The intervention effects of Lactobacillus casei LC2W on Escherichia coli O157:H7-induced mouse colitis

Gungqing Wng,Hongyu Tng,Ying Zhng,Xing Xio,Yongjun Xi,Linzhong Ai,*

a Shanghai Engineering Research Center of Food Microbiology,School of Medical Instrument and Food Engineering,University of Shanghai for Science and Technology,Shanghai 200093,China

b School of Food and Biological Engineering,Jiangsu University,Zhenjiang 212013,China

ABSTRACT This study investigated the intervention effects of Lactobacillus casei LC2W in murine(SPF C57BL/c)challenge infection models induced by Escherichia coli O157: H7.Mice were fed streptomycin with water for 3 days prior to intragastric gavage by E.coli O157:H7(Control)or L.casei LC2W together with E.coli O157:H7(Intervention)to explore the role of L.casei LC2W by biochemical indicators,histological evaluation,expression of colonic pro-inflammatory and intestinal barrier factors related to enteritis.Results showed that the administration of L.casei LC2W was able to alleviate the symptoms of colitis induced by E.coli O157:H7,exhibiting lower weight loss as well as more intact colon tissue.Furthermore,L.casei LC2W could down-regulate the expression of pro-inflammatory cytokines(TNF-α,IL-6,and IL-1β)and protect the intestinal barrier function by improving the level of MUC2,ZO-1 and E-cadherin 1 compared to the control group.These results demonstrate that L.casei LC2W can reduce the severity of E.coli O157:H7 infection,and suggest L.casei LC2W may maintain the immune balance and intestinal barrier to reduce colitis.In addition, we found the effect of intervention is similar to that of prevention, which is better than that of treatment.

Keywords:Probiotics Lactobacillus casei LC2W Intervention Escherichia coli O157:H7

1.Introduction

Escherichia coli O157:H7 (E.coli O157:H7) is a major type of foodborne pathogenic bacteria, which can cause acute enteritis,intestinal hemorrhagic colitis,and hemolytic uremic syndrome.It is characterized by a high explosion, strong pathogenic, the high fatality rate.So far,E.coli O157:H7 has posed a great threat to people’s health [1-4].It has become a global public health problem,which has attracted worldwide attention[5].Antibiotics are widely used to control E.coli O157:H7 infection, but their use may cause drug resistance and chronic diarrhea [6,7].Therefore, the world health organization(WHO)advocates the use of probiotics instead of antibiotics.

Probiotics are commonly recognized as alive beneficial microorganisms that colonize in the gut and reproductive system of animals and produce organic acids or other bacteriostatic substances to inhibit the growth of pathogens [8].Many studies have provided evidence: Lactobacillus fermentum ME-3 and Bifidobacterium lactis Bb12 can antagonize Shigella sonnei[9],Bifidobacterium lactis DR10 can limit the adhesion of E.coli to intestinal epithelial cells [10],

Lactobacillus plantarum ZJ008 has Staphylococcus aureus inhibition[11], Lactobacillus acidophilus IBB801 can oppress the invasion of intestinal epithelial cells by Salmonella[12].The antagonism of probiotics on pathogenic bacteria is mainly manifested in the following aspects: production of antibacterial substances [13]; inhibition of adhesion of pathogenic bacteria[14];enhancement of barrier function of intestinal epithelial cells [15]; inhibition of colonization of E.coli O157:H7 in mice[16].These researches majorly focus on the use of probiotics for prevention and treatment,but the strategy for dealing with disease should include prevention, intervention and treatment.Based on the previous research,this experiment was to study the intervention effects of L.casei LC2W on E.coli O157:H7-induced mouse colitis.This study will consummate the roles of L.casei LC2W on the colitis and provide the guidance for taking this strain.

2.Materials and methods

2.1.Strains and culture conditions

L.casei LC2W was anaerobically cultured in de Man,Rogosa and Sharpe(MRS)medium at 37°C for 16 h.E.coli O157:H7 was aerobically cultured in LB medium at 37°C for 16 h.After the bacteria were activated for 3 times successfully, then washed with phosphate buffer solution (PBS) for 2 times and resuspended to the desired concentration(5×109CFU/mL).

2.2.Animals and experimental design

Six-week-old male SPF C57BL6/J mice weighted 18-20 g, purchased from Shanghai Jiesijie Experimental Animal Co., Ltd.The mice were housed at a constant temperature of 23-25°C with a 12 h dark/light cycle and fed standard mouse chow ad libitum with free access to water all times.All mice were housed at least one week to adapt before experiments, of which the protocols were performed according to the ethical guidelines of the European Community guidelines(Directive 2010/63/EU).

The experimental protocols were as follows: the male SPF C57BL6/J mice were randomly divided into three groups (n=8).Mice were treated with reference to the Streptomycin-treated Mouse Model for E.coli O157:H7 Infection[17].At first,mice were fed 5 g/L streptomycin with water for the first three days to kill the normal intestinal flora and increase the body susceptibility of E.coli O157:H7 [18].The normal group was orally administered orally with sterile water per day.While,the control group was intragastrically administered with E.coli O157:H7 suspension from day 4 to day 13.At the same time,the intervention group was administered with E.coli O157:H7 suspension and L.casei LC2W suspension for the next 10 days of the experiment.

2.3.Assessment of EHEC infection in mice

The disease activity index(DAI)can reflect the degree of infection in mice with E.coli O157:H7.It is evaluated by the body weight,stool consistency,occult blood in the feces of the mice,which were measured and scored daily based on a modified scoring system[19].Among this,the occult stool blood test was carried complied with the Occult Blood Kit(Jiancheng,Nanjing).At the end of the experiment,the colon of each group was taken for measuring the length after the mice were euthanized.Then, take 1 cm of the colon for RNA extraction,HE staining for histological examination and measuring the activity of myeloperoxidase (MPO), respectively.MPO is one of the indexes for characterizing the degree of inflammation, which was assessed according to the Myeloperoxidase Test Kit instructions(Jiancheng,Nanjing).As for histological injury,the colon tissue was taken and immersed in formalin solution, fixed at 4°C for 24 h, embedded in paraffin and sliced.Then were subjected to HE staining and placed under a microscope for microscopic examination and images were collected for analysis.The assays for determining the degree of inflammation, mucosal damage, crypt injury, lesions, colonic tissue damage in each group were scored according to the specific standards[19].

2.4.RNA extraction and real-time quantitative polymerase chain reaction

Total RNA was extracted from each colon tissue sample using Trizol reagent(Invitrogen,Carlsbad,CA).1 μg total RNA was usedto generate complementary DNA (cDNA) using the Prime Script RT kit (TaKaRa Bio, Otsu, Japan) and amplified by RT-qPCR using SYBR Premix Ex Taq II(TaKaRa Bio,Otsu,Japan).The primers were designed as shown in Table 1.The conditions were 40 cycles of 95°C for the 30 s,95°C for 5 s,60°C for 30 s.The transcription level of per group is measured by the 2-ΔΔCt,including the ΔCt reflected the difference between for each target gene and ΔCt actin mRNA transcription level.This measure is expressed as fold change relative to this of the control group.

Table 1 Primer sequences used in this study.

2.5.Data analysis

All data were presented as mean±SD(n=8).Statistical significance of the results was analyzed by one-way analysis of variance(ANOVA)followed by a Tukey’s posthoc test.A P-value ＜0.05 was considered significant.Statistical analyses were performed using SPSS software(SPSS Inc,Chicago,IL).

3.Results

3.1.L.casei LC2W alleviates Escherichia coli O157:H7-induced mouse colitis L.Casei LC2W alleviates Escherichia coli O157:H7-induced mouse colitis

Biochemical indexes used in this study were composed of the weight,DAI,colon length,and MPO activity.Mice in control group showed the most severe symptoms after infection, such as diarrhea, gross bleeding and highest weight loss, which were similar to the changes in typical cases of naturally occurring E.coli disease[20](Fig.1A).However,L.casei LC2W could effectively relieve the clinical symptoms caused by E.coli O157:H7.The DAI score of the intervention group was consistently lower than that of the control group(Fig.1A).Colon length was measured after the mice were euthanized(Fig.1B).L.casei LC2W effectively prevented colon shortening induced by E.coli O157:H7, and recover it back to the levels of normal group(6.9±0.32 vs 7.2±0.29,P ＞0.05)(Fig.1B).The MPO activity of colon tissue can reflect the degree of tissue neutrophil infiltration,reflecting the degree of inflammation[21].Our results showed that L.casei LC2W effectively inhibited the increase of MPO activity compared with the control group (0.30±0.02 vs.0.39±0.02, P ＜ 0.05) (Fig.1C), indicating that the MPO correlation degree of neutrophil infiltration in the intervention group is improved.

Fig.1.Lactobacillus casei LC2W alleviates the symptoms of E.coli O157:H7-induced colitis in mice:(A)DAI;(B)colon length;(C)MPO activity(D)Histological examination;(E)Histology scores.All data are expressed as mean±SD(n=8 per group).The means with different letters indicate significant differences between the groups(P ＜0.05).

Images were captured from HE stained of all the colon sections and histopathological injury was scored.Based on our histopathological analysis, the mice in the control group showed the typical characteristics of shortened intestinal villi,partially ruptured epithelial cells,increased goblet cells,and the appearance of inflammatory cell infiltration in the lamina propria and mucous membrane.By contrast,mice gavaged with L.casei LC2W appeared more moderate tissue damage.Although the colon tissue still has inflammatory cell infiltration,most of the intestinal epithelial cells are structurally intact and the crypt structure is relatively intact(Fig.1D).The tissue damage score of the intervention group was 3.8±0.76,which was significantly lower than the control group of 8.4±0.82(Fig.1E).As a result,the symptom of enteritis mice was effectively alleviated.So we infer that L.casei LC2W can play a protective role in intestinal tissue destruction,which is similar to the results of Herias’s[22].

3.2.L.casei LC2W regulates pro-inflammatory cytokine expression in colonic tissue L.Casei LC2W regulates pro-inflammatory cytokine expression in colonic tissue

The degree of inflammation in the body can be expressed by proinflammatory cytokines,typically TNF-α,IL-6,and IL-1β.Compared to the normal group,E.coli O157:H7 infection(Control)resulted in significantly increased transcriptional levels of pro-inflammatory cytokines, which caused immune disorders and increased intestinal inflammation as well as tissue destruction in mice,which was consistent with the study of Zhang[23].As shown in Fig.2A-2C,the mRNA levels of pro-inflammatory cytokines showed a significant increase due to E.coli O157:H7.L.casei LC2W(Intervention)significantly reduced the expression of IL-6(2.95±0.32 vs 1.87±0.39,P ＜0.05)and IL-1β(2.76±0.34 vs 2.08±0.29,P ＜0.05)compared with control group.The mRNA level of TNF-α was also down-regulated.Furthermore, there was no significant difference in mRNA levels of TNF-α, and IL-6 between the normal and intervention group(Fig.2A-2B, P ＜0.05).So, it is known that the intervention of L.casei LC2W has a better effect on the immunomodulation of mouse enteritis caused by E.coli O157:H7 infection.

3.3.L.casei LC2W protects the colonic mucous layer L.Casei LC2W protects the colonic mucous layer

The barrier consists of a mucous layer and a monolayer of epithelial cells,while MUC2 is the main component of the intestinal mucus layer[24].We examined changes in the transcriptional level of colonic mucin MUC2 in each group, it can be seen from Fig.3A, MUC2 was significantly decreased in the control group,while the intervention of L.casei significantly prevented this down-regulation.It indicated that the infection of E.coli O157:H7 destroyed the intestinal mucus layer of mice, and the pathogenic bacteria were in contact with intestinal epithelial cells, which aggravated intestinal inflammation and tissue damage.Zhang’s study [23] indicated that Lactobacillus acidophilus could increase the number of colonic mucin cells and mucin content, and promote the gene expression of MUC2,which was consistent with this study.

The upper cell monolayer is connected by tight junctions(TJs),adhesion junctions (AJs) [25].Well, the TJs can form a ring seal between intestinal epithelial cells,which contributes to the barrier function of intestinal epithelial cells.There are a variety of tight junction-associated proteins,including Claudin-1,E-cadherin,ZO-1,Catenins,and Actin[25,26].By detecting the transcription levels,we found that the mRNA levels of the intestinal epithelial junction genes (Claudin-1, E-cadherin, ZO-1) in the control group were significantly lower than those in normal group (P ＜0.05), indicating that the infection of E.coli O157:H7 affects the tight junction of the mouse colon,leading to destruction of the intestinal epithelial structure,thereby expanding the contact between the pathogenic bacteria and the intestinal epithelium,exacerbating the symptoms of enteritis.The relative mRNA expression of ZO1 in the intervention group exhibited a certain degree of up-regulation compared with the control group, but not significant (P ＞0.05).The mRNA level of E-cadherin 1 was significantly higher than control group(1.29±0.12 vs 0.87±0.18, P ＜0.05), but no significant difference from normal group (1.4±0.14, P ＞0.05).So we could infer that L.casei LC2W played an intervention role by preventing the destruction of colonic tight junctions in mice.

3.4.Comparison of prevention,intervention and treatment effects

Fig.2.Effects of Lactobacillus casei LC2W on TNF-α (A), IL-6 (B), IL-1β (C) mRNA levels in colonic tissue.All data are expressed as mean±SD (n=8 per group).The means with different letters indicate significant differences between the groups(P ＜0.05).

Fig.3.Effects of Lactobacillus casei LC2W on the mucous layer and mRNA expression of TJ-related genes in colon.(A) mRNA expression of MUC2, (B) mRNA expression of ZO-1,(C)mRNA expression of E-cadherin 1.The means with different letters indicate significant differences between the groups(P ＜0.05).

We compared the three effects as follows: In terms of physiological and biochemical indexes, the effects of prevention and intervention of L.casei LC2W can significantly alleviate the colon shortening in mice (prevention: 6.6±0.37 vs 5.7±0.38, P ＜0.05; intervention: 6.9±0.32 vs.5.7±0.37, P ＜0.05; treatment:6.3±0.31 vs 5.6±0.38, P ＞0.05) and avoid the serious damage of E.coli O157:H7.In regard to immunoregulation,the prevention of L.casei LC2W can significantly down-regulate the mRNA levels of the pro-inflammatory factors including TNF-α,IL-6 and IL-1β,while the treatment has no significant effect on IL-1β(prevention:1.6±0.37 vs 2.54±0.26,P ＜0.05;intervention:2.08±0.29 vs 2.76±0.34,P ＜0.05;treatment:1.93±0.29 vs 2.17±0.29,P ＞0.05).As for intestinal barrier function, L.casei LC2W has a similarly positive effect of intervention and prevention on MUC2, ZO-1 and E-cadherin 1 levels,but the treatment has little effect on the mRNA expression of ZO-1(prevention:0.89±0.12 vs 0.69±0.12,P ＜0.05;intervention:0.75±0.19 vs 0.73±0.09,P ＜0.05;treatment:0.75±0.15 vs 0.68±0.08,P ＜0.05).In summary,it can be concluded that L.casei LC2W has a stronger intervening and preventive eff;ects than the therapeutic eff;ect on colitis in mice caused by E.coli O157:H7.

4.Discussion

In recent years, a number of studies have been conducted on the interaction mechanism between probiotics and intestinal cells,probiotics to pathogenic bacteria and probiotics to diseases, and some fundamental achievements have been made.Overall,with the development of research,people will have a deeper understanding of the relationship between probiotics and intestinal disease.However,the mechanism of a probiotic function for a specific intestinal disease still needs to be further studied.

In our study,Lactobacillus casei LC2W exhibited the capacity to alleviate colitis caused by E.coli O157:H7, there may be 3 possible hypotheses to account for this phenomenon:first hypothesis is that probiotics with strong adhesion are competitive to inhibit the adhesion of pathogenic bacteria to intestinal epithelial cells after entering the intestinal tract of mice,including competitive inhibition, exclusive inhibition and displacement inhibition [16].Thus,the opportunity for pathogenic bacteria to bind to adhesion sites of intestinal epithelial cells is reduced.In addition,a series of adherent substances secreted by the probiotics,such as lipopolysaccharide,polysaccharide A, lipoate, and peptidoglycan, can stimulate the growth of the thickness of the intestinal epithelial cells, mainly including the expression of mucin such as MUC2, and also inhibit and stimulate the expression of a plurality of interleukins genes[19].Here,our results have shown that L.casei LC2W could increase the expression of MUC2 gene and inhibited the expression of interleukin gene, thus increasing the adhesion effect of probiotics and resisting the adhesion of pathogenic bacteria,likely this is the key factor to alleviate enteritis caused by E.coli O157:H7.Moreover,our lab studies have proved that when L.casei LC2W can inhibit the colonization of O157:H7 in mice by aff;ecting the adhesion of the intestinal epithelial cells and accelerating the excretion process of E.coli O157:H7[16].Lactobacillus plantarum 299v has also been shown to increase the expression of MUC2 and inhibit the adhesion of pathogenic bacteria[27].

The second hypothesis is that probiotic bacteria can modulate immune responses by regulating the secretion of intestinal cytokines and participating in intestinal inflammation and immune response.Cytokines are small peptides or glycoproteins that are produced during the stages of innate and specific immunity and mediate and regulate immune responses and inflammatory responses.Well, intestinal epithelial cells are the source and target of cytokines.Notably,our data demonstrate that L.casei LC2W have a similar capacity to down-regulate the expression of IL-1β, IL-6, TNF-α.Studies have found that probiotics can prevent the release of various inflammatory factors,such as inhibiting the activity of IL-1β, TNF-α, and NF-κB, and activating and promoting the expression of anti-inflammatory factor IL-10 in colitis mice[28].Roselli also reported that live L.reuteri, L.acidophilus, L.salivarius, L.ulgaricus and L.pantarum can inhibit the expression or secretion of pro-inflammatory cytokines IL-8 or IFN-γ, TNF-α, IL-1 and IL-23 in intestinal epithelial cells induced by pathogenic bacteria or pro-inflammatory factors; and it also prevented the down-regulation of IL-10 induced by E.coli, thereby limiting the pro-inflammatory response of epithelial cells and promoting the restoration of immune balance[29].

Hypothesis 3 is that the probiotics can regulate the physical barrier function of the intestinal mucosa and improve the permeability of the intestinal mucosa.The tight junction (TJ) between intestinal epithelial cells and epithelial cells constitutes a continuous and complete physical barrier, which can mechanically block the invasion of pathogenic microorganisms in the intestinal cavity[30,31].TJ is mainly composed of three kinds of protein including Occludin, Claudins and ZO, when the protein hermetic degree stimulated by external, and physiological and pathological conditions changed,it will increase the intestinal epithelial cell clearance permeability,so that pathogens entered to the cell,causing intestinal infectious diseases [32].Our study provided evidence that L.casei LC2W could increase the expression of ZO-1 and E-cadherin 1,perhaps this is another key factor to alleviate enteritis caused by E.coli O157:H7.Other studies have also shown that probiotics can inhibit changes in intestinal connectivity and permeability due to pathogen infection.As Puthenedam’s study[33]showed that Lactobacillus and Bifidobacteria can enhance the stimulation of epidermal growth factor and repair the destruction caused by Escherichia coli by maintaining the expression of cytoskeleton,ZO-1 and Occludin.

In conclusion, probiotics can enhance the immune function of intestinal epithelial cells by increasing the binding protein of intestinal epithelial cells,increasing the expression of mucin,regulating immune cells and cytokines, and competing for adhesion with pathogens.We found that L.casei LC2W seemingly exhibited the same mechanism to reduce the colitis in the treatment, prevention and intervention.By comparing the three effects with the improvement degree of each index,it can be concluded that L.casei LC2W has the same prevention and intervention effect on colitis caused by E.coli O157:H7,and is better than the treatment effect.

5.Conclusions

In summary,our findings demonstrate that L.casei LC2W could effectively regulate and improve the symptoms of enteritis caused by E.coli O157:H7 through stabilizing intestinal barrier and regulating cytokines.The effect of intervention is similar as the effect of prevention,which is better than the treatment.These findings will lay a foundation for the research of probiotics on enteritis,and provide guidance for clinical applications.It is worthy of further study that probiotics interfere with the intrinsic regulatory mechanisms of enteritis.

Interpretive summary

“The intervention effects of Lactobacillus casei LC2W on Escherichia coli O157:H7 -induced mouse colitis” by Wang et al.,reports a study of the intervention eff ;ects of Lactobacillus casei LC2W on the colitis severity of mice infected by O157: H7 in vivo from the perspective of physical and chemical indexes.The authors further explored the intervention mechanism of L.casei LC2W and provide the guidance for taking this strain.

Declaration of Competing Interest

There are no conflicts of interest to declare.

Acknowledgements

This work was supported by the National Key Research and Development Program of China (No.2018YFD0501600), National Natural Science Foundation of China (No.31972056) and Shanghai Agriculture Applied Technology Development Program, China(Grant No.2019-02-08-00-07-F01152).