The Anti-Inflammatory Effect of a Probiotic Cocktail in Human Feces Induced-Mouse Model

Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract due to altered interaction between the immune system and the gut microbiota. The aim of this study was to investigate the role of a probiotic cocktail in modulating immune dysregulation induced in mice. Mice were divided into 5 groups (n = 5/group), and inflammation was induced in two separate groups by fecal microbiota transplantation (FMT) from the stool of human with IBD and dextran sulfate sodium (DSS). In the other two groups, the cocktail of Lactobacillus spp. and Bifidobacterium spp. (108CFU/kg/day) was administered daily for a total of 28days in addition to inducing inflammation. A group as a contcxsrol group received only water and food. The alteration of the selected genera of gut microbiota and the expression of some genes involved in the regulation of the inflammatory response were studied in the probiotic-treated and untreated groups by quantitative real-time PCR. The selected genera of gut microbiota of the FMT and DSS groups showed similar patterns on day 28 after each treatment. In the probiotic-treated groups, the population of the selected genera of gut microbiota normalized and the abundance of Firmicutes and Actinobacteria increased compared to the DSS and FMT groups. The expression of genes related to immune response and tight junctions was positively affected by the probiotic. Changes in the gut microbiota could influence the inflammatory status in the gut, and probiotics as a preventive or complementary treatment could improve the well-being of patients with inflammatory bowel disease symptoms.


INTRODUCTION
Inflammatory bowel disease (IBD) is a pathological and chronic inflammatory disease that includes Crohn's disease (CD) and ulcerative colitis (UC) [1].In addition to the large and small intestines, Crohn's disease affects other parts of the gastrointestinal tract, including the stomach and anus, whereas ulcerative colitis is limited The Anti-Inflammatory Effect of a Probiotic Cocktail... to the colon and rectum [2].Although the exact cause of IBD is unknown, abnormal immune response to the gut microbiota could be one of causes of IBD, and other factors such as environment and host genetics also play an important role in this disease [3][4][5][6][7].
The gastrointestinal tract has a large population of gut microbiota [8].The gut microbiota contains more than 1000 different bacterial species, most of which belong to four major phyla, including Bacteroidetes, Firmicutes, Actinobacteria, and Proteobacteria [9].It is now well established that the gut microbiota plays a key role in intestinal homeostasis and physiology, immunity, and energy metabolism [10].Obvious differences in the population of the gut microbiota between healthy and IBD patients and between inflammatory and noninflammatory gut segments have been identified [11][12][13][14].The alteration of the gut microbiota in IBD pathology is well established.However, it is unclear whether these alterations are the cause or the consequence of intestinal inflammation and how exactly these bacteria are involved in the pathogenesis of IBD [15,16].
Determining the relationship between gut microbiota and IBD may be effective in treating this disease as well as other diseases [17].Standard treatments such as corticosteroids, aminosalicylates, and immunosuppressants are commonly used in IBD patients [18][19][20][21][22].These current treatments are short-lived and alleviate symptomatic complications but are associated with serious side effects such as loss of immune tolerance and drug resistance [23].The poor therapeutic effect and severe side effects of this treatment have led to the suggestion of other treatments, including the use of prebiotics, probiotics, and symbiotic as complementary or alternative medications for the treatment of IBD and other similar diseases [24].Probiotics are live microorganisms with specific potential activities that, when ingested in sufficient quantities, can provide health benefits to the host [25].Probiotics have several potential mechanisms of action for the prevention and control of IBD, including antimicrobial effects, suppression of pathogenic bacteria, modulation of the immune system, enhancement of antiinflammatory responses, and improvement of intestinal barrier activity [26].In particular, the effect of probiotics such as Lactobacillus spp.and Bifidobacterium spp. in regulating intestinal inflammation has been studied [27,28].Several studies have demonstrated that Bifidobacterium can promote the proliferation of Treg cells, enhance the expression of Foxp3, and strengthen the immunosuppressive function by increasing the anti-inflammatory factors such as IL-10 and TGF-β1, thereby reducing the immune response of IBD [28].Numerous studies have shown that various Lactobacillus species, particularly L. fermentum, L. reuteri, L. paracasei, and L. plantarum, can alleviate ulcerative colitis in animal models and clinical trials [29].
The aim of this study was to investigate the alteration of the selected genera of gut microbiota as a cause or consequence of IBD by fecal transplantation in mice and the effect of Lactobacillus spp.and Bifidobacterium spp. in regulating intestinal inflammation.

Sample Preparation for FMT
In previous study, 45 stool samples were collected from three groups, including IBD patients, cured IBD, and control group and kept at 4 °C and under anaerobic conditions during transport to the laboratory.The specimens were collected from 18 (40%) males and 27 (60%) females at an average age of 19.64 ± 23.51 and 22.66 ± 25.90 years old, respectively.IBD patients were diagnosed by a specialized gastroenterologist based on accepted radiological, clinical symptoms, and paraclinical findings.Cured IBD patients were defined as those who were in the remission phase and the disease in these cases was under control by medication, i.e. mesalazine (for all patients) and thiopurine (for some cases), depending on the severity of the disease.Control group included healthy individuals, with inclusion criteria including normal body mass index, no history of gastrointestinal disease, no use of antibiotics in 4 weeks before sampling, no special diet, and no pregnancy.
DNA was extracted from the stool sample, and realtime PCR was performed based on the analysis of the 16S rRNA gene for the selected genera of gut microbiota population (Table 1).The IBD sample that showed the most changes in microbiota compared with healthy individuals was selected for FMT.The patient sample (No.IBD 11) that had the highest population of Proteobacteria and the lowest population of Firmicutes compared to the healthy group was selected (Fig. 1 supplementary) [30].
Then, the IBD stool (30 mg) was diluted in 1 ml sterile phosphate-buffered saline (PBS) and spun (1000 rpm) for 30 s to allow sedimentation of the particles.A 200 μl of human fecal suspension was given by gavage once a week for four weeks.The control group as a healthy group normally received only water and feed and was given 200 μl PBS.Participants signed an informed consent form, and the study was conducted in accordance with the Ethics Committee of the Pasteur Institute of Iran (number IR.PII.REC.1398.060).

Probiotic Preparations
The probiotic cocktail used in this study included six strains of Lactobacillus and Bifidobacterium previously isolated and described [36,37].We used a probiotic cocktail of Levilactobacillus brevis, Lactiplantibacillus plantarum, and Lacticaseibacillus rhamnosus isolated from the feces of healthy humans, with a strain of B. bifidum, a strain of B. longum, and a strain of B. breve isolated from human breast milk.An equal ratio of specific dilutions of all strains (10^8 CFU) was mixed, and the final concentration of the cocktail was 10^8 CFU.Our previous studies in cell culture model showed that the studied strains have synergistic effects on inflammatory responses [38,39].

Mouse Model of Inflammation
Male BALB/c mice (8 weeks old) were purchased from the Pasteur Institute of Iran (Karaj, Iran).Mice were maintained at room temperature (25-28 °C) and 50 ± 5% relative humidity with a 12-h cycle of light and darkness.Food and water are provided ad libitum (5 mice per cage) and treated as indicated in Table 2.
Group 1 (FMT group): on day 1, the intestines of the mice had to be cleaned to perform FMT.One hour before starting the intestinal cleansing, the mice were provided only water and fasted.After 1 h, the mice were placed in a clean cage and 200μl polyethylene glycol (PEG); 425g/L solution was administered four times at 20-min intervals.Four hours after bowel cleansing, the Stool samples were collected from all mice on days 0 (one day before the induction of inflammation in mice), 4, 21, and 28 and stored at -80 °C until microbiota population analysis was performed.On day 28, blood samples were collected for a determination of inflammatory cytokines in serum.The mice were euthanized on day 28 and their colon was removed.The length of the colon was measured, and then, colon were washed with PBS and cut lengthwise.The distal colon tissue was collected for histopathology and RNA extraction.All experimental protocols were performed in accordance with the Ethics Committee of the Pasteur Institute of Iran (number IR.PII.REC.1398.060).All methods were in accordance with the relevant methods and regulations.

Investigation of Selected Genera of Gut Microbiota Population
DNA was extracted from 200 mg of mouse stool using the FavorPrep stool DNA isolation mini kit (Favorgen, Taiwan).DNA concentration was measured using a NanoDrop 1000 UV-Vis spectrophotometer.Real-time PCR was performed using 2 × SYBR Green Master Mix (Amplicon Bio, Denmark) and ABI Step One Plus detection system (Applied Biosystems, USA).Primers were tested with gradient PCR to obtain an appropriate annealing temperature (Table 1).All reactions were carried out in duplicate.Data were analyzed using the RQ = 2 −ΔΔCt formula, in which readings were normalized with all bacteria gene [40].In this method, the Ct values of the target bacteria were normalized with all bacteria gene, and their comparison was evaluated using the comparative fold change.

Investigation of Inflammatory Markers and Tight Junction Proteins
Total RNA was extracted from mouse colon tissue using the high pure RNA isolation kit (Roche, Germany) according to the manufacturer's instructions, and the quality and quantity of purified RNA were determined using a NanoDrop 1000 UV-Vis spectrophotometer at a wavelength of 260-280 nm.cDNA was synthesized from 0.1-1 µg RNA using the cDNA synthesis kit (Biotechrabbit GmbH, Germany).RT-PCR was performed with the 2 × SYBR Green Master Mix (Amplicon Bio, Denmark) and using specific primers from the online primer bank website (http:// pga.mgh.harva rd.edu/ prime rbank) (Table 3).RT-PCR data were analyzed by the RQ = 2 −ΔΔCt method using glyceraldehyde-3-phosphate dehydrogenase (gapdh) as a normalizer.

Histopathology Analysis
In all groups of mice, the total length of the colon was measured, and a segment was placed in 10% formalin and fixed.They were then placed in paraffin using standard procedures and stained with hematoxylin and eosin after sectioning.The colon tissue was examined and imaged using a Nikon Eclipse E400 microscope and a Canon DS -Fi1 camera (Japan).In this study, the pathology data were measured using the ordinal type.In this method, samples were assigned to a category that show an orderly progression in severity (0: normal, 1: mild, 2: moderate, 3: severe, 4: very severe).

Measurement of Serum IL-1β, IL-6, and TNF-α
The levels of inflammatory cytokines and interleukins in mouse serum were quantified by ELISA.The blood sample was incubated at room temperature for 2 h to form a clot and then centrifuged at 5000 g for 20 m.Serum was analyzed for IL-1β, IL-6, and TNF-α using an ELISA kit (Karmania Pars Gene, Iran).The absorbance of the samples was measured in the ELISA reader at 455 nm.Results were expressed as the concentration of cytokines per milliliter of serum.

Statistical Analysis
All data are expressed as mean ± standard deviation (SD).Differences between the results of the groups of mice were determined using a one-way analysis of variance (ANOVA).Differences were considered statistically significant at a value of p < 0.05 in all comparisons.GraphPad Prism 8.0.2 was used for the analysis.SPSS Statistics 26 software was used to investigate the correlation between the results of changes in the intestinal microbiota population of mice, inflammatory cytokines, inflammatory gene expression, and TJP gene expression.According to the results obtained from the Shapiro-Wilk test, it was found that the data were not normal to a very small extent, and as a result, Kendall's tau-b test was used to check the relationship between the data.In this analysis, if the correlation coefficient = r is positive, it means that there is a direct relationship, and if the number is negative, it means that there is an inverse relationship between the results.

Inflammation Induction by FMT
Inflammation was induced in mice by transplantation of human stool, which was investigated by histopathology and tissue analysis.As shown Fig. 1, the length of the colon is decreased in the FMT and DSS groups compared to the control group.Histological results of the colon in the FMT and DSS groups showed various signs of inflammation.Hypertrophy of the muscle layer, significant architectural distortions, depletion of goblet cells, intra-epithelial neutrophils, and regenerative changes were significantly evident.However, no pathological changes in the colon tissue and only mild hypertrophy of the muscle layer were observed in LacBif-treated mice (Fig. 1).On day 28 after induction of inflammation in the FMT and DSS groups, an increase in serum levels of IL-1 and IL-6 was observed.On the other hand, TNF-α was decreased.Moreover, in the FMT + LacBif and DSS + LacBif groups, the levels of IL-1 and IL-6 were lower than in the FMT and DSS groups.TNF-α levels in these groups were not different from those in the FMT or DSS groups (Fig. 1).

Selected Genera of Gut Microbiota Changes
The changes in some bacterial groups were observed in the mice groups from the fourth day of the study.In the FMT group, on the fourth day of the study, only Actinobacteria, Firmicutes, Clostridium clostridiiforme, and Enterococcus faecalis changed and decreased significantly.In this group, the bacteria belonging to the phyla Actinobacteria and Firmicutes decreased, while the species belonging to the γ-Proteobacteria, especially Enterobacteriaceae, increased on the 28st day.The results showed a sharp decrease in the population of Enterococcus faecalis in the FMT group, which dropped to almost zero on days 21 and 28.Moreover, the bacterial population of Bacteroidetes was higher on the 21st day.However, later, on the 28th day, there was a sudden decrease, so there was no significant difference compared to the control group (Fig. 2).
The results of the changes in selected genera of gut microbiota in the DSS group were similar to the FMT group, but in the DSS group, there were more changes on day 4 (Fig. 3).In the FMT + LacBif group, the relative abundance of Actinobacteria and Firmicutes was decreased in the first days, but the population of Enterobacteriaceae was increased (Fig. 2 supplementary).However, with further treatment on day 21 and thereafter, the population of Lactobacillus, Bifidobacterium, and Roseburia increased, and there was no significant difference compare to the control group (Fig. 4).Also in this group, the population of Faecalibacterium increased and higher than the control group on day 28.But there was no significant difference in the abundance of Enterobacteriaceae compared to the control group.In the DSS + LacBif group, the population of Enterobacteriaceae and E. faecalis was increased on the fourth day of the study.But in this group, there was no significant difference in the abundance of Lactobacillus and Bifidobacterium compared to the control group on day 28 (Fig. 5 and Fig. 3 supplementary).

Expression of Inflammatory Markers
The expression of inflammatory genes in the colon tissue of mice was studied.RT-PCR results in the FMT group showed that the expression of inflammatory genes was significantly increased compared with the control group.In the FMT group, the expression of IRAK4 and MYD88 genes was increased more than in the DSS group (p < 0.05).The expression of inflammatory genes was significantly increased in the DSS group compared with the control group.However, the expression of these genes was lower in the treated groups (LacBif) than in the FMT and DSS groups (Fig. 6).

Expression of Tight Junction Proteins
The results showed that in both groups FMT and DSS, the expression of TJP genes was significantly reduced.The expression of claudin1 and ZO-1 was lower in the FMT group than in the DSS group (p < 0.05).In addition, the expression of claudin1, claudin2, ZO-1, and occludin genes was increased in the FMT + LacBif and DSS + LacBif groups compared with the FMT and DSS groups (Fig. 7).

Correlation Between Selected Genera of Gut Microbiota Changes and Immune Response
The results of Kendall's statistical analysis showed that in this study, there was almost a significant relationship between the changes in the bacterial population of mice and inflammatory cytokines in most of them (Table 1 supplementary).In some cases, a positive relationship was seen, and with the increase in the population of some bacterial groups, the amount of cytokines also increased, and in other cases, there  The Anti-Inflammatory Effect of a Probiotic Cocktail... was an inverse relationship.In this study, a significant relationship between intestinal bacteria and the expression of some TJP proteins was seen (Table 2 supplementary).In fact, with the decrease in the population of Actinobacteria, Firmicutes, and Bacteroidetes, the expression of TJP proteins has also decreased.While there was an inverse relationship between Proteobacteria population and TJP expression, there was a significant correlation between the changes in the bacterial population and the expression of some inflammatory genes in mice, and depending on certain bacteria, this relationship has been seen directly or inversely (Table 3 supplementary).In fact, with the decrease in the population of Actinobacteria, Firmicutes, and Bacteroidetes, the expression of these genes increased and had an inverse relationship.However, the Proteobacteria phyla had a direct relationship with the expression of these genes.

Correlation Between Inflammatory Cytokines and expression of Inflammatory Pathway Genes
The results of this study showed that there is a significant and direct relationship between interleukin-1 and interleukin-6 with the expression of inflammatory genes (Table 4 supplementary).While an inverse relationship was seen between TNF and inflammatory genes.

Correlation Between Inflammatory Cytokines and the Expression of TJP Genes
The results showed that with the increase of interleukins-1 and interleukins-6, the expression of TJP genes decreased, and a significant correlation was reported (Table 5 supplementary), while a direct and significant correlation between TNF and the expression of ZO-1 and Claudin1 genes was observed.

DISCUSSION
In this study, in addition to chemical induction with DSS, we used transplantation of stool to induce inflammation in mice.DSS-induced IBD in mice leads to the destruction of intestinal mucus and causes inflammation by inhibiting the repair of intestinal epithelial cells [41].In our FMT group, bacteria are involved in the development of intestinal inflammation.Human microbiotaassociated (HMA) mice are the best available model to study the role of gut microbiota in diseases [42], and in this study, we used the stool of IBD patient to produce an HMA model.Because germ-free mice have biological limitations and the gut microbiota is essential for the maturation of the gut and host immune system [43][44][45], we used PEG to cleanse the gut microbiota of conventional mice in preparation for transplantation of human stool.Studies have shown that PEG eliminates nearly 90% of intestinal bacteria in mice [10].
The results of the FMT group showed that after transferring human stool to mice, some of the intestinal bacteria were altered on day 4 (Fig. 2).Most of the changes on day 4 involved the phyla of Actinobacteria and Firmicutes and the species of Clostridium clostridioforme and E. faecalis.Following a decline in the Firmicutes and Actinobacteria population, the abundance of Lactobacillus, Bifidobacterium, Faecalibacterium, and Roseburia was significantly reduced on days 21 and 28 of the study.In contrast, the population of γ-Proteobacteria, especially Enterobacteriaceae, increased.On the other hand, the population of Bacterioidetes increased significantly on day 21, but decreased on day 28 and had a significant decrease compared to the control group.
In general, the microbiota population of the FMT group was similar to that of the DSS group on day 28 of the study, and the increased abundance of γ-Proteobacteria, especially Enterobacteriaceae, in the FMT and DSS groups provided the basis for intestinal inflammation in vivo.The overall results suggest a parallel relationship between gut microbiota and IBD, implying that changes in the population of gut microbiota can cause IBD, just as IBD can alter the population of gut microbiota.Our observations suggest that alterations in the gut microbiota may play a critical role in Fig. 3 Changes in the selected genera of gut microbiota population in the DSS group compared to the control group at days 0, 4, 21, and 28.Results were expressed as mean; error bars (standard deviation).Statistical analysis was performed using the one-way test ANOVA (*p < 0.05, **p < 0.001).The Anti-Inflammatory Effect of a Probiotic Cocktail... inflammatory symptoms in the gut and in diseases such as IBD, irritable bowel syndrome, and others [46].
Probiotics as commensals in the gut could modulate the gut microbiota and prevent substantial changes in the microbiome population.In this regard, our results showed that in the FMT + LacBif and DSS + LacBif groups, with an increasing abundance of Lactobacillus and Bifidobacteria, the population of Faecalibacterium and Roseburia also increased, while the population of Enterobacteriaceae and Bactroidetes decreased (Fig. 4, Fig. 5).The role of Lactobacillus spp.has been shown in inhibiting pro-inflammatory cytokines and maintaining the balance of the gut microbiota [47].In this study, the probiotics was simultaneously consumed with the induction of inflammation, and due to the inflammatory conditions and disrupted gut barrier and changes in the population of gut microbiota, the amount of attached Lactobacillus spp.and Bifidobacterium spp.did not increase significantly.
Alteration of the gut microbiota population, characterized by an increase in potentially pathogenic bacteria and a decrease in beneficial bacteria, disrupts normal microbe-host interactions and impairs intestinal homeostasis [48].Intestinal epithelial cells recognize microorganisms by specific molecular regions called pathogenassociated molecular patterns (PAMPs) [49][50][51].One of the important pattern recognition receptors (PRRs) for modulating the cellular immune response to PAMPs is the toll-like receptor (TLR) [48].TLR activates intracellular signaling via two major pathways, the myeloid differentiation factor 88 (MyD88)-dependent pathway, and the MyD88-independent pathway, after pathogen recognition.In the cell, TRAF6, IRAK1, and IRAK4 proteins are activated, and finally, the activation of MAPK increases the production of IL-1, IL-6, and TNF-α [52].The evaluation of inflammatory genes in our study showed that in the FMT and DSS groups, the expression of inflammatory genes increased compared with the control group (Fig. 6).In the FMT group, the expression of MyD88 genes was higher than in the DSS group, so the MyD88dependent pathway was more activated in the FMT group.Also, the expression of the IRAK4 gene, which is one of   the components of the MyD88 signaling pathway, was more expressed in this group than in the DSS group.The expression of Map3k7 gene in the DSS group was higher than in the FMT group, and this is probably the reason for the increase in the serum levels of interleukin-1 and interleukin-6 in this group.
In the FMT + LacBif and DSS + LacBif groups, probiotics were able to inhibit the increase of inflammatory factors, and compared to the DSS and FMT groups, the activity of signaling pathways decreased.Probiotics are able to regulate the host immune response by interacting with immune cells and intestinal epithelial cells [53].The PAMPs of probiotics are recognized by the TLRs of dendritic cells, and unlike pathogenic bacteria that induce inflammation by stimulating TLR, probiotics reduce inflammation by reducing NF-κB [54].One study has shown that Lactobacillus are able to degrade the chemokine IP-10 by producing a protease called lactocepin [55].Another study that examined the effect of probiotics on the expression of TLR4 and TLR5 in mice with colitis showed, similar to our results, that probiotics reduced the expression of TLR4 and TLR5 [56].
The intestinal mucosa plays a key role in the development of inflammatory bowel disease.The normal mucosa of the colon has tight junction proteins, and their functional integrity can protect the colon from bacterial invasion [57].The expression of tight junction proteins was decreased in the DSS and FMT groups compared with the control group (Fig. 7).One study reported that the expression of claudin-1 and claudin-2 in the rat model of IBD reached the highest level on day 14 and then gradually decreased [58].Another study showed that TNF upregulated the expression of claudin-2 [59].The results of our study also showed a decrease in serum TNF level and claudin-2 expression.Accordingly, cytokines are involved in the expression and distribution of tight junction proteins in the inflammatory state.
However, the expression of claudin-1 and ZO-1 was lower in the FMT group than in the DSS group, which was probably due to the effect of the bacteria in the stool sample transferred to the mice.Thus, with the colonization of the gastrointestinal tract with pathogenic bacteria, the intestinal immune system is activated, and inflammatory cytokines and chemokines are produced, and cellular junctions are weakened.However, in the groups treated with probiotics, the expression of tight junction proteins was higher than in the FMT and DSS groups.Probiotics are able to decrease gut permeability caused by pathogens and increase tight junction protein expression [60].The most effective probiotics are Lactobacillus spp.and Bifidobacterium spp.strains, and the effect of combined probiotics, such as the use of multiple bacterial cocktails, is more effective [61].
This study had some limitations: We did not have NGS facilities to compare the gut microbiota population of the FMT group with the gut microbiota of the human donor.In addition, we did not compare our gut cleansing strategy (PEG) with the other models (germ-free or antibiotic treatment).Also due to the lack of facilities and funding for this project, we do not have experimental replicates.

CONCLUSION
In this study, we were able to induce inflammation by transferring stool from humans with IBD to conventional mice.The population of the selected genera of gut microbiota of the FMT group changed on day 4 and was significantly different from the population of the selected genera of gut microbiota on day 0. Probiotic cocktail of Lactobacillus spp.and Bifidobacterium spp.play an important role in balancing the population of the gut microbiota and inhibiting inflammation.Consequently, the daily intake of probiotics was able to keep the gut microbiota population in balance and prevent the proliferation of potentially pathogenic bacteria.

Fig. 2
Fig. 2 Changes in the selected genera of gut microbiota population in the FMT group compared to the control group at days 0, 4, 21, and 28.Results were expressed as mean; error bars (standard deviation).Statistical analysis was performed using the one-way ANOVA test (*p < 0.05, **p < 0.001).

Fig. 4
Fig.4 Changes in the selected genera of gut microbiota population in the FMT + LacBif group compared to the control group at days 0, 4, 21, and 28.Results were expressed as mean; error bars (standard deviation).Statistical analysis was performed using the one-way test ANOVA (*p < 0.05, **p < 0.001).

Fig. 5
Fig. 5 Changes in the selected genera of gut microbiota population in the DSS + LacBif group compared to the control group at days 0, 4, 21, and 28.Results were expressed as mean; error bars (standard deviation).Statistical analysis was performed using the one-way test ANOVA (*p < 0.05, **p < 0.001).

Fig. 6
Fig. 6 Differences in the expression of inflammatory genes in the FMT, DSS, F + P (FMT + LacBif), and D + P (DSS + LacBif) groups compared with the control group.Results were expressed as mean; error bars (standard deviation).Statistical analysis was performed using one-way ANOVA test (*p < 0.05, **p < 0.001).

Fig. 7
Fig. 7 Differences in tight junction protein expression in the FMT, DSS, F + P (FMT + LacBif), and D + P (DSS + LacBif) groups compared with the control group.Results were expressed as mean; error bars (standard deviation).Statistical analysis was performed using the one-way ANOVA test (*p < 0.05, **p < 001).

Table 1
Sequences of Oligonucleotide Primers Used in This Study

Target bacterial Sequence (5′-3′) Amplicon size (bp) References
The Anti-Inflammatory Effect of a Probiotic Cocktail... mice were placed in a clean cage and given 200 μl of a human fecal suspension.FMT was performed once a week for 4 weeks.Group 2 (FMT + LacBif): received probiotics (10 8 CFU/kg/day) daily in addition to performing FMT.Group 3 (DSS): received 200 μl DSS 2% daily for 4 weeks.Group 4 (DSS + LacBif): received DSS as group 3 and also received probiotics (10 8 CFU/ kg/day) daily for 4 weeks.Group 5 (control) as a healthy group receive only water and food and given 200 μl PBS by gavage daily for 4 weeks.D: The days of the study.FMT: Fecal microbiota transplantation.DSS: dextran sulfate sodium.LacBif: The cocktail of Lactobacillus spp.and Bifidobacterium spp.(10 8 CFU/kg/day) was administered only daily for a total of 28 days.

Table 3
Sequences of Oligonucleotide Primers