乙酸叔丁酯与丁酸乙酯对胆固醇结石的溶石作用分析

Effect of tert-butyl acetate and ethyl butyrate on the dissolution of gallbladder cholesterol stones

目的:比较乙酸叔丁酯(TBA)和丁酸乙酯(EB)对胆囊结石的体外溶石效果及生物毒性。方法:选择10份胆囊多发性结石手术患者的结石样本,高效液相色谱分析结石胆固醇含量。以甲基叔丁基醚(MTBE)作为对照,分析TBA和EB对胆固醇结石的体外溶石能力。以正常培养基作为对照,利用细胞增殖实验和流式细胞仪比较MTBE、TBA及EB对人正常肝细胞株LO2的增殖抑制、早期凋亡、晚期凋亡以及细胞活性氧水平的影响。结果:10份胆囊多发性结石样本中6份为胆固醇结石,4份为非胆固醇结石。对胆固醇结石的体外溶石实验表明,MTBE、TBA、EB三组在溶石30 min时的剩余结石分别为(47.83±3.84)%、(58.12±4.53)%、(75.75±4.61)%,溶石90 min时的剩余结石分别为(18.38±6.47)%、(33.82±6.22)%、(56.38±3.91)%,MTBE的溶石效果优于TBA和EB,TBA的溶石效果优于EB,差异均具有统计学意义(均P<0.05)。细胞增殖抑制实验表明对照组、MTBE组、TBA组的细胞活力分别为(100.00±4.46)%、(96.79±4.32)%、(93.72±3.51)%,三组间差异无统计学意义(均P>0.05);但是EB组的细胞活力(87.57±5.29)%低于上述三组,差异均有统计学意义(均P<0.001)。对照组的早期凋亡和晚期凋亡分别为(1.67±0.15)%、(1.27±0.06)%,EB可诱导细胞发生早期凋亡(15.9±0.53)%(P<0.001)及晚期凋亡(5.13±0.76)%(P<0.05),差异有统计学意义;而MTBE及TBA对细胞凋亡均无影响,差异无统计学意义(P>0.05)。与对照组相比,MTBE、TBA、EB均可抑制细胞活性氧水平,其中EB作用又强于另外两组,差异均具有统计学意义(均P<0.05)。结论:TBA对胆囊胆固醇结石具有较好的体外溶石效果与生物安全性,而EB相对较差,TBA具有作为溶石药物的潜力。

Objective To compare the effect and biotoxicity of tert-butyl acetate(TBA)and ethyl butyrate(EB)on stone dissolution in vitro.Methods Ten gallstone samples from patients with multiple gallbladder stones were selected and the cholesterol content was analyzed by HPLC.Stone dissolution tests of TBA and EB were performed on cholesterol gallstone in vitro,and the weight of stone at each time point was recorded,meanwhile,methyl tert-butyl ether(MTBE)was used as the control.The inhibitory effects of MTBE,TBA and EB on proliferation of human normal liver cell line LO2 were analyzed by cell proliferation inhibition assay.Flow cytometry was used to analyze the effects of MTBE,TBA and EB on the early and late apoptosis of LO2 cells,and the changes of reactive oxygen species level in LO2 cells were also analyzed.Results Of the 10 gallbladder gallstones,6 were cholesterol gallstones and 4 were non-cholesterol gallstones.Stone dissolution experiment showed that the remaining stones of MTBE,TBA and EB groups were(47.83±3.84)%,(58.12±4.53)%and(75.75±4.61)%30 minutes later.The remaining stones were(18.38±6.47)%,(33.82±6.22)%and(56.38±3.91)%90 minutes later.MTBE had the best stone dissolution effect in vitro,the stone dissolution effect of TBA was slightly weaker than MTBE,and the stone dissolution effect of EB was relatively weak in all(P<0.05).The cell proliferation inhibition experiment showed that the cell viability of the control group,MTBE group and TBA group were(100.00±4.46)%,(96.79±4.32)%and(93.72±3.51)%,respectively,and there were no significant differences among the three groups(P>0.05).However,the cell viability of EB group(87.57±5.29)%was lower than the above three groups,and the differences were statistically significant(P<0.001).The early apoptosis and late apoptosis of the control group were(1.67±0.15)%and(1.27±0.06)%,respectively.EB induced early apoptosis(15.90±0.53)%(P<0.001)and late apoptosis(5.13±0.76)%(P<0.05).However,MTBE and TBA had no significant effect on cell apoptosis(P>0.05).Compared with control group,MTBE,TBA and EB all significantly inhibited the level of reactive oxygen species(P<0.05),and the inhibitory effect of EB was the most obvious.Conclusions TBA has good stone dissolution effect and biosafety for gallbladder cholesterol stones in vitro,while EB has relatively poor performance.TBA is a potential drug for gallstone dissolution.