Probiotic Cocktail Alleviates Intestinal Inflammation Through Improving Gut Microbiota and Metabolites in Colitis Mice.

The modulation of the gut microbiome has been widely suggested as a promising therapeutic strategy for inflammatory bowel disease (IBD). Here, we established a novel probiotic cocktail to investigate its therapeutic role in acute colitis mice. During dextran sulfate sodium (DSS)-induced colitis, the mice were treated with the probiotic cocktail, fecal microbiota transplantation (FMT) from a healthy mice donor, or 5-aminosalicylic acid (5-ASA), respectively. The inflammatory responses were assessed by symptoms, serum inflammatory factors, and histological scoring. The intestinal barrier function was assessed by detecting tight junction proteins. Gut microbiota and its metabolites were further identified using 16S rDNA sequencing and a liquid chromatograph mass spectrometer (LC-MS/MS). Compared with FMT and 5-ASA treatment, the probiotic cocktail performed better in alleviating symptoms of colitis and decreasing disease activity score and mucosal inflammation. The probiotic cocktail also significantly decreased serum IL-17 level and increased JAM-1 expression in colon. The gut microbiota analysis confirmed that the beneficial effects of the probiotic cocktail were attributed to increasing anti-inflammatory bacteria Akkermansia, Bifidobacterium, and Blautia, while decreasing pro-inflammatory bacteria Parasutterella. The targeted metabolome analysis further indicated a rise in the production of Bifidobacterium-related short-chain fatty acids (SCFAs) such as propanoic acid and isobutyric acid after probiotics treatment. Taken together, the probiotic cocktail effectively alleviated intestinal inflammation through improving gut microbiota and metabolites in colitis mice, suggesting its great potential to be a novel therapeutic approach for IBD patients.

Treatment Response, Survival Benefit and Safety Profile of PD-1 Inhibitor Plus Apatinib Versus Apatinib Monotherapy in Advanced Colorectal Cancer Patients.

Purpose: Programmed cell death protein 1 (PD-1) inhibitor plus apatinib is reported to be a promising strategy for advanced cancers. Moreover, a PD-1 inhibitor or apatinib exerts a certain efficacy in advanced colorectal cancer (CRC), whereas their synergistic effect is unclear. This study aimed to evaluate the treatment efficacy and safety of a PD-1 inhibitor plus apatinib in advanced CRC patients. Methods: In total, 45 advanced CRC patients who received a PD-1 inhibitor plus apatinib (PD-1 inhibitor plus apatinib group, N=20) or apatinib monotherapy (apatinib group, N=25) as third-line therapies were enrolled in the current study. Results: The objective response rate (20.0% vs. 8.0%) (P=0.383) and disease control rate (70.0% vs. 52.0%) (P=0.221) were numerically increased in the PD-1 inhibitor plus apatinib group, respectively, compared with the apatinib group, but no statistical significance was observed. The median progression-free survival (PFS) was 7.5 versus 4.8 months; the 1-year PFS rate was 32.5% versus 9.9%; the median overall survival (OS) was 12.3 versus 8.7 months; and the 1-year OS rate was 50.7% versus 27.0% in the PD-1 inhibitor plus apatinib group versus the apatinib group, respectively. PFS (P=0.038) and OS (P=0.048) were prolonged in the PD-1 inhibitor plus apatinib group compared with the apatinib group. PD-1 inhibitor plus apatinib (versus apatinib) was independently associated with longer PFS (P=0.012) and OS (P=0.009). The majority of the adverse events were of grade 1-2, wherein the incidence was similar between groups, except for the fact that the incidence of capillary proliferation was elevated in the PD-1 inhibitor plus apatinib group compared with the apatinib group (25.5% versus 0.0%) (P=0.013). Conclusion: PD-1 inhibitor plus apatinib presents a potential improvement in efficacy and survival benefit compared with apatinib monotherapy, with tolerable safety in advanced CRC patients.

Long-term and continuous administration of Bacillus subtilis during remission effectively maintains the remission of inflammatory bowel disease by protecting intestinal integrity, regulating epithelial proliferation, and reshaping microbial structure and function.

Gut microbiota takes part in the pathogenesis of inflammatory bowel disease (IBD). Clinical research has found that probiotics have a beneficial effect on active ulcerative colitis, but to date, significant efficacy has rarely been found in the use of probiotics in the remission phase of ulcerative colitis and Crohn's disease. More studies are needed to assess the utilization of probiotics in IBD remission. In this study, we assessed the administration of Bacillus subtilis in remission and its possible mechanism in mice with IBD. Oral administration of B. subtilis was implemented for 6 weeks (dextran sulfate sodium (DSS)-P6w group), 2 weeks (DSS-P2w group) or 0 weeks (DSS-control(CT) group) in the remission phase in rodents with (DSS)-induced IBD. The body weight, colon length and disease activity index (DAI) were recorded, and colon H&E staining was performed. The expression of tight junction proteins (ZO-1 and occludin) mRNA and epithelium proliferation-related Ki67 was detected. Gut microbiota were tested by 16S rRNA sequencing. Administration of B. subtilis in remission effectively increased the body weight and colon length and decreased DAI in the DSS-P6w group compared with the DSS-CT group, but there is no significant difference between the DSS-P2w and DSS-CT groups. The epithelial integrity was improved, and the expression of ZO-1 and occludin increased due to administration of B. subtilis in remission, which was more evident in the DSS-P6w group. The expression of Ki67 increased in the DSS-CT group compared with that in the CT group. The administration of B. subtilis effectively down-regulated the expression of Ki67 in the DSS-P6w and DSS-P2w groups compared with the DSS-CT group. Furthermore, gut microbial structure was improved, with significantly decreased Escherichia/Shigella and Enterococcus, and increased Akkermansia and corresponding microbial function in the DSS-P6w group. Short-term administration of B. subtilis in the remission phase showed no significant improvement in mice with IBD. Long-term and continuous supplementation of B. subtilis in remission could effectively maintain the remission by protecting epithelial integrity, regulating proliferation of intestinal epithelial cells, and improving gut microbiota and the corresponding microbial function.

Gut microbiota dysbiosis signature is associated with the colorectal carcinogenesis sequence and improves the diagnosis of colorectal lesions.

BACKGROUND AND AIM: The gut microbiota is associated with colorectal lesions in cases of precancer and colorectal cancer (CRC). However, there are apparent differences in studies on the gut microbiota in the pathogenic sequence from precancer to cancer. Here, we characterize the gut microbiota signatures of colorectal precancer and cancer and test their utility in detecting colorectal lesions in two independent Chinese cohorts. METHODS: Stool samples collected from patients with precancer and CRC were subjected to 16S ribosomal RNA gene sequencing and metagenomic shotgun sequencing analyses, which revealed the microbial signatures of the two disease stages. RESULTS: In comparison with healthy controls, lower microbial richness and diversity were observed in precancer and intensive interbacterial associations were found in CRC. We identified 41 bacteria that showed gradual increases while 12 bacteria showed gradual decreases at the genus level gradually during the development of CRC. Novel CRC-associated pathogenetic species were identified. Species units that contributed to altered microbial functions were identified in CRC patients and healthy controls. The microbial panel showed a comparable ability to fecal immunochemical test (FIT) in detecting CRC. However, the combination of microbes and FIT significantly improved the detection ability and sensitivity of colon lesions based on 18 genera. Microbial network analysis revealed a significant positive correlation among beneficial microbes and a negative correlation in detrimental phenotypes. CONCLUSIONS: Microbial dysbiosis was revealed in colorectal lesions. The combination of microbial markers and FIT improved the CRC detection ability, which might assist in the early diagnosis of CRC.

Clinical significance and biological role of KLF17 as a tumour suppressor in colorectal cancer.

It has been reported that kruppel­like factor 17 (KLF17) acts as a tumour suppressor in several tissues and cancer cells, however, the molecular roles, the underlying mechanisms and clinical significance of KLF17 in colorectal cancer (CRC) have not been completely elucidated. In the present study, KLF17 protein expression was detected in 140 primary CRCs and paired adjacent non­tumour tissues using immunohistochemistry with tissue microarrays. The KLF17 mRNA expression was determined in 4 CRC cell lines and 20 pairs of the aforementioned tissues using reverse transcription quantitative polymerase chain reaction. The correlation between KLF17 expression and clinicopathologic characteristics was determined. Next, the functions of KLF17 in CRC were examined by cell proliferation, colony formation, adhesion, invasion and mouse xenograft assays. Methylation­specific PCR and bisulfite sequencing PCR were also carried out to investigate the promoter methylation status of KLF17 in CRC cells and tissues and explore the effects of lentiviral­mediated RNAi of UHRF1 on the methylation and expression of KLF17. The results revealed that KLF17 expression was abnormally decreased in CRC and associated with lymph node metastasis and unfavorable overall survival. Moreover, ectopic KLF17 expression suppressed CRC cell growth and invasion in vitro and in vivo. In addition, the downregulation of KLF17 was associated with the hypermethylation of the CpG nucleotides on the KLF17 promoter. The knockdown of the epigenetic regulator UHRF1 reduced the methylation level of the KLF17 promoter and inhibited CRC cell adhesion, invasion and epithelial­mesenchymal transition by upregulating KLF17. The present findings indicated that KLF17 may act as a tumour suppressor gene in CRC and a potential independent prognostic biomarker in CRC patients. UHRF1 can suppress KLF17 expression through the hypermethylation of its promoter in CRC. These results offer insights into the KLF17 expression regulation in CRC and suggest an inhibitory effect of KLF17 on tumourigenesis.

Micro Integral Membrane Protein (MIMP), a Newly Discovered Anti-Inflammatory Protein of Lactobacillus Plantarum, Enhances the Gut Barrier and Modulates Microbiota and Inflammatory Cytokines.

BACKGROUND/AIMS: Recent studies have demonstrated that the manipulation of the gut microbiome represents a promising treatment for inflammatory bowel disease (IBD). We previously identified micro integral membrane protein (MIMP) as the smallest domain of surface layer protein from Lactobacillus Plantarum. However, the therapeutic relevance of MIMP in IBD remains unknown. METHODS: We initially employed a dextran sodium sulphate (DSS)-induced colitis model and evaluated the effect of MIMP on the inflammation response, intestinal barrier and gut microbiota using histological examination, Fluorescein isothiocyanate-Dextran detection and pyrosequencing analysis respectively. We then established peripheral blood mononuclear cells (PBMCs) and an epithelial CaCO-2 co-culture model to investigate the regulatory role of MIMP in inflammatory cytokines. The level changes of inflammatory cytokines were detected using Enzyme-linked immunosorbent and real-time polymerase chain reaction assay. The involved regulatory mechanisms were investigated mainly using dual luciferase reporter and chromatin immunoprecipitation assay. RESULTS: In the DSS-induced colitis model, we observed that MIMP intervention effectively improved the body weight loss, increased the colon length and decreased disease activity index. Consistently, the inflammation scores in the MIMP treatment group were significantly lower than those in the DSS treatment group. Furthermore, MIMP intervention was found to successfully neutralize DSS treatment by decreasing the expression of pro-inflammatory cytokines (IFN-γ, IL-17 and IL-23) and increasing the expression of anti-inflammatory cytokines (IL-4 and IL-10). Notably, the permeability assay demonstrated that the MIMP treatment group was remarkably lower than that in the DSS treatment group. We also showed that MIMP improved gut microbiota dysbiosis caused by DSS-induced inflammation. Additionally, in PBMCs and the CaCO-2 co-culture model, MIMP showed an obvious suppressive effect on lipopolysaccharide-induced inflammation in a time- and dose-dependent manner. Furthermore, we revealed that MIMP could modulate inflammatory cytokine expression through the toll-like receptor 4 pathway and histone acetylation. CONCLUSIONS: Our results suggested that MIMP showed a significant anti-inflammatory effect through regulating the gut barrier, microbiota and inflammatory cytokines. MIMP may have translational relevance as clinically relevant therapy for IBD patients.

Identification of molecular mechanisms of glutamine in pancreatic cancer.

The aim of the present study was to explore the critical genes and molecular mechanisms in pancreatic cancer (PC) cells with glutamine. By analyzing microarray data GSE17632 from the Gene Expression Omnibus database, the DEGs between PC cells treated with glutamine and without glutamine were evaluated. Additionally, function enrichment analyses and protein-protein interaction (PPI) network construction of DEGs were performed. Network module and literature mining analyses were performed to analyze the critical DEGs in PC cells. In total, 495 genes were selected as DEGs between control and glutamine cells in PC. These DEGs were mainly enriched in several Gene Ontology (GO) terms in biological process, cellular components and molecular function. Additionally, they were also enriched in certain pathways, including metabolic pathways and the Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway. MYC, heat shock 70kDa protein 5 (HSPA5), interleukin 8 (IL8), and chemokine (C-X-C motif) receptor 4 (CXCR4) were hub genes in the PPI network. Furthermore, two sub-network modules of PPI network and two co-occurrence networks were obtained. The DEGs of MYC, HSPA5, IL18 and CXCR4 may exert important roles in molecular mechanisms of PC cells with glutamine.

SP1 mediates the link between methylation of the tumour suppressor miR-149 and outcome in colorectal cancer.

Although recent studies indicate that DNA methylation contributes to the down-regulation of microRNAs (miRNAs) in colorectal cancer (CRC), this field remains largely unexplored. To identify methylation-silenced miRNAs and clarify their role in CRC, we performed a microarray analysis and screened for miRNAs that were induced in CRC cells by 5-aza-2'-deoxycytidine treatment or by the knockdown of DNA methyltransferases. The DNA methylation status of the candidate miRNA was analysed by bisulphite sequencing PCR and methylation-specific PCR. We found that miRNA-149 (miR-149) was epigenetically silenced in CRC and down-regulation of miR-149 was associated with hypermethylation of the neighbouring CpG island (CGI). Quantitative RT-PCR analysis demonstrated that the miR-149 level was markedly reduced in 51.6% of the CRC tissues compared with matched non-cancerous tissues. In addition, low expression of miR-149 was associated with a greater depth of invasion (p = 0.012), lower 5-year survival rate (p = 0.025), and was found to be an independent prognostic factor for overall survival (p = 0.016) in a multivariate analysis. Moreover, transfection of miR-149 inhibited cell growth and invasion of CRC cells in vitro. We also identified mRNA for Specificity Protein 1 (SP1, Sp1), a potential oncogenic protein, as a target of miR-149. Our data suggest that, as a methylation-sensitive miRNA, miR-149 may play an important role as a tumour suppressor in CRC, which has prognostic and therapeutic implications.

Effects of Lactobacillus plantarum on gut barrier function in experimental obstructive jaundice.

AIM: To investigate the mechanisms of Lactobacillus plantarum (L. plantarum) action on gut barrier in preoperative and postoperative experimental obstructive jaundice in rats. METHODS: Forty rats were randomly divided into groups of sham-operation, bile duct ligation (BDL), BDL + L. plantarum, BDL + internal biliary drainage (IBD), and BDL + IBD + L. plantarum. Ten days after L. plantarum administration, blood and ileal samples were collected from the rats for morphological examination, and intestinal barrier function, liver function, intestinal oxidative stress and protein kinase C (PKC) activity measurement. The distribution and expression of the PKC and tight junction (TJ) proteins, such as occludin, zonula occludens-1, claudin-1, claudin-4, junction adhesion molecule-A and F-actin, were examined by confocal laser scanning microscopy, immunohistochemistry, Western blotting, real-time fluorescent quantitative polymerase chain reaction assay. RESULTS: L. plantarum administration substantially restored gut barrier, decreased enterocyte apoptosis, improved intestinal oxidative stress, promoted the activity and expression of protein kinase (BDL vs BDL + L. plantarum, 0.295 ± 0.007 vs 0.349 ± 0.003, P < 0.05; BDL + IBD vs BDL + IBD + L. plantarum, 0.407 ± 0.046 vs 0.465 ± 0.135, P < 0.05), and particularly enhanced the expression and phosphorylation of TJ proteins in the experimental obstructive jaundice (BDL vs BDL + L. plantarum, 0.266 ± 0.118 vs 0.326 ± 0.009, P < 0.05). The protective effect of L. plantarum was more prominent after internal biliary drainage ( BDL + IBD vs BDL + IBD + L. plantarum, 0.415 ± 0.105 vs 0.494 ± 0.145, P < 0.05). CONCLUSION: L. plantarum can decrease intestinal epithelial cell apoptosis, reduce oxidative stress, and prevent TJ disruption in biliary obstruction by activating the PKC pathway.

Functional characterization of MIMP for its adhesion to the intestinal epithelium.

The micro integral membrane protein (MIMP), the domain within the integral membrane protein of Lactobacillus plantarum CGMCC 1258, has been shown to adhere to mucin and antagonize the adhesion of enteroinvasive E. coli and enteropathogenic E. coli. To further characterize the functions of MIMP, we investigated its effects on the intestinal permeability, expression of tight junction (TJ) proteins and TJ ultrastructure in vitro and in vivo. We also determined the interaction between MIMP and dendritic cells (DCs). We observed that MIMP reduced intestinal permeability and restored the expression and distribution of TJ proteins in both NCM460 cell monolayers and in IL-10(-/-) mice. MIMP adhered to immature (i) DCs by binding to DC-SIGN, and induced DCs to produce anti-inflammatory cytokines and to mediate Th2 differentiation. Moreover, MIMP stimulated the expression of anti-inflammatory cytokines in colonic mucosa and attenuated colitis in IL-10(-/-) mice. In conclusion, MIMP is the main functional component of L. plantarum that contributes to its protective effects, and thus may be a potential therapeutic agent for intestinal diseases.

Identification of the Lactobacillus SLP domain that binds gastric mucin.

Surface layer proteins (SLPs) of lactobacillus bacteria have some structural regions responsible for adhesion to the intestinal epithelium. To identify the SLP and the smallest domain within the protein that is responsible for the adhesion of the bacterium to the intestinal epithelium, L. plantarum strain CGMCC1258 was investigated in this study. Using bioinformatics and molecular techniques, we first identified and purified a novel protein, integral membrane protein-2 (IMP-2, 33-45 kDa) responsible for adhesion to gastric mucin. Truncated forms of IMP-2 were then constructed and expressed, and the amino acids from 515 to 575 (designated micro IMP, MIMP) was identified as the smallest domain responsible for adhesion to gastric mucin. Competing assay was performed, which further confirmed the ability of MIMP to compete with enteroinvasive E. coli and enteropathogenic E. coli to adhere to cells of a normal colon cell line, NCM460. Furthermore, MIMP could maturate dendritic cells. These findings set a foundation for further investigation on the role of MIMP in the treatment and prevention of inflammation-related diseases of the intestine.

Lactobacillus plantarum surface layer adhesive protein protects intestinal epithelial cells against tight junction injury induced by enteropathogenic Escherichia coli.

Lactobacillus plantarum (LP) has previously been used for the treatment and prevention of intestinal disorders and disease. However, the role of the LP surface layer adhesive protein (SLAP) in inhibition of epithelial cell disruption is not fully understood. The aim of the present study was to investigate the protective effects of purified SLAP on Caco-2 cells infected with enteropathogenic Escherichia coli (EPEC). The role of ERK in LP-mediated inhibition of tight junction (TJ) injury was also evaluated in order to determine the molecular mechanisms underlying the protective effects of LP in epithelial cells. SLAP was extracted and purified from LP cells using a porcine stomach mucin-Sepharose 4B column. SLAP-mediated inhibition of bacterial adhesion was measured using a competition-based adhesion assay. Expression of TJ-associated proteins, maintenance of TJ structure, and levels of extracellular signal regulated kinase (ERK) and ERK phosphorylation were assessed in SLAP-treated cells by a combination of real-time PCR, western blotting, and immunofluorescence microscopy. Cell permeability was analyzed by measurement of trans-epithelial electrical resistance (TER) and dextran permeability. The effect of SLAP on levels of apoptosis in epithelial cells was assessed by flow cytometry. Results from these experiments revealed that treatment with SLAP decreased the level of adhesion of EPEC to Caco-2 cells. SLAP treatment also enhanced expression of TJ proteins at both the mRNA and protein levels and affected F-actin distribution. Although ERK levels remained unchanged, ERK phosphorylation was increased by SLAP treatment. Caco-2 cells treated with SLAP exhibited increased TER and decreased macromolecular permeability, which was accompanied by a decrease in the level of apoptosis. Together, these results suggest that LP-produced SLAP protects intestinal epithelial cells from EPEC-induced injury, likely through a mechanism involving ERK activation.

Protective effects of Lactobacillus plantarum against epithelial barrier dysfunction of human colon cell line NCM460.

AIM: To investigate the effects of Lactobacillus plantarum (L. plantarum) in the intestinal permeability and expression of tight junction (TJ) using the normal human colon cell line NCM460. METHODS: Paracellular permeability of NCM460 monolayers was determined by transepithelial electrical resistance and dextran permeability. Expression of TJ proteins in NCM460 cell monolayers was detected by Western blotting and quantitative real-time polymerase chain reaction. RESULTS: L. plantarum played an important role in increasing transepithelial electrical resistance and decreasing the permeability to macromolecules of NCM460 monolayers against the disruption caused by enteropathogenic Escherichia coli (E. coli) or enteroinvasive E. coli. L. plantarum also prevented the decrease in the expression of TJ proteins and F-actin in NCM460 cells. CONCLUSION: L. plantarum can protect against dysfunction of NCM460 intestinal epithelial barrier caused by enteropathogenic E. coli or enteroinvasive E. coli, and thus can be a potential candidate of therapeutic agents for the treatment of intestinal diseases.

Lactobacillus plantarum consumption increases PepT1-mediated amino acid absorption by enhancing protein kinase C activity in spontaneously colitic mice.

Although probiotic consumption has generally been shown to have many beneficial effects for the prevention and treatment of inflammatory bowel disease, the effects of Lactobacillus plantarum (LP) on intestinal nutrient absorption, particularly oligopeptide transporter 1 (PepT1)-mediated absorption of dietary protein under inflammatory conditions, has not yet been characterized. In this study, we first investigated the effects of LP consumption on plasma amino acid concentrations and PepT1-mediated absorption of cephalexin in the small intestine of wild-type (WT) mice and interleukin-10 knockout (IL-10(-/-)) mice, a model of spontaneous colitis. We then analyzed expression and distribution of PepT1 and protein kinase C (PKC) activity in the jejunum of these mice. LP consumption (10(9) colony-forming units/0.5 mL) delivered by gavage once per day for 4 wk increased the total plasma amino acid concentration and the concentration of plasma cephalexin through enhancement of PepT1-mediated uptake in LP treated IL-10(-/-) mice compared with IL-10(-/-) mice. However, Western blotting and quantitative PCR analysis revealed no significant differences in PepT1 protein and mRNA expression between LP-treated and untreated mice. Additionally, immunofluorescence analysis showed that PepT1 did not appear to be mislocalized in IL-10(-/-) mice. Interestingly, IL-10(-/-) mice had significantly lower PKC activity and expression of phosphorylated PKC compared with WT mice, and these decreases could be prevented by LP treatment. These data suggest that consumption of LP enhances PepT1-mediated amino acid absorption, likely through alterations in PKC activity, as opposed to changes in expression or distribution of PepT1 in the small intestine of IL-10(-/-) mice.

Lactobacillus plantarum ameliorates colonic epithelial barrier dysfunction by modulating the apical junctional complex and PepT1 in IL-10 knockout mice.

Probiotics are efficacious in the treatment of inflammatory bowel disease. However, the precise mechanisms remain unknown. To determine whether probiotic Lactobacillus plantarum (LP) ameliorates colonic epithelial barrier dysfunction present in interleukin-10 knockout (IL-10⁻(/)⁻) mice, IL-10⁻(/)⁻ and wild-type mice received LP or the vehicle for 4 wk. Colitis was assessed by histological scores and clinical manifestation, and gut paracellular permeability was measured by Ussing chamber. Oligopeptide transporter 1 (PepT1)-mediated transepithelial transport was evaluated by measuring the plasma cephalexin concentration. The expression and distribution of apical junctional complex (AJC) proteins and PepT1 were determined by Western blotting and immunofluorescence and their mRNA by reverse transcriptase-PCR. Spontaneous colitis was observed in all IL-10⁻(/)⁻ mice in which paracellular permeability was increased, in conjunction with decreased expression and redistribution of zonula occludens-1, occludin, claudin-1, and ß-catenin. PepT1 expression was increased, accompanied with an enhanced cephalexin transport. Colonic epithelial barrier dysfunction was further confirmed by increased bacterial translocation and proinflammatory cytokine production. Treatment with LP decreased colonic paracellular permeability with restoration of expression and distribution of AJC proteins and partially prevented PepT1 expression and cephalexin transport in IL-10⁻(/)⁻ mice. Moreover, treatment with LP also prevented bacterial translocation and proinflammatory cytokine production in IL-10⁻(/)⁻ mice. Results from this study indicated that treatment with LP may ameliorate colonic epithelial barrier dysfunction in IL-10⁻(/)⁻ mice, by modulating the AJC- and PepT1-mediated transepithelial transport.

HnRNP K and PDI marked response to chemotherapy to human colorectal cancer cells.

UNLABELLED: 5-Fluorouracil has been the chemotherapy agent of first-choice for colorectal cancer for many years, but since there are no proven predictors of a patient's response to therapy, all patients receive similar treatment. Consequently, identification of biomarkers for therapeutic effect is crucial for the development of novel therapeutic strategies. Two human colorectal cancer cell lines of different metastatic potential (LoVo and SW480) were studied. IC50 of 5-FU for both cell lines were measured by 3-(4,5-dimethy-lthiazol-2-yl)-2,5-diphenyltetrazolium assay and validated by cell cycle analysis. Then the cell lines were treated with 5-FU at IC50 concentration and protein was extracted for 2-DE. Differential protein spots were examined by MALDI-TOF/TOF MS. The expression levels of the different proteins were further confirmed by Western blot and immunofluorescence analyses. Eleven proteins were identified. Expression of heterogeneous nuclear ribonucleoprotein K (hnRNP K) in LoVo cells was higher than in SW480 cells, while protein disulfide isomerase (PDI) displayed the opposite trend. After treatment with 5-FU, the expression of hnRNP K in LoVo decreased more significantly than in SW480, while PDI in SW480 increased more significantly than in LoVo cells. CONCLUSION: hnRNP K and PDI in the two cell lines have different expression characteristics. The sensitivity to 5-FU is not consistent in tumor progression. It may assist in development of novel treatment strategies for colorectal cancer metastasis.

Lactobacillus plantarum inhibits intestinal epithelial barrier dysfunction induced by unconjugated bilirubin.

Although a large number of in vitro and in vivo tests have confirmed that taking probiotics can improve the intestinal barrier, few studies have focused on the relationship between probiotics and the intestinal epithelial barrier in hyperbilirubinaemia. To investigate the effects of and mechanisms associated with probiotic bacteria (Lactobacillus plantarum; LP) and unconjugated bilirubin (UCB) on the intestinal epithelial barrier, we measured the viability, apoptotic ratio and protein kinase C (PKC) activity of Caco-2 cells. We also determined the distribution and expression of tight junction proteins such as occludin, zonula occludens (ZO)-1, claudin-1, claudin-4, junctional adhesion molecule (JAM)-1 and F-actin using confocal laser scanning microscopy, immunohistochemistry, Western blotting and real-time quantitative PCR. The present study demonstrated that high concentrations of UCB caused obvious cytotoxicity and decreased the transepithelial electrical resistance (TER) of the Caco-2 cell monolayer. Low concentrations of UCB inhibited the expression of tight junction proteins and PKC but could induce UDP-glucuronosyltransferases 1 family-polypeptide A1 (UGT1A1) expression. UCB alone caused decreased PKC activity, serine phosphorylated occludin and ZO-1 levels. After treatment with LP, the effects of UCB on TER and apoptosis were mitigated; LP also prevented aberrant expression and rearrangement of tight junction proteins. Moreover, PKC activity and serine phosphorylated tight junction protein levels were partially restored after treatment with LP, LP exerted a protective effect against UCB damage to Caco-2 monolayer cells, and it restored the structure and distribution of tight junction proteins by activating the PKC pathway. In addition, UGT1A1 expression induced by UCB in Caco-2 cells could ameliorate the cytotoxicity of UCB.

Effects of oral Lactobacillus plantarum on hepatocyte tight junction structure and function in rats with obstructive jaundice.

Surgery and infection are prominent risk factors for the development of obstructive cholestasis which in turn is associated with failure of the liver barrier. We studied the effects of oral Lactobacillus plantarum (LP) supplementation on endotoxemia, oxidative stress, apoptosis, and tight junctions of hepatocytes in an experimental model of obstructive jaundice. Fifty male Wistar rats were randomly divided into five groups of 10 each: group I, sham-operated; group II, ligation and division of the common bile duct (BDL); group III, BLD followed by oral LP treatment; group IV, BDL followed by internal biliary drainage (IBD); group V, BDL followed by IBD and oral LP treatment. Hepatocyte apoptosis, plasma reduced glutathione (GSH) and oxidized glutathione (GSSG) levels, and portal blood endotoxin levels were measured and changes in tight junction-associated proteins occludin, claudin-1, claudin-4, and ZO-1 were observed. Compared to the sham-operated group I, significant increases in endotoxemia, apoptosis, and GSSG were observed in group II and significant decreases were observed in group V. Tight junctions were destroyed in group II animals but were not in animals treated with oral LP (groups III and V). An increase in occludin, claudin-1, claudin-4, and ZO-1 mRNA and protein levels were detected in livers in LP-treated animals (group V) compared with group II levels. Oral LP treatment of rats with obstructive jaundice assisted in the return of active hepatic barrier function. These results may lead to treatments to prevent the deleterious effects of obstructive jaundice.

[Proteomic research of biomarker of colorectal cancer metastasis].

OBJECTIVE: To explore the potential markers of colorectal cancer metastasis and the influence of 5-FU on differentially expressed proteins by using proteomic technology, and to elucidate the mechanism of colorectal cancer metastasis. METHODS: Human colorectal carcinoma cell lines of different metastatic potential, Lovo and SW480 were conventionally cultured, and the protein was extracted. 50% inhibitory concentration (IC(50)) of 5-FU to these two cell lines was measured by MTT assay. Proteins of these two cell lines after intervention by 5-FU at IC(50) were extracted, then 2-dimensional gel electrophoresis was conducted for the proteins. The differential protein spots were examined by mass spectrometry and analyzed by bioinformatics. Difference of expressed proteins in two cell lines before and after the intervention of 5-FU was validated by Western blot and immunofluorescence. RESULTS: Eleven differentially expressed proteins were identified by 2-dimensional gel electrophoresis and mass spectrometry. The hnRNP K protein and PDI were selected to be examined by Western blot and immunofluorescence. Results revealed that the expression of hnRNP K in Lovo was higher than that in SW480, while the expression of PDI was lower in Lovo. After intervention by 5-FU at IC(50), the expression of hnRNP K in Lovo decreased more as compared to SW480, while the expression of PDI in SW480 increased more as compared to Lovo. CONCLUSION: There are significant differences in expression of hnRNP K and PDI proteins between Lovo and SW480 cell lines, and the proteins alter regularly after 5-FU intervention.

Heterogeneous nuclear ribonucleoprotein A1 is identified as a potential biomarker for colorectal cancer based on differential proteomics technology.

Colorectal cancer (CRC) is the third most common cancer worldwide and has poor prognosis. To identify the proteins involved in colorectal carcinogenesis, we employed 2-DE and MALDI-TOF/TOF-based proteomics approach to study the differentially expressed proteins in tumor and adjacent nontumor tissue samples. Samples from 10 colorectal patients were analyzed. Of the 7 significantly and consistently altered proteins identified, hnRNP A1 was one of the most significantly altered proteins and its overexpression was confirmed using RT-PCR and Western blot analyses. Immunohistochemical examination showed that the enhanced expression of hnRNP A1 was correlated with the increasing severity of colorectal tissue and the progression of the colorectal cancer, as well as UICC (International Union against Cancer) staging, histo-differentiation, recurrence and decreased survival. By developing a highly sensitive immunoassay, hnRNP A1 could be detected in human serum and was significantly elevated in CRC patients compared with healthy volunteers. We proposed that hnRNP A1 could be considered as a novel serum tumor marker for CRC that may have significance in the detection and in the management of patients with this disease. Knockdown of hnRNP A1 expression by RNA interference led to the significant suppression of the cell growth in colorectal cancer SW480 cells in vitro. These data suggested that hnRNP A1 may be a potential biomarker for early diagnosis, prognosis, and monitoring in the therapy of colorectal cancer. Further studies are needed to fully assess the potential clinical value of this biomarker candidate.