Volume 128, Issue 1, Pages 108-120 (January 2005) Inhibition of inhibitor of κB kinases stimulates hepatic stellate cell apoptosis and accelerated recovery from rat liver fibrosis  Fiona Oakley, Muriel Meso, John P. Iredale, Karen Green, Carylyn J. Marek, Xiaoying Zhou, Michael J. May, Harry Millward-Sadler, Matthew C. Wright, Derek A. Mann  Gastroenterology  Volume 128, Issue 1, Pages 108-120 (January 2005) DOI: 10.1053/j.gastro.2004.10.003 Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 1 Sulfasalazine-induced apoptosis of rat and human HSC. (A) Photomicrographs of activated rat HSC stained with acridine orange at 20× magnification. Yellow arrow, normal cell; red arrow, apoptotic cell. (B) Apoptotic cells were counted in 5 fields in duplicate for each concentration of sulfasalazine in 4 independent experiments, and results are expressed as mean ± SEM. P = .002, P = .003, and P = .002 for sulfasalazine 0.5, 1, and 2 mmol/L, respectively. (C) Caspase 3 activity was calculated as picomoles of p-nitroaniline (pNA) liberated per hour per microgram of protein and was expressed as mean ± SEM (n = 4). P = .026, P = .013, and P = .0168 for rat HSC treated for 24 hours with sulfasalazine 0.5, 1, and 2 mmol/L, respectively. (D) Human HSC were untreated or incubated with dimethyl sulfoxide (DMSO) or sulfasalazine for 24 hours. Apoptotic cells were counted in 5 fields in duplicate for each concentration in 2 independent experiments, and results are expressed as mean ± SEM. (E) Activated rat HSC were untreated or incubated with DMSO, sulfapyridine, or mesalamine (ASA) for 24 hours. Apoptotic cells were counted in 5 fields in duplicate for each concentration in 3 independent experiments, and results are expressed as mean ± SEM. Gastroenterology 2005 128, 108-120DOI: (10.1053/j.gastro.2004.10.003) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 2 Effect of sulfasalazine treatment on spontaneous resolution of fibrosis. Rats injured for 5 weeks with CCl4 or vehicle recovered for 24 hours before the administration of either PBS (CCl4-alone and vehicle groups) or sulfasalazine 150 mg/kg (CCl4/sulfasalazine and sulfasalazine groups). After a further 24 hours, rat livers were assessed for fibrosis by examination of Sirius red-stained sections. (A) Fibrosis scores expressed as the average grade ± SEM from 3 animals. (B) Photomicrographs of Sirius red-stained livers at 200× magnification. C, collagen; BH, ballooned hepatocytes; PT, portal tract. Gastroenterology 2005 128, 108-120DOI: (10.1053/j.gastro.2004.10.003) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 3 Reduced expression of procollagen 1, α-SMA, and TIMP1 transcripts and increased MMP2 activity in sulfasalazine-treated livers. Quantitative reverse-transcription polymerase chain reaction for expression of (A) procollagen I, (B) α-SMA, and (C) TIMP1 transcripts in livers of rats injured for 5 weeks with CCl4 followed by recovery for 48 hours with or without 24 hours of treatment with sulfasalazine. The relative level of transcriptional difference between treated and untreated livers was calculated and expressed as an average ± SEM from 3 independent experiments. (D) Endogenous MMP2 activity was measured in whole-liver extracts isolated from either CCl4 or CCl4/sulfasalazine-treated livers at 48 hours of recovery after 5 weeks of CCl4 injury. Gastroenterology 2005 128, 108-120DOI: (10.1053/j.gastro.2004.10.003) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 4 Selective clearance of α-SMA-positive cells by sulfasalazine. Photomicrographs of liver sections from rats treated with CCl4, CCl4/sulfasalazine (24 hours), vehicle, or sulfasalazine and immunohistochemically stained for ED1-positive macrophages (M; top panel), α-SMA-positive cells (A; middle panel), or TUNEL-positive liver sections (T; bottom panel). Photomicrographs are at 200× magnification and are representative of 3 animals. Gastroenterology 2005 128, 108-120DOI: (10.1053/j.gastro.2004.10.003) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 5 Imaging of mitochondrial permeability and polarization; laser scanning confocal images of rat HSC loaded with TMRM and calcein 2 AM. HSC were loaded with dyes for 1 hour before visualization. (A) Control cells were visualized for calcein (green) fluorescence only. (B) Control cells were visualized for TMRM (red) fluorescence only. (C) Merged calcein and TMRM image of (A) and (B). (D) Merged calcein and TMRM image of HSC treated with gliotoxin 1.5 μmol/L for 1 hour. (E) Merged calcein and TMRM image of HSC treated with TNF-α 20 ng/mL and cycloheximide 10 μmol/L for 4 hours. (F) Merged calcein and TMRM image of HSC treated with sulfasalazine 1 mmol/L for 8 hours; the arrow indicates a cell with early morphological signs of cell death. (G) Merged calcein and TMRM image of HSC treated with sulfasalazine 1 mmol/L for 8 hours; the cell shows later signs of cell death with primarily mitochondrial depolarization. (H) Merged image of HSC not loaded with TMRM and calcein dyes. Gastroenterology 2005 128, 108-120DOI: (10.1053/j.gastro.2004.10.003) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 6 Specific inhibition of NF-κB activity by sulfasalazine. Activated rat HSC were transfected with 100 ng of pRLTK and 1 μg of reporter vectors: (A) NF-κB-dependent pIL6-Luc and IκB-α-luc; (B) NF-κB-independent pTIMP-1-luc and p7×AP-1-Luc for 24 hours. Cells were then treated with either dimethyl sulfoxide (DMSO) or sulfasalazine for a further 24 hours. Luciferase activity was determined, normalized to pRLTK activity, and expressed as mean ± SEM of 4 independent triplicate transfections. Gastroenterology 2005 128, 108-120DOI: (10.1053/j.gastro.2004.10.003) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 7 Sulfasalazine-induced apoptosis is associated with reduced expression of Gadd45β and phosphorylation of JNK. (A) Total RNA isolated from activated rHSC treated with either dimethyl sulfoxide (DMSO) or sulfasalazine 1 mmol/L for varying times was used as a template in reverse-transcription polymerase chain reaction reactions for Gadd45β (30 cycles) or β-actin (24 cycles). (B) Activated rat HSC were exposed to sulfasalazine 1 mmol/L for 2, 4, or 8 hours, and the levels of total and phosphorylated JNK relative to cells treated with vehicle control (DMSO) for 8 hours were determined by immunoblotting. (C) Activated rat HSC stained with acridine orange after incubation with DMSO or 1 mmol/L sulfasalazine ± JNK inhibitor SP600125 (added to cells 30 minutes before DMSO or sulfasalazine) for 6 hours. Apoptotic cells were counted in 5 fields in duplicate for each concentration of sulfasalazine ± JNK inhibitor in 3 independent experiments, and results are expressed as mean ± SEM. Gastroenterology 2005 128, 108-120DOI: (10.1053/j.gastro.2004.10.003) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 8 IKKγ (NEMO) peptide-induced HSC apoptosis is mediated via the activation of JNK. (A) Activated rat HSC transfected with the NF-κB reporter pIκB-α-Luc were treated with either dimethyl sulfoxide (DMSO; vehicle) or 50 μmol/L wild-type (■) or mutant (□) NBD peptide for 24 hours before determination of luciferase activities. Luciferase activity was normalized to pRLTK activity and expressed as mean ± SEM of 3 independent triplicate transfections. (B) Activated rat HSC were stained with acridine orange after incubation with DMSO or NBD peptides for 24 hours. Apoptotic cells were counted in 5 fields in duplicate for each concentration of peptide in 3 independent experiments, and results are expressed as mean ± SEM. (C) Activated rat HSC stained with acridine orange after incubation with DMSO or 50 μmol/L mutant or wild-type NBD peptide ± JNK inhibitor SP600125 (added to cultures 30 minutes before DMSO or peptides) for 8 hours. Apoptotic cells were counted in 5 fields in duplicate for each concentration of mutant or wild-type NEMO peptide ± JNK inhibitor in 3 independent experiments, and results are expressed as mean ± SEM. (D) Total RNA isolated from activated rat HSC treated with either DMSO or 50 μmol/L mutant or wild-type NEMO peptide for 1 hour was used to obtain first-strand complementary DNA, which was then used as a template in reverse-transcription polymerase chain reaction for Gadd45β over 24–32 cycles. Gastroenterology 2005 128, 108-120DOI: (10.1053/j.gastro.2004.10.003) Copyright © 2005 American Gastroenterological Association Terms and Conditions