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目的 探讨牛磺酸熊脱氧胆酸(TUDCA)对牛磺酸脱氧胆酸(TDCA)诱导HepG2细胞凋亡阻抑作用及分子机制。 方法 应用Hoechst 3325 8染色、电镜和DNA电泳对细胞凋亡进行定性;应用流式细胞仪对凋亡细胞进行定量;检测TDCA单独孵育及与TUDCA共孵育时,胞浆中细胞色素C含量及Caspase-3、8、9蛋白酶活性的变化。 结果 TDCA 400μmol/L孵育12 h可诱导显著HepG2细胞凋亡,凋亡率为(50.35±2.20)％,细胞经TUDCA与TDCA共孵育后,细胞凋亡率明显下降,为(13.78±0.84)％;TUDCA能显著抑制TDCA引起的细胞色素C释放及Caspase-9、3蛋白酶活性升高,孵育12 h时,Caspase-3活性分别下降54.9％(t=16.88,P<0.01)和52.5％(t≥13.00,P<0.01),轻度降低Caspase-8活性升高,活性下降24.5％(t=1.94,P>0.05)。结论 稳定线粒体膜、阻止细胞色素C释放及随后Caspase-9、Caspase-3活化,是TUDCA抗凋亡的主要机制。抗凋亡作用可能是TUDCA治疗胆汁淤积性肝病取得显著疗效的重要机制之一。
Objective To investigate the inhibitory effect of tauroxy beneoxydecanoate (TUDCA) on the apoptosis of HepG2 cells induced by taurodeoxycholic acid (TDCA) and its molecular mechanism. Methods Hoechst 3325 8 staining, electron microscopy and DNA electrophoresis were used to characterize apoptosis. Flow cytometry was used to quantify apoptotic cells. When incubated with TUDCA alone or in combination with TUDCA, cytochrome C content and Caspase -3,8,9 protease activity changes. Results The apoptosis rate of HepG2 cells induced by TDCA 400μmol / L for 12 h was (50.35 ± 2.20)%, and the apoptotic rate was (13.78 ± 0.84)% after incubated with TUDCA and TDCA. TUDCA could significantly inhibit the release of cytochrome C and the activity of Caspase-9 and 3 protease induced by TDCA. The activity of Caspase-3 decreased by 54.9% (t = 16.88, P <0.01) and 52.5% ≥13.00, P <0.01). The activity of Caspase-8 increased slightly with a slight decrease of 24.5% (t = 1.94, P> 0.05). Conclusion Stabilization of mitochondrial membrane, blocking the release of cytochrome C and subsequent activation of Caspase-9 and Caspase-3 are the main mechanisms of anti-apoptotic TUDCA. Anti-apoptotic effect may be TUDCA treatment of cholestatic liver disease achieved significant effect of one of the mechanisms.