胆酸减轻鹅膏毒肽攻击人肝细胞损伤的作用及机制

Mechanisms of cholic acid for reducing damage to human live cell lines L-O2 induced by amanita toxic pep-tides

目的探讨不同胆酸减轻受鹅膏毒肽攻击人肝细胞（L．02）损伤的作用及机制。方法根据鹅膏毒肽的质量浓度将实验分为0．00g／L、0．26g/L、0．40g／L、1．40g／L、2．80g／L共5个剂量组，将培养至指数生长期的L-02铺于96孔板，每孔1×10^3细胞，24h加入上述浓度的鹅膏毒肽，每组设3个复孔，分别与药物作用24、48、72h采用四唑盐（MTT）比色法检测肝细胞活性，确定肝细胞生存的最小鹅膏菌毒素攻击剂量。实验分为空白对照组、损伤组和胆酸组，每个组均设3个时间点：攻击24、48、72h，在攻击24h后使用牛磺胆酸、鹅脱氧胆酸、甘氨胆酸、甘氨鹅脱氧胆酸和牛磺鹅脱氧胆酸分别保护损伤肝细胞，在倒置相差显微镜下观察细胞形态学改变，MTT法进行活细胞计数，生化法检测培养上清液中AST和ALT。结果肝细胞生存的鹅膏毒肽体外最小攻击剂量为1．40g／L。空白对照组、损伤组、牛磺胆酸组、鹅脱氧胆酸组、甘氨胆酸组、甘氨鹅脱氧胆酸组及牛磺鹅脱氧胆酸组在鹅膏毒肽攻击肝细胞72h时吸光度分别为0．812±0．035、0．345±0．021、0．363±0．018、0．387±0．027、0．431±0．018、0．465±0．015及0．452±0．030。胆酸各组较损伤组攻击72h时吸光度逐渐升高，其中鹅脱氧胆酸组、甘氨胆酸组、甘氨鹅脱氧胆酸组、牛磺鹅脱氧胆酸组与损伤组比较差异有统计学意义（P〈0．05）。胆酸各组AST和ALT活性下降，其中甘氨鹅脱氧胆酸组AST和Au活性最低，与损伤组比较差异有统计学意义（P〈0．01）。结论胆酸对鹅膏毒肽攻击L-02有保护作用，作用机制可能与鹅膏毒肽和胆酸同为钠离子-牛磺酸共同转运多肽和有机阴离子转运多肽底物有关。

Objective To explore the mechanisms of different cholic acid for reducing damage to human liver cells lines L-O2 induced by amanita toxic peptides （ amataxins ）. Methods According to different concentrations of amataxins, the experiment was conducted with different dosages in 5 groups：0.00 g/L,0.26 g/L,0.40 g/L, 1.40 g/L and 2.80 g/L. The human liver cells lines L-O2 in the exponential growth phase were cultured into 96-well plates, 1×10^3 cells per well. After 24 hours ,the concentrations of amanita toxic peptides mentioned above were added. The mini- mum concentration of mushroom toxins keeping the liver cells alive was determined after 24,48 and 72 hours, respec- tively, and Mq＇T method was used to test each group＇s liver cell activity. The experiment included 3 groups：the control group, the damage group, and the cholic acid group. Each group had 3 time points：24,48 and 72 hours after being at- tacked. Twenty four hours after attack,in cholic acid group, cholic acid drugs including taurocholic acid gea, goose de- oxycholic acid,gansu ammonia goose deoxycholic acid and bovine goose deoxycholic acid were given, respectively, to protect the injured liver cells. Ceils＇ morphology changes were observed under the inverted phase contrast microscope, living cells were counted by using MT＇I＇ method, and aspartate aminotransferase （AST） and alanine aminotransferase （ALT） activities in the culture supernatant were tested by the biochemical method. Results The minimum attack con- centration of lamanita toxic peptides keeping liver cell survival in vitro was 1.40 g/L. Seventy-two hours after attack by amanita toxic peptides,the absorbance value was 0. 812±0. 035,0. 345 ±0. 021,0. 363±0. 018,0. 387±0. 027, 0. 431±0.018,0. 465 ± 0. 015 and 0. 452 ± 0. 030, respectively in the control group, the damage group, the taurocholic acid group, the goose deoxycholic acid group, the glycocholic acid group, the glycochenodeoxycholic acid group and the sodium deoxycholic acid group. Compared with the damage group, absorbance value 72 hours after attack in each cholic acid group gradually increased, and compared with damage group, the differences were statistically significant among goose deoxycholic acid group, glycocholic acid group, glycochenodeoxycholic acid group and sodium deoxycholic acid group（P 〈 0. 05）. AST and ALT activity in each cholic acid group declined,and that in glycochenodeoxycholic acid group was the lowest. Compared with the damage group, the difference was statistically signifiCant （P 〈 0.01 ）. Conclu- sions Cholic acid can protect human liver cells from the damage induced by amanita toxic peptides. Such effect may be related to the fact that both amanita toxic peptides and cholic acid are the substrates of NTCP and OATP.