Medical Department II, Klinikum Grosshadern, University of Munich, Munich, Germany.
Biochem Biophys Res Commun. 2012 Aug 10;424(4):758-64. doi: 10.1016/j.bbrc.2012.07.029. Epub 2012 Jul 15.
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.
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.
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.
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.
β-鼠胆酸（βMCA）是一种三羟基胆汁酸，是大鼠和小鼠胆汁中的主要胆汁酸。βMCA 的亲水性强于熊去氧胆酸，并已被评估用于溶解胆固醇性胆囊结石。由于尚不清楚βMCA 是否对肝细胞死亡有有益作用，因此我们确定了牛磺-βMCA（TβMCA）对体外细胞凋亡的影响。
用人 Ntcp 转染的 HepG2 细胞和大鼠及小鼠的原代肝细胞，用促凋亡的甘氨胆酸（GCDCA）和游离脂肪酸棕榈酸孵育，同时存在和不存在 TβMCA。用 caspase 3/7 测定法和 Hoechst 33342 染色后定量细胞凋亡。用 JC-1（5,5',6,6'-四氯-1,1',3,3'-四乙基苯并咪唑碳氰碘化物）荧光法测量线粒体膜电位（MMP）。用免疫印迹法检测促凋亡 Bcl-2 蛋白 Bax。
在 Ntcp-HepG2 细胞中，GCDCA 在 4 小时后明显增加细胞凋亡。同时用 TβMCA 孵育可将细胞凋亡减少至 49%（p<0.01，与 GCDCA 相比，每组 n=6）。GCDCA（100μmol/L）在 6 小时后将 MMP 降低至 34%，而同时用 TβMCA 治疗可在所有时间点将 MMP 恢复至对照水平（n=4）。TβMCA 还可恢复由棕榈酸诱导的 MMP 破裂。GCDCA 诱导 Bax从细胞质向线粒体易位，用等摩尔浓度的 TβMCA 同时处理可抑制这种易位。
TβMCA 通过抑制 Bax 向线粒体易位和维持 MMP，限制了低微摩尔浓度的 GCDCA 或棕榈酸诱导的肝细胞凋亡。因此，需要进一步研究 TβMCA 在改善胆汁淤积性肝损伤中的潜在用途。
