Human Hepatic Stellate Cells LX-2 Research Articles

CXCL6 promotes human hepatocyte proliferation through the CXCR1-NFκB pathway and inhibits collagen I secretion by hepatic stellate cells.

Hepatocyte proliferation and collagen I (COLI) secretion are important processes during liver regeneration. This study aimed to investigate the role of CXCL6 in hepatocyte proliferation and COLI secretion. Serum CXCL6 levels in patients with chronic hepatitis B (CHB) were examined and the effects of CXCL6 on the proliferation of L02 hepatocytes and the secretion of COLI from LX2 human hepatic stellate cells were evaluated. We found that serum CXCL6 levels increased gradually with disease progression of CHB, and there was positive correlation between serum CXCL6 level and alanine transaminase (ALT) and aspartate transaminase (AST). In vitro, CXCL6 promoted L02 proliferation but this was blocked upon CXCR1 knockdown. The level of phospho-IκBα was upregulated by CXCL6 but downregulated by CXCR1 siRNA in L02 cells. CXCL6 inhibited the secretion of COLI by LX2 cells, dependent on CXCR1 and CXCR2. Taken together, these data suggest that increased expression of CXCL6 during CHB could promote hepatocyte proliferation through the CXCR1-NFκB pathway and inhibit the secretion of COLI by hepatic stellate cells.

MicroRNA-122 Inhibits the Production of Inflammatory Cytokines by Targeting the PKR Activator PACT in Human Hepatic Stellate Cells.

MicroRNA-122 (miR-122) is one of the most abundant miRs in the liver. Previous studies have demonstrated that miR-122 plays a role in inflammation in the liver and functions in hepatic stellate cells (HSCs), which reside in the space of Disse. Here, we showed that the transient inhibition of PKR-activating protein (PACT) expression, by miR-122 or siRNA targeting of PACT, suppressed the production of proinflammatory cytokines, such as interleukin (IL)-6, monocyte chemoattractant protein-1 (MCP-1) and IL-1β, in human HSC LX-2. Sequence and functional analyses confirmed that miR-122 directly targeted the 3′-untranslated region of PACT. Immunofluorescence analysis revealed that miR-122 blocked NF-κB-nuclear translocation in LX-2 cells. We also showed that conditioned medium from miR-122-transfected LX-2 cells suppressed human monocyte-derived THP-1 cell migration. Taken together, our study indicates that miR-122 may downregulate cytokine production in HSCs and macrophage chemotaxis and that the targeting of miR-122 may have therapeutic potential for preventing the progression of liver diseases.

LPS-induced TNF-α factor mediates pro-inflammatory and pro-fibrogenic pattern in non-alcoholic fatty liver disease.

Lipopolysaccharide (LPS) is currently considered one of the major players in non-alcoholic fatty liver disease (NAFLD) pathogenesis and progression. Here, we aim to investigate the possible role of LPS-induced TNF-α factor (LITAF) in inducing a pro-inflammatory and pro-fibrogenic phenotype of non-alcoholic steatohepatitis (NASH).We found that children with NAFLD displayed, in different liver-resident cells, an increased expression of LITAF which correlated with histological traits of hepatic inflammation and fibrosis. Total and nuclear LITAF expression increased in mouse and human hepatic stellate cells (HSCs). Moreover, LPS induced LITAF-dependent transcription of IL-1β, IL-6 and TNF-α in the clonal myofibroblastic HSC LX-2 cell line, and this effect was hampered by LITAF silencing. We showed, for the first time in HSCs, that LITAF recruitment to these cytokine promoters is LPS dependent. However, preventing LITAF nuclear translocation by p38MAPK inhibitor, the expression of IL-6 and TNF-α was significantly reduced with the aid of p65NF-ĸB, while IL-1β transcription exclusively required LITAF expression/activity. Finally, IL-1β levels in plasma mirrored those in the liver and correlated with LPS levels and LITAF-positive HSCs in children with NASH.In conclusion, a more severe histological profile in paediatric NAFLD is associated with LITAF over-expression in HSCs, which in turn correlates with hepatic and circulating IL-1β levels outlining a panel of potential biomarkers of NASH-related liver damage. The in vitro study highlights the role of LITAF as a key regulator of the LPS-induced pro-inflammatory pattern in HSCs and suggests p38MAPK inhibitors as a possible therapeutic approach against hepatic inflammation in NASH.

MicroRNA125b-mediated Hedgehog signaling influences liver regeneration by chorionic plate-derived mesenchymal stem cells.

Although chorionic plate-derived mesenchymal stem cells (CP-MSCs) were shown to promote liver regeneration, the mechanisms underlying the effect remain unclear. Hedgehog (Hh) signaling orchestrates tissue reconstruction in damaged liver. MSCs release microRNAs mediating various cellular responses. Hence, we hypothesized that microRNAs from CP-MSCs regulated Hh signaling, which influenced liver regeneration. Livers were obtained from carbon tetrachloride (CCl4)-treated rats transplanted with human CP-MSCs (Tx) or saline (non-Tx). Sonic Hh, one of Hh ligands, increased in CCl4-treated liver, whereas it decreased in CP-MSC-treated liver with CCl4. The expression of Hh-target genes was significantly downregulated in the Tx. Reduced expansion of progenitors and regressed fibrosis were observed in the liver of the Tx rats. CP-MSCs suppressed the expression of Hh and profibrotic genes in co-cultured LX2 (human hepatic stellate cell) with CP-MSCs. MicroRNA-125b targeting smo was retained in exosomes of CP-MSCs. CP-MSCs with microRNA-125b inhibitor failed to attenuate the expression of Hh signaling and profibrotic genes in the activated HSCs. Therefore, these results demonstrated that microRNA-125b from CP-MSCs suppressed the activation of Hh signaling, which promoted the reduced fibrosis, suggesting that microRNA-mediated regulation of Hh signaling contributed to liver regeneration by CP-MSCs.

Effects of small interfering RNA-mediated downregulation of the Krüppel-like factor 4 gene on collagen metabolism in human hepatic stellate cells.

The nuclear transcription factor Krüppel-like factor 4 (KLF4) has an important role in cellular biological processes. However, the influence of KLF4 on collagen metabolism remains to be elucidated. In the present study, the effects and underlying mechanism of action of KLF4 on collagen metabolism was investigated in human hepatic stellate cells (HSC), by downregulating KLF4 expression using small interfering RNA (siRNA). The effects of KLF4 silencing by three predesigned siRNAs (siRNA1‑3) were evaluated using both reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) and western blotting in the human LX2 HSC line. The mRNA expression levels of KLF4 were decreased by ~34, 40, and 69% in the siRNA1, siRNA2, and siRNA3 groups, respectively, as compared with the control group. These results were concordant with the protein expression levels of KLF4, as determined by western blot analysis. In the siRNA3 group, the quantity of type Ⅰ and type III collagen, and the expression levels of collagen metabolism proteins including matrix metalloproteinase‑1 (MMP‑1) and tissue inhibitors of metalloproteinases‑1 (TIMP‑1), were determined using both RT‑qPCR and western blotting. Both the mRNA and protein expression levels of type I and type III collagen were significantly decreased in the siRNA3 group, as compared with the control group. The mRNA and protein expression levels of TIMP‑1 were also significantly reduced in the siRNA3‑treated cells, whereas the mRNA and protein expression levels of MMP‑1 were significantly upregulated. Furthermore, KLF4 gene silencing significantly decreased the expression levels of numerous cytokines, including transforming grow factor‑β1, tumor necrosis factor‑α, and interleukin‑1β. The results of the present study provide evidence of siRNA‑mediated silencing of KLF4 expression, which may promote extracellular matrix (ECM) degradation, and inhibition of ECM synthesis. Therefore, KLF4 may be a promising target for the development of novel antifibrotic therapies.

Upregulation of microRNA-126 in hepatic stellate cells may affect pathogenesis of liver fibrosis through the NF-κB pathway.

Hepatic fibrosis, which results from chronic liver disease, currently lacks effective treatment. MicroRNAs, a group of small noncoding RNA molecules, have been observed to play an essential role in liver diseases, including hepatic fibrosis. In this study, we described the regulation of nuclear factor kappa B (NF-κB) inhibitor alpha (IκBα) and its possible signaling pathway by miR-126 in human hepatic stellate cell (HSC) line LX-2. The 3'-untranslated region (3'-UTR) of IκBα combined with miR-126 was analyzed by using a dual-luciferase reporter assay. Furthermore, the effects of miR-126 on IκBα mRNA and protein and NF-κB protein expression were assessed by real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and western blot analysis in the human HSC LX-2 cell line transfected with miR-126 mimic or inhibitor. Moreover, to understand the molecular mechanism of miR-126 in promoting liver fibrosis through NF-κB signaling pathway, the NF-κB downstream signaling factors expression such as transforming growth factor (TGF)-β1 and collagen I mRNA were detected by real-time qRT-PCR. We identified that IκBα is a potential target gene of miR-126, by directly targeting its 3'-UTR. Endogenous miR-126 and exogenous miR-126 mimic inhibited IκBα expression. Moreover, overexpression of miR-126 reduced total and the cytoplasm IκBα protein expression and increased total and cytoblast NF-κB protein expression of LX-2. Conversely, knockdown of miR-126 could inhibit NF-κB activation by upregulation of IκBα protein expression. Further, miR-126 promoted TNF-a-induced TGF-β1 and collagen I mRNA expression in LX-2 cells. miR-126 may play an important role in hepatic fibrosis by downregulating the expression of IκBα partly through the NF-κB signaling pathway.

The anti-fibrotic effects of epigallocatechin-3-gallate in bile duct-ligated cholestatic rats and human hepatic stellate LX-2 cells are mediated by the PI3K/Akt/Smad pathway.

(-)-Epigallocatechin-3-gallate (EGCG) is one of the most abundant polyphenols in green tea with strong antioxidant activity and various therapeutic effects. In this study, we investigated the anti-fibrotic effects of EGCG and underlying mechanisms in bile duct-ligated (BDL) rats and a liver fibrosis model in vitro. BDL rats were treated with EGCG (25 mg·kg(-1)·d(-1), po) for 14 d, and then the serum, bile and liver samples were collected. Liver fibrosis was assessed by serum, urine and bile biochemistry analyses and morphological studies of liver tissues. TGF-β1-stimulated human hepatic stellate LX-2 cells were used as a liver fibrosis model in vitro. The expression of liver fibrogenic genes and signaling proteins in the PI3K/Akt/Smad pathway was examined using Western blotting and/or real-time PCR. In BDL rats, EGCG treatment significantly ameliorates liver necrosis, inflammation and fibrosis, and suppressed expression of the genes associated with liver inflammation and fibrogenesis, including TNF-α, IL-1β, TGF-β1, MMP-9, α-SMA, and COL1A1. In LX-2 cells, application of EGCG (10, 25 μmol/L) dose-dependently suppressed TGF-β1-stimulated expression of COL1A1, MMP-2, MMP-9, TGF-β1, TIMP1, and α-SMA. Furthermore, EGCG significantly suppressed the phosphorylation of Smad2/3 and Akt in the livers of BDL rats and in TGF-β1-stimulated LX-2 cells. Application of LY294002, a specific inhibitor of PI3K, produced similar effects as EGCG did in TGF-β1-stimulated LX-2 cells, but co-application of EGCG and LY294002 did not produce additive effects. EGCG exerts anti-fibrotic effects in BDL rats and TGF-β1-stimulated LX-2 cells in vitro via inhibiting the PI3K/Akt/Smad pathway.

Total flavonoids of litchi suppress proliferation of LX2 human hepatic stellate cells by upregulating p27 expression

Total flavonoids of litchi suppress proliferation of LX2 human hepatic stellate cells by upregulating p27 expression

Celecoxib derivative OSU-03012 inhibits the proliferation and activation of hepatic stellate cells by inducing cell senescence.

Liver fibrosis may lead to portal hypertension, liver failure or hepatocellular carcinoma, and predominantly results from the proliferation and activation of hepatic stellate cells. OSU‑03012, a non‑cyclooxygenase‑inhibiting celecoxib derivative, has been previously demonstrated to promote apoptosis in certain cell types, however, its function in hepatic fibrosis remains unclear. In the current study, the inhibitory effect of OSU‑03012 on the proliferation of the LX2 human hepatic stellate cell line was evaluated by cell counting kit‑8 assay. Reverse transcription‑quantitative polymerase chain reaction was performed in order to examine the expression of α‑smooth muscle actin and type I collagen, which are representative of LX2 cell activation. The senescence of LX2 cells was measured by senescence‑associated β‑galactosidase staining, and the cell cycle and apoptosis levels were assessed by flow cytometry. The impact of senescence‑associated signaling on protein expression was assessed by western blot analysis. OSU‑03012 was observed to inhibit cell proliferation and prevent the secretion of profibrotic factors in LX2 cells in a dose‑dependent manner. Furthermore, the results demonstrated that OSU‑03012 inhibited the proliferation and activation of LX2 via the induction of cell senescence at the G1 phase, rather than via cell apoptosis. The induction of senescence may be via the upregulation of p16, p21 and p27. In conclusion, the current study provided insight into the pharmacological mechanisms of OSU‑03012 in preventing the proliferation and activation of hepatic stellate cells through cell senescence. The current study supports the theory that OSU‑03012 is a novel agent for potential use against liver fibrosis.

Profiling of concanavalin A-binding glycoproteins in human hepatic stellate cells activated with transforming growth factor-β1.

Glycoproteins play important roles in maintaining normal cell functions depending on their glycosylations. Our previous study indicated that the abundance of glycoproteins recognized by concanavalin A (ConA) was increased in human hepatic stellate cells (HSCs) following activation by transforming growth factor-β1 (TGF-β1); however, little is known about the ConA-binding glycoproteins (CBGs) of HSCs. In this study, we employed a targeted glycoproteomics approach using lectin-magnetic particle conjugate-based liquid chromatography-tandem mass spectrometry to compare CBG profiles between LX-2 HSCs with and without activation by TGF-β1, with the aim of discovering novel CBGs and determining their possible roles in activated HSCs. A total of 54 and 77 proteins were identified in the quiescent and activated LX-2 cells, respectively. Of the proteins identified, 14.3% were glycoproteins and 73.3% were novel potential glycoproteins. Molecules involved in protein processing in the endoplasmic reticulum (e.g., calreticulin) and calcium signaling (e.g., 1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase β-2 [PLCB2]) were specifically identified in activated LX-2 cells. Additionally, PLCB2 expression was upregulated in the cytoplasm of the activated LX-2 cells, as well as in the hepatocytes and sinusoidal cells of liver cirrhosis tissues. In conclusion, the results of this study may aid future investigations to find new molecular mechanisms involved in HSC activation and antifibrotic therapeutic targets.

Fibronectin Peptides as Potential Regulators of Hepatic Fibrosis Through Apoptosis of Hepatic Stellate Cells

The turnover of extracellular matrix (ECM) components can generate signals that regulate several cellular functions such as proliferation, differentiation, and apoptosis. During liver injury, matrix metalloproteases (MMPs) production is enhanced and increased levels of peptides derived from extracellular matrix proteins can be generated. Synthetic peptides with sequences present in extracellular matrix proteins were previously found to induce both stimulating and apoptotic effects on several cell types including the inflammatory cells monocytes/macrophages. Therefore, in inflammatory liver diseases, locally accumulated peptides could be also important in regulating hepatic fibrosis by inducing apoptosis of hepatic stellate cells (HSC), the primary cellular source of extracellular matrix components. Here, we describe the apoptotic effect of fibronectin peptides on the cell line of human hepatic stellate cells LX-2 based on oligonucleosomal DNA fragmentation, caspase-3 and -9 activation, Bcl-2 depletion, and accumulation of Bax protein. We also found that these peptides trigger the activation of Src kinase, which in turn mediated the increase of JNK and p38 activities. By the use of specific inhibitors we demonstrated the involvement of Src, JNK, and p38 in apoptosis induced by fibronectin peptides on HSC. Moreover, fibronectin peptides increased iNOS expression in human HSC, and specific inhibition of iNOS significantly reduced the sustained activity of JNK and the programmed cell death caused by these peptides. Finally, the possible regulatory effect of fibronectin peptides in liver fibrosis was further supported by the ability of these peptides to induce metalloprotease-9 (MMP-9) expression in human monocytes.

Construction and validation of a dual-luciferase reporter gene system for screening and evaluating anti-liver fibrosis drugs that inhibit transcription of the gene encoding collagen I, chain a1

To construct a dual-reporter gene system that will be applicable for use as a tool to screen and evaluate therapeutic drug compounds that inhibit transcription of the gene encoding collagen I, chain at1 (COL1A1). The full-length eDNA of transforming growth factor beta1 (TGFbeta1) was cloned by RT-PCR and inserted into two vectors, pcDNA3.1 and pJW4303, for construction of two eukaryotic expression vectors, pcDNA3.1-TGFbeta1 and pJW4303-TGFbeta1.Next, the promoter region of COL1A1, cloned by PCR using human genome DNA as template, was inserted into the vector pGL4.29 to construct the reporter gene vector, pGL4.29-COL1A1 promoter.All three recombinant vectors were verified by restriction enzyme digestion and DNA sequencing.Either the pcDNA3.1-TGFbeta1 or pJW4303-TGFbeta1 vector along with the pGL4.29-COL1A1 promoter vector or a pRL-null, control reporter, vector were co-transfected into the LX-2 human hepatic stellate cells to establish the transcription-activated dualreporter gene system.This system was used as a cell model for screening anti-liver fibrosis compounds that inhibit the transcription of COL1A1.Dexamethasone, a model drug that is known to inhibit the expression of COL1A1, was used as a control to validate the dual-reporter gene system. The two TGFbeta1-expressing vectors and the reporter gene vector containing the promoter region of COL1A1 were successfully constructed.The results of a dual-reporter gene assay showed that TGFbeta1 co-expression increased the activity of the COL1A1 promoter by above 200-fold (t =21.78, P =0.0001), whereas in the absence of TGF31 co-expression the activity was below 2-fold (t =3.396, P =0.0274).The transcriptionactivated dual-reporter gene system was successfully established.The model drug, dexamethasone, effectively inhibited the activity of the COL 1A1 promoter in dose-dependent manner; the activity decreased 29.6% with 10 mumol/L dexamethasone (t =4.140, P =0.0144) and 53.9% with 100 mumol/L (t =6.193, P =0.0035). The dual-luciferase reporter system of TGFbeta1 and COL1A1 co-expression developed here can be used as a cell model to screen and evaluate anti-liver fibrosis compounds that inhibit activity of the COL1A1.

Proteomic Effects of the Coagulation Proteinase Thrombin on LX-2 Hepatic Stellate Cells

Summary Background: The aim of this study was to characterize the effects of the coagulation proteinase thrombin on proteomic level in human hepatic stellate LX-2 cells. Methods: Proteomic analyses were performed using surface-enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF-MS). The protein profiles obtained from LX-2 cell lysates using strong anion exchanger Q10 ProteinChip arrays were statistically analyzed. Results: The peak intensities of 50 protein/peptide clusters were identified as being different between nonstimulated and LX-2 cells treated with thrombin for 6 h and 24 h, respectively. As the most significantly enhanced single signal in LX-2 cells stimulated with thrombin, a protein with a molecular mass of 13.560 kDa has been identified that corresponds exactly to calcium dependent phospholipase 2 (cPLA2). Thrombin-induced increase in the cPLA2 protein expression in LX-2 cells was confirmed by using the Western blotting technique. Conclusions: Together with the finding that thrombin induced phosphorylating activation of cPLA2 in LX-2 cells, our data point to an important function of the thrombin-mediated modulation of cytosolic phospholipase A2 in hepatic stellate cells.

Brivanib Attenuates Hepatic Fibrosis In Vivo and Stellate Cell Activation In Vitro by Inhibition of FGF, VEGF and PDGF Signaling

Background and AimsBrivanib is a selective inhibitor of vascular endothelial growth factor receptor (VEGFR) and fibroblast growth factor receptor (FGFR) tyrosine kinases, which are both involved in mechanisms of liver fibrosis. We hypothesized that inhibition of VEGFR and FGFR by brivanib would inhibit liver fibrosis. We therefore examined the effect of brivanib on liver fibrosis in three mouse models of fibrosis.Methods In vivo, we induced liver fibrosis by bile duct ligation (BDL), chronic carbon tetrachloride (CCl4), and chronic thioacetamide (TAA) administration. Liver fibrosis was examined by immunohistochemistry and Western immunoblotting. In vitro, we used LX-2 human hepatic stellate cells (HSCs) to assess the effect of brivanib on stellate cell proliferation and activation.ResultsAfter in vivo induction with BDL, CCl4, and TAA, mice treated with brivanib showed reduced liver fibrosis and decreased expression of collagen Iα1 and α-smooth muscle actin in the liver. In vitro, brivanib decreased proliferation of HSCs induced by platelet-derived growth factor (PDGF), VEGF, and FGF. Brivanib also decreased stellate cell viability and inhibited PDGFBB-induced phosphorylation of its cognate receptor.ConclusionBrivanib reduces liver fibrosis in three different animal models and decreases human hepatic stellate cell activation. Brivanib may represent a novel therapeutic approach to treatment of liver fibrosis and prevention of liver cancer.

S-adenosylmethionine inhibits the activated phenotype of human hepatic stellate cells via Rac1 and Matrix metalloproteinases

To investigate the effects of S-adenosylmethionine (SAM) on the proliferation, adhesion, migration, invasion and apoptosis of activated human hepatic stellate cells (HSCs) and to explore the relevant potential mechanisms. Human HSCs LX-2 were cultured with SAM. The proliferation and adhesion were detected by CCK-8. Cell apoptosis rate were analyzed by flow cytometry, and cell migration and invasion were tested by the transwell assay. The expression of Rac1 and MMP-2 was identified by real-time PCR or Western blotting, and activated Rac1 was detected by GST pull-down assay. The activity of MMP-2 and MMP-9 was analyzed by substrate zymography. The proliferation of LX-2 cells was inhibited by SAM, exhibiting a dose-dependent manner. Cell apoptosis rate induced by SAM was remarkably increased. SAM decreased the adhesion, migration and invasion of LX-2 cells. The expression and activation of Rac1 in LX-2 cells were significantly suppressed by SAM. In contrast, the activity of MMP-2 and MMP-9 was enhanced by SAM. SAM attenuated the up-regulated expression of Smad3/4 and Rac1 induced by TGF-β1. SAM inhibits the proliferation, adhesion, migration and invasion of LX-2 cells in vitro probably via attenuating the expression and activation of Rac1 and up-regulating MMP-2 and MMP-9 expression, which possibly provide a molecular basis for potential application of SAM in the therapy of liver fibrosis.

Chronic vitamin C insufficiency aggravated thioacetamide-induced liver fibrosis in gulo-knockout mice

Given the involvement of oxidative stress in liver-disease- or hepato-toxicant-induced hepatic damage and fibrosis, antioxidants are an effective preventive and therapeutic tool. The beneficial results of vitamin C, one of the physiological antioxidants, have been observed both in experimental animals and in humans. However, most of these studies have been concerned with supplementary vitamin C; the effects of under vitamin C insufficiency, which humans sometimes confront, have not been substantially investigated. In the present study, we established a vitamin C-insufficient animal model (half-to-normal serum vitamin C concentration) with gulo-/- mice that cannot synthesize vitamin C, and induced hepatotoxicity by means of thioacetamide (TAA) injections twice a week for 18 weeks. Additionally, we explored the direct effects of vitamin C both on immortalized human hepatic stellate LX-2 cells and on rat primary hepatic stellate cells. Vitamin C insufficiency resulted in a decreased survival rate and increased serum markers for hepatocyte damage, such as alanine aminotransferase and aspartate aminotransferase. Concomitantly, the levels of reactive oxygen species (ROS) and lipid peroxides in the liver were increased. Histological examinations of the vitamin C-insufficient liver revealed increases in collagen fiber deposition and activated-hepatic-stellate-cell number. Vitamin C, when directly applied to the LX-2 cells as well as the rat primary hepatic stellate cells, suppressed not only proliferation but hydrogen peroxide-induced collagen expression as well. In conclusion, vitamin C insufficiency exacerbated TAA-induced hepatotoxicity. These effects seem to be mainly from insufficient scavenging of ROS in the liver, and possibly in part, by directly affecting hepatic stellate cells.

Hepatic Fibrosis Inhibitory Effect of Peptides Isolated from Navicula incerta on TGF-β Induced Activation of LX-2 Human Hepatic Stellate Cells

In this study, novel peptides (NIPP-1, NIPP-2) derived from Navicula incerta (microalgae) protein hydrolysate were explored for their inhibitory effects on collagen release in hepatic fibrosis with the investigation of its underlying mechanism of action. TGF-β1 activated fibrosis in LX-2 cells was examined in the presence or absence of purified peptides NIPP-1 and NIPP-2. Besides the mechanisms of liver cell injury, protective effects of NIPP-1 and NIPP-2 were studied to show the protective mechanism against TGF-β1 stimulated fibrogenesis. Our results showed that the core protein of NIPP-1 peptide prevented fibril formation of type I collagen, elevated the MMP level and inhibited TIMP production in a dose-dependent manner. The treatment of NIPP-1 and NIPP-2 on TGF-β1 induced LX-2 cells alleviated hepatic fibrosis. Moreover, α-SMA, TIMPs, collagen and PDGF in the NIPP-1 treated groups were significantly decreased. Therefore, it could be suggested that NIPP-1 has potential to be used in anti-fibrosis treatment.

Metallothionein Gene Transfection Reverses the Phenotype of Activated Human Hepatic Stellate Cells

Metallothionein (MT) gene therapy leads to resolution of liver fibrosis in mouse model. The present study was undertaken to test the hypothesis that reversal of the phenotype of activated hepatic stellate cells (HSCs) contributes to the fibrinolysis effect of MT. Human HSC LX-2 cells were activated after they were cultured for 24 hours, as indicated by expression of α-smooth muscle actin (α-SMA) and collagen-I and depressed expression of collagenases. Transfection with a plasmid containing human MT-IIA gene in the activated HSCs effectively increased the protein level of MT. The expression of MT was accompanied by the reduction in protein levels of α-SMA and collagen-I and a decrease in their mRNA levels. Of importance, MT gene transfection resulted in upregulation of matrix metalloproteinases 1, 8, and 13, which are involved in the resolution of liver fibrosis. This study demonstrates that reversal of the phenotype of activated HSCs, particularly the upregulation of collagenases, is likely to be involved in the resolution of liver fibrosis observed in MT gene therapy.

TRAIL regulates collagen production through HSF1-dependent Hsp47 expression in activated hepatic stellate cells

Hsp47 is a collagen-specific molecular chaperone, whose activity has been implicated in liver fibrosis. In this study, we showed that TRAIL treatment inhibited Hsp47 expression in dose- and time-dependent manners, subsequently leading to the decrease of collagen production in activated human hepatic stellate LX-2 cells. Overexpression of Hsp47 in LX-2 cells acquired resistance for TRAIL-induced collagen reduction and conversely, siRNA suppression of Hsp47 enhanced the decrease of collagen production due to TRAIL treatment. Moreover, we found that Hsp47 expression was under the transcriptional control of heat shock factor (HSF) 1 which is highly located on nucleus in activated human hepatic stellate LX-2 cells. Treatment of LX-2 cells with TRAIL decreased the active trimer formation of HSF1, increased the dephosphorylation of HSF1 (Ser230), and enhanced the translocation of HSF1 into cytosol. The accumulated HSF1 in cytosol led to downregulation of Hsp47 expression, resulting in the reduction of collagen production. Consistently, HSF1 silencing by siRNA prevented Hsp47 induction and subsequent collagen production, whereas overexpression of HSF1 restored the expression level of Hsp47 as well as collagen production in response to TRAIL treatment in LX-2 cells. Taken together, our data suggested that TRAIL induced HSF1 inactivation, consequently leading to the suppression of Hsp47-dependent collagen production in activated human hepatic stellate cells. Therefore, this study suggests that TRAIL may be an effective strategy for antifibrotic therapy in liver fibrosis.

MicroRNA-21 activates hepatic stellate cells via PTEN/Akt signaling

Activation of hepatic stellate cells is the key event in the liver fibrosis. miRs have been shown to play fundamental role in diverse biological and pathological processes. In the present study, we investigated the fibrogenic role of miR-21 in human hepatic stellate LX-2 cells and explored underlying mechanisms. The results showed that treatment of LX-2 cells with platelet-derived growth factor (PDGF)-BB significantly stimulated α1(I) collagen mRNA synthesis and the protein expression of α-SMA, which are characteristics of activation of hepatic stellate cells and simultaneously increased miR-21 expression. Downregulation of miR-21 expression by transfection of anti-miR-21 into LX-2 cells prevented PDGF-BB-induced LX-2 cell activation. Overexpression of miR-21 expression alone also stimulated LX-2 cell activation, while downregulation of miR-21 expression suppressed LX-2 cell activation. miR-21 also played a role in mRNA expression and activity of matrix metalloproteinase 2 (MMP2) in LX-2 cells. Moreover, overexpression of miR-21 decreased protein expression of PTEN in LX-2 cells, resulting in activation of the Akt. Inhibition of Akt signaling by specific inhibitor LY 294002 blocked miR-21-induced fibrogenic effects in LX-2 cells. In summary, miR-21 is an important mediator in LX-2 cell activation. The fibrogenic effects of miR-21 on LX-2 cell activation are mediated through PTEN/Akt pathway. miR-21 may be a potential novel molecular target for the liver fibrosis.