Impairment of endogenous bilirubin generation aggravates bile-duct ligation injury in rats: role of bilirubin antioxidant effects.

MARTÍN P.L.; RAZORI M.V.; CORBO, M.L.; HARVEY G.B.; SÁNCHEZ POZZI E.J.; ROMA M.G.; BASIGLIO C.L.

Mar del Plata

Congreso; Reunión de Sociedades de Biociencias (SAIC, SAI, SAFIS) 2022; 2022

SAIC, SAI, SAFIS

We showedthat hemeoxygenase-1 (HO1) induction, and the consequent bilirubin (BR)elevation, protects the liver from oxidative stress (OS)-induced cholestasis invivo. Here, we evaluated the effect of HO1 inhibition on the endogenouslevels of BR and its protective action in obstructive cholestasis. For thispurpose, male Wistar rats were subjected to a 7-day bile-duct ligation (BDL, n=7)or sham surgery (Sh, n=5). HO1 was inhibited with Zn(II) protoporphyrinIX (PP, 25 mg/Kg b.w., i.p.), 24h before BDL (PP+BDL, n=10) or Sh(PP+Sh, n=6). At euthanasia, blood and liver samples were obtained.After BDL, plasma BR levels (mg/dl, media±SD) were higher in BDL (8.46±1.70a)than in PP+BDL (3.90±2.80b), confirming HO1 inhibition (ap<0.01 vs Sh and PP+Sh; bp<0.01 vs BDL; Kruskal-Wallis’ test,unless otherwise indicated). A similar result was obtained for plasma bile acidlevels (μmol/L): 116±14a for BDL vs 67±25b forPP+BDL (ap<0.01vs Sh and PP+Sh; bp<0.05 vs BDL). Serum bile acids increasedin BDL due to the obstructive process, but this increase was milder in PP+BDL,likely due to the expected lower BR-mediated induction of the sinusoidalbile-acid extrusion pump, Mrp3. Histopathological analysis ofhematoxylin/eosin-stained liver samples revealed signs of obstructive injury(portal and parenchymal inflammation, ductular injury, canalicularproliferation, proliferation of hepatic stellate cells, and fibrosis), whichwere more noticeable in PP+BDL than in BDL (p<0.05, Fisher’s exact test), thusindicating that BR-generation impairment aggravates obstructive injury; thesame holds true for the fibrotic process, as visualized by trichromic and picrosirius red stainings. Hepatic lipid peroxidation (nmol malondialdehyde /mgprot) was higher in PP+BDL than in BDL (7.1±1.8 vs.5.5±0.7, p<0.05) and plasmatic total antioxidant status (µM Trolox™equivalents, media±SD) was lower in BDL (4.65±1.5a) compared to control group, while PP+BDL groupshowed the lowest values (3.59±1.5b, ap<0.05 vs Sh and PP+Sh; bp<0.05 vs). These results show that,under cholestatic conditions, hepatic oxidative damage is more severe andantioxidant defenses levels are more impaired when BR endogenous production is inhibited.We conclude that the viciouscircle by which OS can induce inflammation and vice versa, a major eventleading toprogressive cellular and tissular damage in cholestasis, is significantly attenuatedby the accumulation of BR during the cholestatic process, via its antioxidanteffects.