Fig. 3.

In vitro determination of toxicities and gallstone dissolubility of each solvent. a Cell viability assay showing the effects of each solvent on the viability of human gallbladder epithelial cells (hGBECs). The viability of GBECs did not considerably change according to the concentration of MTBE or MMP (0.0–1.0 mM) over 24 h. b Western blot analysis showing the effects of MTBE and MMP on the expression of PCNA (proliferation marker), Mcl-1 (anti-apoptosis marker), and BAX (apoptosis marker). [Top] With rising MTBE concentrations, there were sharp rises in the expression of PCNA and BAX, and fall in the expression of Mcl-1. [Bottom] With rising MPP concentrations, the expression of PCNA was less prominently increased than that of MTBE. In addition, unlike to MTBE, the expression of BAX was decreased at higher concentration (1 mM), and the expression of Mcl-1 was increased. c Dissolubility of cholesterol gallstones. [Left] Time-response graph of cholesterol gallstones. MMP dissolved the cholesterol gallstones significantly better than MTBE after 8 h and 24 h (P < 0.05). [Middle] Representative pictures of the residual gallstones at 24 h. [Right] Comparison of dissolubility of each solvent after 8 h and 24 h in cholesterol gallstones. d Dissolubility of mixed gallstones. [Left] Time-response graph of mixed gallstones. The two solvents demonstrated similar dissolutions in mixed gallstones, except for a higher temporal dissolution of MMP at 4 h. [Middle] Representative pictures of the residual gallstones after 24 h. [Right] Comparison of dissolubility of each solvent after 8 h and 24 h in mixed gallstones. e Dissolubility of pigmented gallstones. [Left] Time-response graph of pigmented gallstones. MMP dissolved the pigmented gallstones significantly better than MTBE after 4 h, 8 h, and 24 h (P < 0.05). [Middle] Representative pictures of the residual gallstones after 24 h. [Right] Comparison of dissolubility of each solvent after 8 h and 24 h in pigmented gallstones. Values are presented as mean ± standard deviation of three independent experiments. The in vitro gallstone dissolubility (%) of each solvent was defined as (the average weight of untreated gallstones − the average weight of gallstones after solvent treatment at specific time interval)/the average weights of untreated gallstones. *P < 0.05. BAX, Bcl-2-like protein 4; Mcl-1, myeloid cell leukemia 1; MMP, 2-methoxy-6-methylpyridine; MTBE, methyl tert-butyl ether; PCNA, proliferation cell nuclear antigen