Dietary κ-carrageenan facilitates gut microbiota-mediated intestinal inammation

Background: After nearly half a century, the inammatory effects of carrageenan (CGN), a ubiquitous food additive, remain controversial. Little is known about its impact on the gut microbiota and intestinal homeostasis. Results: Mice fed κ-CGN showed no signicant inammatory symptoms, but showed altered colonic microbiota composition, thereby decreasing bacteria-derived short-chain fatty acids (SCFAs) and increased penetrability of the mucus layer. In mice administered the pathogenic bacterium Citrobacter rodentium, inammation and mucosal damage were further aggravated in the presence of κ-CGN. Mucus layer defects and altered SCFA levels could be reproduced by fecal transplantation from κ-CGN-fed mice, but not from germ-free κ-CGN-fed mice. These symptoms could be partially repaired by administering the probiotics Bidobacterium longum NCC-2705 and Faecalibacterium prausnitzii. Conclusions: We report a novel evidence that κ-CGN may not be directly inammatory, but creates an environment that favors inammation by perturbation of gut microbiota composition and then facilitates expansion of pathogens, which may be partially reversed by the introduction of probiotics.


Introduction
The rise in processed foods during the last half-century has witnessed a steady increase in the consumption of food additives. Many food additives are applied with little or no restriction when they are expanded used [1]. A survey in the Asia-Paci c region found that urbanization is an important correlate of in ammatory bowel disease (IBD) development [2]. In terms of diet, urbanization also leads to an increased adherence to Westernized dietary patterns. Therefore, strict evaluation of food additives in processed foods such as carrageenan (CGN) is appropriate.
CGNs are a family of polysaccharides isolated from red seaweeds. The wide functionality of CGN has led to incorporation of CGN into a myriad of food products. Individuals who consume multiple CGNcontaining foods may ingest several grams on a daily basis [3]. The safety and toxicology of CGN has therefore been strictly examined by various agencies and researchers. However, opinions regarding the overall safety and the potential adverse effects that may lead to gut in ammation and even colon cancer remain con icting. This controversy has been debated for more than half a century, and recent attention by the media has affected the commercial market for CGN [4]. Evidence for the safety of CGN from researches showing that food grade CGN is stable in the gut and shows no harmful effects [5,6]. On the contrary, a series of papers by Dr. Tobacman's group and others suggest that CGN has potent in ammatory properties [7,8]. These con icting ndings appear to stem from scienti cally rigorous experiments, which suggests that the in ammatory potential of CGN could be conditional. We previously showed that a short course (1-2 weeks) of pre-treatment of mice with CGN led to increased in ammatory bowel symptoms caused by oxazolone, trinitrobenzene sulfonic acid and Citrobacter rodentium [9][10][11].
Moreover, in the presence of THP-1 macrophages, CGN can destroy a monolayer of Caco-2 cells at very low concentrations [12]. This leads to the question of whether CGN can affect intestinal epithelial barrier integrity under certain conditions. The gut microbiota is a crucial environmental factor contributing to host health [13]. The gut microbiota composition is affected by diet [14], and certain microbial can degrade the intestinal mucus layer, contributing to severe colitis in mice [15]. Other studies have found that CGN can alter microbiota composition, including decreasing the abundance of the anti-in ammatory bacterium, Akkermansia muciniphila [16]. We therefore hypothesized that CGN may act as a proin ammatory adjuvant, disrupting homeostasis through the alteration of intestinal microbiota, and destruction of mucosal integrity, which may provide a suitable environment for other in ammatory agents or pathogens.
Here, using high molecular weight, non-degraded κ-CGN, we investigate the controversial role of CGN in intestinal in ammation, from the perspective of its impact on intestinal microbiota. We explore the effects of κ-CGN on intestinal microbiota, intestinal mucosal structure, bacterial metabolism and in ammatory changes in mice, and verify these results by fecal microbiota transplantation and germ-free mice.

κ-CGN alters colonic microbiota composition
After 90 days of κ-CGN oral gavage at different concentrations, intestinal microbiota community (based on 16S rRNA analysis) in mice had changed, with a signi cant increase of species richness (operational taxonomic unit [OTU], Chao1 and abundance-based coverage estimator [ACE] indices). Total bacterial load was increased in the high dose group (CGN-H) by approximately 13% compared with the control group (NC) (P < 0.01, Fig. S1a). Shannon and Simpson indices showed no signi cant differences in microbiota diversity after 90 days of treatment. The changes in the composition of the microbiota led to the changes in the enterotype. Mice in the NC group were clustered into the Prevotella (enterotype 2), an intestinal bacteria type are capable of generating short-chain fatty acids (SCFAs) [17]. Mice in the low and medium dose groups (CGN-L and CGN-M) were clustered into Ruminococcus (enterotype 3), with dominant bacteria effectively bind mucin and hydrolyze them into monosaccharides. Mice in the CGN-H group was clustered into Bacteroides (Enterotype 1), containing bacteria with a large amount of glycosidase capable of degrading polysaccharides (Fig. S1b) [18,19].
H&E staining of the colon tissues showed that the intestinal tissue structure of mice in the κ-CGN, CGN-H trans+ , and CGN-H free+ (CGN-H treated germ-free mice) groups was normal, with tightly arranged goblet cells and no in ammation and in ltration in the crypts (Fig. 2d). The CGN/CGN trans+ treatment groups had no sign of hyperemia, edema or ulceration (Fig. 2d, Fig. S2). C. rodentium induced colon tissue damage in conventional mice, which was further aggravated in CGN C. rodentium mice; both groups had submucosal edema in colon tissues, and a signi cantly increase of in ammatory cells in the basal layer.
The colonic mucosa of CGN C. rodentium mice was clearly hyperemic, with edema and hemorrhagic ulcers ( Fig. 2d, Fig. S2), demonstrating a state of heightened mucosal in ammation in CGN C. rodentium mice. κ-CGN alters microbiota composition, leading to changes in mucus degradation genes and SCFA Metagenomics analysis was consistent with the results from 16S rRNA analysis, Chao1 and ACE indices increased signi cantly after 90 days with CGN-H treatment (P < 0.01, Fig. S3a), while Shannon and Simpson indices did not. In addition, the relative abundances of SCFA-producing genera, including Ruminiclostridium_5, Lachnospiraceae and Eubacterium brachy_group [23] were reduced in CGN-Htreated mice. The relative abundance of Bacteroides was increased in CGN-H-treated mice (Fig. S3b, Table S3), and excessive proliferation of Bacteroides may lead to destruction of polysaccharides in the intestinal mucus layer [24]. We therefore sought to determine the abundance of carbohydrate utilization genes in relation to intestinal mucus composition. A total of 28,687 genes encoding carbohydrate active enzymes (CAZymes) were identi ed in mouse fecal samples, with the largest number of genes belonging to the family of glycoside hydrolases (Fig. 3a). CGN-H treatment led to a signi cant increase in genes encoding mucosal polysaccharide binding proteins and mucin degrading enzymes. For example, CBM32 and CBM40, which encode mucosal glycan binding proteins, were increased 12.5 and 2.1-fold, respectively, compared with the NC group (P < 0.05); the genes encoding N-acetyl galactosidase, CE9 and CE11 were increased by 9.8 and 36.1-fold, respectively (P < 0.01); genes encoding mucosal polysaccharide and glycosyltransferase, GH106 and GT4, were increased 29.5 and 26.7-fold, respectively (P < 0.01, Fig. 3b). By performing correlation analysis between CAzymes genes changes and microbial abundance (Fig. 3c, Table S4), we found that the abundance of Bacteroides ovatus (Bacteroides genus) containing CBM32, CE11, GH106, GH109, GH84, and GH85 genes increased 25.35-fold (P < 0.01). The abundances of Bacteroides nordii and Bacteroides thetaiotaomicron containing GH33 were 17.89 and 21.35 folds higher, respectively than that of the NC group (P < 0.01). The abundance of Bacteroides intestinihominis containing GT4 gene was increased 3.45-fold (P < 0.05).
These bacterial can produce large amounts of SCFAs [19,23]. We therefore quanti ed SCFAs and, as expected, fecal SCFA contents were signi cantly altered by κ-CGN treatment. OPLS-DA demonstrated clear separation between CGN-M/CGN-H and NC group, but with limited separation between CGN-L and NC groups (Fig. S4). Among several typical SCFAs, butyric acid, isobutyric acid, valeric acid, and isovaleric acid were all reduced in CGN-M and CGN-H groups, especially butyric and valeric acid, which were signi cantly reduced by 67.4% and 60.6%, respectively in the CGN-H group (P < 0.01). Similar changes in SCFA contents were seen in mice receiving fecal transplants from CGN-H mice. Compared with the NC trans+ group, the CGN-H trans+ group had a 65.3% reduction in butyric and a 69.7% reduction in valeric acid (P < 0.01). In germ-free mice, SCFA contents were very low in general, even after κ-CGN transplantation (Fig. 3d), which indicates that the change in SCFA contents in feces of CGN-treated mice was related to changes in microbiota composition.
Correlation analysis between SCFA abundance and intestinal microbiota (Fig. 3e, Table S1) showed that bacteria affected by κ-CGN treatment, including cellulose degrading bacteria, and the SCFA-producing bacterial genera Ruminiclostridium_5, Lachnospiraceae, and [Eubacterium]_brachy_group [23] were positively correlated with changes in isovaleric acid, caproic acid, and acetic acid (P < 0.05). In addition, positive correlations were also found between Ruminiclostridium_5 and isobutyric acid and butyric acid; and between Lachnospiraceae and butyric acid and valeric acid (P < 0.05); whereas a negative correlation was found between Ruminococcaceae and valeric acid and butyric acid (P < 0.05).

Shifts in microbial composition contribute to intestinal barrier dysfunction
Fluorescent in situ hybridization (FISH) examination revealed both κ-CGN treatment of conventional mice and the corresponding fecal bacteria transplantation caused thinning of the intestinal mucus layer, by reducing 61% and 58%, respectively (Fig. 4a, b; P < 0.01), but not correlated with the expression of the major intestinal mucin Muc2, Krüppel-like factor 4 (Klf4) or goblet cell protein (Tiff3). The expression of these three proteins were not altered by κ-CGN treatment (P > 0.05, Fig. 4c). However, after 90 d of κ-CGN treatment in germ-free mice, the thickness of the mucus layer was almost unchanged. No bacteria were observed in the intestinal mucus layer within 25 µm from the epithelial cells of NC group (Fig. 4d, e). In contrast, bacteria penetrated the mucus layer more frequently in mucosal biopsies obtained from κ-CGN-H treated and fecal transplant groups, compared with NC group, with the average distance reduced by greater than 87% in the CGN-H group and 83% in CGN-H trans+ group (P < 0.01). Such microbiota encroachment correlated with reduced mucus thickness. A classical permeability marker FITC-dextran test revealed a signi cant increase in mucus penetrability in CGN-M, CGN-H and CGN trans+ groups with higher FITC-dextran levels than in NC mice (P < 0.05, Fig. 4f). To determine whether the observed defective mucus layer was caused directly by κ-CGN treatment, we next studied mucus properties in germ-free mice. Compared with NC free+ group (germ-free control mice), no signi cant difference in penetrability was detected in CGN-H free+ mice: mucus thickness, penetrability and expression of Muc2, Klf4, Tiff3 were not affected (Fig. 4a, b, f).
Probiotics attenuate pathogen-induced in ammatory mucosal damage We supplemented mice with two probiotics Bi dobacterium longum NCC-2705 and Faecalibacterium prausnitzii after 90 d of oral gavage of κ-CGN (CGN Pro+ , Fig. 5a). PCoA analysis of 16S rRNA sequencing at genus level showed two distinct clusters between NC or CGN-H mice; after 30 d of subsequent probiotic intervention, the bacterial composition of CGN-H pro+ group was closer to that of the NC group (Fig. 5b).
In addition, the phenomenon of thinning of the mucus layer and increased intestinal permeability caused by exposure to CGN-H was reversed after probiotic intervention. The thickness of the mucus layer recovered by 55.6% (P < 0.01, Fig. 5g, h), and intestinal permeability was dampened by probiotics, approaching levels similar to that of the NC group (P < 0.05, Fig. 5i). Colonic tissue and in ammatory factors were not signi cantly changed by probiotic intervention (Fig. S5). rodentium group, SCFA contents were further reduced in the CGN C. rodentium group (P < 0.05, Fig. 6a). Probiotic intervention signi cantly reversed this reduction in intestinal SCFAs (P < 0.05), with the content of propionic acid and butyric acid higher than that of the C. rodentium group (P < 0.05). Even acetic acid, isobutyric acid, and isovaleric acid were basically restored to normal levels. H&E histochemical analysis produced similar ndings: Probiotic intervention signi cantly reduced intestinal in ammation, eliminated submucosal edema, and signi cantly reduced the in ltration of in ammatory cells in the basal layer (P < 0.05, Fig. 6b, Fig. S6). Similarly, probiotic intervention signi cantly improved C. rodentium-induced increase in in ammatory cytokines, which was further promoted by CGN-H. Compared with the CGN C. rodentium group, TNF-α, IL-6 and MCP-1 were reduced by 26%, 20%, and 18%, respectively in the CGN C. rodentium+pro+ group (P < 0.05, Fig. 6c), suggesting the ability of probiotics to dampen the pro-in ammatory effect of C. rodentium.

Discussion
Some researchers have suggested that CGN may exert chronic low-grade in ammation, de ned by elevated systemic expression of proin ammatory cytokines in the absence of the classical aggregates of immune cell in ltrates [27]. In our data, without increased in ammatory cytokines, this de nition for κ-CGN may be slightly inappropriate. These are consistent with most o cial reports [1,3,4]. However, in the present study, 90 days of κ-CGN gavage promoted colitis-associated gut in ammatory symptoms in C. rodentium-treated mice, which also had been observed in our short-term tests [11]. We therefore hypothesized that κ-CGN provides a favorable environment for in ammation.
Mucosal compartmentalization functions to minimize direct contact between intestinal bacteria and the epithelial cell surface (strati cation). Bhattacharyya et al. reported that 14 weeks of CGN administration led to disruption of the mucosal surface in mice [28]. We previously found that κ-CGN can lead to destruction of the differentiated colonic layer via immune system effects [12], which suggest that the intestinal mucus layer may be a target of κ-CGN. Administration of κ-CGN to germ-free mice did not result in low-grade in ammation, as assessed by fecal Lcn-2 and proin ammatory factors, or any of the parameters related to mucus thinning and altered localization of bacteria, even the alteration of SCFA contents. However, in normal C57BL/6 mice, κ-CGN caused mucosal thinning, increased gut permeability and decreased SCFA levels. These ndings oppose the notion that κ-CGN directly impacts the mucus layer and, rather, suggests that microbial dysbiosis may be a key driver of κ-CGN-dependent in ammatory susceptibility.
Here, high intake of κ-CGN induced changes in gut microbiota composition in mice, with increased relative abundance in several taxa belonging to the phylum Bacteroidetes, and decreased relative abundance of Proteobacteria and Firmicutes taxa. Shang et al. found that CGN profoundly decreased the relative abundance of the anti-in ammatory bacterium A. muciniphila in the gut [16]. Our study supports these ndings where Akkermansia was signi cantly decreased in response to κ-CGN treatment. Surprisingly, we found that κ-CGN exposure did not decrease microbiota diversity, but increased total bacterial abundance. In general, individuals with chronic in ammation have lower bacterial diversity than their healthy counterparts. This may explain why although it caused mucus defection, but did not translate to colitis development compared to other food additives, such as carboxymethylcellulose and polysorbate-80. In their results, gut microbiota diversity was decreased [16]. Thus, κ-CGN's preservation of bacterial diversity may maintain a delicate balance in the intestinal microbiota and its environment. For example, some bacteria associated with in ammation and immunity showed no negative changes. We even observed the decrease of the relative abundance of gut Proteobacteria, which has positive correlation to colitis [29], and the increase of the abundance of Rhodospirillaceae, which can enhance the cellular and humoral immunity of the body [30].
The analysis of the gut metagenomes revealed altered microbial gene pro les in κ-CGN-treated animals. It con rmed the signi cant increase in relative abundance of CAZymes belonging to the genus Bacteroides in response to κ-CGN. A variety of CAZymes encoded by this taxon were regulated, including genes encoding glycoside hydrolases and polysaccharide lyases. For example, increased expression of CBM32 and CBM40, which encode mucous membrane-binding proteins; CE9 and CE11 degradation of mucin O-glycans, and the mucosal polysaccharide lyase and glycosyltransferase genes GH106 and GT4, which may allow switching to utilize host glycans of the intestinal mucus layer [20].
SCFAs contribute to the maintenance of immune homeostasis in the intestine and enhance intestinal epithelial barrier by assembly of tight junctions [22]. We observed a signi cant up-regulation of the genus Ruminococcaceae_unclassi ed, which is capable of inhibiting SCFA synthesis, and decreased abundance of [Ruminococcus]_torques_group, Lachnospiraceae, Ruminiclostridium_5, and E. brachy_group, which can synthesize SCFAs [23]. Among these, R. torques and L. bacterium_10 possess enzymes necessary for the hydrolysis of cellulose, starch and mannan, and contribute to the production of high amounts of SCFAs [31,32], such as GH5, GH42, GH15, GH151, GH36, GH77, GH30, for which the gene contents were down-regulated. Furthermore, the decreased fecal levels of SCFAs, especially butyric acid and valeric acid, was con rmed. However, the expression of Muc2, a building block of colonic mucus; Klf4, a transcription factor involved in barrier function, and Tiff3, a mucosal repairing protein [31], were not affected by κ-CGN treatment. Therefore, based on changes in microbial composition and CAZymes and SCFA synthesis gene contents, we speculate that κ-CGN treatment led to increased bacterial consumption of mucinderived nutrients, which exceeded production of these compounds, hence destroying the integrity of the mucus layer, which led to increased penetrability. We previously reported that once κ-CGN has a conversation with the immune system, mucosal damage is intensi ed [12]; our latest ndings may indicate that κ-CGN increases crosstalk between gut microbiota and the immune system by increased contact with the gut epithelium following mucosal damage.
The observation that germ-free mice receiving fecal transplants from κ-CGN-treated mice also experienced mucosal thinning, decreased production of SCFAs and similar levels of in ammatory factors to conventional mice, suggests relatively stable colonization by transplant microbiota with subsequent inheritance of similar phenotypes.
Various probiotics have demonstrated clinical e cacy in patients with ulcerative colitis. Bi dobacterium strains have several bene cial effects, including strengthening epithelial barrier function in human ulcerative colitis patients [31]. Indeed, administration of the probiotics B. longum NCC-2705 and F. prausnitzii led to a partial recovery of gut microbiota and mucus barrier destruction by ameliorating SCFA de ciency, reinforcing intestinal barrier and dampening intestinal permeability. Probiotic administration further limited the C. rodentium-induced immune reaction, metabolic impairment and in ammation.

Conclusion
Overall, this approach revealed that the consumption of κ-CGN, a ubiquitous food additive, does not induce histopathologically evident in ammation in mice, but does alter gut microbiota in a manner that leads to colonic mucus layer destruction, and disruption of intestinal homeostasis, thereby creating a weakly pro-in ammatory environment (scheme 1). These phenomena can be reproduced by fecal transplantation from κ-CGN-treated mice to germ-free mice. It worth noting that when animals are exposed to pathogens or other in ammatory agents, κ-CGN may exacerbate pathogen-induced intestinal in ammation, as seen here in C. rodentium-treated mice. Most of these κ-CGN-associated alterations were partly reversed by supplementation with probiotics, which therefore has potential therapeutic implications.

Experimental materials
Male C57BL/6 mice (6 weeks old, SCXK 2018-0004) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. Mice were housed in the animal center of Ningbo University Medicine College (Ningbo, China), with four mice per cage, under a 12-h light/12-h dark cycle (lights on at 08:00 AM) in temperatureand humidity-controlled environments (21 ± 2 °C and approximately 60%, respectively) with free access to food and water. Male germ-free mice (genetic background of C57BL/6, 6 weeks old) from the National Laboratory Animal Seed Center (Shanghai, China) were housed with one mouse/Combibloc aseptic package isolator under germ-free conditions. All mice were habituated to their housing environment for 5 days prior to oral gavage. All experimental procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and animal-related experiments were approved by the Ethical Committee of Animal Use and Protection of Ningbo University Health Science Center.
Fresh feces were collected at days 0, 45, and 90. Blood was collected by retrobulbar intraorbital capillary plexus at day 90. Serum was obtained by centrifugation of blood. Mice were then euthanized, and colons were collected.
For germ-free experiments, 16 male germ-free mice were sorted into two groups (n = 8), including a control group (NC free+ ) and CGN-H-treated group (41.7 mg/kg for 90 days, CGN-H free+ ). For the microbiota transplantation experiment, fecal samples were suspended in 30% glycerol. Male germ-free mice were divided into 4 groups (NC trans+ , CGN-L trans+ , CGN-M trans+ , and CGN-H trans+ ; n = 8). They were orally administered 200 µL of fecal suspension (from the feces of mice of CGN or NC group) with a concentration of 400 mg/mL for 3 consecutive days. After 90 days, each group was sampled as described above (Fig. S7).

Macroscopic evaluation and histological analysis
Clinical severity of gut tissues was graded as outlined in Table S2, mice were weighed daily, checked for stool consistency, rectal bleeding and presence of blood in the stool [34].
Colonic tissues were treated for H&E staining. The histological grade and level of in ammation were determined by blinded veterinary pathologists. Colitis severity was quanti ed using the histological activity index (HAI) [35,36], as detailed in Table S8.
Fecal microbiota analysis by metagenome sequencing DNA was extracted from mice fecal samples using standard methods [37]. Paired-end (PE) library was constructed using NEXTFLEX Rapid DNA-Seq (Bioo Scienti c, Austin, TX, USA) and subjected to Illumina

SCFA composition analysis
Lyophilized samples were extracted and analyzed via GC/MS according to Zhao et al [23]. SCFA levels were analyzed by hierarchical clustering with the pheatmap package in R (version 3.6.1). OPLS-DA was used to evaluate the effect of each treatment group. The correlation between SCFA levels and intestinal microbial abundance was analyzed by calculating Spearman's rank correlation coe cient.

Mucin immunostaining and localization of bacteria by FISH
Fixed colonic tissues containing fecal material were washed and para n-embed; 5 µm sections were dewaxed. Hybridization was performed at 50 °C overnight with EUB338 probe (5'-GCTGCCTCCCGTAGGAGT-3', with 5' labeled Alexa 647, Gene Pharma Co., Ltd, Shanghai, China), followed by mucin-2 primary antibody (1:1500, Cell Signaling Technology Inc., Shanghai, China) overnight at 4 °C, and anti-rabbit Alexa 488 secondary antibody (1:1500) at room temperature for 1 h, then washed thrice with TBST. Then, 10 µg/mL DAPI (Sigma-Aldrich, St. Louis, Missouri, US) was applied for 2 h, washed, and observed with a Zeiss LSM 700 confocal microscope (Carl Zeiss AG, Oberkochen, Germany). The distance between bacteria and epithelial cell monolayer, as well as the thickness of the mucus layer, were analyzed by ZEN software 2011.
In vivo epithelial barrier permeability An in vivo assay of intestinal barrier function was performed using a FITC-labeled dextran method, where the serum concentration of FITC-labeled dextran was used as a proxy for paracellular permeability according to the method of Chassaing et al [13].

Statistical analysis
Analyses were performed using the SPSS software, version 16.0 (IBM Corp., Armonk, New York, USA). The results were expressed as means ± SEM. Differences among groups were analyzed using a post-hoc Fisher's LSD test after a one-way ANOVA. P < 0.05 was considered statistically signi cant. The Levene test was used to verify the homogeneity of variance.    Positive and negative regulation of encoding CAZymes between CGN-H/NC comparisons. CAZyme families (x-axis) in which > 2-fold changes (P < 0.05, Student's t test) were observed for all genes in that family in RPKM-normalized fecal community metagenomes (n = 3 mice/group). (d). Heatmap of mean values of SCFA contents in mice feces (μg/g). *P < 0.05, **P < 0.01, compared with the NC group. #P < 0.05, ##P < 0.01, compared with the NCtrans+ group. (e). Spearman's correlation between changes in fecal SCFAs and genus abundances of individual genera after CGN-H treatment. The intensity of the colors represents the degree of association between changes in fecal SCFA concentrations and the relative abundances of individual genera. Scale represents correlation coe cients. *P < 0.05, **P < 0.01.   The histologic activity index scores of colons in Citrobacter rodentium-stimulated mice, reduced by probiotics. (c). Probiotics improve C. rodentium-induced increase in pro-in ammatory cytokines (pg/ml, mean ± SEM). *P < 0.05, **P < 0.01, compared with the negative group, #P < 0.05, ##P < 0.01, compared with the C. rodentium group, &P < 0.05, &&P < 0.01, compared with the CGNC. rodentium group.

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