The synergic impact of lignin and Lactobacillus plantarum on DSS-induced colitis model via regulating CD44 and miR 199a alliance

Chronic or recurrent immune system activation and inflammation inside the gastrointestinal tract is characterized by inflammatory bowel disease (IBD). Due to the lack of safety and efficacy of traditional medications, the use of food supplements for IBD management is on the rise. Numerous studies reported that, certain food supplements have a variety of therapeutic benefits for IBD. In the present study, a mouse model of IBD was used to the anti-colitis effects of lignin supplementation with Lactobacillus plantarum (L. plantarum) on intestinal inflammation. The animal model was treated with dextran sodium sulphate (DSS), the illness index increased, and colon length and body weight declined, but these effects were reversed when lignin and L. plantarum treated groups. In addition, lignin and L. plantarum supplementation inhibited the DSS induced increase in levels of cytokines TNF-α (250 pg/mL), INF-γ (180 pg/mL), IL-1β (70 pg/mL) and TGF- β (72 pg/mL). Gene and protein expression study revealed that Lignin and L. plantarum supplementation restored the expression of E-cad and suppressed the expression of STAT3 in DSS induced colitis model. Lignin and L. plantarum supplementation also suppressed CD44 expression (1.2 fold) by up regulating the expression of miR199a (1 fold) over DSS induced colitis. Our study suggests that Lactobacillus, lignin, and their synergistic treatments have protective roles against inflammatory bowel disease through changes in inflammatory cytokines, and miR 199a expression in DSS-induced colitis.


Introduction
Inflammatory bowel disease (IBD) is an inflammatory irritation in the bowel of host system. It is caused by a multifactorial disease interaction between factors (Microbial, immunological and genetical) in nature, which is progressed to the development of bowel syndrome (Friedrich et al. 2019). IBD is the global autoimmune idiopathic issue of the intestine (Blumberg et al. 1999). In IBD there are two major conditions namely ulcerative colitis (UC) and Crohn's Disease (CD). Under Ulcerative colitis condition, the inflammated mucosal layer of the colon leads to chronic inflammation (Baumgart and Sandborn 2007). Whereas, under Crohn's disease condition the whole gastrointestinal tract was affected. Both the inflammatory bowel disease is a major bottleneck in the healthcare system in developed countries and currently increasing in developing countries (Baumgart and Sandborn 2007). There is a significantly higher risk of neurodegenerative diseases, gastrointestinal tumors and 1 3 233 Page 2 of 13 psychological issues in patients with IBD. These risks make it more important to diagnose the disease earlier and provide appropriate attention in the clinical treatment process (Feng et al. 2022). The intestinal environmental factors, which are complex to the immune response yet not clear in the etiology of IBD (Orel 2014). IBD diagnosis was very challenging in the early period over 60 years before, now both UC and CD are diagnosed with the technology of fiberoptic colonoscopy and ileocolonoscopy (Mulder et al. 2014). The IBD is currently treated with many agents such as 5-aminosalicylic acid, steroids, antibiotics, probiotics, immunosuppressant, etc. These agents are successful in improving acute and chronic inflammation, however they do not prevent/reverse intestinal fibro stenosis (Vetuschi et al. 2022). This drawback of the current treatment strategies has steered research towards more effective treatment methods.
Microflora of the human intestine constitute bacteria, fungi and viruses. Bacteria are dominating 96% of the intestine, and around 5000 species were found in the human bowel. An enormous amount microorganisms are obtained in the large intestine of the human bowel, with 1 × 10 12 microbial cells indexed in the intestinal bowel. The microbiota of intestinal flora could influence host health in both direct and indirect ways by its metabolites. The profile of flora may vary based on host life style, genetic factors and diet (Basso et al. 2019). The beneficial microbes, which positively regulate the host immune system are known as probiotics.
Probiotic is distinct live microorganisms which is helping to protect the host bowel system against inflammations (Borchers et al. 2009). Lactobacillus is a significant microbiota of the digestive system, it is involved in host health improvement and so considered as probiotic. Lactobacillus involved in immunoregulatory properties of the host, which stimulate the intestinal immune response to fight against the inflammation in the bowel system. Probiotic could be classified as a two groups, such as immunostimulating and antiinflammatory (Macho Fernandez et al. 2011). These probiotics secrete short chain fatty acids, protease, butyrate, and vitamins etc., Few reports revealed that, Short chain fatty acid (SCFA) inhibit the adherence of pathogenic microbes in the host gut. Gut epithelium injured in IBD was restore by butyrate, which supplies the essential nutrient to colonocytes and deed as an agent for inactivating intracellular transcriptional factors pathways to synthesis the cytokines factors (Kanauchi et al. 2005). In the binding sites of the mucus layer the pathogenic microbes were dispatched from luminal mucosal (Veerappan et al. 2012). The probiotic enhances the gut barrier and mucous layer to reduce the deleterious microbes and other antigen (Collado et al. 2007).
Probiotics regulates various mRNA transcripts of bowel inflammatory symptoms. The Mucin 3 (MUC3) gene were regulated by Lactobacillus strains, in goblet cells of gut mucosa and tend to increased mucus production (Mack 2003). Probiotic help to produce and secrete more cytokines against inflammation by stimulating the lymphocytes. The probiotic products and metabolites regulate inflammatory cytokines such as Tumor necrosis factor alpha (TNFα), Interleukin 1 beta (IL1β), Transforming growth factor beta (TGFβ), etc., (Caballero-Franco et al. 2007).
Lactobacillus plantarum is a potential probiotics, which improves the host's health and benefits against bowel inflammation. L.plantarum has many health-promoting effects and is hence used frequently in food and pharmaceutical industries. Some of the benefits of L. plantarum are the prevention of irritable bowel disease, IBD, cancer, coronary heart disease, etc., (Le and Yang 2018). L.plantarum which inhibits activation of the Nuclear Factor Kappa B (NF-κB) signaling pathway and reduction of gastrointestinal pro-inflammatory factors (Dubuquoy et al. 2002). Lactobacillus sp. are regulating the cytokines factors to reduce the inflammation in the Bowel diseases. L.plantarum which suppressed the pathogens and regulates the production of cytokines (TNF-α, (IL)-1β, IL-6, IL-10, IL-12), and Interferon gamma (IFN-γ) to modulate the balance between T Helper 1 (Th1) and T Helper 2 (Th2) of the T-cells (Ahrne and Johansson Hagslatt 2011). It also modulates the T-cell maturation. L.plantarum induced Treg cells suppress the effector T-cells and activate anti-inflammatory cytokines such as IL10 and TGF β. The suppression of effector T-cells also activates hyperplasia of the goblet cell population (Mayne and Williams 2013;Kim et al. 2020). Lactobacillus interfere with the progression of macrophages Dextran sodium sulphate (DSS) induced IBD mouse. The M1 macrophage reciprocally regulate the inflammatory cytokines in IBD patients as well as IBD mouse (Liew et al. 2010, Khalifa et al. 2022. Prebiotics are indigestible carbohydrates that improve the health of the host by encouraging the growth and activity of specific bacterial species in the colon (Gibson and Roberfroid 1995;Schrezenmeir and de Vrese 2001). After cellulose, lignin is the second most prevalent natural aromatic polymer in terrestrial ecosystems, accounting for about 30% of organic carbon stored in the biosphere (Ayyachamy et al. 2013). Based on their protective properties against lipid peroxidation caused by oxygen radicals, lignin from various sources have been labeled as antioxidants (Ugartondo et al. 2009). In previous studies, lignin fed chicken improved the beneficial bacteria in ileal content and reduced the Escherichia coli. Broiler chicken with 1% lignin fed significantly increasing populations of lactobacilli and bifidobacteria and lowering Escherichia coli loads after the challenge (Baurhoo et al. 2007;Wang et al. 2015). Although lignin's prebiotic effect is modest, the prebiotic nature of lignin fragments may aid in the eradication of intestinal pathogens in cattle products, protecting people from illness. More research is needed to determine the complete prebiotic value of lignin and lignin, as well 1 3 Page 3 of 13 233 as the best dosage for health benefits, animal welfare, and product safety (Ayyachamy et al. 2013). But no studies have looked into whether probiotic bacteria can use lignin as a prebiotic in an in vivo animal model to cure colitis.
In this study, the effect of Lactobacillus plantarum, lignin and their synergistic effects were investigated against DSS induced C57BL/6J mice. The attenuation of inflammatory markers in IBD mice was evaluated by disease index, body weight, and fecal haem. The microscopic evaluation was done by Hematoxylin and eosin (H&E) staining and inflammatory markers were quantified using Enzyme-linked immunosorbent assay (ELISA), quantitative Real-time polymerase chain (qRT-PCR) and Western blot.

Effect of lignin against Lactobacillus plantarum
The effect of lignin (Sigma Aldrich, India) against L. plantarum (University of Gothenburg, Sweden) was performed by a slightly modified method by Etchepare et al. (2016). The overnight culture of L. plantarum was streak in De Man Rogosa and Sharpe agar (MRS agar) media (Himedia, India) and added 10% Dimethyl sulphoxide (DMSO) as a control and different concentrations of lignin (1 Micrometer (µm), 5 μm, 10 μm, 50 and 100 μm) into culture plate and maintained at 37 °C under continuous stirring [150 Rotation per minute (rpm)/minute (min)] to a total of 4 h. The results were expressed as log Colony forming unit (CFU)/ millilitre (ml).

Animals
The experiment was conducted as per The committee for the purpose of control and supervision of experiments on animals (CPCSEA) guidelines after obtaining approval from the Institute of Animal Ethical Committee (018/IAEC/ KIMS/2019) from Karpaga Vinayaga Institute of Medical Sciences, Chengalpattu, and Tamilnadu, India. Three weeks old male mice (C57BL/6J) were procured from the National Institute of Nutrition, Hyderabad, India and maintained at 25 °C (°C) with 55% (%) humidity and 12 h (Hrs) of cycle Light/Dark in the Polypropylene cage at Karpaga Vinayaga Institute of Medical Sciences. The pellet feed containing sufficient nutrition (Carbohyrates-74%, Protein-22% and Fat 4%) was procured from Agrotech LLP, Pune and used to feed the animals ad libitum. The animals were also provided ad libitum asses to normal drinking water. Treatment was carried out using oral canula.

Colitis induction with treatment
The mice were segregated into five groups and each group holding six mice were used for the experiment. Animals provided with Phosphate buffer solution (PBS) [100 µl (µl) kilogram (kg)/Body Weight (BW)] -Group I (control), animals provided with 2.5% (%) of dextran sodium sulphate (DSS) -Group II (Induced Colitis control), animals provided with 2.5% DSS and Lignin (5 mg kg/BW) for treatment -Group III, and animals provided with 2.5% DSS, L. plantarum strain (1 × 10 8 load/250 µl) and Lignin (5 mg kg/BW) for treatment -Group IV. At the end of the study, the experimental mice were euthanized and sacrificed using Carbon dioxide CO 2 inhalation at the end of the experiment. Immediately, the colon tissues were preserved in 10% buffered formalin for histopathology. While the tissues that were required for other protein related studies and stored at -70 °C immediately upon dissection.

Colitis severity assessment
The preserved colon tissues were washed with Phosphate buffer solution (PBS) and used for assessment of inflammation in the mucous tissue. Macroscopic assessed to identify the damage of tissue and given the scoring as follows: 0 (No inflammation), 1 (Local hyperaemia), 2 (ulceration without hyperaemia), 3 (ulceration and inflammation at one site only), 4 (ulceration and inflammation at more sites) and 5 (ulceration more than 2 cm).

Quantitative real-time RT-PCR (qRT-PCR)
Total Ribonucleic acid (RNA) was extracted from tissues using Tri Isolation Reagent (Thermo Scientific, MA, USA) according to the standard protocol. The extracted RNA was quantified using Nano Drop® 2000 spectrophotometer (Thermo Scientific) and 100 Nano gram (ng)/target was used for Complementary DNA (cDNA) preparation. The upstream forward primers and downstream reverse primers for Signal transducer and activator of transcription 3 (STAT3), Epithelial cadherin (E-cad), Cluster of Differentiation 44 (CD44), and Glyceraldehyde-3-phospate dehydrogenase (GAPDH) are shown in Table 1. Quantitative real-time RT-PCR (qRT-PCR) was performed with template cDNA using the SYBR master mix method. The expression of miRNA was analysed using stem loop RT-PCR assay using U6SnRNA (Table 1) as internal control as described earlier (Ibrahim et al. 2021). Messenger RNA (mRNA) and Micro-RNA (miRNA) expressions were analyzed using quantitative real-time PCR in VII 7 A (Applied Biosystems, Waltham, MA, USA). Results were expressed as relative gene expression using the 2(-Delta Delta C(T)) method (Livak and Schmittgen 2001).

Western blot analysis
Tissues were lysed in Radio immuno precipitation assay (RIPA) lysis buffer (Santa Cruz, Paso Robles, CA, USA) and a 1× protease inhibitor cocktail. The lysate was prepared and preserved at − 80 °C. Protein concentrations of cell lysate were estimated using a Bradford assay at 630 nm (nm). The equivalent of 50 µg (µg) of protein extract was separated by Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes (pore size: 0.45 μm, Bio-Rad, Hercules, CA, USA). Membranes were blocked using 5% non-fat dry milk in TBS buffer, followed by probing with the primary antibodies overnight at 4 °C, according to the manufacturer's protocol. The primary antibodies STAT3 (rabbit monoclonal antibody 1:1000) (Biorybt-CB4 0WY), E-cad (rabbit polyclonal antibody 1:1000) (Biorybt, CB4 0WY), and β-actin (rabbit polyclonal antibody 1:2000) (Cell Signaling Technology, Beverly, MA, USA, 4967 S) were incubated overnight at 4 °C, and then washed with Tris buffered saline with Tween 20 (TBST). Washed blots were incubated with horseradish peroxidase-conjugated primary specific secondary antibody at room temperature for 1 h. The blots were visualized by an enhanced chemiluminescence (ECL) system (Pierce, Life Technologies, Austin, TX, USA) and scanned using a LICOR detection system and expressed bands were analyzed using Image Quant software and quantified by densitometry using ImageJ software v1.8 (Ibrahim et al. 2021).

Statistical analysis
The data are presented as the mean ± SD of a representative experiment from three different trials that yielded similar outcomes. The statistical significance between the variables was determined using one-way Analysis of variance (ANOVA) and a post-doc test was performed between the mean differences of P ≤ 0.05 using SPSS 22.0 ver.

Effect of lignin against Lactobacillus plantarum
To our knowledge, this is the first study to indicate that dietary lignin and Lactobacillus plantarum supplementation, either alone or in combination, can reduce Dextran sodium sulphate (DSS)-induced colitis in mice. Lignin toxicity was evaluated against L.plantarum. Lignin concentration from 1 to 100 micromolar µM was tested against L.plantarum. Significant bacterial toxicity was observed above 5 µM concentration whereas below 5 µM growth of Lactobacillus was not inhibiting (Fig. 1). Lignin concentration 5 µM was not significantly toxic P > 0.05. DMSO was used as a control and it showed a log 2 × 10 7 microbial load was recorded. As shown in Fig. 1, the probiotic effect of lignin on lactic acid bacteria is not significant till 5 µM as test concentration.

To determine the impact of lignin mediated L. plantarum against DSS induced colitis
The disease severity of treated lignin with/without L. plantarum was evaluated using Disease index, body weight and colon length. These parameters were observed from both experimental groups (Fig. 2a, b). The Lactobacillus group had a significant recovery disease index (Fig. 2a), body weight (Fig. 2b) and colon length (Fig. 2c) than DSS induced group (P ≤ 0.05). Meanwhile lignin with L. plantarum treated groups had potent reduction of disease index and improved colon length and body weight (P ≤ 0.05). The individual tested group showed less significane than the combined tested groups. Further, the microscopic score and cytokines were evaluated. The disease index was reduced from 4.5 to 2.2 in the DSS group and Lignin with L. plantarum group prospectively. Interestingly, the colon length was observed in the DSS group 4.1 cm whereas the lignin with Lactobacillus group showed an increased colon length of 7.1 centimeter (cm). The body weight of the DSS induced group was reduced from 22.2 gram (gm) to 15 gm, whereas the Lignin with Lactobacillus group was 24 gm, which showed gaining in the body weight compared with the DSS group.

Lactobacillus plantarum retard DSS induced colitis in mice
The histology examination showed multiple erosive lesions and extensive inflammatory cellular infiltrations were observed in the colon tissue of DSS mice. The macrophages, lymphocytes, neutrophils and a few eosinophils were observed in the infiltrated parts of colon tissue (Fig. 3a,  b). A significant reduction of infiltrating was observed in the lignin and Lactobacillus group (Fig. 3c, d). The corresponding histological severity score was potentially reduced in Lignin with L. plantarum mice (Fig. 3e). Even though the colon tissue of lignin DSS mice showed inflammatory lesions but the severity of inflammation was lesser than DSS induced mice (P ≤ 0.05) (Fig. 3f).

Lignin controls the inflammatory cytokines in colitis mice
The estimation of cytokines was performed in DSS induced colitis mice. After the14th day of DSS induction the distal colon was excised and homogenated for analyzing inflammatory markers. Lignin with and without the treatment of L. plantarum showed a significant decreasing of inflammatory cytokines revealed that the reduction of infiltrated goblet cells in the site of mucus layer of the colon. The inflammatory cytokines Tumor necrosis factor alpha (TNFα) and Interleukin 1 beta (IL1β) were reduced significantly by P ≤ 0.05 in Lignin with L. plantarum mice compared to DSS induced mice. The immuno-modulatory cytokines Interferon gamma (IFN-γ) and Transforming growth factor beta (TGFβ) were negatively augmented in Lignin with and without L. plantarum treated mice compared to DSS induced mice. Lignin with L. plantarum showed a potentially significant P ≤ 0.05 than Lignin alone group (Fig. 4a,  d). The levels of cytokines (TNF-α, INF-γ, IL-1β and TGFβ) increased in DSS-treated mice. The levels of all cytokines studied significantly decreased in Lactobacillus, lignin, and synergistic groups.

Lignin and L. plantarum modulates STAT 3 and E. cad expression in DSS mice
The study evaluates the level of Signal transducer and activator of transcription 3 (STAT3) in lignin treated colitis mice. Lignin treated mice have estimated after the 14th day of DSS induction revealed down regulation of STAT-3 mRNA and protein expression in colitis mice (Fig. 5a, b). The Messenger RNA (mRNA) of STAT 3 was not significantly changed by Lignin associated with L. plantarum treatment P ≥ 0.05, whereas, the protein expression was significantly decreased (P ≤ 0.05) in the treatment. Followed by this treatment cell adhesion epithelial molecule Epithelial cadherin (E.cad) was significantly regulated in inflammatory bowel regions.
The expression of E-cad was down regulated in colitis mice the level of E-cad mRNA and protein expression was increased in lignin with and without L. plantarum treated mice. The expression of the E.cad gene in Lignin with/ without L.plantarum was potentially increased compared to Lignin alone treated mice. These results revealed that the transformation of mesenchymal tissues into normal epithelial cells by reversion of cell adhesion molecule E.cad (Fig. 5c, d).

Micro RNA 199 depletes stem cell modification in DSS induced mice
MicoRNA (miRNA) is single stranded short nucleotide that interferes with the mRNA post transcriptional regulation of the host. CD-44 is an intestinal stem cell marker which is increased in the DSS induced mice, but in lignin with/ without L. plantarum, the marker suppressed their expression due to an increase of E.cad expression in the epithelial cells (Fig. 6a, b and c). The miR-199 level was decreased in colitis mice, whereas Lignin and L. plantarum treatment showed significant P ≤ 0.05 miR-199 progression in colitis mice. These reports conclude that miR-199 is involved in the expression of stem cell marker CD-44 and showed reciprocal regulation in Lignin with/without L. plantarum treated

Discussion
The results explored the biocompatibility of Lignin against Lactobacillus plantarum and the result revealed that the lignin as a biologically compatible prebiotic to L. plantarum was not toxicity upto 5 µM. According to a recent study, less cytotoxicity of lignin makes them suitable candidates for nano system development for cancer medication delivery and therapeutic applications (Imlimthan et al. 2020). In support of this study, (Koh et al. 2013) reported that the prebiotic, tagatose significantly enhanced the growth of probiotic Lactobacillus. The result determined disease index and body weight was reduced and colon length was increased in the lignin with Lactobacillus group. The macroscopic and metabolic regulations were compared with the Dextran sodium sulphate (DSS) group.
Host-probiotic interactions improve gut immune homeostasis and regulate inflammatory markers (Hairul et al. 2014). The reduction in Interleukin (IL)-1, IL-6 and IL-8 levels in Tumor necrosis factor alpha (TNF-α)s-induced cells can be explained by the ability of BA to suppress anti-inflammatory mediators, as further confirmed in an in vivo model. Comparable findings were obtained in many in vitro studies. Lactobacillus acidophilus, Bacillus coagulans and Bacillus subtilis exhibited potential anti-inflammatory effects on colorectal adenocarcinoma cells (Caco2), dendritic and peripheral blood mononuclear (PBM) cells (Khalifa et al. 2022). These results confirm that gut laminal and epithelial cells of mucous play a major role in probiotic interactions via secreting inflammatory mediators such as chemoattractant proteins, interleukins and infiltration signals. Similarly, (Kangwan et al. 2022) reported that Lactobacillus pentosus, which was obtained from fermented tea leaves, improved colon abnormality. Treatment of probiotics significantly protected the colon from being inflamed by mitigating histological and clinical damage traits, enhancing the intestinal barrier integrity, and attenuating inflammation symptoms induced by DSS induction. (Hairul et al. 2011;Khalifa et al. 2022).
The crypt structure was abnormal in the DSS group due to crypt loss, epithelial cell death, and significant inflammatory cell infiltration. Whereas, DSS-induced colon shortening and acute inflammation were reduced by the Lactobacillus, lignin, and syerngistic therapies. A considerable decrease in colon length as a result of intestinal injury is a frequent hallmark of DSS-induced colitis (Rumi et al. 2004;Mizoguchi  and Mizoguchi 2008). Both macroscopic and microscopic observations revealed a decrease in the severity of DSS induced colitis across the treatment groups. Prebiotic fibres that are soluble and non-viscous were reported to alleviate the symptoms of inflammatory bowel diseases (Hardy et al. 2013). In rats, symbiotic Lactobacillus and fructo oligosaccharide supplementation was found to reduce intestinal and systemic inflammation in Crohn's disease (Lindsay 2006;Delcenserie et al. 2008).
Rumen Lactobacillus regulates the immune response and acts as an anti-inflammatory strain and balance the cytokine secretion, whereas the other two active strains were particular inhibition on anti-inflammatory cytokines (Arokiyaraj et al. 2014). Few studies reported that, the Lactobacillus sp. and some rumen bacteria stimulate low levels of proinflammatory cytokines (TNF-α, IL-6 and IL-8), while activating high levels of Interferon gamma (IFN-γ) and IL-12 these two active cytokines to promote cellular immunity and enhance the clearance of pathogens. Probiotic strains might boost host immune status through activation of the particular and nonspecific immune pathways. This also can involve modulating of humoral, cellular, and nonspecific immunity (Hairul et al. 2014;Delcenserie et al. 2008).
Several cytokines generated by lactic acid bacteria serve critical roles in immunological modulation, and they are involved in colitis pathophysiology. These pro-inflammatory cytokines are important in building an immunogenic setting and in the anti-inflammatory response (Kim et al. 2015;Mantovani et al. 1992). An earlier study revealed Lactobacillus and prebiotic (tagatose) have inhibited DSS induced pro-inflammatory cytokines and prevented initiation of the inflammatory response (Son et al. 2019). In this, study the levels of cytokines (TNF-α, INF-γ, IL-1β and TGF-β) increased in DSS-treated mice. The levels of all cytokines studied significantly decreased in Lactobacillus, lignin, and synergistic groups. STAT3 is involved in a number of autoimmune diseases, including inflammatory bowel disease (IBD) (Sugimoto 2008).
In both Ulcerative colitis (UC) and Crohn's disease (CD), there was an increase in total STAT3 protein relative to noninflammatory control cells, and total STAT3 corresponded with greater activated pSTAT3 in tissue sections from both UC and CD (Mudter et al. 2005). Lower expression of E-cadherin on gut epithelial and immune cells resulted in more gut inflammation in DSS-induced colitis (Ooi et al. 2013). In this study the mRNA of STAT 3 was not significantly changed by Lignin associated with L.plantarum treatment, where the protein expression was significantly down regulated. In the E.cad mRNA and protein expression in the lignin with or without L.plantarum were increased potentially which was compared to Lignin alone treated group. CD44 has been identified as a potential therapeutic target for inflammatory disorders such as IBD, rheumatoid arthritis, multiple sclerosis, and other autoimmune diseases (Wittig et al. 2002). The expression of CD44, was stronger in the UC-associated lesions of colon carcinoma (Mikami et al. 2000). It is well known that the expression of CD44 variant isoforms increases in inflammatory infiltrates in mouse and human colitis, and that blocking or deleting CD44 isoforms prevents the development of experimental colitis. Previous investigations have showed that established experimental colitis is cured by short-term administration of anti-CD44 antibody, which prevents T cell extravasation and recruitment to the intestinal mucosa (Farkas et al. 2005). CD44 was identified as a direct target of miR-199a and contributed to a significant role in carcinogenesis (Henry et al. 2010;Gao et al. 2015). As discussed above, aberrant expressions of CD44 induces the infiltration of immune cells into the lamina propria. It is well established that 3′-untranslated region of CD44 mRNA was the direct target of microRNA-199a-3p. Also, the overexpression of miR-199a-3p significantly inhibited CD44 expression (Gao et al. 2015). Thus induction of miR-199 expression targets/reduces the expression of CD44, which subsequently attenuates the infiltration of immune cells and finally plays an important role in the in control of DSS induced colitis. In this study revealed that the E.cad expression increased in the epithelial cells. The lignin with L.plantarum group controls the inflammatory progression in the colitis mice through miR-199.

Conclusion
In conclusion, we used a Dextran sodium sulphate (DSS)induced colitis mouse model to evaluate the synergic effects of Lignin and Lactobacillus plantarum against inflammatory bowel disease (IBD). Our findings suggest that combining Lignin with L.plantarum improves intestinal damage recovery and suppresses the recurrence of experimental colitis, indicating that this new protective or preventative strategy for IBD could be used in the food sector.