Enhancing the apoptotic effect of IL-24/mda-7 on the human hepatic stellate cell through RGD peptide modification.

Induction of apoptosis or quiescent hepatic stellate cells (HSCs) can be an attractive molecular strategy due to the importance of activation of HSCs during hepatic fibrogenesis. Interleukin-24/melanoma differentiation-associated gene-7 (IL-24/mda-7) is a cytokine that has attracted a great deal of attention in the tumor killing as well as pathophysiology of the diseases. In this study, the Pro-apoptotic and senescence inductive properties of IL-24/mda-7 were assessed in human-derived HSCs. Three plasmids expressing natural mda-7, peptide modified version, mda-7-RGD genes beside a recombinant IL-24 protein, were added or transfected into activated LX-2 cells. Cell viability and the amount of apoptosis were analyzed using MTT and Annexin V staining method, respectively. Hence, the expression levels of apoptotic genes and PPARγ in different groups were also compared by real-time PCR analysis. Furthermore, the senescence effect of IL-24/mda-7 by a ß-galactosidase (SA-ß-gal) senescence assay, was evaluated. The viability assessment showed that pmda-7-RGD had the most significant growth inhibitory effect when compared to the control group, pcDNA3.1 (P = 0.0002). The apoptosis analysis also revealed a significant impact of different mda-7 forms in apoptosis induction. The measuring of cell senescence also indicated that IL-24/mda-7 in plasmid and protein forms exhibited a senescence inductive activity as determined by an increase in PPARγ gene expression and beta-galctosidase activity. In conclusion, our findings demonstrated that both endogenous and soluble forms of IL-24/mda-7 induced apoptosis and senescence in activated LX-2 cells and more importantly, fusion of RGD peptide to this cytokine enhanced these activities. So, RGD-modified IL-24/mda-7 could be a suitable candidate for further molecular therapy of fibrosis.

Ammonia produces pathological changes in human hepatic stellate cells and is a target for therapy of portal hypertension.

BACKGROUND & AIMS: Hepatic stellate cells (HSCs) are vital to hepatocellular function and the liver response to injury. They share a phenotypic homology with astrocytes that are central in the pathogenesis of hepatic encephalopathy, a condition in which hyperammonemia plays a pathogenic role. This study tested the hypothesis that ammonia modulates human HSC activation in vitro and in vivo, and evaluated whether ammonia lowering, by using l-ornithine phenylacetate (OP), modifies HSC activation in vivo and reduces portal pressure in a bile duct ligation (BDL) model. METHODS: Primary human HSCs were isolated and cultured. Proliferation (BrdU), metabolic activity (MTS), morphology (transmission electron, light and immunofluorescence microscopy), HSC activation markers, ability to contract, changes in oxidative status (ROS) and endoplasmic reticulum (ER) were evaluated to identify effects of ammonia challenge (50 µM, 100 µM, 300 µM) over 24-72 h. Changes in plasma ammonia levels, markers of HSC activation, portal pressure and hepatic eNOS activity were quantified in hyperammonemic BDL animals, and after OP treatment. RESULTS: Pathophysiological ammonia concentrations caused significant and reversible changes in cell proliferation, metabolic activity and activation markers of hHSC in vitro. Ammonia also induced significant alterations in cellular morphology, characterised by cytoplasmic vacuolisation, ER enlargement, ROS production, hHSC contraction and changes in pro-inflammatory gene expression together with HSC-related activation markers such as α-SMA, myosin IIa, IIb, and PDGF-Rß. Treatment with OP significantly reduced plasma ammonia (BDL 199.1 µmol/L±43.65 vs. BDL+OP 149.27 µmol/L±51.1, p<0.05) and portal pressure (BDL 14±0.6 vs. BDL+OP 11±0.3 mmHg, p<0.01), which was associated with increased eNOS activity and abrogation of HSC activation markers. CONCLUSIONS: The results show for the first time that ammonia produces deleterious morphological and functional effects on HSCs in vitro. Targeting ammonia with the ammonia lowering drug OP reduces portal pressure and deactivates hHSC in vivo, highlighting the opportunity for evaluating ammonia lowering as a potential therapy in cirrhotic patients with portal hypertension.

Characterization and sub-cellular localization of GalNAc-binding proteins isolated from human hepatic stellate cells.

Although the expression levels of total GalNAc-binding proteins (GNBPs) were up-regulated significantly in human hepatic stellate cells (HSCs) activated with transforming growth factor-ß1(TGF-ß1), yet little is known about the precise types, distribution and sub-cellular localization of the GNBPs in HSCs. Here, 264 GNBPs from the activated HSCs and 257 GNBPs from the quiescent HSCs were identified and annotated. A total of 46 GNBPs were estimated to be significantly up-regulated and 40 GNBPs were estimated to be significantly down-regulated in the activated HSCs. For example, the GNBPs (i.e. BTF3, COX17, and ATP5A1) responsible for the regulation of protein binding were up-regulated, and those (i.e. FAM114A1, ENO3, and TKT) responsible for the regulation of protein binding were down-regulated in the activated HSCs. The motifs of the isolated GNBPs showed that Proline residue had the maximum preference in consensus sequences. The western blotting showed the expression levels of COX17, and PRMT1 were significantly up-regulated, while, the expression level of CLIC1(B5) was down-regulated in the activated HSCs and liver cirrhosis tissues. Moreover, the GNBPs were sub-localized in the Golgi apparatus of HSCs. In conclusion, the precision alteration of the GNBPs referred to pathological changes in liver fibrosis/cirrhosis may provide useful information to find new molecular mechanism of HSC activation and discover the biomarkers for diagnosis of liver fibrosis/cirrhosis as well as development of new anti-fibrotic strategies.

Establishment and characterization of an immortalized human hepatic stellate cell line for applications in co-culturing with immortalized human hepatocytes.

BACKGROUND AND OBJECTIVE: The liver-specific functions of hepatocytes are improved by co-culturing hepatocytes with primary hepatic stellate cells (HSC). However, primary HSC have a short lifespan in vitro, which is considered a major limitation for their use in various applications. This study aimed to establish immortalized human HSC using the simian virus 40 large T antigen (SV40LT) for applications in co-culturing with hepatocytes and HSC in vitro. METHODS: Primary human HSC were transfected with a recombinant retrovirus containing SV40LT. The immortalized human HSC were characterized by analyzing their gene expression and functional characteristics. The liver-specific functions of hepatocytes were evaluated in a co-culture system incorporating immortalized human hepatocytes with HSC-Li cells. RESULTS: The immortalized HSC line, HSC-Li, was obtained after infection with a recombinant retrovirus containing SV40LT. The HSC-Li cells were longitudinally spindle-like and had numerous fat droplets in their cytoplasm as shown using electron microscopy. Hepatocyte growth factor (HGF), VEGF Receptor 1(Flt-1), collagen type Iα1 and Iα2 mRNA expression levels were observed in the HSC-Li cells by RT-PCR. Immunofluorescence staining showed that the HSC-Li cells were positive for α-smooth muscle actin (α-SMA), platelet-derived growth factor receptor-beta (PDGFR-ß), vimentin, and SV40LT protein expression. The HSC-Li cells produced both HGF and transforming growth factor-beta1 (TGF-ß1) in a time-dependent manner. Real-time PCR showed that albumin, CYP3A5, CYP2E1, and UGT2B7 mRNA expression generally increased in the co-culture system. The enzymatic activity of CYP1A2 under the co-culture conditions also generally increased as compared to the monoculture of immortalized human hepatocytes. CONCLUSIONS: We successfully established the immortalized human HSC cell line HSC-Li. It has the specific phenotypic and functional characteristics of primary human HSC, which would be a useful tool to develop anti-fibrotic therapies. Co-culturing with the HSC-Li cells improved the liver-specific functions of hepatocytes, which may be valuable and applicable for bioartificial liver systems.

Study on the molecular mechanism of dihydromyricetin in alleviating liver cirrhosis based on network pharmacology.

Dihydromyricetin (DHM) is a bioactive flavonoid extracted from Hovenia dulcis, which has various activities. In the present study, the molecular mechanism of dihydromyricetin (DHM) in relieving liver cirrhosis was investigated through network pharmacology and experimental verification. The cell model was induced by TGF-ß1 activating the human hepatic stellate cell line (HSC; LX-2). The protein levels of α-SMA, collagen I, and collagen III and pathway-related proteins within LX-2 cells were detected using Western blot. EdU staining was conducted to detect cell proliferation. Immunofluorescence staining was performed to detect the expression levels of α-SMA and collagen I. Next, the drug targets of DHM were screened from the PubChem database. The differentially expressed genes in the liver cirrhosis dataset GSE14323 were identified. The expression of the identified drug targets in LX-2 cells was verified using qRT-PCR. The results showed that TGF-ß1 treatment notably increased LX-2 cell viability, promoted cell proliferation, and elevated α-SMA, collagen I, and collagen III protein contents. DHM treatment could partially eliminate TGF-ß1 effects, as evidenced by the inhibited cell viability and proliferation and reduced α-SMA, collagen I, and collagen III contents. After network pharmacology analysis, nine differentially expressed target genes (MMP2, PDGFRB, PARP1, BCL2L2, ABCB1, TYR, CYP2E1, SQSTM1, and IL6) in liver cirrhosis were identified. According to qRT-PCR verification, DHM could inhibit the expression of MMP2, PDGFRB, PARP1, CYP2E1, SQSTM1, and IL6, and enhance ABCB1 expression levels within LX-2 cells. Moreover, DHM inhibited mTOR and MAPK signaling pathways in TGF-ß1-induced HSCs. In conclusion, DHM could inhibit HSC activation, which may be achieved via acting on MMP2, PDGFRB, PARP1, CYP2E1, SQSTM1, IL6, and ABCB1 genes and their downstream signaling pathways, including mTOR and MAPK signaling pathway.

Intrahepatic IL-8 producing Foxp3⁺CD4⁺ regulatory T cells and fibrogenesis in chronic hepatitis C.

BACKGROUND & AIMS: Regulatory CD4(+) T cells (Tregs) are considered to affect outcomes of HCV infection, because they increase in number during chronic hepatitis C and can suppress T-cell functions. METHODS: Using microarray analysis, in situ immunofluorescence, ELISA, and flowcytometry, we characterised functional differentiation and localisation of adaptive Tregs in patients with chronic hepatitis C. RESULTS: We found substantial upregulation of IL-8 in Foxp3(+)CD4(+) Tregs from chronic hepatitis C. Activated GARP-positive IL-8(+) Tregs were particularly enriched in livers of patients with chronic hepatitis C in close proximity to areas of fibrosis and their numbers were correlated with the stage of fibrosis. Moreover, Tregs induced upregulation of profibrogenic markers TIMP1, MMP2, TGF-beta1, alpha-SMA, collagen, and CCL2 in primary human hepatic stellate cells (HSC). HSC activation, but not Treg suppressor function, was blocked by adding a neutralizing IL-8 antibody. CONCLUSIONS: Our studies identified Foxp3(+)CD4(+) Tregs as an additional intrahepatic source of IL-8 in chronic hepatitis C acting on HSC. Thus, Foxp3(+)CD4(+) Tregs in chronic hepatitis C have acquired differentiation as regulators of fibrogenesis in addition to suppressing local immune responses.

Human Hepatic Stellate Cells: Isolation and Characterization.

Liver fibrosis of different etiologies is characterized by activation of hepatic stellate cells (aHSCs) into collagen type I secreting myofibroblasts, which produce fibrous scar and make the liver fibrotic. aHSCs are the major source of myofibroblasts and, therefore, the primary targets of anti-fibrotic therapy. Despite extensive studies, targeting of aHSCs in patients provides challenges. The progress in anti-fibrotic drug development relies on translational studies but is limited by the availability of primary human HSCs. Here we describe a perfusion/gradient centrifugation-based method of the large-scale isolation of highly purified and viable human HSCs (hHSCs) from normal and diseased human livers and the strategies of hHSC cryopreservation.

Aramchol downregulates stearoyl CoA-desaturase 1 in hepatic stellate cells to attenuate cellular fibrogenesis.

BACKGROUND & AIMS: Aramchol is a fatty acid-bile acid conjugate that reduces liver fat content and is being evaluated in a phase III clinical trial for non-alcoholic steatohepatitis (NASH). Aramchol attenuates NASH in mouse models and decreases steatosis by downregulating the fatty acid synthetic enzyme stearoyl CoA desaturase 1 (SCD1) in hepatocytes. Although hepatic stellate cells (HSCs) also store lipids as retinyl esters, the impact of Aramchol in this cell type is unknown. METHODS: We investigated the effects of Aramchol on a human HSC line (LX-2), primary human HSCs (phHSCs), and primary human hepatocytes (phHeps). RESULTS: In LX-2 and phHSCs, 10 µM Aramchol significantly reduced SCD1 mRNA while inducing PPARG (PPARγ) mRNA, with parallel changes in the 2 proteins; ACTA2, COL1A1, ß-PDGFR (bPDGFR) mRNAs were also significantly reduced in LX-2. Secretion of collagen 1 (Col1α1) was inhibited by 10 µM Aramchol. SCD1 knockdown in LX-2 cells phenocopied the effect of Aramchol by reducing fibrogenesis, and addition of Aramchol to these cells did not rescue fibrogenic gene expression. Conversely, in LX-2 overexpressing SCD1, Aramchol no longer suppressed fibrogenic gene expression. The drug also induced genes in LX-2 that promote cholesterol efflux and inhibited ACAT2, which catalyses cholesterol synthesis. In phHeps, Aramchol also reduced SCD1 and increased PPARG mRNA expression. CONCLUSIONS: Aramchol downregulates SCD1 and elevates PPARG in HSCs, reducing COL1A1 and ACTA2 mRNAs and COL1A1 secretion. These data suggest a direct inhibitory effect of Aramchol in HSCs through SCD1 inhibition, as part of a broader impact on both fibrogenic genes as well as mediators of cholesterol homeostasis. These findings illustrate novel mechanisms of Aramchol activity, including potential antifibrotic activity in patients with NASH and fibrosis. LAY SUMMARY: In this study, we have explored the potential activity of Aramchol, a drug currently in clinical trials for fatty liver disease, in blocking fibrosis, or scarring, by hepatic stellate cells, the principal collagen-producing (i.e. fibrogenic) cell type in liver injury. In both isolated human hepatic stellate cells and in a human hepatic stellate cell line, the drug suppresses the key fat-producing enzyme, stearoyl CoA desaturase 1 (SCD1), which leads to reduced expression of genes and proteins associated with hepatic fibrosis, while inducing the protective gene, PPARγ. The drug loses activity when SCD1 is already reduced by gene knockdown, reinforcing the idea that inhibition of SCD1 is a main mode of activity for Aramchol. These findings strengthen the rationale for testing Aramchol in patients with NASH.

Exogenous Liposomal Ceramide-C6 Ameliorates Lipidomic Profile, Energy Homeostasis, and Anti-Oxidant Systems in NASH.

In non-alcoholic steatohepatitis (NASH), many lines of investigation have reported a dysregulation in lipid homeostasis, leading to intrahepatic lipid accumulation. Recently, the role of dysfunctional sphingolipid metabolism has also been proposed. Human and animal models of NASH have been associated with elevated levels of long chain ceramides and pro-apoptotic sphingolipid metabolites, implicated in regulating fatty acid oxidation and inflammation. Importantly, inhibition of de novo ceramide biosynthesis or knock-down of ceramide synthases reverse some of the pathology of NASH. In contrast, cell permeable, short chain ceramides have shown anti-inflammatory actions in multiple models of inflammatory disease. Here, we investigated non-apoptotic doses of a liposome containing short chain C6-Ceramide (Lip-C6) administered to human hepatic stellate cells (hHSC), a key effector of hepatic fibrogenesis, and an animal model characterized by inflammation and elevated liver fat content. On the basis of the results from unbiased liver transcriptomic studies from non-alcoholic fatty liver disease patients, we chose to focus on adenosine monophosphate activated kinase (AMPK) and nuclear factor-erythroid 2-related factor (Nrf2) signaling pathways, which showed an abnormal profile. Lip-C6 administration inhibited hHSC proliferation while improving anti-oxidant protection and energy homeostasis, as indicated by upregulation of Nrf2, activation of AMPK and an increase in ATP. To confirm these in vitro data, we investigated the effect of a single tail-vein injection of Lip-C6 in the methionine-choline deficient (MCD) diet mouse model. Lip-C6, but not control liposomes, upregulated phospho-AMPK, without inducing liver toxicity, apoptosis, or exacerbating inflammatory signaling pathways. Alluding to mechanism, mass spectrometry lipidomics showed that Lip-C6-treatment reversed the imbalance in hepatic phosphatidylcholines and diacylglycerides species induced by the MCD-fed diet. These results reveal that short-term Lip-C6 administration reverses energy/metabolic depletion and increases protective anti-oxidant signaling pathways, possibly by restoring homeostatic lipid function in a model of liver inflammation with fat accumulation.

The Effect of Ferroptosis on the Activation of Human Hepatic Stellate Cells Induced by Inorganic Arsenic / 医学研究杂志

Objective To investigate the effect of NaAsO2 on ferroptosis in human hepatic stellate cells(LX-2).Methods LX-2 cells were cultured in vitro,and different concentrations of NaAsO2(5μmol/L,10μmol/L,15μmol/L)were infected with LX-2 cells for 24h in a group design to construct the activation model of LX-2 induced by NaAsO2 in vitro,and a control group was set up.Mitochon-drial structure of LX-2 cells treated with NaAsO2 was observed by transmission electron microscopy(TEM).Fe2+levels were detected by fluorescence microscope and fluorescent enzyme label.The content of malondialdehyde(MDA)was determined by the colorimetric meth-od.The protein expression levels of SLC7A11,GPX4,and α-smooth muscle actin(α-SMA)were detected by Western blot.Results TEM showed that mitochondrial membrane integrity was damaged and mitochondrial ridges were reduced and disappeared in the NaAsO2group.In addition,compared with the control group,Fe2+levels in NaAsO2 treatment groups were increased(P＜0.05).There were statistically significant differences in MDA content among different doses of NaAsO2groups(F=7.18,P＜0.05).Compared with the control group,MDA content of LX-2 cells in 5 and 15μmol/L NaAsO2groups was higher than that in the control group(P＜0.05).At the translation level,the expression of fibrosis index α-SMA protein level was up-regulated with the increase of NaAsO2dose(P＜0.05),the protein expression levels of ferroptosis index SLC7A11 and GPX4decreased in a dose-dependent manner with the increase of the dose of NaAsO2(P＜0.05).Conclusion Ferroptosis is involved in the activation of LX-2 cells induced by NaAsO2.

Mesenchymal stem cells cultured under hypoxic conditions had a greater therapeutic effect on mice with liver cirrhosis compared to those cultured under normal oxygen conditions.

BACKGROUND: Mesenchymal stem cells (MSCs) can be easily expanded. They can be acquired from medical waste such as adipose and umbilical cord tissues, are influenced by culturing conditions, and exert anti-inflammatory, antioxidant, anti-fibrotic, and angiogenic effects. We analyzed the multi-directional effects of MSCs cultured under hypoxic conditions and their underlying mechanisms in the treatment of liver cirrhosis in a mouse model. METHODS: Human bone marrow-derived MSCs cultured under hypoxic (5% O2; hypoMSCs) and normoxic (21% O2; norMSCs) conditions were compared by cap analysis of gene expression (CAGE) with or without serum from liver cirrhosis patients. The therapeutic effects of MSCs, including serum liver enzyme induction, fibrosis regression, and hepatic oxidative stress, were evaluated by injecting 1 × 106, 2 × 105, or 4 × 104 MSCs/mouse into the tail veins of mice with carbon tetrachloride (CCl4)-induced liver cirrhosis. Intravital imaging was performed with a two-photon excitation microscope to confirm the various MSC migration paths to the liver. RESULTS: CAGE analysis revealed that the RNA expression levels of prostaglandin E synthase (Ptges) and miR210 were significantly higher in hypoMSCs than in norMSCs. In vivo analysis revealed that both hypoMSCs and norMSCs reduced serum alanine aminotransferase, oxidative stress, and fibrosis compared to that in control mice in a dose-dependent manner. However, hypoMSCs had stronger therapeutic effects than norMSCs. We confirmed this observation by an in vitro study in which hypoMSCs changed macrophage polarity to an anti-inflammatory phenotype via prostaglandin E2 (PGE2) stimulation. In addition, miR210 reduced the rate of hepatocyte apoptosis. Intravital imaging after MSC administration showed that both cell types were primarily trapped in the lungs. Relatively a few hypoMSCs and norMSCs migrated to the liver. There were no significant differences in their distributions. CONCLUSION: The therapeutic effect of hypoMSCs was mediated by PGE2 and miR210 production and was greater than that of norMSCs. Therefore, MSCs can be manipulated to improve their therapeutic efficacy in the treatment of liver cirrhosis and could potentially serve in effective cell therapy. MSCs produce several factors with multidirectional effects and function as "conducting cells" in liver cirrhosis.

Screening of DNA methylation sites and construction of differential methylation profiles in human hepatic stellate cells fibrosis and autophagy induced by sodium arsenite / 中华地方病学杂志

Objective:To analyze DNA methylation sites related to fibrosis and autophagy in human hepatic stellate cells (LX-2 cells) induced by sodium arsenite (NaAsO 2), and to screen specific methylation genes related to fibrosis and autophagy. Methods:Genome-wide DNA detection was performed using Illumina Infinium Methylation EPIC BeadChips (850K methylation chip) to derive differential methylation sites in LX-2 cells (control group) and the fibrosis and autophagy models of LX-2 cells induced by NaAsO 2(low, medium and high dose groups: the final concentrations were 5, 10, 15 μmol/L NaAsO 2, respectively, after 48 h intervention). Gene ontology (GO) function enrichment analysis and Kyoto encyclopedia of genes and genomes (KEGG) signaling pathway enrichment analysis were used to explore gene function. Results:The model of cell fibrosis and autophagy was established successfully in high dose group. The results of 850K methylation chip detection showed that there were 25 817 significant different methylation sites between the high dose group and the control group, including 12 083 hypermethylation sites and 13 734 hypomethylation sites. GO function enrichment analysis showed that the molecular functions of differentially methylated genes mainly included protein binding, ion binding, catalytic activity, enzyme binding. KEGG signaling pathway enrichment analysis showed that the pathways involved in differentially methylated genes mainly included metabolic pathway, cancer pathway, phosphatidylinositol-3-kinase-protein kinase B (PI3K-Akt) signaling pathway, endocytosis, and mitogen activated protein kinase (MAPK) signaling pathway. In the promoter region, 11 and 29 differentially methylated genes related to fibrosis and autophagy were screened, respectively.Conclusions:A large number of differential methylation sites exist in the process of NaAsO 2 induced fibrosis and autophagy of LX-2 cells. Specific methylation genes related to fibrosis and autophagy are screened out.

Recombinant NS3 Protein Induced Expression of Immune Modulatory Elements in Hepatic Stellate Cells During Its Fibrotic Activity.

There is a growing body of studies that show the important role of NS3 protein from hepatitis C virus in fibrosis. However, mechanisms of the effects of this protein on immune modulation of stellate cells remain to be investigated. Herein, the effect of NS3 protein on the expression level of suppressor of cytokine signaling (SOCS)1/3 and interleukin-24 (IL-24)-related genes was investigated in hepatic stellate cell (HSC), LX-2. Recombinant NS3 protein was added to LX-2 HSC culture. Leptin and standard medium treatments were also included in experiments as positive and negative controls, respectively. Total RNA was extracted from each well at 6, 12, and 24 h after NS3 addition. The expression levels of the fibrotic (transforming growth factor beta 1 [TGF-ß], alpha-smooth muscle actin [α-SMA], and COL1A1), inflammatory (IL-6 and IL-24), IL-20R, IL-22R, and immunosuppressive genes (SOCS1 and SOCS3) were evaluated by real-time polymerase chain reaction (PCR). Recombinant NS3 protein induced activated phenotypes of LX-2 with a significant increase in the expression level of α-SMA COL1A1 (p < 0.0001) and TGF-ß. Moreover, this exposure led to a meaningful elevation in the expression of IL-6. Furthermore, compared with leptin (control), after the stellate cell treatment with NS3, SOCS1 and SOCS3 gene expression induced at a comparable level. Compared with the control sample, the NS3 protein significantly increased the expression level of IL-24 and its related receptors, IL-20R and IL-22R. This study not only confirmed the previously proved inflammatory and fibrotic effect of this protein but also indicated that high expression levels of SOCS1, SOCS3, and IL-24 have a significant effect on HSC activation. Therefore, these two molecules can be used as a potential therapeutic target candidate.

Human hepatic stellate cell isolation and characterization.

The hepatic stellate cells (HSCs) localize at the space of Disse in the liver and have multiple functions. They are identified as the major contributor to hepatic fibrosis. Significant understanding of HSCs has been achieved using rodent models and isolated murine HSCs; as well as investigating human liver tissues and human HSCs. There is growing interest and need of translating rodent study findings to human HSCs and human liver diseases. However, species-related differences impose challenges on the translational research. In this review, we focus on the current information on human HSCs isolation methods, human HSCs markers, and established human HSC cell lines.

Aucubin and its hydrolytic derivative attenuate activation of hepatic stellate cells via modulation of TGF-ß stimulation.

Eucommia ulmoides is an important traditional Chinese medicine and has been used as a tonic with a long history. Aucubin is an active component extracted from Eucommia ulmoides, which has liver-protection effects. However the mechanisms are still unclear. To investigate the inhibitory effects and the underlying mechanisms of aucubin on TGF-ß1-induced activation of hepatic stellate cells and ECM deposition, Human hepatic stellate cells (LX-2 cells) were incubated with TGF-ß1 to evaluate the anti-fibrotic effect of aucubin. Western blot was used to investigate the expression of α-SMA, Col I, Col III, MMP-2 and TIMP-1. ROS production was monitored using DCFH-DA probe, and NOX4 expression was detected by Real-time PCR. Results indicated that TGF-ß1 stimulated the activation and ECM deposition of LX-2 cells. Compared with the control group, aucubin and aucubigenin both reduced the protein expression of α-SMA, Col I, Col III and MMP-2 in LX-2 cells. Aucubin and aucubigenin also suppressed the generation of ROS and down-regulated the NOX4 mRNA expression. Taken together, aucubin and aucubigenin both inhibit the activation and ECM deposition of LX-2 cells activated by TGF-ß1. Aucubin and aucubigenin are potential therapeutic candidate drugs for liver fibrosis.

Reactive gamma-ketoaldehydes as novel activators of hepatic stellate cells in vitro.

AIMS: Products of lipid oxidation, such as 4-hydroxynonenal (4-HNE), are key activators of hepatic stellate cells (HSC) to a pro-fibrogenic phenotype. Isolevuglandins (IsoLG) are a family of acyclic γ-ketoaldehydes formed through oxidation of arachidonic acid or as by-products of the cyclooxygenase pathway. IsoLGs are highly reactive aldehydes which are efficient at forming protein adducts and cross-links at concentrations 100-fold lower than 4-hydroxynonenal. Since the contribution of IsoLGs to liver injury has not been studied, we synthesized 15-E2-IsoLG and used it to investigate whether IsoLG could induce activation of HSC. RESULTS: Primary human HSC were exposed to 15-E2-IsoLG for up to 48h. Exposure to 5µM 15-E2-IsoLG in HSCs promoted cytotoxicity and apoptosis. At non-cytotoxic doses (50 pM-500nM) 15-E2-IsoLG promoted HSC activation, indicated by increased expression of α-SMA, sustained activation of ERK and JNK signaling pathways, and increased mRNA and/or protein expression of cytokines and chemokines, which was blocked by inhibitors of JNK and NF-kB. In addition, IsoLG promoted formation of reactive oxygen species, and induced an early activation of ER stress, followed by autophagy. Inhibition of autophagy partially reduced the pro-inflammatory effects of IsoLG, suggesting that it might serve as a cytoprotective response. INNOVATION: This study is the first to describe the biological effects of IsoLG in primary HSC, the main drivers of hepatic fibrosis. CONCLUSIONS: IsoLGs represent a newly identified class of activators of HSC in vitro, which are biologically active at concentrations as low as 500 pM, and are particularly effective at promoting a pro-inflammatory response and autophagy.

Combining Vγ9Vδ2 T Cells with a Lipophilic Bisphosphonate Efficiently Kills Activated Hepatic Stellate Cells.

Activated hepatic stellate cells (aHSCs) are now established as a central driver of fibrosis in human liver injury. In the presence of chronic or repeated injury, fibrosis, cirrhosis, and hepatocellular carcinoma (HCC) can occur, so there is interest in down-regulating aHSCs activity in order to treat these diseases. Here, we report that Vγ9Vδ2 T cells are reduced in patients with liver cirrhosis, stimulating us to investigate possible interactions between Vγ9Vδ2 T cells and aHSCs. We find that Vγ9Vδ2 T cells kill aHSCs and killing is enhanced when aHSCs are pretreated with BPH-1236, a lipophilic analog of the bone resorption drug zoledronate. Cytotoxicity is mediated by direct cell-to-cell contact as shown by Transwell experiments and atomic force microscopy, with BPH-1236 increasing the adhesion between aHSCs and Vγ9Vδ2 T cells. Mechanistically, BPH-1236 functions by inhibiting farnesyl diphosphate synthase, leading to accumulation of the phosphoantigen isopentenyl diphosphate and recognition by Vγ9Vδ2 T cells. The cytolytic process is largely dependent on the perforin/granzyme B pathway. In a Rag2-/-γc-/- immune-deficient mouse model, we find that Vγ9Vδ2 T cells home-in to the liver, and when accompanied by BPH-1236, kill not only orthotopic aHSCs but also orthotopic HCC tumors. Collectively, our results provide the first proof-of-concept of a novel immunotherapeutic strategy for the treatment of fibrosis-cirrhosis-HCC diseases using adoptively transferred Vγ9Vδ2 T cells, combined with a lipophilic bisphosphonate.

The pattern of IL-24/mda-7 and its cognate receptors expression following activation of human hepatic stellate cells.

Activation of hepatic stellate cells (HSCs) is the pivotal event during liver fibrosis. Interleukin (IL)-24/melanoma differentiation-associated gene-7 (mda-7) has attracted attention in the pathophysiology of some diseases, while its role in activation/suppression of human HSCs is still unclear. It is important to elucidate whether the expression levels of the IL-24/mda-7 protein and its receptors in HSC cells are changed following activation. LX-2 cells, a human hepatic stellate cell line were activated by a combination of leptin and serum starvation. The activation state was evaluated through measuring the mRNA expression of profibrotic molecules, collagen-I, TIMP metalloproteinase inhibitor-1 and transforming growth factor-ß. The expression of IL-24/mda-7 was assessed in mRNA and protein levels by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and ELISA methods, respectively. Hence, the amount of IL-22R1 and IL-20R2 subunit expression was also compared in activated and normal LX-2 cells by RT-qPCR. The expression level of IL-24/mda-7 and its cognate receptors was detectable both in the normal and activated LX-2 cell line. Furthermore, in activated LX-2, a significant increase of IL24 expression either on IL-22R1 and IL-20R2 subunits was also noticeable in comparison to normal cells. The activation state of LX-2 cells caused significant changes of IL-24/mda-7 and its receptors expression. In addition, the elevation in IL-24/mda-7 during LX-2 cell activation, suggested that IL-24/mda-7 and its cognate receptors serve a possible role in the development of the fibrosis process. Therefore, IL-24/mda-7 and relevant signaling pathways may be employed as a target for fibrosis treatment.

Mechanism of total C-21 steroidal glucosides from root of Cynanchum auriculatum in activation of human hepatic stellate cells via TGF-β / 中国药理学通报

Aim To investigate the inhibitory effect of total C-21 steroidal glucosides (TCSG) from the root of Cynanchum auriculatum on activation of human hepatic stellate cells and the underlying mechanism. Methods The fibrosis model in vitro was established by treating LX-2 cells with TGF-β

Determination and Characterization of Tetraspanin-Associated Phosphoinositide-4 Kinases in Primary and Neoplastic Liver Cells.

Accumulating evidence implicates phosphoinositide 4-phosphate as a regulatory molecule in its own right recruiting specific effector proteins to cellular membranes. Here, we describe biochemical and immunocytochemical methods to evaluate tetraspanin-associated phosphoinositide-4 kinases activity in primary human hepatic stellate cells (hHSC) and neoplastic hepatoblastoma cells.