P141 GREEN TEA POLYPHENOL EPIGALLOCATECHIN-3-GALLATE DIFFERENTIALLY MODULATES ENDOPLASMIC RETICULUM STRESS-MEDIATED WOUND HEALING PROCESS IN SUBEPITHELIAL MYOFIBROBLASTS

Epigallocatechin-3-gallate (EGCG) has multifaceted roles in the preclinical treatment of diseases including liver and lung fibrosis. EGCG has also been tested with fewer reported side effects than therapeutic drugs. Previously we showed that increase in endoplasmic reticulum (ER) stress response in subepithelial myofibroblasts (SEMF) contributes to activation of TGF-β1 and resultant intestinal fibrosis in patients with fibrostenotic Crohn’s disease. Moreover, different migratory potential of myofibroblasts isolated from inflamed, fibrostenotic, or fistulized Crohn’s disease mucosa could be an explanation for impaired or excess wound healing and subsequently for fistula or fibrosis in patients with Crohn’s disease. To investigate the effect of EGCG on ER stress-mediated wound healing process, SEMF were isolated from normal ileum and affected ileum in the same patient with Crohn’s disease. Cells were cultured and treated with EGCG (10 μg/ml), AG1024 (a tyrosine kinase inhibitor, 6 μM), U1026 (an ERK inhibitor, 6 μM), LY294002 (a PI3K inhibitor, 10 μM), SB202190 (a p38 inhibitor, 50 nM), 4-PBA (a chemical chaperone, 10 μg/ml), and MG132 (a NF-KB inhibitor, 3 μM) for 2 hours in serum free medium. Cell lysates were obtained for Western blot analysis. An ER stress agonist tunicamycin (5 μg/ml) was incubated with SEMF for different time points. Wound healing assay was used in a cell monolayer, capturing the images at the beginning and at regular intervals during cell migration to close the wound, and comparing the images to quantify the migratory potential of the cells. In vivo effect of EGCG was tested in a murine TNBS colitis model and observed by Storz Coloview standard operating procedures. Here we showed that EGCG further decreased endogenous GRP78 protein expression by 18∼29±1.5% in SEMF compared to that treated with different inhibitors targeting other non-ER stress signals. EGCG prevented tunicamycin-induced migratory potential of SEMF isolated from normal ileum by 40±2.5%, 65±3.3% after 48 and 72 hours, as well as cell proliferation by 85±3.3%, 120±6.1% after 48 and 72 hours, respectively. Moreover, EGCG also further decreased cell migratory potential of SEMF isolated from affected ileum by 15±1.2% and 50±1.8% compared to the control group after 48 and 72 hours, respectively. Coloview showed that EGCG decreased inflammatory activity in the mice colon compared to TNBS colitis group after 8-week treatment. Ongoing study includes methylene blue staining of the colonic mucosa during endoscopy, endoscopic scoring of inflammation activity, and trichrome staining of collagen production in a colonic biopsy. Taken together, EGCG alleviates ER Stress response, leads to greater inhibition of migratory potential of SEMF, and decreases TGF-β1 and collagen productions, which is the major molecular feature of fibrosis.

RICK/RIP2 is a NOD2-independent nodal point of gut inflammation

Previous studies have shown that inhibition of receptor-interacting serine/threonine kinase (RICK) (also known as RIP2) results in amelioration of experimental colitis. This role has largely been attributed to nucleotide-binding oligomerization domain 2 (NOD2) signaling since the latter is considered a major inducer of RICK activation. In this study, we explored the molecular mechanisms accounting for RICK-mediated inhibition of inflammatory bowel disease (IBD). In an initial series of studies focused on trinitrobenzene sulfonic acid (TNBS)-colitis and dextran sodium sulfate (DSS)-colitis we showed that down-regulation of intestinal RICK expression in NOD2-intact mice by intra-rectal administration of a plasmid expressing RICK-specific siRNA was accompanied by down-regulation of pro-inflammatory cytokine responses in the colon and protection of the mice from experimental colitis. Somewhat surprisingly, intra-rectal administration of RICK-siRNA also inhibited TNBS-colitis and DSS-colitis in NOD2-deficient and in NOD1/NOD2-double deficient mice. In complementary studies of humans with IBD we found that expression of RICK, cellular inhibitor of apoptosis protein 2 (cIAP2) and downstream signaling partners were markedly increased in inflamed tissue of IBD compared to controls without marked elevations of NOD1 or NOD2 expression. In addition, the increase in RICK expression correlated with disease activity and pro-inflammatory cytokine responses. These studies thus suggest that NOD1- or NOD2-independenent activation of RICK plays a major role in both murine experimental colitis and human IBD.

Protective vascular effects of quercitrin in acute TNBS-colitis in rats: the role of nitric oxide

Quercitrin (quercetin 3-rhamnoside) is a bioflavonoid with anti-inflammatory activity in experimental colitis.