Effect and mechanism of Cyclosporin A and cholic acid on reducing the amanitin-induced human liver cell damage

Abstract

Objective To explore the effect and mechanism of Cyclosporin A(CsA) and cholic acid on redu-cing the human liver cell damage induced by α-amanitin(AMA). Methods According to the previous research results, the minimum hepatocellular survival concentration against α-AMA (1.4 g/L), the experiment was conducted in 5 groups: control group, damage group, glycochenodeoxycholic acid group, CsA group, and CsA combined with cholic acid group (CsA+ taurocholic acid, CsA+ chenodeoxycholic acid, CsA+ glycocholic acid, CsA+ glycochenodeoxycholic acid, and CsA+ taurochenodeoxycholic acid). For each group, there were 3 time points for observation (24 h, 48 h and 72 h after attacking), CsA and CsA+ glycochenodeoxycholic acid was used to protect hepatocytes, respectively, and morphological changes in cells were observed with inverted phase contrast microscope, and the live cells were counted by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide(MTT)method, and aspertate aminotransferase (AST) and alanine aminotransferase (ALT) in the culture supernatant were detected by biochemical method. Results Compared with the control group, hepatocellular growth in the injury group was obviously suppressed, with progressive cellular apoptosis and significantly decreased absorbance value of MTT(0.345±0.021); the activity of AST and ALT increased gradually, reaching the highest after 72 h [(98.4±6.7) U/L and (116.2±9.5) U/L, respectively]. Compared with injury group, broken organelles decreased significantly and absorbance value elevated in glycochenodeoxycholic acid group, CsA group and CsA combined with cholic acid group, and at each time point, the highest absorbance value in the CsA+ taurochenodeoxycholic acid group[the highest was (0.656±0.014), P<0.05]; at the same time, the activity of AST and ALT didn't increase obviously, at each time point, the lowest in CsA+ taurochenodeoxycholic acid group[the lowest was (22.3±6.2) U/L and (20.2±5.4) U/L, P<0.05, respectively]. Conclusions CsA, as well as cholic acid, can protect human liver cells from the attack of α-AMA.The mechanism may be CsA, as an organic anion transfer peptide in humans (OATP1B1 and OATP1B3) suppressant, inhibits the absorption of α-AMA.The joint application of CsA and taurochenodeoxycholic acid is superior to the single OATP substrate or inhibitor. Key words: Cyclosporine A; Cholic acid; Human liver cell; Amanitin; Damage