Abstract
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.
DOP16 Endoplasmic reticulum stress in subepithelial myofibroblasts increases the TGF-β1 activity that regulates fibrosis in Crohn’s disease
