Hepatic Stellate Cells Collagen Synthesis Research Articles

Treatment-damaged hepatocellular carcinoma promotes activities of hepatic stellate cells and fibrosis through GDF15

The aim of this study was to investigate whether treatment-damaged hepatocellular carcinoma (HCC) would accelerate liver cirrhosis through promoting the activities of hepatic stellate cells (HSCs). HCC cells were exposed to chemotherapeutic agent or hypoxia to mimic the transarterial chemoembolization (TACE)-like treatment. Growth differentiation factor 15 (GDF15) expression was increased in cisplatin- or hypoxia-treated HCC cells. Treatment-induced GDF15 increase in HCC cells was mediated by p38MAPK, JNK, ERK1/2 activation. GDF15 from treatment-damaged HCC cells enhanced the proliferation and collagen synthesis of HSCs through ERK1/2- and Smad3-dependent pathways. Metformin significantly reduced the GDF15 production from treatment-damaged HCC cells by targeting JNK. The use of metformin could attenuate the in vivo fibrotic activities of HSCs promoted by treatment-damaged HCC cells and inhibit GDF15 expression. In conclusion, treatment-damaged HCC accelerates fibrosis by promoting the activities of HSCs via GDF15 secretion, which could be reversed by metformin. This provides a potential therapeutic target for alleviating TACE-aggravated liver cirrhosis.

Estradiol inhibits hepatic stellate cell area and collagen synthesis in the chicken liver.

Hepatic stellate cells (HSCs) are the main collagen-producing cells in the liver. The HSC area and amount of collagen fibers are different between male and female chickens. This study was performed to confirm the effect of estradiol on collagen synthesis in the growing chicken liver. Blood estradiol levels in chicks were compared at 4 and 8weeks of age, and the collagen fibril network in liver tissue was observed at 8weeks by scanning electron microscopy. Intraperitoneal administrations of estradiol and tamoxifen to male and female chicks, respectively, were performed daily from 5 to 8weeks of age. The areas of HSCs and collagen contents were measured in the liver tissue. The blood estradiol level was higher in females than in males, and the collagen fibril network was denser in males than in females at 8weeks of age. Estradiol administration in males induced decreases in the HSC area and collagen content of the liver. Conversely, tamoxifen administration in females induced an increase in the HSC area but did not facilitate collagen synthesis. Based on these results, estradiol inhibits the area and collagen synthesis of HSCs in the growing chicken liver under normal physiological conditions.

Suppression of collagen synthesis by Dicer gene silencing in hepatic stellate cells

MicroRNAs (miRNAs) have emerged as important mediators of hepatic stellate cells (HSCs) and are pivotal in the pathogenesis of liver fibrosis. Dicer, the key enzyme in the RNA interference (RNAi) pathway, is involved in cutting precursor miRNAs to functionally mature forms. Emerging evidence has demonstrated that Dicer expression is dysregulated in embryo development and tumors. In the present study, we aimed to address whether Dicer expression was correlated with the activity of HSCs. We used a recombinant lentivirus to generate short hairpin RNAs (shRNAs) targeting Dicer. The mRNA and protein expression of Dicer was effectively inhibited by three pairs of Dicer shRNA vectors, of which the shRNA1 vector exhibited the strongest inhibitory effect. The shRNA1 vector demonstrated a marked inhibitory effect on the activity of HSCs, resulting in the reduction of cell proliferation and the decrease of fibrosis-related genes, including type Ⅰ collagen (Col1A1), α-smooth muscle actin (α-SMA) and tissue inhibitor of metalloproteinases (TIMP). The mRNA expression of Col1A1, α-SMA and TIMP were decreased by 60, 56 and 49%, respectively. The protein expression was reduced by 56, 52 and 42%, respectively. Additionally, the inhibition of Dicer resulted in a decrease of miR-138, -143, -140 and -122 levels, of which miR-138 exhibited the strongest decline. The firefly luciferase reporter experiments and RT-PCR indicated that phosphatase and tension homolog deleted on chromosome 10 (PTEN), Ras GTPase activating-like protein 1 (RASAL1), acyl-CoA synthetase long-chain family member 1 (ACSL1) and p27 3' untranslated region sequences were targeted by miR-138, -143, -140 and -122, respectively. Taken together, the present study contributes important new findings for the role of Dicer-mediated miRNA processing in collagen synthesis of HSCs, which may serve as a foundation for RNAi study of liver fibrosis in vivo.

Effect of the protease inhibitor MG132 on the transforming growth factor-β/Smad signaling pathway in HSC-T6 cells

The activation of hepatic stellate cells (HSCs) and their transformation to myofibroblasts are the key steps in the pathological progress of liver fibrosis. The transforming growth factor-β (TGFβ)/Smad pathway is involved in the proliferation and collagen synthesis of HSCs. This study aimed to examine the effect of the protease inhibitor MG132 on the signaling pathway of TGFβ/Smad in HSC-T6 cells and seek a novel therapeutic approach for liver fibrosis. The HSC-T6 cells were treated with MG132 at different concentrations (0-10 μmol/L). Cell proliferation was detected by MTT method. The mRNA and protein expression levels of TGFβ1, Smad3 and Smad7 were determined in HSC-T6 cells by real-time PCR and Western blotting, respectively, after treatment with MG132 at different concentrations (1, 2, 3 μmol/L) or RPMI1640 alone (serving as control). The results showed that MG132 could inhibit the proliferation of HSC-T6 cells in a dose-dependent manner, and the IC(50) of MG132 was 6.84 μmol/L. After treatment with MG132 at 1, 2 or 3 μmol/L for 24 h, the mRNA expression levels of TGF-β1 and Smad3 were significantly decreased (P<0.05), but the Smad7 mRNA expression had no significant change (P>0.05). There was also a significant decrease in the protein expression level of TGF-β1 and Smad3 (P<0.05). However, the expression of Smad7 protein was substantially increased when compared with the control group (P<0.05). It was concluded that the inhibition of TGFβ/Smad pathway in HSC-T6 cells by MG132 can reduce the production of profibrosis factors (TGFβ1, Smad3) and promote the expression of anti-fibrosis factor (Smad7), suggesting that MG132 may become a potential therapeutic alternative for liver fibrosis.

The CYP2E1 inhibitor DDC up-regulates MMP-1 expression in hepatic stellate cells via an ERK1/2- and Akt-dependent mechanism

DDC (diethyldithiocarbamate) could block collagen synthesis in HSC (hepatic stellate cells) through the inhibition of ROS (reactive oxygen species) derived from hepatocyte CYP2E1 (cytochrome P450 2E1). However, the effect of DDC on MMP-1 (matrix metalloproteinase-1), which is the main collagen degrading matrix metalloproteinase, has not been reported. In co-culture experiments, we found that DDC significantly enhanced MMP-1 expression in human HSC (LX-2) that were cultured with hepatocyte C3A cells either expressing or not expressing CYP2E1. The levels of both proenzyme and active MMP-1 enzyme were up-regulated in LX-2 cells, accompanied by elevated enzyme activity of MMP-1 and decreased collagen I, in both LX-2 cells and the culture medium. H2O2 treatment abrogated DDC-induced MMP-1 up-regulation and collagen I decrease, while catalase treatment slightly up-regulated MMP-1 expression. These data suggested that the decrease in ROS by DDC was partially responsible for the MMP-1 up-regulation. ERK1/2 (extracellular signal-regulated kinase 1/2), Akt (protein kinase B) and p38 were significantly activated by DDC. The ERK1/2 inhibitor (U0126) and Akt inhibitor (T3830) abrogated the DDC-induced MMP-1 up-regulation. In addition, a p38 inhibitor (SB203580) improved MMP-1 up-regulation through the stimulation of ERK1/2. Our data indicate that DDC significantly up-regulates the expression of MMP-1 in LX-2 cells which results in greater MMP-1 enzyme activity and decreased collagen I. The enhancement of MMP-1 expression by DDC was associated with H2O2 inhibition and coordinated regulation by the ERK1/2 and Akt pathways. These data provide some new insights into treatment strategies for hepatic fibrosis.

Suppressive effects of YiGanKang, a combination of Chinese herbs, on collagen synthesis in hepatic stellate cell

Clinical practice and animal research demonstrated that YiGanKang, a combination of Chinese herbs, has anti-fibrosis effects in chronic liver diseases. However, the mechanism is not clear. The present study is to investigate the inhibiting mechanism of YiGanKang on collagen type I synthesis induced by Interleukin-1β(IL-1 β) in rat hepatic stellate cells (HSCs). Cultured rat HSCs were divided into 4 groups, control, IL-1β treated group, IL-1β+YiGanKang group and IL-1β+SB203580 (the p38 mitogen-activated protein kinase inhibitor) treated group. The expression of p38 mitogen-activated protein kinase (p38 MAPK), was evaluated by Western blot, collagen type I synthesis was examined by (3)H-Pro incorporation. Type I collagen synthesis in HSCs increased significantly under the stimulation of IL-1β for 24h, YiGanKang could inhibit p38 expression and type I collagen synthesis, SB203580, a p38 inhibitor, can significantly reduce type I collagen synthesis. IL-1β could stimulate the synthesis of type I collagen in rat HSCs, p38 mediate signal pathway between IL-1β and was type I collagen production. YiGanKang inhibits HSCs collagen synthesis induced by IL-1β via p38 inhibition.

New insights into the antifibrotic effects of sorafenib on hepatic stellate cells and liver fibrosis

During the process of liver fibrosis, hepatic stellate cells (HSCs) play a critical role in the excessive production of extracellular matrix (ECM). We evaluated the therapeutic effects of sorafenib, a multiple receptor tyrosine kinase inhibitor, targeting platelet-derived growth factor (PDGF) receptor and the Raf/extracellular-signal-regulated kinase (ERK) signaling pathway, on liver fibrosis and HSC proliferation. The in vivo effects of sorafenib were monitored in the livers of rats with liver fibrosis, and simultaneously proliferation assays, apoptosis induction studies, and collagen synthesis measurement were conducted in vitro in rat and human HSCs and primary HSCs. Sorafenib treatment attenuated liver fibrosis and was associated with a significant decrease in intrahepatic fibrogenesis, hydroxyproline accumulation and collagen deposition. Sorafenib reduced HSC proliferation and resulted in significantly higher levels of apoptosis. Moreover, sorafenib downregulated Cyclin D1 and Cyclin-dependent kinase 4 (Cdk-4), simultaneously increased expression of Fas, Fas-L, and Caspase-3, and decreased the ratio of Bcl-2 to Bax. Sorafenib treatment increased the ratio of matrix metalloproteinases (MMPs) to tissue inhibitor of matrix metalloproteinases (TIMPs) and reduced collagen synthesis in HSCs. Sorafenib inhibited the phosphorylation of ERK, Akt and 70-kDa ribosomal S6 kinase (p70S6K), both in vitro and in vivo. Sorafenib induces the suppression of collagen accumulation and HSC growth warranting the use of sorafenib as a potential therapeutic agent in the treatment of liver fibrosis.

Role of FAK-ERK signal transduction pathway in hepatic stellate cells collagen synthesis inhibited by FRNK plasmid transfection

Role of FAK-ERK signal transduction pathway in hepatic stellate cells collagen synthesis inhibited by FRNK plasmid transfection

Preventive effects of ME3738 on hepatic fibrosis induced by bile duct ligation in rats

The aim of this study was to examine the preventive effects of ME3738 on hepatic fibrosis induced by bile duct ligation (BDL) in rats. ME3738 (20 mg/day) was administered orally for 21 days immediately after BDL. Fibrosis was assessed by measuring hepatic hydroxyproline (Hyp) content. Activated hepatic stellate cells (HSCs) were assessed by alpha-smooth muscle actin (alpha-SMA) immunostaining. Hepatic thiobarbituric acid-reactive substance (TBARS), 4-hydroxy-2-nonenal (4-HNE) and 8-hydroxy-2-deoxyguanosine (8-OHdG) immunostaining were used to analyze oxidative stress. The gene expressions of collagen-I, transforming growth factor-beta1 (TGF-beta1), tissue inhibitor of metalloproteinases-1 (TIMP-1), interleukin-6 (IL-6) and heme oxygenase-1 (HO-1) in the liver were examined by real-time reverse transcriptase polymerase chain reaction (RT-PCR). Hepatic Hyp content and the area of hepatic fibrosis in BDL rats treated with ME3738 were reduced by 24% and 39% compared with non-treated BDL rats (hepatic Hyp, 9.40 +/- 2.85 vs. 12.39 +/- 3.91 mg/liver; P = 0.036; area of hepatic fibrosis, 13.1 +/- 3.8 vs. 21.5 +/- 10.9; P = 0.045). Furthermore, alpha-SMA-positive cells were significantly reduced by 40% (22.3 +/- 14.8 vs. 37.6 +/- 14.2; P = 0.011), collagen-I mRNA by 83% (6.5 +/- 2.2 vs. 38.3 +/- 9.1; P = 0.002), HO-1 mRNA by 58% (4.13 +/- 1.22 vs. 9.73 +/- 1.80; P = 0.018) and hepatic HO-1 content by 26% (2.13 +/- 0.80 vs. 2.87 +/- 0.19; P = 0.01) following ME3738 treatment. The hepatic expression of TBARS, 4-HNE, 8-OHdG and mRNA levels of TGF-beta1, TIMP-1 and IL-6 in the liver were unchanged by ME3738 treatment. Oral ME3738 administration may prevent the progression of hepatic fibrosis in BDL rats through suppression of the activation and collagen synthesis of HSC and, in part, oxidative stress. ME3738 has potential as a therapeutic drug for cholestatic liver fibrosis.

Effect of fetuin, a TGFβ antagonist and pentoxifylline, a cytokine antagonist on hepatic stellate cell function and fibrotic parameters in fibrosis

We have previously shown that monocyte conditioned medium (MCM) from patients with liver fibrosis stimulated proliferation of hepatic stellate cells (HSCs), the major cell involved in hepatic fibrosis. To investigate the potential role of fetuin and pentoxifylline in fibrosis we used MCM samples obtained from patients with biopsy proven hepatic fibrosis related to Hepatitis C (HCV). Our results indicate that the MCM obtained from patients with HCV-related liver fibrosis significantly stimulated collagen synthesis in HSCs as assessed by tritiated proline incorporation into a collagenase sensitive trichloroacetic acid (TCA) precipitate. Collagen synthesis was also stimulated in HSCs using transforming growth factor beta (TGFbeta) and this effect was neutralized using TGFbeta antibody. Incubation of HSCs with fetuin (but not TGFbeta antibody) significantly inhibited collagen synthesis in HSCs that were stimulated by HCV MCM samples. Patient MCM samples would also stimulate proliferation of HSCs as assessed by tritiated thymidine uptake but this effect was not attenuated by fetuin. Likewise the significant stimulatory effect of platelet derived growth factor (PDGF) on HSC proliferation and collagen synthesis was not inhibited by fetuin but could be significantly reduced by 70% and 40% respectively, when treated with pentoxifylline. We also investigated the ability of samples obtained from patients with hepatic fibrosis to inhibit HSC apoptosis, as determined by okadaic acid-induced 4-hydroxynonenal immunocytochemistry in HSCs. We have previously reported that okadaic acid induces apoptosis in HSCs as assessed by Hoescht and TUNEL. Okadaic acid treatment produced a positive 4-hydroxynonenal (4-HNE) immunoreactivity in HSCs and treatment with HCV patient MCM or TGFbeta decreased the 4-HNE positive immunoreactivity in HSCs treated with okadaic acid. Our results suggest that fetuin may be beneficial in hepatic fibrosis and suggest that combination of fetuin and pentoxifylline may target the two key events in hepatic fibrosis by modifying the effects of TGFbeta and PDGF, the two major growth factors in fibrosis.

15d-Deoxy-Δ 12,14-prostaglandin J 2 modulates collagen type I synthesis in human hepatic stellate cells by inducing oxidative stress

15 deoxy-Δ 12,14-prostaglandin 2 (15d-PGJ 2) is known to inhibit the proliferation of hepatic stellate cells (HSCs), major cellular components that cause hepatic fibrosis, in vitro. It also induces oxidative stress, which results in hepatic myofibroblast death. On the other hand, oxidative stress generally induces HSC proliferation and collagen synthesis in vitro, and liver fibrogenesis in vivo. In this study, we evaluated the effects of 15d-PGJ 2 at various concentrations on the viability and collagen synthesis of HSCs. 15d-PGJ 2 increased intracellular reactive oxygen species (ROS), and reduced the viability of human HSCs at concentrations ⩾5 μM by inducing apoptotic cell death. In addition, the antioxidants α-tocopherol and N-acetylcysteine (NAC) blocked 15d-PGJ 2-induced HSC death. Collagen I synthesis was increased 1.5-fold by 0.5 μM 15d-PGJ 2 treatment, but was reduced to 30% of the control level by 10 μM 15d-PGJ 2, and NAC pretreatment prevented these changes in collagen production by 15d-PGJ 2. We conclude that 15d-PGJ 2 may either induce or prevent hepatic fibrogenesis depending on its concentration.

Effect of caffeic acid phenethyl ester on proliferation and apoptosis of hepatic stellate cells in vitro.

To investigate the role of nuclear factor-kappaB (NF-kappaB) inhibitor caffeic acid phenethy1 ester (CAPE) in the proliferation, collagen synthesis and apoptosis of hepatic stellate cells (HSCs) of rats. The HSCs from rats were isolated and cultured in Dulbecco's Modified Eagle's Medium (DMEM) and treated with CAPE. The proliferation and collagen synthesis of HSCs were determined by (3)H-TdR and (3)H-proline incorporation respectively, and the expression of type I, III procollagen genes was further explored by in situ hybridization. Apoptosis cell indices (AIs) were examined using terminal deoxynucleotidyl transferase- mediated DIG-dUTP nick end labeling (TUNEL). In activated HSC in culture, CAPE significantly inhibited (3)H-TdR and (3)H-proline incorporation by HSCs at concentrations of 5 micromol/L and 10 micromol/L respectively. CAPE also reduced the type I procollagen gene expression (P<0.05) at higher concentration. Apoptosis of HSC was induced by CAPE and the AIs were time-and dose-dependently increased from 2.82+/-0.73 % to 7.66+/-1.25 % at 12 h (P<0.01) and from 3.15+/-0.88 % to 10.61+/-2.88 % at 24 h (P<0.01). CAPE inhibits proliferation and collagen synthesis of HSC at lower concentration and induces HSC apoptosis at higher concentration.

Inhibition of hepatic stellate cell collagen synthesis by N-(methylamino)isobutyric acid

The increased deposition of extracellular matrix by hepatic stellate cells following liver injury, in a process known as activation, is considered a key mechanism for increased collagen content of liver during the development of liver fibrosis. We report that N-(methylamino)isobutyric acid (MeAIB), a specific inhibitor of System A-mediated amino acid uptake, reduces the accumulation of collagen in CFSC-2G hepatic stellate cell cultures and in a rat model of liver injury and fibrosis. Rat CFSC-2G cells were cultured in 0–5 mM MeAIB, and the accumulation and synthesis of collagen were measured by binding to Sirius red F3B and pulse-labeling with [ 3 H ]-proline, respectively. The effect of MeAIB on collagen accumulation in vivo was evaluated utilizing a rat model of hepatic fibrosis. MeAIB inhibited collagen accumulation in CFSC-2G cultures in a concentration-dependent manner with 5 mM MeAIB reducing collagen 44.6±1.2% compared with the control. In CFSC-2G cultures, MeAIB selectively inhibited the incorporation of proline into cellular macromolecules by 43±4%, while the synthesis of proteins containing leucine was not affected. In vivo, oral administration of 160 mg MeAIB/kg body weight per day to rats significantly reduced the hepatic collagen accumulation in response to 1 week of CCl 4-induced liver injury. MeAIB reduces the accumulation of collagen in CFSC-2G hepatic stellate cell cultures and in a CCl 4-induced rat model of liver injury and fibrosis.

The regulatory role of AT 1 receptor on activated HSCs in hepatic fibrogenesis:effects of RAS inhibitors on hepatic fibrosis induced by CCl(4).

AIM:To assess the effect of ACE inhibitor and Ang II type 1 (AT1) receptor antagonist in preventing hepatic fibrosis caused by CCl(4) administration in rats;to investigate whether or not there are expression of AT 1 receptors on hepatic stellate cells; and to observe the effect of Ang II on proliferation and ECM synthesis of cultured HSCs.METHODS:Studies were conducted in male Sprague-Dawley rats. Except for the hepatofibrotic model group and the control group, in three treated groups, either enalapril (5mg/kg), or losartan (10mg/kg), or enalapril + losartan were given to the fibrotic rats by daily gavage, and saline vehicle was given to model and normal control rats. After 6 weeks, liver fibrosis was assessed directly by hepatic morphometric analysis, which has been considered the gold standard for the quantification of fibrosis. The expressions of AT 1 receptors and (alpha-mooth muscle actin,alpha-SMA) in liver tissue or isolated hepatic stellate cells (HSCs) were detected by immunohistochemical techniques. The effect of Ang II on HSC proliferation was determined by MTT method. Effect of Ang II on collagen synthesis of HSCs was determined by (3)H-proline incorporation.RESULTS:Contrasted to the fibrosis in rats of the model group, groups of rats treated with either enalapril or losartan, or a combination of two drugs showed a limited expansion of the interstitium (4.23 plus minus 3.70 vs 11.22 plus minus 4.79, P<0.05), but no difference was observed among three treated groups (5.38 plus minus3.43, 4.96 plus minus 2.96, 4.23 plus minus 2.70, P>0.05). Expression of AT 1 receptors was found in fibrotic interstitium of fibrotic rats, whereas in normal control rats they were limited to vasculature only to a very slight degree. AT 1 receptors were also expressed on activated HSCs in the culture. At concentrations from 10(-9) to 10(-5)mol/L, Ang II stimulated HSC proliferation in culture in a dose dependent manner. Increasing Ang II concentrations produced corresponding increases in (3)H-proline incorporation. Differences among groups were significant.CONCLUSION:Angiotensin converting enzyme inhibitors and AT 1 blocker may slow the progression of hepatic fibrosis;activated HSCs express AT 1 receptors, and Ang II can stimulate the proliferation and collagen synthesis of HSCs in a dose-dependent manner; and activation of RAS may be related to hepatic fibrogenesis induced by CCl(4).