coagulans R11, which may change the microbiota structure. Thus, B. coagulans R11 has the potential to be developed and considered as the probiotic that protects the host gut against villi damage and gut microbiota structure and function disorders during lead exposure. © 2019 Elsevier Ltd. All rights reserved.

Keywords: Bacillus coagulans R11 Lead Microbiota structure microRNA Intestinal villus

1. Introduction Lead is one of the toxic heavy metals in the earth, and lead ions can be consumed by humans and animals through the food chain due to a variety of utilizations in anthropic activities (Florea et al., 2005; Hezbullah et al., 2016; Wu et al., 2018; Zoghi et al., 2014). It is difficult to prevent lead contamination from the food chain and

* This paper has been recommended for acceptance by Wen Chen. * Corresponding author. College of Animal Science, South China Agricultural University, Guangzhou 510642, China. E-mail addresses: [email protected] (S.-C. Xing), [email protected] (C.-B. Huang), [email protected] (J.-D. Mi), [email protected] (Y.-B. Wu), [email protected] (X.-D. Liao). 1 These authors contributed equally to the paper as first authors.

https://doi.org/10.1016/j.envpol.2019.113139 0269-7491/© 2019 Elsevier Ltd. All rights reserved.

living environment because the source of lead is abundant, and most lead contamination occurs in unpremeditated conditions, for example, in feed production mechanical engineering, such as painting dope dropout (Adamse et al., 2017; Farmer and Farmer, 2000; Nicholson et al., 1999). Therefore, some techniques that could decrease lead adsorption and harmful activity in animals and even the human body should be discussed and studied. In recent years, the application of probiotics has always been a popular topic, and some studies employed several probiotics as tools for heavy metal detoxification and investigated possible factors to explain the underlying mechanisms (Jahromi et al., 2017; Tian et al., 2012; Yi et al., 2017; Zhai et al., 2013). The main process via which probiotics reduce the toxic effects of lead in the body is adsorption (Tian et al., 2012; Zhai et al., 2013), and the most important barrier to the entry of lead ions into the body is intestinal

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cells. Thus, one of the key points for host health maintenance during lead exposure is lead ion adsorption prevention. However, previous studies investigated only the effects of these probiotics on model animals exposed to heavy metals, and they failed to elucidate why these probiotics could maintain animal health and prevent metal ion adsorption by intestinal cells. In recent studies, the intestinal microbiota is regarded as one of the organs of the host (Nicholson et al., 2012; Round and €ckhed, 2012), and the enteric Mazmanian, 2009; Tremaroli and Ba microbiota stability and the ratio of probiotics are tightly related to nchez et al., 2017). the health of the host (Gareau et al., 2010; Sa Previous studies found that heavy metal exposure could influence and decrease gut microbiota diversity and that lead could also significantly decrease the abundance of some bacterial genera, such as unclassified and uncultured Ruminococcaceae, unclassified Lachnospiraceae, and Ruminiclostridium_9 (Gao et al., 2017). Based on these results, keeping the normal microbiota structure and the ratio of probiotic enhancement is another point to maintain the health of the lead-exposed host. In addition, a previous study also found that the faecal miRNAs of the host could adjust the structure of the gut microbiota (Liu et al., 2016). This effect was due to miRNAs entering bacteria and regulating bacterial gene expression and growth, and their loss resulted in imbalanced microbiota and exacerbated colitis; Liu also found that these miRNAs were predominantly produced by gut epithelial cells and Hopxþ cells (Liu et al., 2016). Therefore, understanding the effects of probiotics on the structure and function of the gut microbiota of lead-exposed hosts could provide references for future applications as heavy metal detoxification tools in the animal even human intestine. In the present study, we fed Bacillus coagulans R11 (B. coagulans R11), which was isolated from a lead mine (Xing et al., 2018) to lead-exposed mice with the aims to examine: 1) whether B. coagulans R11 could be considered as a probiotic potentially, 2) how does B. coagulans R11 affect the intestinal cells; 3) the effects of B. coagulans R11 on the gut microbiota function and structure of lead-exposed mice; 4) the influences of B. coagulans R11 on the faecal microRNA expression level in lead-exposed mice; and 5) the potential relationship among B. coagulans R11, the gut microbiota and microRNAs.

culture medium residue. RPMI-1640 culture medium was added to the culture plate, and the cells were cultured in a CO2 incubator at 37  C for 12 h to ensure that all cells were homogeneous. After 12 h, lead and B. coagulans R11 were added to the cell culture plates based on the experimental design procedure. The experimental group details are shown in Table 1. The lead injection concentration in the cell culture plate was 200 mM, and this concentration was determined by a previous study of the maximum lead toxic concentration for MODE-K cells. In all B. coagulans R11 addition groups, the bacteria amount was 1  106 colony-forming units (CFU)/L for the final concentration, and the bacterial pellets were removed by washing three times with PBS to ensure that no bacteria remained before the next experimental step. The cells from all experimental groups were collected for cell proliferation, apoptosis and intracellular reactive oxygen species (ROS) testing. Cell Counting Kit-8 (CCK-8) stained the cells for the following proliferation test (Sang et al., 2018); the Annexin V-FITC/ PI staining method was used for the assessment of the apoptosis ratio, as previously mentioned (Shen et al., 2018); and fluorescent probe DCFH-DA staining was also used for the testing of ROS (Pan et al., 2019). The staining steps followed the kit manufacturer procedures (CCK-8, Dojindo, Japan; Annexin V-FITC/PI, Beibo, Beijing, China; DCFH-DA, Beibo, Beijing, China). After staining, a microplate spectrophotometer (Multiskan FC, ThermoFisher Scientific, USA) was used to assess the absorbance for proliferation. The apoptosis ratio of cells was tested by flow cytometry (FCM) (FACS Aria II, BD, USA), ROS was viewed by using a laser scanning confocal microscope (LSCM, Leica TCS SP8, Germany), and the ROS ratio was estimated by using a fluorescence microplate reader (SpectraMax Gemini XPS, Molecular Devices, USA). 2.2. Animal ethics statement All the animal experiments were approved by the Animal Experimental Committee of South China Agricultural University (SYXK2014-0136). All experimental steps were considered to decrease animals suffering as much as possible. After the experiment, the bodies of mice were incinerated. 2.3. Mouse lead exposure experiment

2. Materials and methods 2.1. Mouse intestinal MODE-K cell experiment The model cell line of mouse intestinal cells, MODE-K, was purchased from a commercial company (Shanghai Hongshun Biological Technology Co., Ltd). Before formal experimentation, MODEK cells were recovered in T25 bottles by using RPMI-1640 culture medium (Gibco USA) with 20% foetal calf serum (Excell Bio Shanghai). The cell incubation conditions were in a CO2 incubator at 37  C (incubation for 48 h for each generation). B. coagulans R11 was inoculated into de Man, Rogosa, and Sharpe (MRS) broth (HuanKai Microbiology, Guangzhou, China) at 37  C for 18 h in anaerobic jars containing an AnaeroPack (Mitsubishi Gas Chemical Company, Inc. Tokyo, Japan) and harvested for the following experiment. To assess whether B. coagulans R11 has the potential to be a probiotic and the effects of B. coagulans R11 on the intestinal cells of lead-exposed mice, MODE-K cells were employed to mimic the mouse intestine conditions at the laboratory scale to obtain initial results. The MODE-K cells were inoculated in the culture medium as described above and placed in a CO2 incubator at 37  C for 6 h to allow cell adherence on the bottom of the culture plate. Then, the whole culture medium (RPMI-1640 þ 20% foetal calf serum) was removed from the culture plate, and phosphate-buffered saline (PBS) was used to wash the culture plate to remove any whole

Before a mouse feeding experiment, B. coagulans R11 lyophilized powder was prepared by harvesting bacterial pellets from MRS broth, as mentioned above. B. coagulans R11 pellets were mixed with 12% skim milk for lyophilization, and the lyophilized bacterial powder was stored at 4  C for mouse feeding experiments. The viable bacterial count was determined by plating. To obtain the feed concentration of B. coagulans R11, the bile and stomach acid tolerance ability was determined with reference to a previous study by Yi et al. (2017), the final concentration of the bacteria in the mouse intestines was approximately 1  108 CFU. Five-week-old specific pathogen-free (SPF) female Kunming mice were purchased from Guangdong Medical Laboratory Animal Center (qualification certification SCXK (Yue) 2013-0002). There were 60 mice randomly assigned into 4 experimental groups, and each group contained 15 mice (three cages for each group, with five mice bred in one cage). The control group (without lead and B. coagulans R11 treatment) was labelled KB; the group that consumed only B. coagulans R11 was labelled B11; the group that consumed lead (200 mg/L) dissolved in water was labelled C2; and the group fed both lead (200 mg/L) water solution and B. coagulans R11 was labelled B2, the group details were showed in Table 2. B. coagulans R11 was administered orally every morning at 8:00 for the B. coagulans R11 consumption groups. In addition, the mice in the KB and C2 groups were also fed the same dose of pure 12% skim

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Table 1 MODE-K cell experimental group details. Group

Pb addition time

B. coagulans R11 addition time

Total culture time (h)

Control B. coagulans R11 B. coagulans R11 þ Pb Pb

NA NA after cell homogeneous after cell homogeneous

NA after cell homogeneous after cell homogeneous NA

24 24 24 24

“NA” means not applicable.

Table 2 The mouse feeding experiment groups details. Group

Pb addition concentration

B. coagulans R11 addition concentration

KB B11 C2 B2

e e 200 mg/L 200 mg/L

e approximately 1  108 CFU in small intestine e approximately 1  108 CFU in small intestine

milk as the B. coagulans R11 intake groups, and the influence of skim milk could be avoided by this step. All the mice were housed under conditions of 55 ± 5% humidity and a 12-h light cycle at 23 ± 2  C, and feed was provided ad libitum. To mimic chronic lead intoxication, the mice were allowed to consume water ad libitum. Before the formal experiment, one week allowed for mice to adapt to the environment. The formal experimental period was 4 weeks, and the mice were killed according to the requirements of the animal ethics statement of the Animal Experimental Committee of South China Agricultural University (SYXK2014-0136). The small intestine was immediately stored in paraformaldehyde for the following intestine section observation, and caecum intestinal contents were collected from each mouse and stored in liquid nitrogen for DNA and microRNA extraction. The intestinal content was collected in tube from every mouse, after DNA and microRNA extraction, the DNA and RNA extracted from five mice was mixed by equal concentration as one omics testing repeat. 2.4. Small intestine section observation experiment To investigate the intestinal cell changes with and without B. coagulans R11 under lead exposure conditions, the small intestine (ileum) was used for section observation. Briefly, the intestine was taken from paraformaldehyde and washed with PBS solution for several hours to remove any residual paraformaldehyde. After rinsing, the intestine was gradually dehydrated with 70%, 80%, and 90% ethanol, and the intestine was placed in a solution of isopycnic 100% ethanol and xylene for 15 min, xylene I for 15 min and xylene II for 15 min (until the intestine became translucent). After the intestine was translucent, it was saturated in a solution of xylene and paraffin for 15 min and saturated in paraffin I and paraffin II for 50 min and 60 min, respectively. Haematoxylin-eosin (HE) staining was performed after paraffin sectioning and the villi length was assessed by image pro software (MediaCybernetics MD USA).

modification, which referenced the method introduced by Liu et al. (2016), microRNA sample preparation was the same as the DNA mixture method mentioned above; the microRNA was also stored at 80  C for microRNA sequencing. 2.6. Illumina high-throughput sequencing for metagenomics and microRNA Before total DNA sequencing, agarose gel electrophoresis (AGE) analysis of DNA purity and integrity was used to determine the quality of DNA, and the DNA concentration was precisely quantitative by using Qubit. Then, the DNA sample was randomly interrupted into fragments using a Covaris ultrasonic disruptor, and the length was approximately 350 bp. These fragments were repaired on the tail end, and an A tail and sequencing connectors were added. After the purification and PCR amplification steps were finished, the whole library preparation was completed. Illumina PE150 was applied for sequencing after the quality of the library was qualified. Briefly, a Small RNA Sample Pre Kit was used for library building based on the special structure of small RNA and 30 and 50 adaptors were added to the small RNA to compound cDNA for the library building. 2.7. Statistical analysis All experiments were performed in triplicate. Except for genomics data, the basic data were analysed with analysis of variance (ANOVA) using Statistical Package for the Social Sciences (SPSS) software, version 17.0. Significant differences between the means were determined by Tukey's test. Differences were considered significant when P < 0.05. 3. Results

2.5. Total DNA and microRNA extraction from intestinal contents

3.1. Effect of Bacillus coagulans R11 on a mouse MODE-K cell during lead exposure

Total DNA extraction was performed following the procedure of the QIAamp Fast DNA Stool Mini Kit (QIAGEN Germany). The total caecum intestinal content DNA of five mice from one cage was mixed in isoconcentration, and the mixture of pure DNA was stored at 80  C for metagenomics sequencing. MicroRNA extraction followed the mirVana™ miRNA Isolation Kit (Ambion USA), and with

Fig. 1 shows the results of cell apoptosis in all experimental groups. Fig. 1AeD shows the FCM results of every replicate of all groups. Fig. 1E shows the statistical results of FCM. The cell apoptosis test showed that without the protection of Bacillus coagulans R11, the viable/apoptotic cell ratio was significantly increased by 24.48% in the lead exposure group compared with that

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Fig. 1. Effects of B. coagulans R11 on model intestinal cells in lead exposure. Figure AeE were the cell apoptosis results of flow cytometry, A-D were group control, B. coagulans R11, B. coagulans R11 þ Pb and Pb respectively, E was the cell apoptosis quantification result; F was the result of proliferation ratio. Different letters between experiment groups means significantly difference (P < 0.05).

of the control group, and the normal cell ratio was significantly decreased by 17.37%. However, the data did not show a significant difference between the control group and the group with the addition of Bacillus coagulans R11 during lead exposure (Bacillus coagulans R11 þ Pb). In addition, the normal cell ratio in the Bacillus coagulans R11 group was obviously lower than that in the control group, but there was no obvious difference in the viable/apoptotic cell ratio between these two groups. The cell proliferation ratio can reflect the vitality of cells; Fig. 1F shows the MODE-K cell proliferation ratio with and without Bacillus coagulans R11 treatment during lead exposure. Within the Bacillus coagulans R11 treatment group, the cell proliferation ratio was not significantly decreased by lead toxicity, but without the protection of Bacillus coagulans R11, the cell proliferation ratio was obviously decreased by 34.9% in comparison with that of the control group. In addition, based on the results of the Bacillus coagulans R11 treatment group, which was not exposed to lead (Bacillus coagulans R11 group), the proliferation ratio showed no significant difference from that of the control group. The ratio of ROS can indicate the potential injury level of cells. In the present study, the ROS ratio in all experimental groups is shown in Fig. 2. Fig. 2AeD shows the LSCM observations in the control, B. coagulans R11, B. coagulans R11 þ Pb, and Pb groups. The green spot is the ROS, and it was clear that there were more green spots in the Pb group than in the other three groups. Fig. 2E shows the ROS

quantification results, and the ROS ratio was significantly higher in the Pb group than in the other three groups, and there was no significant difference between the control and B. coagulans R11 groups. In general, the results of LSCM and ROS quantification were similar, both results indicated that B. coagulans R11 could decrease the ROS ratio in lead-exposed cells, and B. coagulans R11 also did not enhance the ROS ratio in cells without lead exposure. 3.2. Small intestine section observation Based on the results of the cell model experiment, to further investigate the function of B. coagulans R11 on intestinal cells of lead-exposed mice, the small intestine of experimental mice was used for the section observation. Fig. 3AeD shows the results of paraffin sectioning, and Fig. 3E shows the statistics of intestinal villi length. Fig. 3A and B represent groups KB and B11, respectively. The intestinal villi were damaged in the lead-exposed groups (C2 and B2, Fig. 3C and D) by the comparison with the KB and B11 groups. In addition, the intestinal villi in the C2 group were more injured than those in the B2 group. The statistical results also showed that the intestinal villi length of the C2 group was significantly shorter than that in the B2 group, and the present result also showed that the intestinal villi length of the B11 group was obviously longer than that of the KB group. In addition, the submucosa layers of C2 (Fig. 3C) are thicker than other three groups. In summary,

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3.3. Gut microbiome structure and function of mice with and without B. coagulans R11 feeding during lead exposure

Fig. 2. ROS ratio result. A-D were the result of laser scanning confocal microscope of group control, B. coagulans R11, B. coagulans R11 þ Pb and Pb respectively, green spots were the ROS, E was the quantification result of ROS ratio. Different letters between experiment groups means significant difference (P < 0.05).

B. coagulans R11 feeding could enhance the length of intestinal villi and decrease the intestinal villi damage caused by lead exposure.

The mouse caecum intestinal contents were used for metagenomics analysis to assess the effects of B. coagulans R11 on the mouse gut microbiome structure and function changes in lead exposure conditions. Fig. 4A shows the relative abundance of caecum microbiota constituents at the phylum level. Bacteroidetes, Firmicutes and Verrucomicrobia were the three major bacterial phyla in all experimental groups. There was no obvious difference among all experiment groups in Bacteroidetes, but the abundances of Firmicutes and Verrucomicrobia were significantly decreased and increased, respectively, in the B2 group compared with those in the KB and C2 groups. In addition, lead exposure and B. coagulans R11 feeding could significantly increase the abundance of Verrucomicrobia compared with that in the KB group. To better understand the differences between C2 and B2 based on the normal level (KB group data), the greatest difference in taxa from class to species level was identified via linear discriminant analysis (LDA) score (among the KB, C2 and B2 groups) (Fig. 4B), and the structure and predominant bacteria of the caecum microbiota in the KB, C2 and B2 groups are represented in a cladogram (Fig. 4C). Based on further analysis of the microbiota structure at the genus level, we found that the following bacterial genera were involved in intestinal probiotics and pathogens: Alistipes, Akkermansia, Alloprevotella, Lachnospiraceae, Parabacteroides, Oscillibacter, Ruminiclostridium and Rikenella. Furthermore, the relative abundance of these bacterial genera in all experimental groups was also quantified (Fig. 4D). The Alistipes relative abundance was significantly lower in group C2 than in groups B11 and B2, but no obvious difference was found between groups KB and C2. The relative abundance of Akkermansia was significantly higher in groups C2 and B2 than in groups KB and B11. In addition, the relative abundance in B2 was also obviously higher than that in group C2. Alloprevotella, Lachnospiraceae_NK4A136_group and Ruminiclostridium were all significantly higher in group C2 than in the other three experimental groups, Parabacteroides was obviously

Fig. 3. The influences of B. coagulans R11 on intestinal villi under lead exposure. Figure AeD were the intestine section images of KB, B11, C2 and B2 respectively. E was the fluff length quantification. Different letters between experiment groups means significant difference (P < 0.05).

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Fig. 4. Metagenomics test on gut microbiota. A was the quantification of major three phylum in each experiment groups, B and C were LEfSe analysis, D was the quantification of major probiotics and pathogens genus in each experiment groups, E was the major functional proteins of the caecum microbiota of all experimental groups (warm color means high relative abundance), F was the proteins which were absence in lead exposure group C2, white color means none. Different letters between experiment groups means significant difference (P < 0.05).

higher in group C2 than in the other two B. coagulans R11 feeding groups but not difference than in the control group. Oscillibacter was obviously higher in group C2 than in the no lead exposure groups, but there was no significant difference among groups KB, B11 and C2 by the comparison with group B2. The major functional proteins of the caecum microbiota of all experimental groups were shown in Fig. 4E. In addition, this present study also found that the protein ureAB existed in only group B2, and other functional proteins were absent in only group C2 (Fig. 4F). The Kyoto Encyclopedia of Genes and Genomes (KEGG) database indicated that ureAB was involved in the following pathways: arginine biosynthesis, purine metabolism, atrazine degradation, microbial metabolism in diverse environments, epithelial cell signalling in Helicobacter pylori infection and metabolic pathways. The proteins MSH6 (DNA mismatch repair protein), PTEN (phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase), dltD (D-alanine transfer protein), ARF1 (ADP-ribosylation factor 1) and EIF2AK3 (eukaryotic translation initiation factor 2-alpha kinase 3) were absent in group C2 (Fig. 4F).

3.4. Faecal microRNA expression level and the potential relation between the host and bacteria In the present study, the microRNA in the faeces, which was secreted by the mouse intestine, was sequenced, and the KEGG

functional annotation of the most enriched microRNAs of groups B11, C2 and B2 by the comparison with those of group KB is shown in Fig. 5AeC. Based on the results, the most enriched microRNA was different among all experimental groups, indicating that lead and B. coagulans R11 feeding could all influence the excretion of faecal microRNA. In further analysis, we found that some microRNA could target 16S rRNA and 23S rRNA of the bacterial genera mentioned above (Fig. 4D), and the gene target result is shown in Fig. 5D. We referenced the method mentioned by Liu to find that each bacterial nucleic acid sequence was predicted to be targeted by microRNAs (Griffiths-Jones et al., 2008; Liu et al., 2016). The Akkermansia bacteria genus could be targeted by more microRNAs than the other five bacterial genera. Alloprevotella, Ruminiclostridium and Oscillibacter were all targeted by microRNA on only 16S rRNA, while Parabacteroides and Alistipes were both targeted by microRNA on only 23S rRNA.

4. Discussion 4.1. The effect of B. coagulans R11 on mouse MODE-K cells during lead exposure B. coagulans R11 was isolated from a lead mine by our laboratory (Xing et al., 2018), but whether this strain could be a potential prebiotic required further assessment. Therefore, the cell proliferation, ROS rate and apoptosis rate were tested after incubation with

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Fig. 4. (continued).

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Fig. 5. microRNA sequencing of mouse faecal. A-B were the most enrichment function analysis of microRNA in group B11, C2 and B2 by the comparison of KB respectively, D was the gene target result.

B. coagulans R11 in the present mouse MODE-K cell experiment. Based on the results, lead exposure could decrease proliferation and increase the ratio of apoptosis and ROS, as a previous study mentioned that lead exposure could hurt intestinal cells (Tomaszewska et al., 2015), and the present study showed similar results. In addition, the cells showed a similar proliferation status in the B. coagulans R11 treatment groups (B. coagulans R11 and B. coagulans R11 þ Pb) compared with that in the control group, which indicated that B. coagulans R11 had no negative effect on the proliferation of the MODE-K cells and had potential as a probiotic. The results also showed that B. coagulans R11 could decrease the apoptosis ratio of the MODE-K cells with and without lead exposure, possibly because B. coagulans R11 secreted some compounds that weakened cell apoptosis, but this hypothesis needs further study. Thus, B. coagulans R11 could be considered a probiotic that could protect cells from lead toxicity damage. To our surprise, B. coagulans R11 could increase the ROS ratio in the MODE-K cells incubated without lead addition, and this result could be caused by the stimulation of B. coagulans R11; in vitro cell incubation should be more sensitive to the bacteria than living host tissue, but the result also needs further assessment.

4.2. B. coagulans R11 feeding modified the gut microbiota and decreased intestinal villi damage in lead-exposed mice In the present study, we mimicked chronic lead exposure, and lead was added to daily drinking water. Although 200 mg/L was higher than the Chinese water safety standard concentration, to test and understand the function of B. coagulans R11 in leadexposed mice, such a high lead concentration was used. The

small intestine section observations revealed that B. coagulans R11 feeding could decrease the damage caused by lead exposure and that B. coagulans R11 could also enhance the length of intestinal villi without lead exposure. This characteristic may support the use of B. coagulans R11 as a probiotic in the future. Section observation results were also similar to the cell model experiment results; in addition, submucosa layers of C2 were thicker than normal tissue and B. coagulans feeding groups, possibly due to inflammation and infiltration of immune cells which caused by lead exposure, thus, B. coagulans R11 decreased the lead damage due to an increase in or maintenance of intestinal cell proliferation and reduced the apoptosis and ROS ratio. The major two bacterial phyla of the mouse gut microbiota were Bacteroidetes and Firmicutes, and this result was similar to previous studies (Ba et al., 2017). In the present study, the relative abundance of Firmicutes was influenced by B. coagulans R11. In particular, B. coagulans R11 feeding decreased the relative abundance of Firmicutes significantly in lead-exposed mice. However, the relative abundance of Verrucomicrobia was significantly increased in this group (B2). This result elucidated that B. coagulans R11 feeding may adjust the ratio of Firmicutes and Verrucomicrobia in lead-exposed mice, which may help reduce the damage caused by lead exposure. Based on this hypothesis, further analysis also showed that Firmicutes was the major phylum that had a significant influence on group C2 by comparison with its influence on group B2. Alistipes is a probiotic that could cure intestinal function disorders such as Clostridium difficile infection and colorectal cancer (Dziarski et al., 2016; Milani et al., 2016; Wang et al., 2012), and this bacterial genus belongs to Bacteroidetes. Our results showed that the relative abundance of Alistipes was significantly decreased in

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group C2 compared with that in the other three groups. The present result also indicated that the relative abundance of Akkermansia was highest in group B2. Previous studies have reported that this bacterial genus is a native probiotic of the host gut, and Akkermansia muciniphila exists in the intestinal mucus layer (Belzer and de Vos, 2012; Derrien et al., 2004; Everard et al., 2013). Akkermansia is also involved in inflammation anesis and curbing (Caesar et al., 2015; Everard et al., 2013; Shin et al., 2014). It is worth noting that Akkermansia belongs to Verrucomicrobia, and the relative abundance of Akkermansia and Verrucomicrobia were both the highest in B2. This result suggested that B. coagulans R11 feeding could enhance the Akkermansia abundance to help healthy gut environment building in lead-exposed mice. In addition, the relative abundance of Akkermansia was also higher in C2 than in KB and B11, which was in contrast with Zhai's study (Zhai et al., 2017), possibly due to the dose of lead exposure. The present study also found that the major pathogen genus was in the highest relative abundance in group C2 compared with that in other three groups. Many studies have reported that Alloprevotella, Lachnospiraceae, Parabacteroides and Ruminiclostridium are in high abundance in a host that has intestinal function disorder, colorectal cancer and other intestine diseases (Xia et al., 2000; Awadel-Kariem et al., 2010; McLellan et al., 2013; SchulzeSchweifing et al., 2014; Coit et al., 2016). In addition Lachnospiraceae and Ruminiclostridium belong to Firmicutes, and this result also similar as that of the LDA score analysis; thus these results could elucidate that lead exposure could increase the relative abundance of Firmicutes pathogens in the gut but B. coagulans R11 feeding could reduce the abundance of these pathogens in leadexposed mouse intestines. We also found that Rikenella, which is regarded as an inflammation indicator (Breton et al., 2013), showed an obvious difference between only B11 and B2. This result could suggest that B. coagulans R11 feeding could reduce the intestinal damage caused by lead, but the good benefit of B. coagulans R11 feeding on intestinal cells cannot function as daily feeding of normal mice. The abundance of Oscillibacter was reported to be involved in central nervous system diseases in the human gut, and the abundance of Oscillibacter was increased in stroke and depression patient intestines (Jiang et al., 2015; Yin et al., 2015). In this study, the relative abundance of Oscillibacter was the highest in group C2, and the LDA score analysis also showed that Oscillibacter provided the major influence in the mouse gut of group C2 compared with that of B2. The major effect of lead toxicity was nervous system damage. This result may suggest that mice exposed to lead had a trend towards nervous system damage, and the relative abundance of Oscillibacter was increased. This result also elucidated that B. coagulans R11 feeding could reduce such negative facts. Based on the analysis of microbiota function, five functional proteins were absent in only group C2, and it is worth noting that the protein dltD may help to protect against dextran sulfate sodium-induced ulcerative colitis in mouse models (Lee et al., 2015). This result may elucidate that lead exposure could damage the gut microbiota normal function and that B. coagulans R11 may help to maintain the normal function of the gut microbiota. In summary, B. coagulans R11 feeding could reduce intestinal villi damage by regulating the gut microbiota structure and function for the lead-exposed mice. 4.3. Faecal microRNA expression and gut microbiota structure As observed in a previous characterization, faecal microRNA was secreted by gut epithelial cells and Hopxþ cells (Liu et al., 2016), and this microRNA could adjust the growth and structure of the gut microbiota. Therefore, in the present work, we tested faecal

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microRNA expression and compared the microRNA function difference in all experimental groups to determine the relationship among the host, B. coagulans R11, lead and the gut microbiota. We found that lead and B. coagulans R11 all could influence the function of the most enriched microRNA. This result could be due to the intestinal cells being influenced by lead exposure and B. coagulans R11 effects. In addition, there were more microRNAs targeting Akkermansia in our study, but the function of these microRNAs on these bacterial genera needs further experiments. 5. Conclusion B. coagulans R11 feeding for lead-exposed mice could increase the relative abundance of Akkermansia and Alistipes and reduce the relative abundance of Alloprevotella, Lachnospiraceae, Parabacteroides and Ruminiclostridium, which helped to maintain intestinal villi health and reduced the damage caused by lead exposure. Furthermore, B. coagulans R11 feeding also maintained the beneficial function of the gut microbiota during lead exposure. Host faecal microRNAs may be influenced by lead and B. coagulans R11, which may change the microbiota structure. Thus, B. coagulans R11 has the potential to be developed and considered as the probiotic that protects the host gut against villi damage and gut microbiota structure and function disorders during lead exposure. Declarations of interest None. Funding This work was supported by the earmarked fund for the Modern Agro-industry Technology Research System (CARS-41). Role of the funding source Modern Agro-industry Technology Research System (CARS-41) belongs to the Chinese Ministry of Agriculture. Data availability De Man, Rogosa, Sharpe (MRS), Phosphate-buffered saline (PBS), Reactive oxygen species (ROS), Flow cytometry (FCM), Specificpathogen-free (SPF), Laser scanning confocal microscope (LSCM), Haematoxylin-eosin (HE), Statistical Package for the Social Sciences (SPSS). Conflict of interest This manuscript has not been submitted for publication elsewhere and the content and authorship have been approved by all authors. The authors wish to confirm that: (1) all of the reported work is original, (2) all authors have seen and approved the final version submitted, (3) all prevailing local, national and international regulations and normal scientific ethical practices have been respected, and (4) consent is given for publication in Environmental Pollution, if accepted. All authors confirm that no conflict of interest. Acknowledgements We thank professor Xi-quan Zhang provided helps in cell model experiments.

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S.-C. Xing et al. / Environmental Pollution 255 (2019) 113139

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