Volume 118, Issue 2, Pages 380-389 (February 2000) Genipin, a metabolite derived from the herbal medicine Inchin-ko-to, and suppression of Fas-induced lethal liver apoptosis in mice  Masahiro Yamamoto, Naoko Miura, Nobuhiro Ohtake, Sakae Amagaya, Atsushi Ishige, Hiroshi Sasaki, Yasuhiro Komatsu, Kazunori Fukuda, Takashi Ito, Katsutoshi Terasawa  Gastroenterology  Volume 118, Issue 2, Pages 380-389 (February 2000) DOI: 10.1016/S0016-5085(00)70220-4 Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 Effects of hepatoprotective drugs on Fas-mediated lethality. Five groups of mice were injected IV with 6.7 μg Jo2 pretreated with vehicle (•), ICKT (2 g/kg PO; ■), GLN (100 mg/kg IP; ◊), ursodeoxycholic acid (2 g/kg PO; ▿), or Sho-saiko-to (2 g/kg PO; Δ) as described in Materials and Methods. n = 20 for each group. The data represent survival curves of 1 of 4 experiments. Gastroenterology 2000 118, 380-389DOI: (10.1016/S0016-5085(00)70220-4) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 Apoptotic index in mouse liver after Jo2 injection. Mice given vehicle, ICKT (2 g/kg), or GLN (100 mg/kg) were IV injected with 3.3 μg Jo2. Liver tissue was obtained 1.75 hours after Jo2 injection, and apoptotic index was calculated as described in Materials and Methods. Data represent means ± SD (n = 6) from 1 representative of 3 experiments. *P < 0.0001, **P < 0.0005 vs. vehicle. Gastroenterology 2000 118, 380-389DOI: (10.1016/S0016-5085(00)70220-4) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 3 Fluorescent observation of H&E-stained liver sections from Jo2-injected mice that received vehicle, ICKT, or GLN pretreatment. Mice given vehicle, ICKT (2 g/kg), or GLN (100 mg/kg) were IV injected with 3.3 μg Jo2. Mice were randomly assigned to be killed at various time points. Liver sections obtained at indicated times were stained with H&E and observed with an Olympus BH-2 microscope equipped with a filter set. Gastroenterology 2000 118, 380-389DOI: (10.1016/S0016-5085(00)70220-4) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 4 Changes in (A) AST and (B) ALT in Jo2-injected mice that received vehicle, ICKT, or GLN pretreatment. Mice given vehicle (○), ICKT (•), or GLN (■) were treated with 3.3 μg Jo2 and killed at various time points (4–6 mice/group) as described in Figure 3. Blood samples were collected, and AST and ALT levels were measured. Each value represents the mean ± SD. *P < 0.005, **P < 0.05 vs. vehicle. Gastroenterology 2000 118, 380-389DOI: (10.1016/S0016-5085(00)70220-4) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 5 Effects of geniposide and genipin on Fas-mediated apoptosis in primary cultured mouse hepatocytes. Cells were treated with (•) or without (○) 100 ng/mL Jo2 in the presence of 50 μg/mL cycloheximide and various concentrations of geniposide or genipin. At 24 hours of culture, (A) LDH activity of the supernatant or (B) MTT reduction by the cells was measured as described in Materials and Methods. After Jo2 treatment, LDH release increased to 84.9% ± 0.9% vs. 3.10% ± 1.2% control culture, and MTT reduction decreased to 18.3% ± 0.5% vs. control. Data represent means ± SD (n = 3) from 1 of 4 experiments. Gastroenterology 2000 118, 380-389DOI: (10.1016/S0016-5085(00)70220-4) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 6 Time course of caspase 3 activation after Jo2 administration. BALB/c mice with genipin (■) or vehicle (•) pretreatment were injected with 3.3 μg Jo2. Control mice (○) were pretreated with vehicle and injected with saline. Livers were excised at the indicated times. Extracts were prepared as described in Materials and Methods. Rate of acetyl-DEVD-AMC cleavage at each time (mean ± SD of triplicate determinations) is shown. This experiment was repeated once with similar results. Gastroenterology 2000 118, 380-389DOI: (10.1016/S0016-5085(00)70220-4) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 7 Time course of caspase 8 activation after Jo2 administration. BALB/c mice with genipin or vehicle pretreatment were injected with 3.3 μg Jo2. Livers were excised at the indicated times. Protein in the liver homogenate was separated on 5%–20% sodium dodecyl sulfate–polyacrylamide gels and electroblotted onto nylon membranes. The blot was probed with anti–caspase 8 rabbit polyclonal antibody (H-134; Santa Cruz Biotechnology) as described in Materials and Methods. Actin protein detected by antiactin monoclonal antibody (clone AC-15; Sigma) was used as an internal control for relative protein amount loaded in each lane. This experiment was repeated on another occasion with similar results. Gastroenterology 2000 118, 380-389DOI: (10.1016/S0016-5085(00)70220-4) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 8 Ex vivo determination of ΔΨm change in hepatocytes. BALB/c mice were pretreated with genipin or vehicle; at 30 minutes after the final drug treatment, 3.3 μg Jo2 or saline was injected. At 0 or 15 minutes after injection, the hepatocytes were isolated and purified within 30 minutes. Cells were incubated with 1 μg/mL rhodamine 123 for 15 minutes, and flow cytometric analysis was performed. Times shown are when incubation with rhodamine 123 started. A negative control (cells treated with mClCCP) was included. Data are representative of 4 experiments. Gastroenterology 2000 118, 380-389DOI: (10.1016/S0016-5085(00)70220-4) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 9 Effect of genipin-treatment of mice on Ca2+-induced MPT ex vivo. Liver mitochondria were purified from vehicle- (○) and genipin- (•) administered mice, and MPT was induced by addition of 300 μmol/L Ca2+. Mitochondrial swelling was monitored at 540 nm at 50-second intervals for 600 seconds. Data represent means ± SE (n = 4). Gastroenterology 2000 118, 380-389DOI: (10.1016/S0016-5085(00)70220-4) Copyright © 2000 American Gastroenterological Association Terms and Conditions