Inhibition of inhibitor of κB kinases stimulates hepatic stellate cell apoptosis and accelerated recovery from rat liver fibrosis
2005; Elsevier BV; Volume: 128; Issue: 1 Linguagem: Inglês
10.1053/j.gastro.2004.10.003
ISSN1528-0012
AutoresFiona 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,
Tópico(s)Liver Disease Diagnosis and Treatment
ResumoBackground & Aims: Resolution of liver fibrosis is associated with clearance of hepatic myofibroblasts by apoptosis; development of strategies that promote this process in a selective way is therefore important. The aim of this study was to determine whether the inhibitor of κB kinase suppresser sulfasalazine stimulates hepatic myofibroblast apoptosis and recovery from fibrosis. Methods: Hepatic myofibroblasts were generated by culture activation of rat and human hepatic stellate cells. Fibrosis was established in rat livers by chronic injury with carbon tetrachloride followed by recovery with or without sulfasalazine (150 mg/kg) treatment. Results: Treatment of hepatic stellate cells with sulfasalazine (0.5–2.0 mmol/L) induced apoptosis of activated rat and human hepatic stellate cells. A single in vivo administration of sulfasalazine promoted accelerated recovery from fibrosis as assessed by improved fibrosis score, selective clearance of smooth muscle α-actin-positive myofibroblasts, reduced hepatic procollagen I and tissue inhibitor of metalloproteinase 1 messenger RNA expression, and increased matrix metalloproteinase 2 activity. Mechanistic studies showed that sulfasalazine selectively blocks nuclear factor-κB-dependent gene transcription, inhibits hepatic stellate cell expression of Gadd45β, stimulates phosphorylation of Jun N-terminal kinase 2, and promotes apoptosis by a mechanism that is prevented by the Jun N-terminal kinase inhibitor SP600125. As further evidence for a survival role for the inhibitor of κB kinase/nuclear factor-κB pathway in activated hepatic stellate cells, a highly selective cell-permeable peptide inhibitor of κB kinase activation also stimulated hepatic stellate cell apoptosis via a Jun N-terminal kinase-dependent mechanism. Conclusions: Inhibition of the inhibitor of κB kinase/nuclear factor-κB pathway is sufficient to increase the rate at which activated hepatic stellate cells undergo apoptosis both in vitro and in vivo, and drugs that selectively target inhibitor of κB kinase have potential as antifibrotics. Background & Aims: Resolution of liver fibrosis is associated with clearance of hepatic myofibroblasts by apoptosis; development of strategies that promote this process in a selective way is therefore important. The aim of this study was to determine whether the inhibitor of κB kinase suppresser sulfasalazine stimulates hepatic myofibroblast apoptosis and recovery from fibrosis. Methods: Hepatic myofibroblasts were generated by culture activation of rat and human hepatic stellate cells. Fibrosis was established in rat livers by chronic injury with carbon tetrachloride followed by recovery with or without sulfasalazine (150 mg/kg) treatment. Results: Treatment of hepatic stellate cells with sulfasalazine (0.5–2.0 mmol/L) induced apoptosis of activated rat and human hepatic stellate cells. A single in vivo administration of sulfasalazine promoted accelerated recovery from fibrosis as assessed by improved fibrosis score, selective clearance of smooth muscle α-actin-positive myofibroblasts, reduced hepatic procollagen I and tissue inhibitor of metalloproteinase 1 messenger RNA expression, and increased matrix metalloproteinase 2 activity. Mechanistic studies showed that sulfasalazine selectively blocks nuclear factor-κB-dependent gene transcription, inhibits hepatic stellate cell expression of Gadd45β, stimulates phosphorylation of Jun N-terminal kinase 2, and promotes apoptosis by a mechanism that is prevented by the Jun N-terminal kinase inhibitor SP600125. As further evidence for a survival role for the inhibitor of κB kinase/nuclear factor-κB pathway in activated hepatic stellate cells, a highly selective cell-permeable peptide inhibitor of κB kinase activation also stimulated hepatic stellate cell apoptosis via a Jun N-terminal kinase-dependent mechanism. Conclusions: Inhibition of the inhibitor of κB kinase/nuclear factor-κB pathway is sufficient to increase the rate at which activated hepatic stellate cells undergo apoptosis both in vitro and in vivo, and drugs that selectively target inhibitor of κB kinase have potential as antifibrotics. Liver fibrosis is caused by a variety of etiologic agents, including chronic viral hepatitis, alcohol toxicity, autoimmune disease, and hereditary metabolic disorders. For all of these diseases, there is a common pathologic mechanism that leads to fibrosis: the generation and proliferation of smooth muscle α-actin (α-SMA)-positive myofibroblasts of periportal and perisinusoidal origin. By far the best understood of these wound-healing cells is the perisinusoidal-derived myofibroblast that arises as a consequence of the activation of hepatic stellate cells (HSC). 1Friedman S.L. Molecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury.J Biol Chem. 2000; 275: 2247-2250Crossref PubMed Scopus (1905) Google Scholar HSC exist in the normal liver as quiescent retinoid-storing cells, and in response to injury, they activate to become proliferative, profibrogenic cells. This event can be recapitulated in a culture model in which isolated HSC are cultured on plastic in serum-containing media. The activated HSC is a rich source of fibrillar type I and III collagens and also secretes high levels of the tissue inhibitor of metalloproteinase 1 (TIMP1). 2Benyon R.C. Arthur M.J. Extracellular matrix degradation and the role of hepatic stellate cells.Semin Liver Dis. 2001; 21: 373-384Crossref PubMed Scopus (453) Google Scholar Consequently, the persistence of activated HSC in the chronically injured liver leads to qualitative and quantitative alterations of the hepatic extracellular matrix. Net deposition of fibrillar collagens causes both structural and functional perturbation of the liver, which, unless the cause of the underlying disease can be treated, can lead to death. 3Friedman S.L. Liver fibrosis—from bench to bedside.J Hepatol. 2003; 38: S38-S53Abstract Full Text Full Text PDF PubMed Google Scholar Accumulating evidence from clinical and experimental studies indicates that liver fibrosis is reversible. 4Arthur M.J. Reversibility of liver fibrosis and cirrhosis following treatment for hepatitis C.Gastroenterology. 2002; 122: 1525-1528Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar Experimental models of reversible liver fibrosis have provided evidence that clearance of activated HSC by apoptosis is a key event that leads to the removal of collagen- and TIMP1-producing cells. 5Iredale J.P. Benyon R.C. Pickering J. McCullen M. Northrop M. Pawley S. Hovell C. Arthur M.J. Mechanisms of spontaneous resolution of rat liver fibrosis. Hepatic stellate cell apoptosis and reduced hepatic expression of metalloproteinase inhibitors.J Clin Invest. 1998; 102: 538-549Crossref PubMed Scopus (946) Google Scholar, 6Iredale J.P. Cirrhosis new research provides a basis for rational and targeted treatments.BMJ. 2003; 327: 143-147Crossref PubMed Google Scholar, 7Wright M.C. Issa R. Smart D.E. Trim N. Murray G.I. Primrose J.N. Arthur M.J. Iredale J.P. Mann D.A. Gliotoxin stimulates the apoptosis of human and rat hepatic stellate cells and enhances resolution of liver fibrosis in rats.Gastroenterology. 2001; 121: 685-698Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar This in turn leads to restitution of normal baseline matrix metalloproteinase (MMP) activity and remodeling of the hepatic extracellular matrix to a near-normal state. More recently, we have shown in a proof-of-concept study that experimental stimulation of HSC apoptosis promotes accelerated resolution of liver fibrosis in rats. 7Wright M.C. Issa R. Smart D.E. Trim N. Murray G.I. Primrose J.N. Arthur M.J. Iredale J.P. Mann D.A. Gliotoxin stimulates the apoptosis of human and rat hepatic stellate cells and enhances resolution of liver fibrosis in rats.Gastroenterology. 2001; 121: 685-698Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar The fungal metabolite gliotoxin was shown to selectively stimulate HSC apoptosis in culture via a caspase-dependent mechanism possibly involving stimulation of the opening of the membrane permeability transition pore and inhibition of the antiapoptotic transcription factor nuclear factor-κB (NF-κB). 7Wright M.C. Issa R. Smart D.E. Trim N. Murray G.I. Primrose J.N. Arthur M.J. Iredale J.P. Mann D.A. Gliotoxin stimulates the apoptosis of human and rat hepatic stellate cells and enhances resolution of liver fibrosis in rats.Gastroenterology. 2001; 121: 685-698Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar, 8Elsharkawy A.M. Wright M.C. Hay R.T. Arthur M.J.P. Hughes T. Bahr M.J. Degitz K. Mann D.A. Persistent activation of nuclear factor-kappa B in cultured rat hepatic stellate cells involves the induction of potentially novel Rel-like factors and prolonged changes in the expression of IκB family proteins.Hepatology. 1999; 30: 761-769Crossref PubMed Scopus (123) Google Scholar, 9Kweon Y.O. Paik Y.H. Schnabl B. Qian T. Lemasters J.J. Brenner D.A. Gliotoxin-mediated apoptosis of activated human hepatic stellate cells.J Hepatol. 2003; 39: 38-46Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar The aim of this study was to provide defining experimental evidence that the NF-κB signal transduction pathway promotes the survival of activated HSC and that inhibition of components of this pathway is a potential therapeutic strategy for promoting recovery from fibrosis. Sulfasalazine is a drug that has been used on humans for decades for the treatment of chronic inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease. 10Box S.A. Pullar T. Sulfasalazine in the treatment of rheumatoid arthritis.Br J Rheumatol. 1997; 36: 382-386Crossref PubMed Google Scholar, 11Hanauer S.B. Present D.H. The state of the art in management of inflammatory bowel disease.Rev Gastroenterol Disord. 2003; 3: 81-92PubMed Google Scholar Sulfasalazine is a selective inhibitor of NF-κB activation via its ability to block the activity of the inhibitor of κB (IκB) kinases α and β (IKKα and IKKβ). 12Wahl C. Liptay S. Adler G. Schmid R.M. Sulfasalazine a potent and specific inhibitor of nuclear factor kappa B.J Clin Invest. 1998; 101: 1163-1174Crossref PubMed Scopus (639) Google Scholar, 13Weber C.K. Liptay S. Wirth T. Adler G. Schmid R.M. Suppression of NF-κB activity by sulfasalazine is mediated by direct inhibition of IκB kinases α and β.Gastroenterology. 2000; 119: 1209-1218Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar Activated HSC express persistently increased levels of NF-κB and also express constitutively high levels of classic NF-κB-dependent genes such as intercellular adhesion molecule 1 and interleukin (IL)-6. 8Elsharkawy A.M. Wright M.C. Hay R.T. Arthur M.J.P. Hughes T. Bahr M.J. Degitz K. Mann D.A. Persistent activation of nuclear factor-kappa B in cultured rat hepatic stellate cells involves the induction of potentially novel Rel-like factors and prolonged changes in the expression of IκB family proteins.Hepatology. 1999; 30: 761-769Crossref PubMed Scopus (123) Google Scholar In this study we show that both sulfasalazine and a peptide inhibitor of IKK/NF-κB signaling promote HSC apoptosis without the need for any additional stimulation. We also show that in vivo administration of sulfasalazine accelerates the rate at which hepatic myofibroblasts are cleared from the liver and the rate at which fibrosis is resolved. These results implicate the IKK/NF-κB pathway in the regulation of HSC survival and indicate that the IKK complex is a therapeutic target in liver disease. HSC were isolated from normal livers of 350-g adult male Sprague-Dawley rats by sequential perfusion with collagenase and pronase, followed by discontinuous density centrifugation in 11.5% Optiprep (Life Technologies, UK). HSC were cultured on plastic in Dulbecco’s modified Eagle medium supplemented with penicillin 100 U/mL, streptomycin 100 μg/mL, l-glutamine 2 mmol/L, and 16% fetal calf serum and were maintained at 37°C in an atmosphere of 5% CO2. Activated HSC were generated by continuous culture of freshly isolated cells on plastic for 7 days. Human HSC were isolated with pronase and collagenase (as described for rat HSC) from the livers of adult male patients after partial hepatectomy as approved by the UK South and West Local Research Ethics Committee and subject to patient consent. Sulfasalazine, mesalamine, and sulfapyridine were all dissolved in dimethyl sulfoxide at a stock concentration of 0.1 mol/L. The cell-permeable NF-κB essential modulator (NEMO)-binding domain (NBD) peptide inhibitor and its control peptide have been described elsewhere. 14May M.J. D’Acquisto F. Madge L.A. Glockner J. Pober J.S. Ghosh S. Selective inhibition of NF-κB activation by a peptide that blocks the interaction of NEMO with the IκB kinase complex.Science. 2000; 289: 1550-1554Crossref PubMed Scopus (624) Google Scholar The Jun N-terminal kinase (JNK) inhibitor SP600125 was purchased from Calbiochem (UK). Rat liver tissue was fixed in 10% formalin in phosphate-buffered saline (PBS), and liver sections were stained with either Sirius red or H&E as previously described. 7Wright M.C. Issa R. Smart D.E. Trim N. Murray G.I. Primrose J.N. Arthur M.J. Iredale J.P. Mann D.A. Gliotoxin stimulates the apoptosis of human and rat hepatic stellate cells and enhances resolution of liver fibrosis in rats.Gastroenterology. 2001; 121: 685-698Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar Immunohistochemical staining for α-SMA and the macrophage marker ED1 (Serotec, UK) in formalin-fixed tissue was performed by dewaxing slides in xylene and dehydrating in alcohol. Antigen retrieval was achieved by microwaving in citric saline for 15 minutes. Endogenous peroxidase activity was blocked by hydrogen peroxide pretreatment for 10 minutes and was then further blocked by using the avidin/biotin blocking kit (Vector Laboratories, UK). The monoclonal mouse anti-rat ED1 or monoclonal mouse anti-rat α-SMA primary antibodies (Serotec) were diluted 1:160 and incubated for 1.5 hours at room temperature; secondary and anti-immunoglobulin G horseradish peroxidase-conjugated tertiary antibody was incubated for 20 minutes (Vector Laboratories). ED1 and α-SMA expression was visualized by 3,3′-diaminobenzidine tetrahydrochloride staining. Slides were counterstained with Mayers hematoxylin for 30 seconds, dehydrated, and mounted in p-xylene-bis-(N-pyridinium bromide). Formalin-fixed liver sections were stained for terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL)-positive cells by using the in situ cell death kit (Roche) according to the manufacturer’s instructions. Antigen retrieval was achieved by pronase pretreatment, and TUNEL-positive cells were visualized with 3,3′-diaminobenzidine tetrahydrochloride. Whole-cell protein extracts were prepared in radioimmunoprecipitation buffer containing a cocktail of protease and phosphatase inhibitors (Sigma, UK), and 30 μg of each was fractionated by electrophoresis through a 9% sodium dodecyl sulfate-polyacrylamide gel before transfer onto a nitrocellulose membrane. Membranes were blocked for nonspecific antibody binding by incubation for 1 hour at room temperature in Tris-buffered saline/Tween 20 (0.1%) containing 5% BSA. Blots were then incubated overnight at 4°C with either rabbit anti-stress-activated protein kinase/JNK (1:1000) or anti-phospho stress-activated protein kinase/JNK (1:500). Blots were then washed 3 times in Tris-buffered saline/Tween 20 before incubation with a 1:2000 dilution of goat anti-rabbit horseradish peroxidase-conjugated antibody (Sigma). After extensive washing, the blots were processed to distilled water for detection of antigen using the enhanced chemiluminescence system (Amersham). Liver fibrosis was generated by 5-week treatment of adult male Sprague-Dawley (200–225 g) rats with CCl4 (CCl4/olive oil, 1:1 [vol/vol] per kg body weight by intraperitoneal injection twice weekly). Vehicle control animals were treated intraperitoneally with 1 mL of olive oil per kg body weight. Twenty-four hours after the final CCl4 administration, animals were treated with either 150 mg of sulfasalazine per kg body weight by intraperitoneal injection (from 50 mg/mL PBS, 137 mmol/L NaCl, 2.7 mmol/L KCl, and 10 mmol/L phosphate [pH 7.4] stock) or PBS alone. After a further 24 hours, animals were killed by CO2 asphyxiation, and liver and serum samples were prepared. Serum liver enzyme activities were determined essentially as previously described. 7Wright M.C. Issa R. Smart D.E. Trim N. Murray G.I. Primrose J.N. Arthur M.J. Iredale J.P. Mann D.A. Gliotoxin stimulates the apoptosis of human and rat hepatic stellate cells and enhances resolution of liver fibrosis in rats.Gastroenterology. 2001; 121: 685-698Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar Culture-activated rat HSCs were incubated with 500 nmol/L tetramethylrhodamine methylester (TMRM) and 1 μmol/L calcein 2 acetoxymethyl ester diacetate over 1 hour before laser scanning confocal microscopy. A medium change was made after dye loading without repeated addition of treatments except for gliotoxin, which was only added at this medium change because its effects are rapid. At the required time points, the culture medium was discarded, and the cells were washed extensively with HEPES/Hank’s balanced salt solution buffer before visualization of cells with an Olympus BX50WI microscope fitted with a Bio-Rad μRadiance confocal scanning system. TMRM was excited at 543 nm, and fluorescence emission was collected at wavelengths greater than 570 nm. Calcein was excited at 488 nm, and fluorescence emission was collected between 515 and 530 nm. The MMP2 activity assay was purchased from Amersham Pharmacia. Whole liver was placed into ice-cold MMP2 tissue assay buffer (1 mmol/L Tris [pH 7.4] and 1 mmol/L monothioglycerol [Sigma]). Liver samples were homogenized by being sequentially passed through 19- and 21-gauge needles and were then put through a QIAshredder (Qiagen, UK). The protein concentration of liver homogenates was assayed with the Bradford DC assay kit (Bio-Rad). Whole-liver protein 100 μg was used to measure endogenous MMP2 activity according to the manufacturer’s instructions, and the endogenous MMP2 activity was calculated by using the following equation: (Abst=4 hours−Abst=0 hours/hours2)×1000 Plasmid DNA was prepared with a commercial DNA extraction and isolation kit (Maxiprep; Qiagen). The IL-6 and IκB-α promoter reporter constructs have been described elsewhere. 15Mann J. Oakley F. Johnson P.W. Mann D.A. CD40 induces interleukin-6 gene transcription in dendritic cells regulation by TRAF2, AP-1, NF-kappaB and CBF1.J Biol Chem. 2002; 277: 17125-17138Crossref PubMed Scopus (79) Google Scholar TIMP1 promoter activity was determined by using a TIMP1 promoter/luciferase reporter constructed from a previously described TIMP1-chloramphenical acetyl transferase (CAT) reporter. 16Bahr M.J. Vincent K.J. Arthur M.J.P. Fowler A.V. Smart D.E. Wright M.C. Clark I.M. Benyon R.C. Iredale J.P. Mann D.A. Control of the tissue inhibitor of metalloproteinases-1 promoter in culture-activated rat hepatic stellate cells regulation by activator protein-1 DNA binding proteins.Hepatology. 1999; 29: 839-848Crossref PubMed Scopus (78) Google Scholar, 17Trim J.E. Samra S. Arthur M.J.P. Wright M.C. McAuley M. Beri R. Mann D.A. Upstream tissue inhibitor of metalloproteinase-1 (TIMP-1) element-1, a novel and essential regulatory DNA motif in the human TIMP-1 gene promoter, directly interacts with a 30-kDa nuclear protein.J Biol Chem. 2000; 275: 6657-6663Crossref PubMed Scopus (57) Google Scholar Activator protein (AP)-1-dependent gene transcription was measured by using a commercial 7×AP-1-Luc vector (Stratagene, UK). HSC were transfected by the nonliposomal Effectene protocol (Qiagen) with 1 μg of reporter plasmid DNA and 10 ng of the control Renilla plasmid pRLTK. Twenty-four hours after transfection, HSC were treated for 24 hours with sulfasalazine, and a reporter gene activity assay was performed with a dual luciferase kit (Promega, UK). Apoptotic HSC were stained with a 1 μg/mL solution of acridine orange (Sigma) in 10 mmol/L HEPES buffer (pH 7.4). Apoptotic cells in 5 random fields were counted in duplicate wells at 20× magnification with a fluorescein isothiocyanate filter. Cells were counted in 4 independent experiments. Caspase 3 activity was determined by using the caspACE 3 (DEVDase) colorimetric assay and calculated as described by the manufacturer (Promega). Total RNA was isolated from approximately 200 mg of frozen livers by using the Total RNA Purification Kit (Qiagen). First-strand complementary DNA was generated by using 1 μg of deoxyribonuclease-treated RNA, 1 μL of random hexamer primer [p(dN)6], and ribonuclease-free water (Qiagen); heated at 70°C for 5 minutes; and then placed on ice. RNasin (ribonuclease inhibitor), 100 U of Moloney murine leukemia virus reverse transcriptase, 1× Moloney murine leukemia virus buffer, and 0.4 mmol/L deoxynucleoside triphosphates were added, and the mix was incubated at 42°C for 1 hour. 18S ribosomal RNA Taqman primers and probe were purchased from Applied Biosystems (UK). For rat procollagen I, forward primer 5′-ttcacctacagcacgcttgtg-3′, reverse primer 5′-gatgactgtcttgccccaagtt-3′, probe 5′-atggctgcacgagtcacaccg-3′, quencher-N,N,N’,N’-Tetra methyl-6-carboxy Rhodamine (TAMRA), and flourophore-6-carboxy fluorescein (FAM) were used. For rat α-SMA, forward 5′-cgaagcgcagagcaagaga-3′, reverse 5′-catgtcgtcccagttggtgat-3′, and probe 5′-tcctgaccctgaagtatccgatag-3′ were used. For rat TIMP1, forward 5′-agcctgtagctgtgccccaa-3′, reverse 5′-aactcctcgctgcggttctg-3′, and probe 5′-agaggctctccatggctgggggtgta-3′ were used. Taqman quantitative reverse-transcription polymerase chain reactions were composed of complementary DNA; 0.3 μmol/L of forward, reverse, and probe primers; and 12.5 μL of Taqman master mix (Applied Biosystems) in a final volume of 25 μL. Reaction conditions were 50°C for 2 minutes and 95°C for 10 minutes, followed by denaturing for 15 seconds at 95°C and annealing and extension at 60°C for 1 minute for 40 cycles. The relative level of transcriptional difference between CCl4 and CCl4/sulfasalazine livers was calculated with the equation [1/(2A)] × 100, where A indicates the cycle threshold (ct) of the CCl4 group minus the ct of the CCl4/sulfasalazine group after the 18S RNA ct value had been deducted from the target gene for each animal. Amplification of β-actin and Gadd45β was performed by using specific oligonucleotide primers selected within the gene coding regions. Rat-specific β-actin primers used were 5′-agagggaaatcgtgcgtgaca-3′ and 5′-acatctgctggaaggtggaca-3′, designed to produce a 500-base pair product. Rat-specific Gadd45β primers were 5′-ctggtgacggtaagagac-3′ and 5′-tccctacacaagtcaaaa-3′, which produced a 541-base pair product. Sulfasalazine induced a dose-dependent increase in HSC apoptosis as visualized by acridine orange staining (Figure 1). Apoptotic cells were identified by nuclear condensation/blebbing (Figure 1 A). Incubation of HSC with 0.5, 1, and 2 mmol/L sulfasalazine stimulated 28%, 43%, and 50% apoptosis, respectively, compared with dimethyl sulfoxide-treated HSC (Figure 1 B). Sulfasalazine treatment also induced a dose-dependent increase in caspase 3 activity (Figure 1 C). We also confirmed that sulfasalazine stimulates apoptosis of human HSC (Figure 1 D). The sulfasalazine constituent moieties mesalamine and sulfapyridine also have anti-inflammatory properties, although they do not block IKK or NF-κB activity. 12Wahl C. Liptay S. Adler G. Schmid R.M. Sulfasalazine a potent and specific inhibitor of nuclear factor kappa B.J Clin Invest. 1998; 101: 1163-1174Crossref PubMed Scopus (639) Google Scholar, 13Weber C.K. Liptay S. Wirth T. Adler G. Schmid R.M. Suppression of NF-κB activity by sulfasalazine is mediated by direct inhibition of IκB kinases α and β.Gastroenterology. 2000; 119: 1209-1218Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar Mesalamine was without effect on HSC apoptosis, whereas sulfapyridine had only a minor proapoptotic effect at 1 and 2 mmol/L (Figure 1 E). Apoptosis of HSC is mechanistically implicated in the resolution of liver fibrosis. 5Iredale J.P. Benyon R.C. Pickering J. McCullen M. Northrop M. Pawley S. Hovell C. Arthur M.J. Mechanisms of spontaneous resolution of rat liver fibrosis. Hepatic stellate cell apoptosis and reduced hepatic expression of metalloproteinase inhibitors.J Clin Invest. 1998; 102: 538-549Crossref PubMed Scopus (946) Google Scholar, 6Iredale J.P. Cirrhosis new research provides a basis for rational and targeted treatments.BMJ. 2003; 327: 143-147Crossref PubMed Google Scholar, 7Wright M.C. Issa R. Smart D.E. Trim N. Murray G.I. Primrose J.N. Arthur M.J. Iredale J.P. Mann D.A. Gliotoxin stimulates the apoptosis of human and rat hepatic stellate cells and enhances resolution of liver fibrosis in rats.Gastroenterology. 2001; 121: 685-698Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar Therefore, we determined whether sulfasalazine treatment attenuates the rate of recovery from fibrosis induced in rats by twice-weekly administration of CCl4 for 5 weeks. After the final injection of CCl4, the animals were allowed a 24-hour period of recovery before intraperitoneal administration of sulfasalazine (150 mg/kg) or PBS control. After a further 24-hour period of recovery, the degree of liver fibrosis was determined histologically. Sirius red-stained sections were graded according to the degree of fibrosis from 4 (cirrhosis) to 0 (normal) on the basis of the extent of collagen deposition (Figure 2 A). Livers from injured animals treated with sulfasalazine displayed marked improvements in terms of the fibrosis pathology score (1.5 compared with 3.0 for CCl4-only livers). As shown in the representative Sirius red-stained sections (Figure 2 B), livers not treated with sulfasalazine (CCl4 alone) retained the characteristic thick bands of collagen that form bridging tracts between hepatic blood vessels that are absent in control (sulfasalazine alone and vehicle) livers. By contrast, the livers of CCl4/sulfasalazine-treated animals displayed thin fibrotic bands of which most did not bridge vessels. These data indicate that the single administration of sulfasalazine stimulated accelerated recovery from that occurring spontaneously upon withdrawal of injury. In support of this conclusion, sulfasalazine treatment also reduced the hepatic expression of 3 classic profibrogenic genes (procollagen I, α-SMA, and TIMP1) and increased the activity of at least 1 key collagen-degrading enzyme, MMP2 (Figure 3).Figure 3Reduced 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.View Large Image Figure ViewerDownload (PPT) To determine the underlying cellular events responsible for the improved recovery of sulfasalazine-treated animals, we performed further histological studies. Reduced hepatic α-SMA staining was associated with CCl4/sulfasalazine-treated animals compared with CCl4 controls (Figure 4). Counting of α-SMA-stained cells showed that sulfasalazine treatment generated a significant decline in numbers of activated HSC/myofibroblasts (Table 1). In contrast to a 64% decrease in numbers of α-SMA-positive cells, we observed only a 17% reduction in numbers of macrophages in CCl4/sulfasalazine-treated livers, and this did not reach significance (Table 1; Figure 4). TUNEL staining was performed to determine the effects of sulfasalazine on liver cell apoptosis (Table 1; Figure 4). No appreciable differences were observed in total TUNEL-positive cells per field between sulfasalazine-treated and untreated livers, thus indicating that sulfasalazine is unlikely to significantly influence hepatocyte apoptosis. Furthermore, analysis of liver enzyme (alanine aminotransferase and aspartate aminotransferase) activities further supports a lack of effect of the single administration of sulfasalazine on hepatocyte viability (Table 2). At an early 24-hour recovery time point (8 hours of recovery followed by 16 hours with or without sulfasalazine) generated as part of a pilot study, we observed no trends or significant differences in enzyme activities induced by the drug. At the later 48-hour time point there was an apparent trend toward a higher aspartate aminotransferase value for livers of animals treated with sulfasalazine; however, this was not a statistically significant effect. By contrast, when TUNEL-positive cells were counted in association with fibrotic bands (location of activated HSC), we observed a significant difference (P = .0088) between CCl4/sulfasalazine-treated (34% TUNEL positive) and CCl4-only-treated (21% TUNEL positive) livers. Hence, sulfasalazine seems to selectively promote the clearance of activated HSC from recovering livers.Table 1Cell Counts for Macrophages (EDI Stained), Myofibroblasts (α-SMA Positive), and TUNEL-Positive CellsCells counte
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