Silibinin (SIL) prevents estradiol 17 beta-glucuronide (E217G)-induced bile salt excretory failure and internalisation of the canalicular bile salt export pump, Bsep, in the isolated rat hepatocyte couplet (IRHC) model: Possible involvement of cAMP

CROCENZI FA; PORTESIO MS; BASIGLIO CL; COLEMAN R; ROMA MG

Genova

Congreso; 38th Annual Meeting of the European Association for the Study of the Liver; 2003

EASL

Silymarin has beneficial effects in estrogen-induced cholestasis in vivo (Hepatology 2001,34:329). Since Eâ17G-induced internalization of Bsep (J Hepatol 2002;36(Supp 1):6), we studied in IRHCs, wether Sil, the active component of silymarin, prevents changes in Bsep localization/function. Eâ17G (50 µM) diminished the % of IRHCs accumulating apically the fluorescent bile salt, cholyl-lysylfluorescein (CLF), and Sil (2.5 µM, 30 min) fully prevented this effect. Sil benefical effect was abolished by the intracellular Ca2+ chelating agent, BAPTA/AM, but not by PKA inhibitor, KT5720 (% of IRHCs accumulating CLF, referred to controls: Eâ17G: 43 ± 2*; Eâ17G +Sil: 93 ± 1; Eâ17G+Sil+BAPTA/AM: 50 ± 4*; Eâ17G +Sil+KT5720: 92 ± 3#; *p<0.05 vs control, #p<0.05 vs Eâ17G). Eâ17G induced internalization of Bsep into intracellular vesicles, visualized by immunofluorescence, and this effect was prevented by Sil (% of canalicular Bsep content: Control: 69 ± 2; Eâ17G: 38 ± 3*; Sil+ Eâ17G: 59 ± 3*#; *p<0.05 vs control; #p<0.05 vs Eâ17G). Since Sil is a potent inhibitor of cAMP phosphodiesterase, and cAMP protects against Eâ17G-induced internalization of another major canalicular pump, Mrp2 (Hepatology 2002;35:1409), we analized wether Sil increases cAMP in hepatocytes, and wether the cAMP analogue, DBcAMP, is able to mimic Sil effects. Sil increased hepatocellular cAMP levels by 36 ± 6 % (p<0.05). DBcAMP fully mimicked Sil ability to prevent Eâ17G-induced impairment in CLF apical accumulation and Bsep internalization. Furthermore, the beneficial effect of DBcAMP was also Ca2+, but not PKA-dependent. Taken together, these results suggest that Sil prevents Eâ17G-induced bile salt excretory failure by counteracting internalization of Bsep, probably by a mechanism involving cAMP-induced cytosolic Ca2+ elevations.