比较猪去氧胆酸与牛磺熊去氧胆抗神经细胞缺氧缺糖再给氧损伤的作用

Protective effects of hyodeoxycholic acid versus tauroursodesoxycholic acid on the injury induced by hypoxia/hypoglycemia and reoxygenation in nerve cells

目的:研究猪、熊胆粉的主要成分猪去氧胆酸及牛磺熊去氧胆对神经细胞缺氧缺糖再给氧损伤的保护作用。方法:实验于2000-07/2002-08在北京中医药大学中医内科学教育部重点实验室完成。培养人神经母细胞瘤细胞,以含连二亚硫酸钠的无糖Earle液模拟造成缺氧缺糖再给氧损伤,随机分为正常培养对照组,缺氧缺糖再给氧组,猪去氧胆酸3.125,1.563,0.781μmol/L浓度组,牛磺熊去氧胆3.125,1.563,0.781μmol/L浓度组。应用锥虫蓝染色测定细胞死亡率,应用比色法测定乳酸脱氢酶漏出率,四甲基偶氮唑蓝比色法测定细胞存活率,应用以Hoechst33342和碘化丙啶原位双染法荧光显微镜检测细胞坏死率和凋亡率。结果:①细胞死亡率:猪去氧胆酸、牛磺熊去氧胆3.125,1.563,0.781μmol/L浓度组细胞死亡率均明显低于缺氧缺糖再给氧组犤(34.1±7.1)%,(32.8±8.2)%,(29.4±9.8)%,(27.7±6.9)%,(26.8±9.4)%,(26.2±11.1)%,(71.2±9.2)%,P<0.001犦。②乳酸脱氢酶漏出率:猪去氧胆酸、牛磺熊去氧胆3.125,1.563,0.781μmol/L浓度组乳酸脱氢酶漏出率均明显低于缺氧缺糖再给氧组犤(46.6±5.6)%,(49.8±5.3)%,(47.0±4.0)%,(48.5±2.7)%,(44.1±4.3)%,(40.2±7.5)%,(66.4±7.6)%,P<0.001犦。③细胞存活率:猪去氧胆酸、牛磺熊去氧胆3.125,1.563,0.

AIM:To study the protective effects of the main cholic acid like components of porcine gall powder hyodeoxycholic acid and bear gall powder tauroursodesoxycholic acid on the injury induced by hypoxia/hypoglycemia and reoxygenation in nerve cells.METHODS:The experiment was finished in the Internal Medicine of Traditional Chinese Medicine,Beijing University of Chinese Medicine from July 2000 to August 2002.The human neuroblastoma was cultured,and models of hypoxia/hypoglycemia was established with Earle solution of sodium dithionate without glucose.The models were divided randomly into control group,hypoxia/hypoglycemia and reoxygenation group,3.125,1.563,0.781 μ mol/L hyodeoxycholic acid groups,3.125,1.563,0.781 μ mol/L tauroursodesoxycholic acid groups.The cellular mortality was detected with trypan blue stain;the cell survival rate was detected with MTT assay;the cellular lactate dehydrogenase release rate was detected with chromometry;the cellular necrosis rate and the cellular apoptosis rate were observed with fluorescence microscope of Hoechst 33342 and PI double staining in situ. RESULTS: ① The cell survival rate in the groups of 3.125, 1.563, 0.781 μ mol/L hyodeoxycholic acid and tauroursodesoxycholic acid increased significantly as compared with that in hypoxia /hypoglycemia and reoxygenation group[(34.1± 7.1)% , (32.8± 8.2)% , (29.4± 9.8)% , (27.7± 6.9)% , (26.8± 9.4)% , (26.2± 11.1)% , (71.2± 9.2)% ,P< 0.001]. ② The cellular lactate dehydrogenase release rate in the groups of 3.125, 1.563, 0.781 μ mol/L hyodeoxycholic acid and tauroursodesoxycholic acid decreased significantly as compared with that in hypoxia /hypoglycemia and reoxygenation group[(46.6± 5.6)% , (49.8± 5.3)% , (47.0± 4.0)% , 48.5± 2.7] % , (44.1± 4.3)% , (40.2± 7.5)% , (66.4± 7.6)% , P< 0.001]. ③ The cell survival rate in the groups of 3.125, 1.563, 0.781 μ mol/L hyodeoxycholic acid and tauroursodesoxycholic acid increased significantly as compared with that in hypoxia /hypoglycemia and reoxygenation group[(44.5± 3.2)% , (45.3± 2.0)% , (41.5± 1.6)% , (41.6± 2.4)% , (37.6± 2.8)% , (40.1± 1.8)% ,(25.6± 3.7)% ,P< 0.01- 0.05]. ④ With double staining in situ, the cellular necrosis rate in the groups of 3.125, 1.563, 0.781 μ mol/L hyodeoxycholic acid and tauroursodesoxycholic acid decreased significantly as compared with that in hypoxia /hypoglycemia and reoxygenation group[(19.7± 6.2)% , (23.9± 7.0)% , (26.0± 6.9)% , (21.8± 5.6)% , (18.7± 6.1)% , (25.4± 5.8)% , (46.8± 10.4)% , P< 0.01]; ⑤ The cellular apoptosis rate in the groups of 3.125, 1.563 μ mol/L hyodeoxycholic acid and 3.125, 1.563, 0.781 μ mol/L tauroursodesoxycholic acid decreased significantly as compared with that in hypoxia/hypoglycemia and reoxygenation group[(7.4± 2.7)% , (8.3± 4.0)% , (6.9± 3.8)% , (9.2± 4.5)% ,(8.7± 3.6)% ,(16.0± 4.8)% ,PCONCLUSION: Both hyodeoxycholic acid and tauroursodesoxycholic acid can inhibit the necrosis and apoptosis of nerve cells after the hypoxia/hypoglycemia and reoxygenation, and the difference of the protective effects between hyodeoxycholic acid and tauroursodesoxycholic acid is insignificant.