Hailong Cao

Berberine promotes recovery of colitis and inhibits inflammatory responses in colonic macrophages and epithelial cells in DSS-treated mice

Inflammatory bowel disease (IBD) results from dysregulation of intestinal mucosal immune responses to microflora in genetically susceptible hosts. A major challenge for IBD research is to develop new strategies for treating this disease. Berberine, an alkaloid derived from plants, is an alternative medicine for treating bacterial diarrhea and intestinal parasite infections. Recent studies suggest that berberine exerts several other beneficial effects, including inducing anti-inflammatory responses. This study determined the effect of berberine on treating dextran sulfate sodium (DSS)-induced intestinal injury and colitis in mice. Berberine was administered through gavage to mice with established DSS-induced intestinal injury and colitis. Clinical parameters, intestinal integrity, proinflammatory cytokine production, and signaling pathways in colonic macrophages and epithelial cells were determined. Berberine ameliorated DSS-induced body weight loss, myeloperoxidase activity, shortening of the colon, injury, and inflammation scores. DSS-upregulated proinflammatory cytokine levels in the colon, including TNF, IFN-γ, KC, and IL-17 were reduced by berberine. Berberine decreased DSS-induced disruption of barrier function and apoptosis in the colon epithelium. Furthermore, berberine inhibited proinflammatory cytokine production in colonic macrophages and epithelial cells in DSS-treated mice and promoted apoptosis of colonic macrophages. Activation of signaling pathways involved in stimulation of proinflammatory cytokine production, including MAPK and NF-κB, in colonic macrophages and epithelial cells from DSS-treated mice was decreased by berberine. In summary, berberine promotes recovery of DSS-induced colitis and exerts inhibitory effects on proinflammatory responses in colonic macrophages and epithelial cells. Thus berberine may represent a new therapeutic approach for treating gastrointestinal inflammatory disorders.

Systematic Review: Adverse Events of Fecal Microbiota Transplantation

Background Fecal microbiota transplantation (FMT) is a microbiota-based therapy that shows therapeutic potential in recurrent or refractory Clostridium difficile infections and other intestinal or extra-intestinal disorders. Nonetheless, adverse events (AEs) remain a major challenge in the application of FMT. Aim To review the AEs of FMT and to address the concerns of safety during the procedure. Methods Publications were retrieved in the databases of Medline, Embase and Cochrane Library. AEs were classified according to their causality with FMT or their severity. Results A total of 7562 original articles about FMT were identified in this study, 50 of them fulfilled the inclusion criteria. Totally 78 kinds of AEs were revealed enrolled in these 50 selected publications. The total incidence rate of AEs was 28.5%. Among the 42 publications, 5 kinds were definitely and 38 kinds were probably related to FMT. The commonest FMT-attributable AE was abdominal discomfort, which was reported in 19 publications. For upper gastrointestinal routes of FMT, 43.6% (89/204) patients were compromised by FMT-attributable AE, while the incidence dropped to 17.7% (76/430) for lower gastrointestinal routes. In contrast, the incidences of serious adverse events (SAEs) were 2.0% (4/196) and 6.1% (40/659) for upper and lower gastrointestinal routes, respectively. A total of 44 kinds of SAEs occurred in 9.2% patients, including death (3.5%, 38/1089), infection (2.5%, 27/1089), relapse of inflammatory bowel diseases (0.6%, 7/1089) and Clostridium difficile infection (0.9%, 10/1089). Conclusion Consequently, both AEs and SAEs are not rare and should be carefully monitored throughout FMT. However, high quality randomized controlled trials are still needed for the more definite incidence of AEs of FMT.

Activation of the Epidermal Growth Factor Receptor in Macrophages Regulates Cytokine Production and Experimental Colitis

Macrophages regulate innate immunity to maintain intestinal homeostasis and play pathological roles in intestinal inflammation. Activation of the epidermal growth factor receptor (EGFR) promotes cellular proliferation, differentiation, survival, and wound closure in several cell types. However, the impact of EGFR in macrophages remains unclear. This study was to investigate whether EGFR activation in macrophages regulates cytokine production and intestinal inflammation. We found that EGFR was activated in colonic macrophages in mice with dextran sulfate sodium (DSS)–induced colitis and in patients with ulcerative colitis. DSS-induced acute colitis was ameliorated, and recovery from colitis was promoted in Egfrfl/flLysM-Cre mice with myeloid cell–specific deletion of EGFR, compared with LysM-Cre mice. DSS treatment increased IL-10 and TNF levels during the acute phase of colitis, and increased IL-10 but reduced TNF levels during the recovery phase in Egfrfl/flLysM-Cre mice. An anti–IL-10 neutralizing Ab abolished these effects of macrophage-specific EGFR deletion on DSS-induced colitis in Egfrfl/flLysM-Cre mice. LPS stimulated EGFR activation and inhibition of EGFR kinase activity enhanced LPS-stimulated NF-κB activation in RAW 264.7 macrophages. Furthermore, induction of IL-10 production by EGFR kinase-blocked RAW 264.7 cells, in response to LPS plus IFN-γ, correlated with decreased TNF production. Thus, although selective deletion of EGFR in macrophages leads to increases in both pro- and anti-inflammatory cytokines in response to inflammatory stimuli, the increase in the IL-10 level plays a role in suppressing proinflammatory cytokine production, resulting in protection of mice from intestinal inflammation. These results reveal an integrated response of macrophages regulated by EGFR in intestinal inflammatory disorders.

Berberine Inhibits Proliferation and Down-Regulates Epidermal Growth Factor Receptor through Activation of Cbl in Colon Tumor Cells

Berberine, an isoquinoline alkaloid, is an active component of Ranunculaceae and Papaveraceae plant families. Berberine has been found to suppress growth of several tumor cell lines in vitro through the cell-type-dependent mechanism. Expression and activation of epidermal growth factor receptor (EGFR) is increased in colonic precancerous lesions and tumours, thus EGFR is considered a tumour promoter. The aim of this study was to investigate the effects and mechanisms of berberine on regulation of EGFR activity and proliferation in colonic tumor cell lines and in vivo. We reported that berberine significantly inhibited basal level and EGF-stimulated EGFR activation and proliferation in the immorto Min mouse colonic epithelial (IMCE) cells carrying the APC min mutation and human colonic carcinoma cell line, HT-29 cells. Berberine acted to inhibit proliferation through inducing G1/S and G2/M cell cycle arrest, which correlated with regulation of the checkpoint protein expression. In this study, we also showed that berberine stimulated ubiquitin ligase Cbl activation and Cbls interaction with EGFR, and EGFR ubiquitinylation and down-regulation in these two cell lines in the presence or absence of EGF treatment. Knock-down Cbl expression blocked the effects of berberine on down-regulation of EGFR and inhibition of proliferation. Furthermore, berberine suppressed tumor growth in the HT-29 cell xenograft model. Cell proliferation and EGFR expression level was decreased by berberine treatment in this xenograft model and in colon epithelial cells of APC min/+ mice. Taken together, these data indicate that berberine enhances Cbl activity, resulting in down-regulation of EGFR expression and inhibition of proliferation in colon tumor cells.

Secondary bile acid‐induced dysbiosis promotes intestinal carcinogenesis

The gut microbiota plays an important role in maintaining intestinal homeostasis. Dysbiosis is associated with intestinal tumorigenesis. Deoxycholic acid (DCA), a secondary bile acid increased by a western diet, correlates with intestinal carcinogenesis. However, evidence relating bile acids, intestinal microbiota and tumorigenesis are limited. In our study, we investigated the effect of DCA on induction of intestinal dysbiosis and its roles in intestinal carcinogenesis. Alteration of the composition of the intestinal microbiota was induced in DCA‐treated APCmin/+ mice, which was accompanied by impaired intestinal barrier, gut low grade inflammation and tumor progression. The transfer of fecal microbiota from DCA‐treated mice to another group of Apcmin/+ mice increased tumor multiplicity, induced inflammation and recruited M2 phenotype tumor‐associated macrophages. Importantly, the fecal microbiota transplantation activated the tumor‐associated Wnt/β‐catenin signaling pathway. Moreover, microbiota depletion by a cocktail of antibiotics was sufficient to block DCA‐induced intestinal carcinogenesis, further suggesting the role of dysbiosis in tumor development. Our study demonstrated that alteration of the microbial community induced by DCA promoted intestinal carcinogenesis.

Dysbiosis contributes to chronic constipation development via regulation of serotonin transporter in the intestine

Chronic constipation is a prevalent functional gastrointestinal disorder accompanied with intestinal dysbiosis. However, causal relationship between dysbiosis and constipation remains poorly understood. Serotonin transporter (SERT) is a transmembrane transport protein which re-uptakes excessive 5-hydroxytryptamine (5-HT) from effective location to terminate its physiological effects and involves in regulating gastrointestinal motility. In this study, fecal microbiota from patients with constipation and healthy controls were transplanted into the antibiotic depletion mice model. The mice which received fecal microbiota from patients with constipation presented a reducing in intestinal peristalsis and abnormal defecation parameters including the frequency of pellet expulsion, fecal weight and fecal water content. After fecal microbiota transplantation, the SERT expression in the colonic tissue was significantly upregulated, and the content of 5-HT was decreased which negatively correlated with the gastrointestinal transit time. Moverover, fecal microbiota from the mice which received fecal microbiota from patients with constipation also upregulated SERT in Caco-2 cells. Besides, this process accompanied with the decreased abundance of Clostridium, Lactobacillus, Desulfovibrio, and Methylobacterium and an increased tend of Bacteroides and Akkermansia, which also involved in the impairment of intestinal barrier after FMT. Taken together, intestinal dysbiosis may upregulate the SERT expression and contribute to the development of chronic constipation.

Is surveillance colonoscopy necessary for patients with sporadic gastric hyperplastic polyps

Background Gastric polyps, such as adenomas and hyperplastic polyps, can be found in various colonic polyposis syndromes. Unlike in sporadic gastric adenomas, in which the increased risk of colorectal neoplasia has been well characterized, information in sporadic gastric hyperplastic polyps was limited. Aim To evaluate the association of sporadic gastric hyperplastic polyps with synchronous colorectal neoplasia in a large cohort. Methods Patients with sporadic gastric hyperplastic polyps who underwent colonoscopy simultaneously or within six months were consecutively enrolled. Each patient was compared with two randomly selected age and sex matched controls without gastric polyps who also underwent colonoscopy in the same period. Data of patients’ demographics and characteristics of the gastrointestinal polyps were documented. Results A total of 261 cases in 118,576 patients who underwent esophagogastroduodenoscopy were diagnosed as sporadic gastric hyperplastic polyps, and 192 of 261 (73.6%) patients underwent colonoscopy. Colorectal neoplasias were identified in 46 (24.0%) of 192 cases and in 40 (10.4%) of 384 controls (P＜0.001). The mean size and distribution of colorectal neoplasias were not significantly different between the two groups. There was a significantly higher rate of colorectal adenoma (odds ratio [OR] 3.2, 95% confidence interval [CI] 1.9–5.3) in the gastric hyperplastic polyps group than in the control group, while the prevalence of colorectal cancer was similar in the two groups. Logistic regression analysis also suggested that the presence of gastric hyperplastic polyps (OR 2.5, 95% CI 1.5–4.0) was an independent risk factor for colorectal neoplasias. Conclusion The risk of colorectal adenoma increases in patients with sporadic gastric hyperplastic polyps, and surveillance colonoscopy for these patients should be considered.

Mo1674 Upregulation of Mucin Production by a Probiotic-Derived Protein Through Activation of EGF Receptor

Hassan Brim, Edward Lee, Scot Dowd, Adeyinka Laiyemo, Hassan Ashktorab Background: Linkage of specific bacterial markers to colorectal pathogenesis has been hampered by limited knowledge of the colonic microbiota and changing bacterial classification schemes. More than 80% of the colonic microbiota is not cultivatable and can only be assessed through metagenomic analysis. Aim: To perform a metagenomic analysis of 10 colon cancer tumors and their matched normal tissues. Methods: Metagenome sequencing was conducted on the illumina 2x150bp sequencing platform. Briefly DNA was prepared and libraries created using nextera library preparation kits. Sequencing was performed on each sample. For all samples an average of 20 million reads were obtained. Paired Sequencing reads were joined and entered into MG-RAST for metagenome analysis. The data was compared to M5NR using a maximum e-value of 1e-5, a minimum identity of 60 %, and a minimum alignment length of 15 measured in amino acids for protein databases. Results: Metagenomes were compared using best hit classification in two groups (normal and tumor). Based upon this best hit classification at the kingdom level in MG-RAST it was found, using one-way ANOVA, that tumor samples had a significantly higher classification related to Microviridae (bacteriophage) specifically related to Enterobacteria phage with a P= 0.0028. The other notable significant difference was the archaea. It was found at the class level that methanobacteria had higher classification hits in the normal samples (p = 0.03). Based upon functional gene abundance classification it was found that phage (P=0.007) as well as iron acquisition and metabolism genes (p= 0.05) were significantly different between the groups. Further analysis showed that as expected from the taxonomic classification data that tumor samples had higher levels of phage related functional classifications primarily related to phage capsid proteins. Other notable findings consisted of bacterial protein acetylation and deacetylation genes that were found to be significantly higher in normal tissue compared to tumor.

T1158 Ameliorating Intestinal Inflammation and Preventing Colon Tumor Development by the Plant Extract, Berberine

Background. Berberine, an isoquinoline alkaloid derived from plants, has been used as a traditional medicine for several diseases, including bacterial diarrhea. Recently, Berberine has been shown to suppress tumor cell growth and regulate several signaling pathways involved in apoptosis and cell cycle arrest In Vitro. However information is limited regarding mechanisms of Berberines action in diseases. This study was designed to investigate Berberines anti-tumor and anti-inflammatory effects. Methods. 7 patients with familial adenomatous polyposis history and recurrent colon adenomatous polyps were enrolled for oral Berberine treatment (0.1 g, 3 times/day) immediately following polypectomy and patients followed-up up to two years. Animal studies were performed on C57BL/6 mice administered 3% DSS in drinking water, or water only, for 7 days. Mice were given Berberine (200 μg/ g body weight, once daily) via gavage from day 4-7. Colon sections were prepared for H&E staining to determine intestinal tissue injury score (0: normal, 18: severe colitis). Mouse colon epithelial cells derived from wt Immorto (YAMC), Immorto-Min mice carrying APC min mutation (IMCE), and IMCE cells over-expression of Ras (IRas) were treated with Berberine (25 μM 200 μM) in the presence or absence of TNF (100 ng/ml), EGF (10 ng/ ml) or caspase inhibitor for detecting apoptosis using Annexin staining and TUNEL assay, signaling by Western blot analysis, and colony formation in agar. Results. 6 (7 total) patients showed no recurrence of polyps during the 2-year Berberine treatment. Berberine induced apoptosis in IMCE and IRas, but not YAMC cells and inhibited IRas cell colony formation in a concentration-dependent manner. Berberine stimulated p53 activation and blocked TNF-induced NFκB activation. In addition, Berberine inhibited EGF-stimulated EGF receptor activation and cell proliferation. However, Berbeine did not activate caspase 3 or 9 nor did the caspase inhibitor prevent Berberine-induced apoptosis. DSS-induced colitis (score: 15±0.9) was significantly reduced by Berberine treatment (score: 10±1.3, p<0.05). Conclusion. Berberine exerts the anti-tumor effect, which may be through induction of tumor cell apoptosis and inhibition of the action of tumor-promoting factors, including EGF and TNF. In addition, anti-inflammatory effects of Berberine may contribute to prevention of colitisassociated tumor development. The role of p53 activation and NFκB inhibition by Berberine in prevention of tumor development and inflammation is under investigation. Thus, Berberine may serve as a potential alternative approach for treating intestinal inflammatory diseases and tumor development.