Tauro-β-muricholic acid restricts bile acid-induced hepatocellular apoptosis by preserving the mitochondrial membrane potential.
OBJECTIVE
β-Muricholic acid (βMCA) is a trihydroxylated bile acid that constitutes the major bile acid in rat and mouse. βMCA is more hydrophilic than ursodeoxycholic acid and has been evaluated for dissolution of cholesterol gallstones. Since it is unknown if βMCA has beneficial effects on hepatocyte cell death we determined the effect of tauro-βMCA (TβMCA) on apoptosis in vitro.


METHODS
Human Ntcp-transfected HepG2 cells and primary hepatocytes from rat and mouse were incubated with the proapoptotic glycochenodeoxycholic acid (GCDCA) as well as the free fatty acid palmitate in the absence and presence of TβMCA. Apoptosis was quantified using caspase 3/7-assays and after Hoechst 33342 staining. The mitochondrial membrane potential (MMP) was measured fluorometrically using JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazol-carbocyaniniodide). Immunoblotting was performed against the proapoptotic Bcl-2-protein Bax.


RESULTS
In Ntcp-HepG2 cells, GCDCA markedly increased apoptosis after 4h. Co-incubation with TβMCA reduced apoptosis to 49% (p<0.01 vs. GCDCA, each; n=6). While GCDCA (100μmol/L) reduced the MMP to 34% after 6h, combination treatment with TβMCA restored the MMP to control levels at all time points (n=4). TβMCA also restored breakdown of the MMP induced by palmitate. GCDCA induced a translocation of Bax from the cytosol to mitochondria that was inhibited by simultaneous treatment with TβMCA in eqimolar concentrations.


CONCLUSIONS
TβMCA restricts hepatocellular apoptosis induced by low micromolar concentrations of GCDCA or palmitate via inhibition of Bax translocation to mitochondria and preservation of the MMP. Thus, further studies are warranted to evaluate a potential use of TβMCA in ameliorating liver injury in cholestasis.
Medical Department II, Klinikum Grosshadern, University of Munich, Munich, Germany.
Gerald Ulrich
GU
Denk
Carl Philipp
CP
Kleiss
Ralf
R
Wimmer
Timo
T
Vennegeerts
Florian Paul
FP
Reiter
Sabine
S
Schulz
Hans
H
Zischka
Christian
C
Rust
D017209:Apoptosis / Q000187:drug effects*; D001647:Bile Acids and Salts / Q000494:pharmacology; D019610:Cytoprotection; D005999:Glycochenodeoxycholic Acid / Q000037:antagonists & inhibitors* / Q000494:pharmacology; D056945:Hep G2 Cells; D022781:Hepatocytes / Q000187:drug effects*; D006801:Humans; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects*; D008930:Mitochondria, Liver / Q000187:drug effects / Q000378:metabolism; D013656:Taurocholic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D051028:bcl-2-Associated X Protein / Q000378:metabolism
C494826:BAX protein, human; D001647:Bile Acids and Salts; D051028:bcl-2-Associated X Protein; C037351:tauromuricholic acid; D013656:Taurocholic Acid; D005999:Glycochenodeoxycholic Acid