Tocotrienols have potent antifibrogenic effects in human intestinal fibroblasts.
BACKGROUND
Excessive fibroblast expansion and extracellular matrix (ECM) deposition are key events for the development of bowel stenosis in Crohn's disease (CD) patients. Tocotrienols are vitamin E compounds with proven in vitro antifibrogenic effects on rat pancreatic fibroblasts. We aimed at investigating the effects of tocotrienols on human intestinal fibroblast (HIF) proliferation, apoptosis, autophagy, and synthesis of ECM.


METHODS
HIF isolated from CD, ulcerative colitis (UC), and normal intestine were treated with tocotrienol-rich fraction (TRF) from palm oil. HIF proliferation was quantified by (3) H-thymidine incorporation, apoptosis was studied by DNA fragmentation, propidium iodide staining, caspase activation, and poly(ADP-ribose) polymerase cleavage, autophagy was analyzed by quantification of LC3 protein and identification of autophagic vesicles by immunofluorescence and production of ECM components was measured by Western blot.


RESULTS
TRF significantly reduced HIF proliferation and prevented basic fibroblast growth factor-induced proliferation in CD and UC, but not control HIF. TRF enhanced HIF death by promoting apoptosis and autophagy. HIF apoptosis, but not autophagy, was prevented by the pan-caspase inhibitor zVAD-fmk, whereas both types of cell death were prevented when the mitochondrial permeability transition pore was blocked by cyclosporin A, demonstrating a key role of the mitochondria in these processes. TRF diminished procollagen type I and laminin γ-1 production by HIF.


CONCLUSIONS
Tocotrienols exert multiple effects on HIF, reducing cell proliferation, enhancing programmed cell death through apoptosis and autophagy, and decreasing ECM production. Considering their in vitro antifibrogenic properties, tocotrienols could be useful to treat or prevent bowel fibrosis in CD patients.
Luna
Jeroni
J
Department of Gastroenterology, Hospital Clı´nic i Provincial / IDIBAPS, CIBER EHD, Barcelona, Catalunya, Spain.. msans@clinic.ub.es
Masamunt
Maria Carme
MC
Rickmann
Mariana
M
Mora
Rut
R
España
Carolina
C
Delgado
Salvadora
S
Llach
Josep
J
Vaquero
Eva
E
Sans
Miquel
M
D000975:Antioxidants / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D001343:Autophagy; D015153:Blotting, Western; D020169:Caspases / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects*; D002478:Cells, Cultured; D003093:Colitis, Ulcerative / Q000378:metabolism / Q000473:pathology; D005109:Extracellular Matrix / Q000187:drug effects; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D005347:Fibroblasts / Q000187:drug effects*; D005455:Fluorescent Antibody Technique; D006801:Humans; D007422:Intestines / Q000166:cytology / Q000187:drug effects*; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D033681:Mitochondrial Membrane Transport Proteins / Q000187:drug effects; D000083162:Mitochondrial Permeability Transition Pore; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D024508:Tocotrienols / Q000494:pharmacology*
D000975:Antioxidants; D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D024508:Tocotrienols; D016222:Fibroblast Growth Factor 2; D011065:Poly(ADP-ribose) Polymerases; D020169:Caspases