Collagen peptides derived from Alaska pollock skin protect against TNFα‐induced dysfunction of tight junctions in Caco‐2 cells

Abstract

Dysfunction of tight junctions in the intestinal barrier promotes enhanced antigen uptake, bacteria translocation, and induction of abnormal immune responses, which contribute to the pathogenesis of inflammatory bowel disease and necrotizing enterocolitis. Tumor necrosis factor‐α (TNFα) is a key proinflammatory cytokine regulating the function and structure of tight junctions. With their immunomodulatory actions, collagens and their derived bioactive peptides may protect against TNFα‐induced dysfunction in tight junctions. Fish skins generated during fish processing may be a valuable by‐product useful in the development of functional foods as they contain a large amount of collagen. Thus, we examined the effect of Alaska pollock skin‐derived collagen (SDC) and its 3 collagen peptides (CP) derived from tryptic hydrolysis, CP1 (MW >6 kDa), CP2 (3–6 kDa), and CP3 (<3 kDa), on TNFα‐induced dysfunction in tight junctions in Caco‐2 cell monolayers. After being cultured in a Transwell® system for 21 d, Caco‐2 cells were treated with or without 2 mg/mL SDC or CPs for 24 h, followed by treatment with 10 ng/mL TNFα for 24 h. TNFα impaired monolayer integrity, as reflected by a 26% reduction in trans‐epithelial electrical resistance (TEER) from 454 ± 16 to 340 ± 10 Ω·cm2 and by a 58% increase in FITC‐Dextran 4 kDa (FD‐4) transmembrane flux from 186 ± 17 to 293 ± 28 μg/mL. The pre‐treatment of SDC or CPs protected against TNFα‐induced destruction of tight junction integrity to different degrees. CP3 was the most protective, maintaining TEER at 438 ± 14 Ω·cm2 and FD‐4 flux at 195 ± 14 μg/mL, values not different from the control. The phosphorylation of myosin light chain (pMLC) mediated by myosin light chain kinase (MLCK) induces F‐actin rearrangement and disruption of tight junction assembly; TNFα treatment augmented pMLC and MLCK expression by 37 and 47%, respectively, compared to controls (P ≤0.05). SDC and CPs down‐regulated TNFα‐induced pMLC expression to levels comparable to controls, and CPs completely blocked TNFα‐induced MLCK expression. TNFα activated NFκB and ERK1/2 pathways, two key transcription factors in the regulation of tight junctions, as illustrated by increases in nuclear NFκB‐p65 content from 6.9 ± 0.8 to 10.1 ± 1.2 nmol/mg protein and in nuclear ELK‐1 content from 0.9 ± 0.1 to 1.3 ± 0.2 ng/mg protein (P ≤0.05). SDC and CPs completely inhibited their activations. TNFα decreased expression of tight junction proteins ZO‐1 by 35% from 496 ± 72 to 321 ± 42 ng/mg protein and occludin by 43% from 2.0 ± 0.2 to 1.1 ± 0.1 ng/mg protein compared to controls (P ≤0.05). Of the 4 treatments, only CP3 maintained the expression of these 2 tight junction proteins comparable to controls. In conclusion, Alaska pollock skin‐derived peptides, particularly CP3, protected against TNFα‐induced dysfunction in tight junctions via the NFκB‐ and ERK1/2‐mediated MLCK‐pMLC pathway.Support or Funding InformationNational Science Foundation China, China Scholarship Council & USDA