The Na+/H+ exchanger modulates the fibrogenic effect of oxidative stress in rat hepatic stellate cells.
OBJECTIVE
Oxidative stress is associated with liver fibrosis in vivo and with hepatic stellate cell (HSC) activation in vitro, but the intracellular mechanisms mediating these effects are mostly unknown. The Na+/H+ exchanger plays a key role in regulating the cell cycle, and is involved in HSC proliferation. Its role in different HSC features, such as collagen accumulation, is still unknown. We thus evaluated if the Na+/H+ exchanger modulates the fibrogenic effect of oxidative stress in rat HSC.


METHODS
HSC were incubated with 0.1 mM ferric nitrilotriacetate complex (FeNTA). Intracellular hydroperoxides and malonildialdehyde (MDA) levels in the culture media were measured by the dichlorofluorescein and TBARS method, respectively. Intracellular pH and Na+/H+ exchanger activity were measured using the fluorescent dye BCECF. Cell proliferation was measured by immunohistochemistry for bromodeoxyuridine incorporation. Collagen type I accumulation in the culture media was measured by ELISA.


RESULTS
HSC incubation with FeNTA resulted in a significant production of intracellular hydroperoxides and MDA, associated with increased Na+/H+ exchange activity and baseline intracellular pH (pHi). Exposure of HSC to FeNTA significantly enhanced the number of proliferating HSC and collagen type I levels in the culture medium. All these effects were reversed by the antioxidant resveratrol and by the Na+/H+ exchanger inhibitor amiloride.


CONCLUSIONS
This study indicates that the Na+/H+ exchanger might represent a common mediator of the different effects induced by oxidative stress on HSC. The reduction in cell proliferation and collagen synthesis induced by amiloride could represent a new therapeutic challenge in liver fibrosis.
Svegliati-Baroni
G
G
Department of Gastroenterology, University of Ancona, Italy. clingastro@popcsi.unian.it
Di Sario
A
A
Casini
A
A
Ferretti
G
G
D'Ambrosio
L
L
Ridolfi
F
F
Bolognini
L
L
Salzano
R
R
Orlandi
F
F
Benedetti
A
A
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D002273:Carcinogens / Q000494:pharmacology; D002453:Cell Cycle; D002455:Cell Division; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism; D003470:Culture Media; D005290:Ferric Compounds / Q000494:pharmacology; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007700:Kinetics; D008099:Liver / Q000166:cytology / Q000473:pathology / Q000502:physiology*; D008106:Liver Cirrhosis, Experimental / Q000473:pathology / Q000503:physiopathology / Q000517:prevention & control; D008315:Malondialdehyde / Q000378:metabolism; D009571:Nitrilotriacetic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology*; D051381:Rats; D000077185:Resveratrol; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*; D013267:Stilbenes / Q000494:pharmacology; D017392:Thiobarbituric Acid Reactive Substances / Q000032:analysis
D000975:Antioxidants; D002273:Carcinogens; D003470:Culture Media; D005290:Ferric Compounds; D005452:Fluoresceins; D005456:Fluorescent Dyes; D017923:Sodium-Hydrogen Exchangers; D013267:Stilbenes; D017392:Thiobarbituric Acid Reactive Substances; C057433:2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester; D008315:Malondialdehyde; D003094:Collagen; D009571:Nitrilotriacetic Acid; D000077185:Resveratrol; C020326:ferric nitrilotriacetate