Anticholestatic mechanisms of ursodeoxycholic acid in inflammatory cholestasis induced by lipopolysacharide

RAZORI M.V.; MAIDAGAN P.M.; ANDERMATTEN R.; RUIZ M.L.; ROMA M. G.

París

Congreso; The International Liver Congress - 53th Annual Meeting of the European Association for the Study of the Liver (EASL); 2018

European Association for the Study of the Liver (EASL)

Background and Aims: Sepsis-induced cholestatic is causally associated with the release of lipoplysacaride (LPS) from Gram (-) bacteria; LPS stimulates the production of inflammatory cytokines, and the further impairment in expression/localization of transporters involved in bile formation, such as Bsep (bile salt transporter) and Mrp2 (bilirubin/GSH transporter). There is no established therapy for sepsis-induced cholestasis. Ursodeoxycholic acid (UDCA) is the most widely used therapeutic agent for human cholestatic diseases, but its action mechanisms in inflammatory cholestasis are largelyunknown. Therefore, we addressed this issue in the rat model of LPS-induced cholestasis.Method: Male Wistar rats were randomized in 4 experimental groups: Control, UDCA (25 mg/Kg/day, i.p., 5 days), LPS (10 mg/Kg, i.p., over the last 2 days), and UDCA + LPS. On the 6th day, we assessed serum alkaline phosphatase (ALP, a membrane enzyme induced/removed by bile salts accumulated in cholestasis), bile salt output (BSO), total glutathione output (GSHtO), Bsep expression by realtime RT-PCR and Western blotting, and Bsep localization by immunofluorescence staining followed by confocal microscopy, image analysis, and statistical comparison of the densitometric profiles along a line perpendicular to the canaliculus by Mann- Whitney test. Circulating levels of the inflammatory cytokines TNF-α and IL-6 were measured by ELISA.Results: (expressed as % of control; *p < 0.05 vs. control; #p < 0.05 vs. LPS). Administration of UDCA to LPS-treated rats reduced serum ALP (199 ± 16*# vs. 280 ±  16%*), and increased BSO (85 ± 20%*# vs. 58 ± 10%*), but not GSHtO. This was associated with an improvement in the proportion of Bsep in the apical membrane, assessed by western blot (85 ± 20% vs. 66 ± 9*). Improved membrane localization was confirmed by comparing Bsep densitometric profiles in confocal images (see figure); LPS decreased the fluorescence intensity in the canalicular area, associated with an increase in the  pericanalicular region (p < 0.001 vs. control), and this was counteracted by UDCA. UDCA also normalized Bsep mRNA levels (119 ± 40# vs.15 ± 3*), without attenuating LPS-induced serum TNF-α and IL-6 elevations. Conclusion: UDCA improves bile salt biliary excretion by mechanisms unrelated to immunosuppressive effects. Its beneficial effects rather involve improvement in gene expression and canalicular membrane localization of Bsep. These effects are likely due to the well-recognized properties of UDCA as a nuclear-receptor ligand and as a stimulator of Bsep canalicular trafficking from its synthesis place,respectively, thus affording proper localization to the newly synthesized transporters.