Hepatic stellate cells (HSCs) transdifferentiation and subsequent inflammation are important pathological processes involved in the formation of cirrhotic portal hypertension. This study characterizes the pathogenetic mechanisms leading to cholestatic liver fibrosis and portal hypertension, and focuses on mammalian target of rapamycin (mTOR) pathway as a potential modulator in the early phase of cirrhotic portal hypertension.
Early cirrhotic portal hypertension was induced by bile duct ligation (BDL) for three weeks. One week after operation, sham-operated (SHAM) and BDL rats received rapamycin (2 mg/kg/day) by intraperitoneal injection for fourteen days. Vehicle-treated SHAM and BDL rats served as controls. Fibrosis, inflammation, and portal pressure were evaluated by histology, morphometry, and hemodynamics. Expressions of pro-fibrogenic and pro-inflammatory genes in liver were measured by RT-PCR; alpha smooth muscle actin (α-SMA) and antigen Ki67 were detected by immunohistochemistry; expressions of AKT/mTOR signaling molecules, extracellular-signal-regulated kinase 1/2 (ERK1/2), p-ERK1/2, and interleukin-1 beta (IL-1β) were assessed by western blot.
The AKT/mTOR signaling pathway was markedly activated in the early phase of cirrhotic portal hypertension induced by BDL in rats. mTOR blockade by rapamycin profoundly improved liver function by limiting inflammation, fibrosis and portal pressure. Rapamycin significantly inhibited the expressions of phosphorylated 70KD ribosomal protein S6 kinase (p-P70S6K) and phosphorylated ribosomal protein S6 (p-S6) but not p-AKT Ser473 relative to their total proteins in BDL-Ra rats. Those results suggested that mTOR Complex 1 (mTORC1) rather than mTORC2 was inhibited by rapamycin. Interestingly, we also found that the level of p-ERK1/2 to ERK1/2 was significantly increased in BDL rats, which was little affected by rapamycin.
The AKT/mTOR signaling pathway played an important role in the early phase of cirrhotic portal hypertension in rats, which could be a potential target for therapeutic intervention in the early phase of such pathophysiological progress.
Over-activation of TGFβ signaling pathway and uncontrolled cell proliferation of hepatic stellate cells (HSCs) play pivotal roles in liver fibrogenesis, while the protein serine/threonine phosphatase PP2Cα was reported to negatively regulate TGFβ signaling pathway and cell cycle. Our study aimed to investigate the role of PP2Cα in liver fibrogenesis.
The effects of PP2Cα activation on liver fibrosis were investigated in human HSCs and primary rat HSCs in vitro using western blotting, real-time PCR, nuclear translocation, cell viability and cell cycle analyses. The antifibrogenic effects in carbon tetrachloride (CCl4)- and bile duct ligation (BDL)-induced mice in vivo were assessed using biochemical, histological and immunohistochemical analyses. The results demonstrated that activation of PP2Cα by overexpression or the new discovered small molecular activator NPLC0393 terminated TGFβ-Smad3 and TGFβ-p38 signaling pathways, induced cell cycle arrest in HSCs and decreased α-smooth muscle actin (α-SMA) expression, collagen deposition and hepatic hydroxyproline (HYP) level in CCl4- and BDL-induced mice.
Our findings suggested that PP2Cα activation might be an attractive new strategy for treating liver fibrosis while the small molecular activator NPLC0393 might represent a lead compound for antifibrogenic drug development. Moreover, our study might provide the first evidence for the role of PP2C family members in the fibrotic disease.
Kupffer cells (KCs), the liver resident macrophages accounting for 80–90% of the total population of fixed tissue macrophages in the body, not only play a key role in host defense via removing particulate materials from the portal circulation, but may also contribute to the pathogenesis of various liver diseases. We have previously demonstrated that KCs play an important role in controlling portal hypertension and hepatocellular injury via releasing thromboxane A2 (TXA2) in early fibrosis induced by one-week bile duct ligation (BDL). Production of TXA2 is controlled by cytosolic phospholipase A2 (cPLA2) that is activated by the interaction of entothelin-1 (ET-1) with its G-protein coupled ET receptor B (ETBR). However, the signaling pathways that contribute to the ET-1-induced activation of cPLA2 and production of TXA2 in KCs in the normal healthy or injured livers are not yet clear, which are investigated in the present study using isolated KCs from one-week BDL or sham rats. The pharmacological inhibition of cPLA2 or chelation of intracellular calcium abrogated the ET-1 induction of TXA2 from KCs. Compared to those from sham rats, KCs from BDL animals displayed a significantly enhanced responsiveness of p38 MAPK to ET-1, increased ETBR and Gαi subunit but decreased Gαq and Gα11 expression. Inhibition of ERK1/2 or Gq signaling abrogated significantly the ET-1 induction of TXA2 in sham KCs but only slightly in BDL KCs. In contrast, inhibition of p38 MAPK and Gi signaling markedly attenuated the ET-1 induction of TXA2 in BDL KCs but had no effect in sham KCs. Lastly, inhibition of PLC or PKC abrogated ET-1 induction of TXA2 in KCs from both sham and BDL groups. The hepatic stress (such as BDL) induces significant modifications in the receptor and intermediates of ET-1 signaling in KC and subsequently alters ET-1 signaling mechanisms, particularly a shift from Gq induced signaling to Gi induced signaling, in the activation of cPLA2 and production of TXA2 in response to ET-1.
Kupffer cell; endothelin-1; cytosolic phospholipase A2; MAPK; TXA2; bile duct ligation
Background & Aims
c-Jun N-terminal kinase (JNK) is activated by multiple profibrogenic mediators; JNK activation occurs during toxic, metabolic, and autoimmune liver injury. However, its role in hepatic fibrogenesis is unknown.
JNK phosphorylation was detected by immunoblot analysis and confocal immunofluorescent microscopy in fibrotic livers from mice after bile duct ligation (BDL) or CCl4 administration and in liver samples from patients with chronic hepatitis C and non-alcoholic steatohepatitis. Fibrogenesis was investigated in mice given the JNK inhibitor SP600125 and in JNK1- and JNK2-deficient mice following BDL or CCl4 administration. Hepatic stellate cell (HSC) activation was determined in primary mouse HSCs incubated with pan-JNK inhibitors SP600125 and VIII.
JNK phosphorylation was strongly increased in livers of mice following BDL or CCl4 administration as well as in human fibrotic livers, occurring predominantly in myofibroblasts. In vitro, pan-JNK inhibitors prevented transforming growth factor (TGF)β-, platelet-derived growth factor (PDGF)-, and angiotensin II-induced murine HSC activation and decreased PDGF and TGFβ signaling in human HSCs. In vivo, pan-JNK inhibition did not affect liver injury but significantly reduced fibrosis after BDL or CCl4. JNK1-deficient mice had decreased fibrosis after BDL or CCl4 whereas JNK2-deficient mice displayed increased fibrosis after BDL but fibrosis was not changed after CCl4. Moreover, patients with chronic hepatitis C who displayed decreased fibrosis in response to the angiotensin receptor type 1 blocker losartan showed decreased JNK phosphorylation.
JNK is involved in HSC activation and fibrogenesis and represents a potential target for antifibrotic treatment approaches.
Two important signaling pathways in liver fibrosis are the PDGF- and TGFβ pathway and compounds inhibiting these pathways are currently developed as antifibrotic drugs. Testing antifibrotic drugs requires large numbers of animal experiments with high discomfort. Therefore, a method to study these drugs ex vivo was developed using precision-cut liver slices from fibrotic rat livers (fPCLS), representing an ex vivo model with a multicellular fibrotic environment. We characterized the fibrotic process in fPCLS from rat livers after 3 weeks of bile duct ligation (BDL) during incubation and tested compounds predominantly inhibiting the TGFβ pathway (perindopril, valproic acid, rosmarinic acid, tetrandrine and pirfenidone) and PDGF pathway (imatinib, sorafenib and sunitinib). Gene expression of heat shock protein 47 (Hsp47), α smooth muscle actin (αSma) and pro-collagen 1A1 (Pcol1A1) and protein expression of collagens were determined. During 48 hours of incubation, the fibrosis process continued in control fPCLS as judged by the increased gene expression of the three fibrosis markers, and the protein expression of collagen 1, mature fibrillar collagen and total collagen. Most PDGF-inhibitors and TGFβ-inhibitors significantly inhibited the increase in gene expression of Hsp47, αSma and Pcol1A1. Protein expression of collagen 1 was significantly reduced by all PDGF-inhibitors and TGFβ-inhibitors, while total collagen was decreased by rosmarinic acid and tetrandrine only. However, fibrillar collagen expression was not changed by any of the drugs. In conclusion, rat fPCLS can be used as a functional ex vivo model of established liver fibrosis to test antifibrotic compounds inhibiting the PDGF- and TGFβ signalling pathway.
Histamine regulates many functions by binding to four histamine G-coupled receptor proteins (H1R, H2R, H3R and H4R). As H3R exerts their effects by coupling to Gαi/o proteins reducing adenosine 3′, 5′-monophosphate (cAMP) levels (a key player in the modulation of cholangiocyte hyperplasia/damage), we evaluated the role of H3R in the regulation of biliary growth. We posed the following questions: (1) Do cholangiocytes express H3R? (2) Does in vivo administration of (R)-(α)-(−)-methylhistamine dihydrobromide (RAMH) (H3R agonist), thioperamide maleate (H3R antagonist) or histamine, in the absence/presence of thioperamide maleate, to bile duct ligated (BDL) rats regulate cholangiocyte proliferation? and (3) Does RAMH inhibit cholangiocyte proliferation by downregulation of cAMP-dependent phosphorylation of protein kinase A (PKA)/extracellular signal-regulated kinase 1/2 (ERK1/2)/ets-like gene-1 (Elk-1)? The expression of H3R was evaluated in liver sections by immunohistochemistry and immunofluorescence, and by real-time PCR in cholangiocyte RNA from normal and BDL rats. BDL rats (immediately after BDL) were treated daily with RAMH, thioperamide maleate or histamine in the absence/presence of thioperamide maleate for 1 week. Following in vivo treatment of BDL rats with RAMH for 1 week, and in vitro stimulation of BDL cholangiocytes with RAMH, we evaluated cholangiocyte proliferation, cAMP levels and PKA, ERK1/2 and Elk-1 phosphorylation. Cholangiocytes from normal and BDL rats express H3R. The expression of H3R mRNA increased in BDL compared to normal cholangiocytes. Histamine decreased cholangiocyte growth of BDL rats to a lower extent than that observed in BDL RAMH-treated rats; histamine-induced inhibition of cholangiocyte growth was partly blocked by thioperamide maleate. In BDL rats treated with thioperamide maleate, cholangiocyte hyperplasia was slightly higher than that of BDL rats. In vitro, RAMH inhibited the proliferation of BDL cholangiocytes. RAMH inhibition of cholangiocyte growth was associated with decreased cAMP levels and PKA/ERK1/2/Elk-1 phosphorylation. Down-regulation of cAMP-dependent PKA/ERK1/2/Elk-1 phosphorylation (by activation of H3R) is important in the inhibition of cholangiocyte growth in liver diseases.
cholestasis; intrahepatic biliary epithelium; proliferation; G-coupled proteins; MAPK
Background and aims
Integrin αvβ6 is highly expressed on certain activated epithelia, where it mediates attachment to fibronectin and serves as coreceptor for the activation of latent TGFβ1. Since its role in liver fibrosis is unknown, we studied αvβ6 function in vitro and explored the antifibrotic potential of the specific αvβ6 antagonist EMD527040.
Experimental liver fibrosis was studied in rats after bile duct ligation (BDL) and in MDR2(abcb4)-/- mice. Different doses of EMD527040 were given to rats from week 2 to 6 after BDL and to Mdr2-/- mice from week 4 to 8. Liver collagen was quantified and expression of αvβ6 and fibrosis related transcripts were determined by quantitative RT-PCR. αvβ6 expressing cells, bile duct proliferation and apoptosis were assessed histologically. The effect of EMD527040 on cholangiocyte adhesion, proliferation, apoptosis and TGFβ1 activation was studied in vitro.
αvβ6 was highly expressed on proliferating bile duct epithelia in fibrosis, with 100-fold increased transcript levels in advanced fibrosis. EMD527040 attenuated bile ductular proliferation and peribiliary collagen deposition by 40-50%, induced downregulation of fibrogenic and upregulation of fibrolytic genes, and improved liver architecture and function. In vitro αvβ6 inhibition reduced activated cholangiocyte proliferation, their adhesion to fibronectin and endogenous activation of TGFβ1 by 50% but did not affect bile duct apoptosis.
Integrin αvβ6 is strongly upregulated in proliferating bile duct epithelia and drives fibrogenesis via adhesion to fibronectin and auto/paracrine TGFβ1 activation. Pharmacological inhibition of αvβ6 potently inhibits the progression of primary and secondary biliary fibrosis.
Abcb4; animal model; antagonist; bile duct; antifibrotic therapy; bile duct ligation; biliary epithelial cell; cell adhesion; cirrhosis; CTGF; fibrogenesis; fibrolysis; fibronectin; fibrosis; gene deletion; gene expression; inhibitor; integrin; liver; MDR2; MMP; nonpeptide; procollagen; proliferation; smooth muscle actin; TGFbeta; TIMP-1
Nogo-B, also known as Reticulon 4B, plays important roles in vascular injuries. Its function in the liver is not understood. The aim of this study was to characterize Nogo-B in liver fibrosis and cirrhosis. Nogo-B distribution was assessed in normal and cirrhotic human liver sections. We also determined the levels of liver fibrosis in wild-type (WT) and Nogo-A/B knockout (NGB KO) mice after sham operation or bile duct ligation (BDL). To investigate the mechanisms of Nogo-B’s involvement in fibrosis, hepatic stellate cells were isolated from WT and NGB KO mice and transformed into myofibroblasts. Portal pressure was measured to test whether Nogo-B gene deletion could ameliorate portal hypertension. In normal livers, Nogo-B expression was found in nonparenchymal cells, whereas its expression in hepatocytes was minimal. Nogo-B staining was significantly elevated in cirrhotic livers. Fibrosis was significantly increased in WT mice 4 weeks after BDL compared with NGB KO mice. The absence of Nogo-B significantly reduced phosphorylation of Smad2 levels upon transforming growth factor β (TGF-β) stimulation. Reconstitution of the Nogo-B gene into NGB KO fibroblasts restored Smad2 phosphorylation. Four weeks after BDL, portal pressure was significantly increased in WT mice by 47%, compared with sham-operated controls (P = 0.03), whereas such an increase in portal pressure was not observed in NGB KO mice (P = NS).
Nogo-B regulates liver fibrosis, at least in part, by facilitating the TGFβ/Smad2 signaling pathway in myofibroblasts. Because absence of Nogo-B ameliorates liver fibrosis and portal hypertension, Nogo-B blockade may be a potential therapeutic target in fibrosis/cirrhosis.
Tetrathiomolybdate (TM), a potent copper-chelating drug, was initially developed for the treatment of Wilson’s disease. Our working hypothesis is that the fibrotic pathway is copper-dependent. Because biliary excretion is the major pathway for copper elimination, a bile duct ligation (BDL) mouse model was used to test the potential protective effects of TM. TM was given in a daily dose of 0.9 mg/mouse by means of intragastric gavage 5 days before BDL. All the animals were killed 5 days after surgery. Plasma liver enzymes and total bilirubin were markedly decreased in TM-treated BDL mice. TM also inhibited the increase in plasma levels of tumor necrosis factor (TNF)-α and transforming growth factor (TGF)-β1 seen in BDL mice. Cholestatic liver injury was markedly attenuated by TM treatment as shown by histology. Hepatic collagen deposition was significantly decreased, and it was paralleled by a significant suppression of hepatic smooth muscle α-actin and fibrogenic gene expression in TM-treated BDL mice. Although the endogenous antioxidant ability was enhanced, oxidative stress as shown by malondialdehyde and 4-hydroxyalkenals, hepatic glutathione/oxidized glutathione ratio, was not attenuated by TM treatment, suggesting the protective mechanism of TM may be independent of oxidative stress. In summary, TM attenuated BDL-induced cholestatic liver injury and fibrosis in mice, in part by inhibiting TNF-α and TGF-β1 secretion. The protective mechanism seems to be independent of oxidative stress. Our data provide further evidence that TM might be a potential therapy for hepatic fibrosis.
Background: Progression of liver fibrosis is characterized by synthesis and degradation of extracellular matrix (ECM). Matrix-metalloproteinases (MMP) cleave collagen fibers at a specific site and thereby generate soluble fragments of ECM (neo-epitopes). The levels of these neo-epitopes might reflect the stage of liver fibrosis and may allow monitoring of anti-fibrotic therapies. Here we analyzed these neo-epitopes as read-out for a liver directed therapy with statins.
Methods: Bile duct ligation (BDL) was performed on wild type rats, which received atorvastatin (15 mg/kg*d) for 1 week starting at 1, 2, 3, 4 and 5 weeks after BDL (T1–T5), while controls remained untreated. Hepatic fibrosis was analyzed by immunohistochemistry and hepatic hydroxyproline content. TGFβ levels were measured by RT-PCR. Proteolytic activity of MMP-2 was examined by zymography. Levels of degradation MMP driven type I, III, IV and VI collagen degradation (C1M, C3M, C4M, and C6M) and type III and IV collagen formation (PRO-C3 and P4NP7S) markers were assessed by specific ELISAs in serum probes.
Results: Serum markers of ECM neo-epitopes reflected significantly the deposition of ECM in the liver and were able to distinguish between early (T1–T3) and severe fibrosis (T4–T5). Statin treatment resulted in reduction of neo-epitope markers, especially when therapy was started in the stage of severe fibrosis (T4–T5). Furthermore, these markers correlated with hepatic expression of profibrotic cytokines TGFβ1 and TGFβ2. Formation markers of type III and IV collagen (PRO-C3 and P4NP7S) and degradation markers C4M and C6M correlated significantly with hepatic MMP-2 activity in rats with severe fibrosis.
Conclusion: Determination of ECM remodeling turnover markers in serum allowed a distinction between mild and severe fibrosis. With respect to statin therapy, the markers may serve as read-out for efficacy of anti-fibrotic treatment.
fibrosis; statins; markers; antifibrotic; ECM (extracellular matrix); remodeling
The vitronectin receptor integrin alpha v beta 3 (αvβ3) promotes angiogenesis by mediating migration and proliferation of endothelial cells, but also drives fibrogenic activation of hepatic stellate cells (HSC) in vitro. Expecting antifibrotic synergism, we studied the effect of αvβ3 inhibition in two in vivo models of liver fibrogenesis. Liver fibrosis was induced in rats by bile duct ligation (BDL) for 6 weeks or by thioacetamide (TAA) injections for 12 weeks. A specific αvβ3 (αvβ5) inhibitor (Cilengitide) was given i.p. twice daily at 15 mg/kg during BDL or after TAA-administration. Liver collagen was determined as hydroxyproline and gene expression was quantified by quantitative PCR. Liver angiogenesis, macrophage infiltration and hypoxia were assessed by CD31, CD68 and HIF-1α immunostaining.
Cilengitide decreased overall vessel formation. This was significant in portal areas of BDL and septal areas of TAA fibrotic rats, and was associated with a significant increase of liver collagen by 31% (BDL) and 27% (TAA), and upregulation of profibrogenic genes and matrix metalloproteinase-13. Treatment increased GGT in both models, while other serum markers remained unchanged. αvβ3 inhibition resulted in mild liver hypoxia, as evidenced by upregulation of hypoxia inducible genes. Liver infiltration by macrophages/Kupffer cells was not affected, although increases in TNF-α, IL-18 and COX-2 mRNA indicated modest macrophage activation.
Specific inhibition of integrin αvβ3 (αvβ5) in vivo decreased angiogenesis but worsened biliary (BDL) and septal (TAA) fibrosis, despite its antifibrogenic effect on HSC in vitro. Angiogenesis inhibitors should be used with caution in patients with hepatic fibrosis. (248 words).
Angiogenesis; collagen; liver fibrosis; rat model; vitronectin receptor
AIM: To investigate the effect of meloxicam on the gut-liver axis after cirrhotic liver resection.
METHODS: Forty-four male Wistar rats were assigned to three groups: (1) control group (CG); (2) bile duct ligation with meloxicam treatment (BDL + M); and (3) bile duct ligation without meloxicam treatment (BDL). Secondary biliary liver cirrhosis was induced via ligature of the bile duct in the BDL + M and BDL groups. After 2 wk, the animals underwent a 50% hepatectomy. In the BDL + M group 15 min prior to the hepatectomy, one single dose of meloxicam was administered. Parameters measured included: microcirculation of the liver and small bowel; portal venous flow (PVF); gastrointestinal (GI) transit; alanine aminotransferase (ALT); malondialdehyde; interleukin 6 (IL-6), transforming growth factor beta 1 (TGF-β1) and hypoxia-inducible factor 1 alpha (HIF-1α) levels; mRNA expression of cyclooxigenase-2 (COX-2), IL-6 and TGF-β1; liver and small bowel histology; immunohistochemical evaluation of hepatocyte and enterocyte proliferation with Ki-67 and COX-2 liver expression.
RESULTS: Proliferative activity of hepatocytes after liver resection, liver flow and PVF were significantly higher in CG vs BDL + M and CG vs BDL group (P < 0.05), whereas one single dose of meloxicam ameliorated liver flow and proliferative activity of hepatocytes in BDL + M vs BDL group. COX-2 liver expression at 24 h observation time (OT), IL-6 concentration and mRNA IL-6 expression in the liver especially at 3 h OT, were significantly higher in BDL group when compared with the BDL + M and CG groups (P < 0.01, P < 0.001, P < 0.01, respectively). Liver and small bowel histology, according to a semi quantitative scoring system, showed better integrity in BDL + M and CG as compared to BDL group. ALT release and HIF-1α levels at 1 h OT were significantly higher in BDL + M compared to CG and BDL group (P < 0.001 and P < 0.01, respectively). Moreover, ALT release levels at 3 and 24 h OT were significantly higher in BDL group compared to CG, P < 0.01. GI transit, enterocyte proliferative activity and number of goblet cells were in favor of meloxicam treatment vs BDL group (P < 0.05, P < 0.001, P < 0.01, respectively). Additionally, villus length were higher in BDL + M as compared to BDL group.
CONCLUSION: One single dose of meloxicam administered after cirrhotic liver resection was able to cause better function and integrity of the remaining liver and small bowel.
Liver cirrhosis; Liver resection; Gut-liver axis; Meloxicam; Cyclooxigenase-2; Microcirculation
Hepatic stellate cell (HSC) activation is a pivotal event in initiation and progression of hepatic fibrosis and a major contributor to collagen deposition driven by transforming growth factor beta (TGFβ). microRNAs (miRs), small non-coding RNAs modulating mRNA and protein expression, have emerged as key regulatory molecules in chronic liver disease. We investigated differentially expressed miRs in quiescent and activated HSCs to identify novel regulators of profibrotic TGFβ signaling. miR microarray analysis was performed on quiescent and activated rat HSCs. Members of the miR-17-92 cluster (19a, 19b, 92a) were significantly down-regulated in activated HSCs. Since miR 19b showed the highest fold-change of the cluster members, activated HSCs were transfected with miR 19b mimic or negative control and TGFβ signaling and HSC activation assessed. miR 19b expression was determined in fibrotic rat and human liver specimens. miR 19b mimic negatively regulated TGFβ signaling components demonstrated by decreased TGFβ receptor II (TGFβRII) and SMAD3 expression. Computational prediction of miR 19b binding to the 3’UTR of TGFβRII was validated by luciferase reporter assay. Inhibition of TGFβ signaling by miR 19b was confirmed by decreased expression of type I collagen and by blocking TGFβ-induced expression of α1(I) and α2(I) procollagen mRNAs. miR 19b blunted the activated HSC phenotype by morphological assessment and decreased αSMA expression. Additionally, miR 19b expression was markedly diminished in fibrotic rat liver compared to normal liver; similarly, miR 19b expression was markedly down-regulated in fibrotic compared to normal human livers.
miR 19b is a novel regulator of TGFβ signaling in HSCs suggesting a potential therapeutic approach for hepatic fibrosis.
Transforming growth factor β; fibrosis; miR 19b; biomarker; hepatic stellate cell
AIM: To investigate whether extracellular signal-regulated kinase 1 (ERK1) is activated and associated with hepatic stellate cell (HSC) proliferation in fibrotic rat liver tissue.
METHODS: Rat hepatic fibrosis was induced by bile duct ligation (BDL). Histopathological changes were evaluated by hematoxylin and eosin staining, and Masson’s trichrome method. ERK1 mRNA in rat liver tissue was determined by reverse transcription-polymerase chain reaction, while the distribution of ERK1 was assessed by immunohistochemistry. ERK1 protein was detected by Western blotting analysis. The number of activated HSCs was quantified after alpha smooth muscle actin (α-SMA) staining.
RESULTS: With the development of hepatic fibrosis, the positive staining cells of α-SMA increased obviously, and mainly resided in the portal ducts. Fiber septa and perisinuses were accompanied with proliferating bile ducts. The positive staining areas of the rat livers in model groups 1-4 wk after ligation of common bile duct (12.88% ± 2.63%, 22.65% ± 2.16%, 27.45% ± 1.86%, 35.25% ± 2.34%, respectively) were significantly larger than those in the control group (5.88% ± 1.46%, P < 0.01). With the development of hepatic fibrosis, the positive cells of ERK1 increased a lot, and were mainly distributed in portal ducts, fiber septa around the bile ducts, vascular endothelial cells and perisinusoidal cells. Western blotting analysis displayed that the expression of ERK1 and ERK2 protein was up-regulated during the model course, and its level was the highest 4 wk after operation, being 3.9-fold and 7.2-fold higher in fibrotic rat liver than in controls. ERK1 mRNA was expressed in normal rat livers as well, which was up-regulated two days after BDL and reached the highest 4 wk after BDL. The expression of ERK1 was positively correlated with α-SMA expression (r = 0.958，P < 0.05).
CONCLUSION: The expression of ERK1 protein and mRNA is greatly increased in fibrotic rat liver tissues, which may play a key role in HSC proliferation and hepatic fibrogenesis.
Extracellular signal-regulated kinase; Hepatic fibrosis; Hepatic stellate cells; Proliferation
AIM: To investigate the anti-hepatofibrotic effects of Gardenia jasminoides in liver fibrosis.
METHODS: Male Sprague-Dawley rats underwent common bile duct ligation (BDL) for 14 d and were treated with Gardenia jasminoides by gavage. The effects of Gardenia jasminoides on liver fibrosis and the detailed molecular mechanisms were also assessed in human hepatic stellate cells (LX-2) in vitro.
RESULTS: Treatment with Gardenia jasminoides decreased serum alanine aminotransferase (BDL vs BDL + 100 mg/kg Gardenia jasminoides, 146.6 ± 15 U/L vs 77 ± 6.5 U/L, P = 0.0007) and aspartate aminotransferase (BDL vs BDL + 100 mg/kg Gardenia jasminoides, 188 ± 35.2 U/L vs 128 ± 19 U/L, P = 0.005) as well as hydroxyproline (BDL vs BDL + 100 mg/kg Gardenia jasminoides, 438 ± 40.2 μg/g vs 228 ± 10.3 μg/g liver tissue, P = 0.004) after BDL. Furthermore, Gardenia jasminoides significantly reduced liver mRNA and/or protein expression of transforming growth factor β1 (TGF-β1), collagen type I (Col I) and α-smooth muscle actin (α-SMA). Gardenia jasminoides significantly suppressed the upregulation of TGF-β1, Col I and α-SMA in LX-2 exposed to recombinant TGF-β1. Moreover, Gardenia jasminoides inhibited TGF-β1-induced Smad2 phosphorylation in LX-2 cells.
CONCLUSION: Gardenia jasminoides exerts antifibrotic effects in the liver fibrosis and may represent a novel antifibrotic agent.
Gardenia jasminoides; Liver fibrosis; Collagen type I; Transforming growth factor-β1/Smad2 pathway; α-smooth muscle actin
Cholestasis is a significant risk factor for immediate hepatic failure due to ischemia reperfusion (I/R) injury in patients undergoing liver surgery or transplantation. We recently demonstrated that inhibition of Hedgehog (Hh) signaling with cyclopamine (CYA) before I/R prevents liver injury. In this study we hypothesized that Hedgehog (Hh) signaling may modulate I/R injury in cholestatic rat liver. Cholestasis was induced by bile duct ligation (BDL). Seven days after BDL, rats were exposed to either CYA or vehicle for 7 d daily before subjecting them to 30 min of ischemia and 4 h of reperfusion. Expression of Hh ligands (sonic hedgehog, Patched-1 and Glioblastoma-1), assessment of liver injury, neutrophil infiltration, cytokines, lipid peroxidation, cell proliferation and apoptosis were determined. Significant upregulation of Hh ligands was seen in vehicle treated BDL rats. I/R injury superimposed on these animals resulted in markedly elevated serum alanine transaminase (ALT), aspartate transaminase (AST), total bilirubin accompanied with increased neutrophil recruitment and lipid peroxidation. Preconditioning with CYA reduced the histological damage and serum liver injury markers. CYA also reduced neutrophil infiltration, proinflammatory cytokines such as TNF-α and IL-1β expression of α-smooth muscle actin and Type 1 collagen resulting in reduced fibrosis. Furthermore CYA treated animals showed reduced cholangiocyte proliferation, and apoptosis. Hepatoprotection by CYA was conferred by reduced activation of protein kinase B (Akt) and extracellular signal regulated kinase (ERK). Endogenous Hh signaling in cholestasis exacerbates inflammatory injury during liver I/R. Blockade of Hh pathway represents a clinically relevant novel approach to limit I/R injury in cholestatic marginal liver.
Preconditioning; bile duct ligation; partial ischemia reperfusion; hepatocellular injury
Background and Purpose
Activation of hepatic stellate cells (HSCs) is a crucial step in the pathogenesis of hepatic fibrosis. Histone deacetylase (HDAC) is an attractive target in liver fibrosis because it plays a key role in gene expression and cell differentiation. We have developed a HDAC inhibitor, N-hydroxy-7-(2-naphthylthio)heptanomide (HNHA), and investigated the anti-fibrotic activity of HNHA in vitro and in vivo.
We investigated the anti-fibrotic effect of HNHA on mouse and human HSC activation in vitro and in the liver of bile duct-ligated (BDL) rats in vivo using cell proliferation assays, cell cycle analysis, biochemical assay, immunohistochemistry and Western blots. Liver pathology was assessed with histochemical techniques.
HNHA inhibited proliferation and arrested the cell cycle via p21 induction in HSCs. In addition, HNHA induced apoptosis of HSCs, which was correlated with reduced COX-2 expression, NF-κB activation and cell death signals. HNHA restored liver function and decreased the accumulation of extracellular matrix in the liver via suppression of HSC activation in BDL rats in vivo. HNHA administration also increased survival in BDL rats.
Conclusions and Implications
HNHA improved liver function, suppressed liver fibrosis and increased survival of BDL rats, accompanied by reduction of cell growth, activation and survival of HSCs. These findings show that HNHA may be a potent anti-fibrosis agent against hepatic fibrosis because of its multi-targeted inhibition of HSC activity in vivo and in vitro.
AIM: To explore the anti-fibrotic effect of Haobie Yangyin Ruanjian Decoction (HYRD) on CCl4-induced hepatic fibrosis in rats and its modulation on the transforming growth factor (TGF) β-Smad signaling pathway.
METHODS: Fifty-six healthy Wistar rats were randomly divided into five groups: normal control group (n = 6), CCl4-induced hepatic fibrosis group (n = 14) and three treatment groups (the treated rats received HYRD via oral administration at daily dosages of 8.2, 2.5 and 0.82 g/kg, respectively) of HYRD (n = 12, respectively). Experimental hepatic fibrosis was induced by subcutaneous injection of carbon tetrachloride solution (CCl4 dissolved in peanut oil, 4:6, V/V) with 0.5 mL/100 g body weight for the first time, and then 0.3 mL/100 g body weight twice a week for 8 wk. In the former 2 wk, rats were raised by feedstuff I (80% corn meal, 20% lard, 0.5% cholesterol). After 2 wk, they were raised by feedstuff II (corn meal and 0.5% cholesterol). Except for the control group, 30% alcohol solution was given orally to each rat every other day from the beginning, 1 mL for each rat. Liver function parameters and hepatic hydroxyproline content were detected by chromatometry. Serum levels of hyaluronic acid (HA), type IV collagen (CIV), type III precollagen (PCIII) and laminin (LN) were assayed with radioimmunoassay. Deposition of collagen was observed with hematoxylin-eosin staining and collagen staining. Gene expression of TGFβ1 and Smad3 were detected with real-time reverse transcriptase-polymerase chain reaction and Western blotting, respectively.
RESULTS: The serum levels of alanine transaminase and aspartate transaminase were increased in the model group compared with the control group (P < 0.01), and they were decreased in the three treatment groups compared with the model group. The serum levels of total protein and albumin were decreased in the model group and increased in the three treatment groups. The hepatic hydroxyproline content and serum levels of PCIII, HA, LN and CIV were markedly increased in the model group compared with the control group, and decreased in the treatment groups. The gene expression of TGFβ1 and Smad3 was enhanced in the model group compared with the control group, and HYRD could down regulate their expression.
CONCLUSION: HYRD can inhibit hepatic fibrosis induced by CCl4 in rats, which is probably associated with its down-regulation on fibrogenic signal transduction of TGFβ-Smad pathway.
Haobie Yangyin Ruanjian Decoction; Hepatic fibrosis; Transforming growth factor β-Smad signaling; Rat model; Carbon tetrachloride
AIM: To assess the antioxidant and antifibrotic effects of long-term Ginkgo biloba administration on liver fibrosis induced by biliary obstruction in rats.
METHODS: Liver fibrosis was induced in male Wistar albino rats by bile duct ligation and scission (BDL). Ginkgo biloba extract (EGb 761, 50 mg/kg·per d) or saline was administered for 28 d. At the end of the treatment period, all rats were killed. Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) levels were determined to assess liver functions and tissue damage, respectively. Tumor necrosis factor-α (TNF-α) was also assayed in serum samples. Liver tissues were taken for determination of the hepatic malondialdehyde (MDA) and glutathione (GSH) levels, myeloperoxidase (MPO) activity and collagen content. Production of reactive oxidants was monitored by chemilu-minescence (CL) assay. Serum AST, ALT, LDH, and TNF-α levels were elevated in the BDL group as compared to control group and were significantly decreased by EGb treatment.
RESULTS: Hepatic GSH level, depressed by BDL, was elevated back to control level in EGb-treated BDL group. Increase in tissue MDA level, MPO activity and collagen content due to BDL were also attenuated by EGb treatment. Furthermore, luminol and lucigenin CL values in BDL group increased dramatically compared to control and reduced by EGb treatment.
CONCLUSION: Our results suggest that Ginkgo biloba protects the liver from oxidative damage following BDL in rats. This effect possibly involves the inhibition of neutrophil infiltration and lipid peroxidation; thus, restoration of oxidant and antioxidant status in the tissue.
Ginkgo biloba; Bile duct ligation; Hepatic fibrosis; Oxidant
Background and Aim
Platelet-derived growth factor (PDGF)-B is a potent profibrogenic mediator expressed by bile duct epithelial cells (BDECs) that contributes to liver fibrosis after bile duct ligation. However, the mechanism of PDGF-B induction in BDECs during cholestasis is not known. Transforming growth factor β (TGFβ) and lipopolysaccharide (LPS) also contribute to the profibrogenic response after bile duct ligation. We tested the hypothesis that LPS and TGFβ1 synergistically induce PDGF-B expression in BDECs.
Transformed human BDECs (MMNK-1 cells) and primary rat BDECs were stimulated with LPS and/or TGFβ1, and signaling pathways through which LPS potentiates TGFβ1-induced PDGF-B mRNA expression were investigated.
Stimulation of MMNK-1 cells with LPS alone did not significantly induce PDGF-B mRNA expression. However, LPS cotreatment enhanced TGFβ1 induction of PDGF-B mRNA in MMNK-1 cells and also in primary rat BDECs. Importantly, cotreatment of MMNK-1 cells with LPS and TGFβ1 also significantly increased PDGF-BB protein expression. Interestingly, LPS did not affect TGFβ1 activation of a SMAD-dependent reporter construct. Rather, stimulation of MMNK-1 cells with LPS, but not TGFβ1, increased JNK1/2 phosphorylation. Expression of dominant negative JNK2, but not dominant negative JNK1, inhibited the LPS potentiation of TGFβ1-induced PDGF-B mRNA expression in MMNK-1 cells. In addition, LPS treatment caused IκBα degradation and activation of a NFκB-dependent reporter construct. Expression of an IκBα super repressor inhibited activation of NFκB and attenuated LPS potentiation of TGFβ1-induced PDGF-B mRNA.
The results indicate that LPS activation of NFκB and JNK2 enhances TGFβ1-induced PDGF-B expression in BDECs.
cholestasis; liver; gene expression; fibrosis; bile ducts
AIM: To investigate the role of oxidative injury and the effect of exogenous melatonin administration on liver damage induced by bile duct ligation (BDL), and second, to evaluate the role of nitric oxide (NO), a free oxygen radical, in oxidative injury.
METHODS: Thirty-two Sprague-Dawley rats were assigned to four groups: sham operation (SO), BDL, BDL+melatonin, and BDL+vehicle. Cholestasis was achieved by double ligature of the common bile duct. Melatonin was injected intraperitoneally 500 µg/(kg·d) for 8 d. Hepatic oxidative stress markers were evaluated by changes in the amount of lipid peroxides, measured as malondialdehyde (MDA), and reduced GSH. Total nitrite (NOX) concentrations were determined in hepatic homogenates. Histopathological examination was performed using a histological scoring system.
RESULTS: The histopathological changes including portal inflammation, necrosis, apoptosis, focal inflammation and fibrosis were severe in the BDL and BDL+vehicle groups. There were numerous large areas of coagulation necrosis. Histological Activity Index scores of these groups were significantly higher than that of the SO group. Treatment with melatonin reduced these alterations significantly. The degree of necro-inflammation and fibrosis showed significant difference between the BDL and BDL+melatonin groups. BDL was accompanied by a significant increase in MDA and NOX, and a significant decrease in GSH levels. Mean±SE values of MDA, GSH and NOX levels of SO group were 147.47±6.69, 0.88±0.33 µmol/g and 180.70±6.58 nm/g, respectively. The values of BDL group were 200.14±21.30, 0.65±0.02 µmol/g, and 400.46±48.89 nm/g, respectively, whereas the values of BDL+melatonin group were 115.93±6.8, 0.74±0.02 µmol/g, and 290.38±32.32 nm/g, respectively. Melatonin treatment was associated with a significant recovery of MDA, GSH and NOX levels.
CONCLUSION: We have concluded that oxidative stress is associated with the pathogenesis of cholestatic liver damage and NO contributes to oxidative damage. Melatonin, even at low dose, is an efficient agent in reducing negative parameters of cholestasis.
Cholestasis; Melatonin; Oxidative stress; Free radicals; Hepatic injury
AIM: To investigate the mechanisms of Lactobacillus plantarum (L. plantarum) action on gut barrier in preoperative and postoperative experimental obstructive jaundice in rats.
METHODS: Forty rats were randomly divided into groups of sham-operation, bile duct ligation (BDL), BDL + L. plantarum, BDL + internal biliary drainage (IBD), and BDL + IBD + L. plantarum. Ten days after L. plantarum administration, blood and ileal samples were collected from the rats for morphological examination, and intestinal barrier function, liver function, intestinal oxidative stress and protein kinase C (PKC) activity measurement. The distribution and expression of the PKC and tight junction (TJ) proteins, such as occludin, zonula occludens-1, claudin-1, claudin-4, junction adhesion molecule-A and F-actin, were examined by confocal laser scanning microscopy, immunohistochemistry, Western blotting, real-time fluorescent quantitative polymerase chain reaction assay.
RESULTS: L. plantarum administration substantially restored gut barrier, decreased enterocyte apoptosis, improved intestinal oxidative stress, promoted the activity and expression of protein kinase (BDL vs BDL + L. plantarum, 0.295 ± 0.007 vs 0.349 ± 0.003, P < 0.05; BDL + IBD vs BDL + IBD + L. plantarum, 0.407 ± 0.046 vs 0.465 ± 0.135, P < 0.05), and particularly enhanced the expression and phosphorylation of TJ proteins in the experimental obstructive jaundice (BDL vs BDL + L. plantarum, 0.266 ± 0.118 vs 0.326 ± 0.009, P < 0.05). The protective effect of L. plantarum was more prominent after internal biliary drainage ( BDL + IBD vs BDL + IBD + L. plantarum, 0.415 ± 0.105 vs 0.494 ± 0.145, P < 0.05).
CONCLUSION: L. plantarum can decrease intestinal epithelial cell apoptosis, reduce oxidative stress, and prevent TJ disruption in biliary obstruction by activating the PKC pathway.
Lactobacillus plantarum; Protein kinase C; Intestinal mucosal barrier; Phosphorylation; Obstructive jaundice
Ischemic injury by hepatic artery ligation (HAL) during obstructive cholestasis induced by bile duct ligation (BDL) results in bile duct damage, which can be prevented by administration of VEGF-A. The potential regulation of VEGF and VEGF receptor expression and secretion by bile acids in BDL with HAL is unknown.
We evaluated whether taurocholic acid (TC) can prevent HAL-induced cholangiocyte damage via the alteration of VEGFR-2 and/or VEGF-A expression.
Utilizing BDL, BDL+TC, BDL+HAL, BDL+HAL+TC, and BDL+HAL+wortmannin+TC treated rats, we evaluated cholangiocyte apoptosis, proliferation, and secretion as well VEGF-A and VEGFR-2 expression by immunohistochemistry. In vitro, we evaluated the effects of TC on cholangiocyte secretion of VEGF-A and the dependence of TC-induced proliferation on the activity of VEGFR-2.
In BDL rats with HAL, chronic feeding of TC prevented HAL-induced loss of bile ducts and HAL-induced decreased cholangiocyte secretion. TC also prevented HAL-inhibited VEGF-A and VEGFR-2 expression in liver sections and HAL-induced circulating VEGF-A levels, which were blocked by wortmannin administration. In vitro, TC stimulated increased VEGF-A secretion by cholangiocytes, which was blocked by wortmannin and stimulated cholangiocyte proliferation that was blocked by VEGFR-2 kinase inhibitor.
TC prevented HAL-induced biliary damage by upregulation of VEGF-A expression.
Apoptosis; cAMP; intrahepatic biliary epithelium; mitosis; VEGF
AIM: To assess the effect of iron reduction after phlebotomy in rats with “normal” hepatic iron concentration (HIC) on the progression of hepatic fibrosis, as a result of bile duct ligation (BDL).
METHODS: Rats underwent phlebotomy before or after sham operation or BDL. Animals undergone only BDL or sham operation served as controls. Two weeks after surgery, indices of hepatic damage and fibrosis were evaluated.
RESULTS: Phlebotomy lowered HIC. Phlebotomy after BDL was associated with body weight increase, lower hepatic weight, less portal hypertension, less periportal necrosis, less portal inflammation, lower hepatic activity index score and higher albumin levels. On the other hand, phlebotomy before BDL was associated with body weight decrease and hepatic activity index score increase. Phlebotomy after sham operation was not associated with any hepatic or systemic adverse effects.
CONCLUSION: Reduction of HIC after induction of liver damage may have beneficial effects in BDL rats. However, iron deficiency could induce impairment of liver function and may make the liver more susceptible to insults like BDL.
Iron; Phlebotomy; Bile duct ligation; Hepatic activity index; Rat.
Epithelial-mesenchymal transitions (EMTs) play an important role in tissue construction during embryogenesis, and evidence suggests that this process may also help to remodel some adult tissues after injury. Activation of the hedgehog (Hh) signaling pathway regulates EMT during development. This pathway is also induced by chronic biliary injury, a condition in which EMT has been suggested to have a role. We evaluated the hypothesis that Hh signaling promotes EMT in adult bile ductular cells (cholangiocytes). In liver sections from patients with chronic biliary injury and in primary cholangiocytes isolated from rats that had undergone bile duct ligation (BDL), an experimental model of biliary fibrosis, EMT was localized to cholangiocytes with Hh pathway activity. Relief of ductal obstruction in BDL rats reduced Hh pathway activity, EMT, and biliary fibrosis. In mouse cholangiocytes, coculture with myofibroblastic hepatic stellate cells, a source of soluble Hh ligands, promoted EMT and cell migration. Addition of Hh-neutralizing antibodies to cocultures blocked these effects. Finally, we found that EMT responses to BDL were enhanced in patched-deficient mice, which display excessive activation of the Hh pathway. Together, these data suggest that activation of Hh signaling promotes EMT and contributes to the evolution of biliary fibrosis during chronic cholestasis.