Int Immunopharmacol., 2018., Aug 23;64:42-51. doi: 10.1016/j.intimp.2018.08.012.

Linoorbitides and enterolactone mitigate inflammation-induced oxidative stress and loss of intestinal epithelial barrier integrity.

Almousa AA, Meurens F, Krol ES, Alcorn J.


Barrier integrity dysfunction and oxidative stress are considered hallmarks of inflammatory bowel disease (IBD) pathogenesis. Their mitigation continues to be a drug discovery target in IBD. Natural products may aid treatment of chronic inflammatory diseases, but their use in IBD requires a better understanding of whether individual bioactives may positively modulate disease course. This study investigated the ability of flax linoorbitides (LOBs) and enterolactone (ENL), to mitigate inflammation-induced loss of intestinal epithelial barrier integrity and oxidative stress in vitro. TNF-α with INF-γ and lipopolysaccharide (LPS) induced an inflammatory response in HCT-8 monoculture and Caco-2/RAW-264.7 coculture, respectively. Trans-Epithelial Electrical Resistance (TEER) and Lucifer Yellow rejection for barrier permeability were assessed in differentiated monolayers in the presence and absence of LOBs and ENL. Additionally, RAW 264.7 cells were used to assess protective effects upon induction of oxidative stress. In HCT-8 model, 200 nM of LOB-J, LOB-A, and ENL mitigated the inflammation-induced reduction in TEER with relative TEER values of 108.6%, 63.2%, and 64.2%, respectively, at 24 h relative to time zero. Similarly, at 24 h Caco-2/RAW-264.7 coculture TEER values ranged from ~200% – 243.4% for LOB-A, LOB-J, LOB-ACEJ, and ENL relative to TEER values of untreated cells. ENL and LOBs reduced malondialdehyde (MDA) lipid peroxidation in RAW 264.7 cells upon induction with lipopolysaccharide (LPS). ENL, but not LOBs, caused an increase in zona occludins 1 (ZO-1) protein expression in HCT-8 cells exposed to an inflammatory stimulus to levels comparable to negative control. Our results demonstrate after an inflammatory insult that ENL and the tested LOBs protect intestinal barrier integrity and reduce oxidative stress damage. In conclusion, use of different flax bioactives in the treatment of IBD warrants further investigation.

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Key Findings

Inflammatory bowel disease (IBD) includes Ulcerative colitis (UC) and Crohn’s disease (CD) and is characterized by idiopathic chronic inflammation along the intestine with varying locations and clinical presentations. Prolonged production of reactive oxygen species (ROS) signaling might magnify the inflammatory process and, thus, reduce barrier integrity to eventually lead to the pathogenesis of IBD. After impairment of intestinal barrier integrity, noxious molecules and pathogens leak to the lymphatic system to initiate a cascade of inflammatory reactions. Flaxseed contains high levels of the bioactive compounds, lignans, and linoorbitides (LOBs). These bioactives, cyclic peptide LOBs, and enterolignan ENL, have evidence of ability to mitigate inflammation and oxidative stress or elicit immune modulating effects. In the present study, flaxseed LOB-A, LOB-J, LOB-ACEJ, and the enterolignan, ENL, based on their known physicochemical and pharmacokinetic properties were assessed for potential use to protect against loss of intestinal epithelial barrier integrity. The study results demonstrated that these flaxseed bioactives imparted antioxidant and effective intestinal epithelial barrier protection. Flaxseed LOBs and ENL provide significant protection of epithelial barrier integrity in a mono- and co-culture model of the intestinal epithelium. Protection of barrier integrity was supported by LOB- and ENL-mediated reduction in oxidative stress, an effect likely the result of different mechanisms of action between these flax bioactives. Further assessment of ENL and LOBs in a colitis animal model is warranted to confirm their in vivo effect on barrier integrity, oxidative damage, and immune system modulation to mitigate these hallmarks of IBD pathogenesis.