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Lipopolysaccharide (LPS) from Gram (-) bacteria induces inflammatory cholestasis by impairing the expression/localization of transporters involved in bile formation (e.g., Bsep, Mrp2). Therapeutic options for this disease are lacking. Ursodeoxycholic acid (UDCA) is the first choice therapy in cholestasis, but its anticholestatic efficacy in this hepatopathy remains to be evaluated. To asses it, male Wistar rats received UDCA for 5 days (25 mg/Kg/day, i.p.) with or without LPS, administered at 8 a.m. of the last 2 days (4 mg/Kg/day, i.p.), plus half of this dose at 8 p.m. of the last day. Then, plasma alkaline phosphatase (ALP), bile flow, basal and taurocholate-stimulated bile acid output, total glutathione output, and total/plasma membrane liver protein expression of Bsep and Mrp2 by confocal microscopy were assessed. mRNA levels of both transporters were assessed by Real-time PCR. Plasma pro-inflammatory cytokines (IL-6 and TNF-α) were measured by ELISA. Our results showed that, UDCA attenuated LPS-induced ALP plasma release and the impairment in the excretion of the Bsep substrate, taurocholate. This was associated with an improved Bsep expression at both mRNA and protein levels, and by an improved localization of Bsep in plasma membrane. UDCA failed to reduce the increase in plasma pro-inflammatory cytokines induced by LPS and Mrp2 expression/function. In conclusion, UDCA protects the hepatocyte against the damaging effect of bile acids accumulated by the LPS-induced secretory failure. This involved an enhanced synthesis of Bsep and an improved membrane stability of the newly synthesized transporters.

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Estradiol-17β-D-glucuronide (E17G), through the activation of different signaling proteins, induces acute endocytic internalization of canalicular transporters in rat, including multidrug resistance-associated protein 2 (Abcc2) and bile salt export pump (Abcb11), generating cholestasis. Insulin-like growth factor 1 receptor (IGF-1R) is a membrane-bound tyrosine kinase receptor that can potentially interact with proteins activated by E17G. The aim of this study was to analyze the potential role of IGF-1R in the effects of E17G in isolated perfused rat liver (IPRL) and isolated rat hepatocyte couplets. In vitro, IGF-1R inhibition by tyrphostin AG1024 (TYR, 100 nM), or its knock-down with siRNA, strongly prevented E17G-induced impairment of Abcc2 and Abcb11 function and localization. The protection by TYR was not additive to that produced by wortmannin (PI3K inhibitor, 100 nM), and both protections share the same dependency on microtubule integrity, suggesting that IGF-1R shared the signaling pathway of PI3K/Akt. Further analysis of the activation of Akt and IGF-1R induced by E17G indicated a sequence of activation GPR30-IGF-1R-PI3K/Akt. In IPRL, an intraportal injection of E17G triggered endocytosis of Abcc2 and Abcb11, and this was accompanied by a sustained decrease in the bile flow and the biliary excretion of Abcc2 and Abcb11 substrates. TYR did not prevent the initial decay, but it greatly accelerated the recovery to normality of these parameters and the reinsertion of transporters into the canalicular membrane. In conclusion, the activation of IGF-1R is a key factor in the alteration of canalicular transporter function and localization induced by E17G, and its activation follows that of GPR30 and precedes that of PI3K/Akt.

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