Ethyl acetate fraction of Amomum xanthioides improves bile duct ligation-induced liver fibrosis of rat model via modulation of pro-fibrogenic cytokines.
ABSTRACT: We investigated anti-hepatofibrotic effects of ethyl acetate fraction of Ammomum xanthoides (EFAX) using bile duct ligation (BDL)-induced hepatic fibrosis in a rat model. Male SD rats (6 weeks old) underwent BDL followed by 15 days of orall administration of EFAX (12.5, 25 or 50 mg/kg) or ursodeoxycholic acid (25 mg/kg). BDL caused animal death, ascites formation, alterations in serum biochemistries, and severe hepatic injury with excessive collagen deposition, whereas EFAX treatment significantly attenuated these effects. BDL markedly increased the pro-fibrogenic cytokines (TGF-?, PDGF-?, and CTGF) and the extracellular matrix indicators ?-SMA, TIMP-1 and collagen type 1 in hepatic proteins and gene expression levels, which were notably normalized by EFAX treatment. EFAX also markedly normalized pro-fibrogenic signaling molecules including Smad2/3, Smad7, Akt, p44/42, and p38. We further explored EFAX mechanisms of actions using LX-2 cells (human derived hepatic stellate cell line). Pre-treatment with EFAX drastically attenuated the activation of ?-SMA and Smad2/3, which are downstream molecules of TGF-?. These findings suggest that EFAX may be a potent anti-hepatofibrotic agent, and its corresponding mechanisms primarily involve the modulation of pro-fibrogenic cytokines.
Project description:<h4>Background and purpose</h4>Chronic hepatic inflammation results in liver fibrosis. As effective anti-fibrogenic agents are lacking, we investigated ursodeoxycholyl lysophosphatidylethanolamide (UDCA-LPE), a synthetic bile acid-phospholipid conjugate with anti-inflammatory and anti-apoptotic properties for tis effects on hepatic fibrogenesis.<h4>Experimental approach</h4>To stimulate fibrogenesis, LX2 hepatic stellate cells were cultured with conditioned medium from CL48 liver cells after exposure to stress-inducing conditions - methionine-choline-deficient (MCD) medium or TNF?/cycloheximide (CHX) - with or without UDCA-LPE preincubation. Anti-fibrogenic effects of UDCA-LPE were further studied in CL48 and LX2 cells and in primary human hepatic stellate cells (HHStec) directly exposed to TGF-?1. To test UDCA-LPE?in vivo, C57BL/6 mice were fed a MCD diet for 11 weeks followed by 30?mg·kg(-1) UDCA-LPE 3× per week for 2.5 weeks.<h4>Key results</h4>Expression of ?-smooth muscle actin (?-SMA), ?1-collagen, vimentin and TGF-?1 was down-regulated by up to 93% by UDCA-LPE in LX-2 cells cultured with conditioned medium. Also, UDCA-LPE inhibited Smad3 phosphorylation in CL48 cells incubated with MCD medium or TNF?/CHX and in LX2 cells exposed to conditioned medium. UDCA-LPE also decreased phosphorylated Smad3 and Smad2 directly induced by TGF-?1. Inhibition of TGF-?1/Smad2/3 signalling with down-regulation of target genes was confirmed in HHStec. In vivo, UDCA-LPE decreased hepatic ?-SMA, ?1-collagen and TGF-?1 expression and markedly lowered ?-SMA protein and collagen deposition in MCD mice.<h4>Conclusions and implications</h4>By blocking TGF-?1/Smad2/3 signalling, UDCA-LPE suppressed key mediators of hepatic fibrogenesis. Thus, UDCA-LPE could be suitable for prevention of fibrotic progression of chronic liver disease.
Project description: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 ?g/g vs 228 ± 10.3 ?g/g liver tissue, P = 0.004) after BDL. Furthermore, Gardenia jasminoides significantly reduced liver mRNA and/or protein expression of transforming growth factor ?1 (TGF-?1), collagen type?I?(Col?I) and ?-smooth muscle actin (?-SMA). Gardenia jasminoides significantly suppressed the upregulation of TGF-?1, Col?I?and ?-SMA in LX-2 exposed to recombinant TGF-?1. Moreover, Gardenia jasminoides inhibited TGF-?1-induced Smad2 phosphorylation in LX-2 cells. CONCLUSION:Gardenia jasminoides exerts antifibrotic effects in the liver fibrosis and may represent a novel antifibrotic agent.
Project description:Transforming growth factor ?1 (TGF-?1) is the pivotal pro-fibrogenic cytokine in hepatic fibrosis. Reducing the over-produced expression of TGF-?1 or blocking its signaling pathways is considered to be a promising therapeutic strategy for hepatic fibrosis. In this study, we evaluated the feasibility of attenuating hepatic fibrosis by vaccination against TGF-?1 with TGF-?1 kinoids. Two TGF-?1 kinoid vaccines were prepared by cross-linking TGF-?1-derived polypeptides (TGF-?1(25)-[41-65] and TGF-?1(30)-[83-112]) to keyhole limpet hemocyanin (KLH). Immunization with the two TGF-?1 kinoids efficiently elicited the production of high-levels of TGF-?1-specific antibodies against in BALB/c mice as tested by enzyme-linked immunosorbent assay (ELISA) and Western blotting. The antisera neutralized TGF-?1-induced growth-inhibition on mink lung epithelial cells (Mv1Lu) and attenuated TGF-?1-induced Smad2/3 phosphorylation, ?-SMA, collagen type 1 alpha 2 (COL1A2), plasminogen activator inhibitor-1 (PAI-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1) expression in the rat hepatic stellate cell (HSC) line, HSC-T6. Vaccination against TGF-?1 with the kinoids significantly suppressed CCl4-induced collagen deposition and the expression of ?-SMA and desmin, attenuated hepatocyte apoptosis and accelerated hepatocyte proliferation in BALB/c mice. These results demonstrated that immunization with the TGF-?1 kinoids efficiently attenuated CCl4-induced hepatic fibrosis and liver injury. Our study suggests that vaccination against TGF-?1 might be developed into a feasible therapeutic approach for the treatment of chronic fibrotic liver diseases.
Project description:Hepatic stellate cells (HSC) play a central role in the pathogenesis of liver fibrosis, transdifferentiating in chronic liver disease from "quiescent" HSC to fibrogenic myofibroblasts. Transforming growth factor-beta (TGF-beta), acting both directly and indirectly, is a critical mediator of this process. To characterize the function of the TGF-beta signaling intermediates Smad2 and Smad3 in HSC, we infected primary rat HSC in culture with adenoviruses expressing wild-type and dominant negative Smads 2 and 3. Smad3-overexpressing cells exhibited increased deposition of fibronectin and type 1 collagen, increased chemotaxis, and decreased proliferation compared with uninfected cells and those infected with Smad2 or either dominant negative, demonstrating different biological functions for the two Smads. Additionally, coinfection experiments suggested that Smad2 and Smad3 signal via independent pathways. Smad3-overexpressing cells as well as TGF-beta-treated cells demonstrated more focal adhesions and increased alpha-smooth muscle actin (alpha-SMA) organization in stress fibers, although all cells reached the same level of alpha-SMA expression, indicating that Smad3 also regulates cytoskeletal organization in HSC. We suggest that TGF-beta, signaling via Smad3, plays an important role in the morphological and functional maturation of hepatic myofibroblasts.
Project description:Activation of hepatic stellate cells (HSCs) plays a crucial role in liver fibrogenesis. armepavine (Arm, C19H23O3N), an active compound from Nelumbo nucifera, has been shown to exert immunosuppressive effects on T lymphocytes and on lupus nephritic mice. The aim of this study was to investigate whether Arm could exert anti-hepatic fibrogenic effects in vitro and in vivo. A cell line of rat HSCs (HSC-T6) was stimulated with tumor necrosis factor-alpha (TNF-alpha) or lipopolysaccharide (LPS) to evaluate the inhibitory effects of Arm. An in vivo therapeutic study was conducted in bile duct-ligated (BDL) rats. BDL rats were given Arm (3 or 10 mg/kg) by gavage twice daily for 3 weeks starting from the onset of BDL. Liver sections were taken for fibrosis scoring, immuno-fluorescence staining and quantitative real-time mRNA measurements. In vitro, Arm (1-10 microM) concentration-dependently attenuated TNF-alpha- and LPS-stimulated alpha-SMA protein expression and AP-1 activation by HSC-T6 cells without adverse cytotoxicity. Arm also suppressed TNF-alpha-induced collagen collagen deposition, NFkappaB activation and MAPK (p38, ERK1/2, and JNK) phosphorylations. In vivo, Arm treatment significantly reduced plasma AST and ALT levels, hepatic alpha-SMA expression and collagen contents, and fibrosis scores of BDL rats as compared with vehicle treatment. Moreover, Arm attenuated the mRNA expression levels of col 1alpha2, TGF-beta1, TIMP-1, ICAM-1, iNOS, and IL-6 genes, but up-regulated metallothionein genes. Our study results showed that Arm exerted both in vitro and in vivo antifibrotic effects in rats, possibly through anti-NF-kappaB activation pathways.
Project description:Dihydrotanshinone I (DHI), a lipophilic component of traditional Chinese medicine Salvia miltiorrhiza Bunge, has various therapeutic effects. We investigated the anti-fibrotic effect of DHI and its underlying mechanisms in vitro and in vivo.Rats subjected to bile duct ligation (BDL) were treated with DHI (25 mg·kg-1 ·day-1 , i.p.) for 14 days. Serum biochemical and liver tissue morphological analyses were performed. The human hepatic stellate cell line LX-2 served as a liver fibrosis model in vitro. Liver fibrogenic genes, yes-associated protein (YAP) downstream genes and autophagy markers were examined using western blot and real-time PCR analyses. Similar analyses were done in rat primary hepatic stellate cells (pHSCs). Autophagy flux was assessed by immunofluorescence.In BDL rats, DHI administration attenuated liver necrosis, bile duct proliferation and collagen accumulation and reduced the expression of genes associated with fibrogenesis, including Tgfb1, Mmp-2, Acta2 and Col1a1. DHI (1, 5, 10 ?mol·L-1 ) time- and dose-dependently suppressed the protein level of COL1A1, TGF?1 and ?-SMA in LX-2 cells and rat pHSCs. Furthermore, DHI blocked the nuclear translocation of YAP, which inhibited the YAP/TEAD2 interaction and its downstream fibrogenic genes, connective tissue growth factor, SOX4 and survivin. This stimulated autophagic flux and accelerated the degradation of liver collagen.DHI exerts anti-fibrotic effects in BDL rats, LX-2 cells and rat pHSCs by inhibiting the YAP and TEAD2 complex and stimulating autophagy. These findings indicate that DHI may be a potential therapeutic for the treatment of liver fibrosis.
Project description:Transforming growth factor-? (TGF-?) is a potent cytokine that promotes the development of fibrogenic cells, stimulates the expression of fibrosis-related genes, and consequently results in hepatic fibrogenesis. The involvement of miRNAs in this process remains largely unknown. We showed that miR-122 was substantially expressed in hepatic stellate cells (HSCs) and fibroblasts, the major sources of fibrogenic cells in liver tissues. Notably, exposure to TGF-? led to significant downregulation of miR-122. Furthermore, reintroduction of miR-122 suppressed TGF-?-induced expression of fibrosis-related genes, including alpha smooth muscle actin (?-SMA), fibronectin 1 (FN1) and ?1 type I collagen (COL1A1), in HSCs and fibroblasts. Subsequent mechanism investigations revealed that miR-122 directly inhibited FN1 expression by binding to its 3'-untranslated region and indirectly reduced the transcription of ?-SMA and COL1A1 by inhibiting the expression of serum response factor (SRF), a key transcription factor that mediated the activation of fibrogenic cells. Further in vivo studies disclosed that intravenous injection of miR-122-expressing lentivirus successfully increased miR-122 level and reduced the amount of collagen fibrils, FN1 and SRF in the livers of CCl4-treated mice. These findings disclose a novel TGF-?-miR-122-FN1/SRF signaling cascade and its implication in hepatic fibrogenesis, and suggest miR-122 as a promising molecular target for anti-fibrosis therapy.
Project description:Inflammation has an important role in the development of liver fibrosis in general and the activation of hepatic stellate cells (HSCs) in particular. It is known that HSCs are themselves able to produce cytokines and chemokines, and that this production may be a key event in the initiation of fibrogenesis. However, the direct involvement of cytokines and chemokines in HSC (self-)activation remains uncertain. In this study, the effects of pro-inflammatory cytokines IL-1? and ?, TNF-?, and IL-8 on the activation state of HSCs were examined, in comparison to the pro-fibrogenic mediator TGF-?1. LX-2 cells were stimulated for 24 or 48 hours with recombinant human form of the pro-inflammatory cytokines IL-1? and ?, TNF-?, and IL-8, and also the pro-fibrogenic mediator TGF-?1. Two drugs were also evaluated, the anti-TNF-? monoclonal antibody infliximab and the IL-1 receptor antagonist anakinra, regarding their inhibitory effects. In LX-2 human HSC, treatment with TGF-?1 are associated with downregulation of the metalloproteinase (MMP)-1 and MMP-3, with upregulation of tissue inhibitor of metalloproteinase (TIMP)-1, collagen type I ?1, collagen type IV ?1, ?-SMA, endothelin-1 and PDGF-BB. Cytokines and chemokines expression were found to be downregulated, excepting IL-6. In contrast, we observed that LX-2 exposure to IL-1, TNF-? and IL-8 can reverse the phenotype of pro-fibrogenic activated cells. Indeed, MMP-1, MMP-3 and MMP-9 were found elevated, associated with downregulation of ?-SMA and/or PDGF-BB, and a greater expression of IL-1?, IL-6, IL-8, CXCL1 and CCL2. Lastly, we found that infliximab and anakinra successfully inhibits effects of TNF-? and IL-1 respectively in LX-2 cells. Infliximab and anakinra may be of value in preclinical trials in chronic liver disease. Overall, our results suggest that (i) pro-inflammatory mediators exert complex effects in HSCs via an MMP/TIMP imbalance, and (ii) targeting IL-1 signaling may be a potentially valuable therapeutic strategy in chronic liver diseases.
Project description:UNLABELLED:Cholestasis leads to liver cell death, fibrosis, cirrhosis, and eventually liver failure. Despite limited benefits, ursodeoxycholic acid (UDCA) is the only Food and Drug Administration-approved treatment for cholestatic disorders. Retinoic acid (RA) is a ligand for nuclear receptors that modulate bile salt homeostasis. RA also possesses immunomodulatory effects and is used to treat acute promyelocytic leukemia and inflammatory disorders such as psoriasis, acne, and rheumatoid arthritis. To test whether the supplementation of RA with UDCA is superior to UDCA alone for treating cholestasis, male Sprague-Dawley rats underwent common bile duct ligation (BDL) for 14 days and were treated with phosphate-buffered saline (PBS), UDCA, all-trans retinoic acid (atRA), or UDCA and atRA by gavage. Treatment with UDCA and atRA substantially improved animal growth rates, significantly reduced liver fibrosis and bile duct proliferation, and nearly eliminated liver necrosis after BDL. Reductions in the bile salt pool size and liver hydroxyproline content were also seen with treatment with atRA or atRA and UDCA versus PBS and UDCA. Furthermore, atRA and UDCA significantly reduced liver messenger RNA and/or protein expression of transforming growth factor ?1 (Tgf-?1), collagen 1a1 (Col1A1), matrix metalloproteinase 2 (Mmp2), cytokeratin 19, ?-smooth muscle actin (?-SMA), cytochrome P450 7A1 (Cyp7a1), tumor necrosis factor ?, and interleukin-?1. The molecular mechanisms of this treatment were also assessed in human hepatocytes, hepatic stellate cells, and LX-2 cells. atRA alone or in combination with UDCA greatly repressed CYP7A1 expression in human hepatocytes and significantly inhibited COL1A1, MMP2, and ?-SMA expression and/or activity in primary human hepatic stellate cells and LX-2 cells. Furthermore, atRA reduced TGF-?1-induced Smad2 phosphorylation in LX-2 cells. CONCLUSION:Our findings indicate that the addition of RA to UDCA reduces the bile salt pool size and liver fibrosis and might be an effective supplemental therapy with UDCA for cholestatic diseases.
Project description: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)-alpha and transforming growth factor (TGF)-beta1 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 alpha-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-alpha and TGF-beta1 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.