Protection against Cholestasis and Ischemia in vitro by Ursodeoxycholyl Lysophosphatidylethanolamide (UDCA-LPE) is Associated with Upregulation of Anti-apoptotic Proteins

Introduction: An increase of toxic bile acids such as glycochenodeoxycholic acid (GCDCA) frequently occurs in an early phase of liver transplantation. Ischemia during transplantation can cause hepatic cholestasis and damage of intrahepatic biliary epithelial cells. Both stresses contribute to the failure in liver transplantation. We aim to test the effectiveness of UDCA-LPE in inhibition of apoptosis induced by GCDCA and ischemia. Methods: Mouse hepatocytes were isolated and treated with 50µM GCDCA for 15h. As an experimental model of ischemia, CoCl₂ at 500–750µM was used to treat the intrahepatic biliary epithelial cancer cell line MzChA1 for 24h. Protein expression of pro- and anti-apoptosis proteins and signalling molecules was measured by Western blot. The effect of UDCA-LPE on protein turnover was studied by immunoprecipitation and immunoblotting. Caspase 3 and 8 activities were measured by luminescence spectrometry. Results: In mouse hepatocytes and MzChA1, apoptosis was induced as measured by cleaved PARP–1, caspase 3 and 8 proteins and activities. Co-treatment with UDCA-LPE markedly inhibited GCDCA- and CoCl₂-induced apoptosis. Expression of anti-apoptotic protein Mcl–1, cIAP2 and cFlip was decreased with CoCl₂ treatment, and UDCA-LPE co-treatment prevented this decrease. By measuring ubiquitinated proteins, we found that UDCA-LPE inhibited the degradation of Mcl–1, but not of cIAP2 and cFlip proteins. This inhibition by UDCA-LPE was concomitant with phosphorylation of GSK–3α/β, which is a known mediator for Mcl–1 stabilization. Conclusion: The effect of UDCA-LPE on inhibition of Mcl–1 ubiquitination via phosphorylation of GSK–3α/β is one of the mechanisms for UDCA-LPE protection against apoptosis during bile acid and ischemic stresses. Further work is needed to investigate transcriptional control of cFlip and cIAP2 proteins by UDCA-LPE. UDCA-LPE may thus be used as an agent to prevent complications associated with liver transplantation.

Mcl-1 stabilization - apoptosis - cytoprotective agent - ischemia - liver transplantation