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Ursodeoxycholic Acid May Inhibit Deoxycholic Acid-Induced Apoptosis by Modulating Mitochondrial Transmembrane Potential and Reactive Oxygen Species Production

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Abstract

Background

The hydrophilic bile salt ursodeoxycholate (UDCA) inhibits injury by hydrophobic bile acids and is used to treat cholestatic liver diseases. Interestingly, hepatocyte cell death from bile acid-induced toxicity occurs more frequently from apoptosis than from necrosis. However, both processes appear to involve the mitochondrial membrane permeability transition (MPT). In this study, we determined the inhibitory effect of UDCA on deoxycholic acid (DCA)-induced MPT in isolated mitochondria by measuring changes in transmembrane potential (ΔΨm) and production of reactive oxygen species (ROS). In addition, we examined the expression of apoptosis-associated proteins in mitochondria isolated from livers of bile acid-fed animals.

Materials and Methods

Adult male rats were maintained on standard diet supplemented with DCA and/or UDCA for 10 days. Mitochondria were isolated from livers by sucrose/percoll gradient centrifugation and MPT was measured using spectrophotometric and fluorimetric assays. ΔΨm and ROS generation were determined by FACScan analysis. Cytoplasmic and mitochondrial protein abundance were determined by Western blot analysis.

Results

DCA increased mitochondrial swelling 25-fold over controls (p < 0.001); UDCA reduced the swelling by >40% (p < 0.001). Similarly, UDCA inhibited DCA-mediated release of calcein-loaded mitochondria by 50% (p < 0.001). ΔΨm was significantly decreased in mitochondria incubated with DCA but not with UDCA. ΔΨm disruption was followed closely by increased superoxide anion and peroxides production (p < 0.01). Coincubation of mitochondria with UDCA significantly inhibited the changes associated with DCA (p < 0.05). In vivo, DCA feeding was associated with a 4.5-fold increase in mitochondria-associated Bax protein levels (p < 0.001); combination feeding with UDCA almost totally inhibited this increase (p < 0.001).

Conclusion

UDCA significantly reduces DCA-induced disruption of ΔΨm, ROS production, and Bax protein abundance in mitochondria, suggesting both short- and long-term mechanisms in preventing MPT. The results suggest a possible role for UDCA as a therapeutic agent in the treatment of both hepatic and nonhepatic diseases associated with high levels of apoptosis.

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Acknowledgements

The authors thank Jeffrey Galecke for technical assistance with mitochondrial isolation and Julie Pribyl for her assistance with FACS analysis. This work was supported in part by a grant from the Minnesota Medical Foundation (to C. J. S.) and by Postdoctoral Fellowship BPD/6082/95 from Junta Nacional de Investigação Científica e Tecnológica, Lisbon, Portugal (to C. M. P. R.).

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Authors and Affiliations

  1. Department of Medicine, University of Minnesota Medical School, Box 36 UMHC, 420 Delaware Street S.E., Minneapolis, Minnesota, 55455, USA

    Cecilia M. P. Rodrigues, Guangsheng Fan, Phillip Y. Wong, Betsy T. Kren &  Clifford J. Steer

  2. Department of Cell Biology, University of Minnesota Medical School, Minneapolis, Minnesota, USA

    Clifford J. Steer

  3. Instituto Superior de Ciências da Saúde-Sul, Monte da Caparica, Portugal

    Cecilia M. P. Rodrigues

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  1. Cecilia M. P. Rodrigues
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  2. Guangsheng Fan
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Correspondence to Clifford J. Steer.

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Communicated by B. O’Malley.

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Rodrigues, C.M.P., Fan, G., Wong, P.Y. et al. Ursodeoxycholic Acid May Inhibit Deoxycholic Acid-Induced Apoptosis by Modulating Mitochondrial Transmembrane Potential and Reactive Oxygen Species Production. Mol Med 4, 165–178 (1998). https://doi.org/10.1007/BF03401914

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