Suppression of hepatic stellate cell activation by microRNA-29b

MicroRNAs (miRNAs) participate in the regulation of cellular functions including proliferation, apoptosis, and migration. It has been previously shown that the miR-29 family is involved in regulating type I collagen expression by interacting with the 3′UTR of its mRNA. Here, we investigated the roles of miR-29b in the activation of mouse primary-cultured hepatic stellate cells (HSCs), a principal collagen-producing cell in the liver. Expression of miR-29b was found to be down-regulated during HSC activation in primary culture. Transfection of a miR-29b precursor markedly attenuated the expression of Col1a1 and Col1a2 mRNAs and additionally blunted the increased expression of α-SMA, DDR2, FN1, ITGB1, and PDGFR-β, which are key genes involved in the activation of HSCs. Further, overexpression of miR-29b led HSCs to remain in a quiescent state, as evidenced by their quiescent star-like cell morphology. Although phosphorylation of FAK, ERK, and Akt, and the mRNA expression of c-jun was unaffected, miR-29b overexpression suppressed the expression of c-fos mRNA. These results suggested that miR-29b is involved in the activation of HSCs and could be a candidate molecule for suppressing their activation and consequent liver fibrosis.     

Hepatic fibrosis--role of hepatic stellate cell activation

Hepatic fibrosis is a reversible wound healing response characterized by accumulation of extracellular matrix (ECM), or "scar," that follows chronic but not self-limited liver disease. The ECM components in fibrotic liver are similar regardless of the underlying cause. Activation of hepatic stellate cells is the central event in hepatic fibrosis. These perisinusoidal cells orchestrate an array of changes including degradation of the normal ECM of liver, deposition of scar molecules, vascular and organ contraction, and release of cytokines. Not only is hepatic fibrosis reversible, but it is also increasingly clear that cirrhosis may be reversible as well. The exact stage at which fibrosis/cirrhosis becomes truly irreversible is not known. Antifibrotic therapies will soon be a clinical reality. Emerging therapies will be targeted to those patients with reversible disease. The paradigm of stellate cell activation provides an important framework for defining therapeutic targets.     

Osthole ameliorates hepatic fibrosis and inhibits hepatic stellate cell activation

Background

Hepatic fibrosis is a dynamic process which ultimately leads to cirrhosis in almost patients with chronic hepatic injury. However, progressive fibrosis is a reversible scarring response. Activation of hepatic stellate cells (HSCs) is the prevailing process during hepatic fibrosis. Osthole is an active component majorly contained in the fruit of Cnidium monnieri (L.) Cusson. This present study investigated the therapeutic effects of osthole on rat liver fibrosis and HSC activation.

Results

We established the thioacetamide (TAA)-model of Sprague–Dawley (SD) rats to induce hepatic fibrosis. Rats were divided into three groups: control, TAA, and TAA + osthole (10 mg/kg). In vivo, osthole significantly reduced liver injury by diminishing levels of plasma AST and ALT, improving histological architecture, decreasing collagen and α-SMA accumulation, and improving hepatic fibrosis scores. Additionally, osthole reduced the expression of fibrosis-related genes significantly. Osthole also suppressed the production of fibrosis-related cytokines and chemokines. Moreover, nuclear translocation of p65 was significantly suppressed in osthole-treated liver. Osthole also ameliorated TAA-induced injury through reducing cellular oxidation. Osthole showed inhibitory effects in inflammation-related genes and chemokines production as well. In vitro, we assessed osthole effects in activated HSCs (HSC-T6 and LX-2). Osthole attenuated TGF-β1-induced migration and invasion in HSCs. Furthermore, osthole decreased TNF-α-triggered NF-κB activities significantly. Besides, osthole alleviated TGF-β1- or ET-1-induced HSCs contractility.

Conclusions

Our study demonstrated that osthole improved TAA-caused liver injury, fibrogenesis and inflammation in rats. In addition, osthole suppressed HSCs activation in vitro significantly.

Electronic supplementary material

The online version of this article (doi:10.1186/s12929-015-0168-5) contains supplementary material, which is available to authorized users.     

Metformin ameliorates activation of hepatic stellate cells and hepatic fibrosis by succinate and GPR91 inhibition

Background

Chronic liver disease is becoming a major cause of morbidity and mortality worldwide. During liver injury, hepatic stellate cells (HSCs) trans-differentiate into activated myofibroblasts, which produce extracellular matrix.Succinate and succinate receptor (G-protein coupled receptor91, GPR91) signaling pathway has now emerged as a regulator of metabolic signaling. A previous study showed that succinate and its specific receptor, GPR91, are involved in the activation of HSCs and the overexpression of α-smooth muscle actin (α-SMA).Metformin, a well-known anti-diabetic drug, inhibits hepatic gluconeogenesis in the liver. Many studies have shown that metformin not only prevented, but also reversed, steatosis and inflammation in a nonalcoholic steatohepatitis (NASH) animal model. However, the role of metformin in HSC activation and succinate-GPR91 signaling has not been clarified.

Autophagy: a new player in hepatic stellate cell activation

Hepatic stellate cell (HSC) activation, the transition from a resident quiescent HSC to a myofibroblastic collagen-producing HSC, is a fundamental feature of liver fibrosis. Autophagy has been implicated in major liver pathologies, such as HCV infection and hepatocarcinoma. However, its role in HSC biology is largely unknown. Recently, we were able to demonstrate that HSC activation is followed by an increased autophagic flux and that its inhibition can partially inhibit the HSC myofibroblastic transition. These results point to autophagy as a possible target in the prevention of HSC activation.     

The hepatic stellate cell in the post-genomic era

The draft human genome sequence was published on February 15, 2001, which will provide a huge amount of information on human genetics, human disease, and human cell biology. Now, medical scientists and cell biologists are turning their attention to illustrating gene expression pattern using gene microarray and to identifying the functions and the expression patterns of proteins encoded by the genes. Hepatic stellate cell is one of the sinusoid-constituent cells that play multiple roles in the liver pathophysiology. Transformation of stellate cells from the vitamin A-storing phenotype to the "myofibroblastic" one closely correlates to hepatic fibrosis during chronic liver trauma. Analyses of the molecular mechanisms of stellate cell activation have made a great progress, in particular, in the field of intracellular signal transduction of transforming growth factor-beta and platelet-derived growth factor, integrin signaling related to cell-adhesion, and cell motility-associated Rho and focal-adhesion kinase. Accumulation of the information on the stellate cell activation would shed light on the establishment of a novel therapeutic strategy against fibrosis of human liver disease.     

Essential role of matrix metalloproteinases in interleukin-1-induced myofibroblastic activation of hepatic stellate cell in collagen

Located within the perisinusoidal space and surrounded by extracellular matrix, hepatic stellate cells (HSC) undergo phenotypic trans-differentiation called "myofibroblastic activation" in liver fibrogenesis. This study investigated the regulation of interleukin-1 (IL-1alpha) on expression of matrix metalloproteinases (MMPs) by HSC grown in three-dimensional extracellular matrix and the role of MMPs in HSC activation. To recapitulate the in vivo "quiescent" state of HSC, the isolated rat HSC were grown in three-dimensional Matrigel or type I collagen. Stimulation with IL-1alpha caused robust induction of pro-MMP-9 (the precursor of matrix metalloproteinase-9) when HSC were cultured in these matrices. IL-1alpha induced a conversion of the pro-MMP-9 to the active form only when the cells were in type I collagen. In collagen lattices, IL-1alpha provoked activation of HSC with induction of MMP-13, MMP-3, and breakdown of the matrix. The HSC activation was completely prevented by a treatment of the cells with tissue inhibitor of metalloproteinase-1 or deprivation of MMP-9. Once fully activated, HSC failed to express MMP-9 and showed attenuated induction of MMP-13 and MMP-3. Further, we demonstrated colocalization of alpha-smooth muscle actin and MMP-9 in a subpopulation of HSC in human fibrotic liver tissues. Thus, this study provides a novel model to enlighten the role of MMPs, particularly that of MMP-9, in HSC activation regulated by a specific cytokine in liver fibrogenesis.     

L-cysteine administration prevents liver fibrosis by suppressing hepatic stellate cell proliferation and activation

Recent studies showed that the function of some amino acids is not only nutritional but also pharmacological. However, the effects of amino acids on liver fibrosis and hepatic stellate cell (HSC) remain unclear. In this research, as a result of screening of amino acids using liver fibrosis induced by DMN administration, L-cysteine was selected as a suppressor of liver fibrosis. Furthermore, the number of activated HSCs, which increased in the fibrotic liver after DMN administration, was decreased in L-cysteine-fed rats. Treatment of freshly isolated HSCs with L-cysteine resulted in inhibition of the increase in smooth muscle alpha-actin (alphaSMA) expression by HSCs and BrdU incorporation into the activated HSCs. These findings suggest that L-cysteine is effective against liver fibrosis. The mechanism of inhibition of fibrosis in the liver is surmized to be direct inhibition of activated HSC proliferation and HSC transformation by L-cysteine.     

Over-expressed microRNA-27a and 27b influence fat accumulation and cell proliferation during rat hepatic stellate cell activation

Hepatic stellate cells (HSCs) activation is an initial event in liver fibrosis. MicroRNAs (miRNAs) have been found to play essential roles in cell differentiation, proliferation, and fat metabolism. In this study, we showed that down-regulation of two over-expressed miRNAs, miR-27a and 27b allowed culture-activated rat HSCs to switch to a more quiescent HSC phenotype, with restored cytoplasmic lipid droplets and decreased cell proliferation. Mechanistically, retinoid X receptor α was confirmed to be the target of miR-27a and 27b. These results indicated a new role and mechanism of miR-27a and 27b in regulating fat metabolism and cell proliferation during HSCs activation.     

MiR-542-3p controls hepatic stellate cell activation and fibrosis via targeting BMP-7

There has been an increasing number of studies about microRNAs as key regulators in the development of hepatic fibrosis. Here, we demonstrate that miR-542-3p can promote hepatic fibrosis by downregulating the expression of bone morphogenetic protein 7 (BMP-7), which is known to antagonize transforming growth factor β1 (TGFβ1)-mediated fibrogenesis effect. The expression of miR-542-3p is increased in activated hepatic stellate cells (HSCs). Downregulation of MiR-542-3p by antisense inhibitors can inhibit HSCs activation markers, including α-smooth muscle actin (α-SMA) and collagen as well as TGFβ signaling pathways. MiR-542-3p was significantly upregulated in carbon tetrachloride (CCl₄)-induced hepatic fibrosis in mice, and downregulation of miR-542-3p by lentivirus could prevent the development of hepatic fibrosis. In addition, miR-542-3p can directly bind to the 3′-untranslated region of BMP-7 mRNA, indicating that its profibrotic effect appears to be caused by its inhibition of BMP-7. Our results suggest that downregulation of miR-542-3p prevents liver fibrosis both in vitro and in vivo, highlighting its potential as a novel biomarker or therapeutic target for hepatic fibrosis.     

Lipopolysaccharide mediates hepatic stellate cell activation by regulating autophagy and retinoic acid signaling

Bacterial translocation and lipopolysaccharide (LPS) leakage occur at a very early stage of liver fibrosis in animal models. We studied the role of LPS in hepatic stellate cell (HSC) activation and the underlying mechanisms in vitro and in vivo. Herein, we demonstrated that LPS treatment led to a dramatic increase in autophagosome formation and autophagic flux in LX-2 cells and HSCs, which was mediated through the AKT-MTOR and AMPK-ULK1 pathway. LPS significantly decreased the lipid content, including the lipid droplet (LD) number and lipid staining area in HSCs; pretreatment with macroautophagy/autophagy inhibitors or silencing ATG5 attenuated this decrease. Furthermore, lipophagy was induced by LPS through the autophagy-lysosomal pathway in LX-2 cells and HSCs. Additionally, LPS-induced autophagy further reduced retinoic acid (RA) signaling, as demonstrated by a decrease in the intracellular RA level and Rar target genes, resulting in the downregulation of Bambi and promoting the sensitization of the HSC's fibrosis response to TGFB. Compared with CCl₄ injection alone, CCl₄ plus LPS injection exaggerated liver fibrosis in mice, as demonstrated by increased Col1a1 (collagen, type I, α 1), Acta2, Tgfb and Timp1 mRNA expression, ACTA2/α-SMA and COL1A1 protein expression, and Sirius Red staining area, which could be attenuated by injection of an autophagy inhibitor. LPS also reduced lipid content in HSCs in vivo, with this change being attenuated by chloroquine (CQ) administration. In conclusion, LPS-induced autophagy resulted in LD loss, RA signaling dysfunction, and downregulation of the TGFB pseudoreceptor Bambi, thus sensitizing HSCs to TGFB signaling.     

Role of reactive oxygen species in zinc deficiency-induced hepatic stellate cell activation

We previously reported that zinc deficiency caused a reduction in intracellular glutathione at 8 h after the addition of zinc chelator, diethylenetriamine pentaacetic acid (DTPA), compared with control levels in rat hepatic stellate cells. In this study, we investigated the role of reactive oxygen species and glutathione on the mechanism of zinc deficiency-induced hepatic stellate cell activation, via assessing collagen synthesis. Isolated hepatic stellate cells were incubated with or without DTPA. Type I collagen expression in hepatic stellate cells was detected by immunohistochemistry, and then quantification of the intensity of type I collagen expression was analyzed using a computer with NIH image. Intracellular glutathione was measured using HPLC. H(2)O(2) release from hepatic stellate cells into the overlying medium was assayed using a fluorimetric method. H(2)O(2) release by DTPA-treated hepatic stellate cells significantly increased from 4 h, but returned to control levels after zinc supplementation. When catalase was added to the culture at 6 h after the addition of DTPA, the staining for type I collagen was as weak as at control levels. Diphenyliodonium chloride, the inhibitor of NADPH oxidase, produced a marked reduction in zinc deficiency-induced H(2)O(2) release. The results of this study show that the depletion of intracellular glutathione levels triggers a progression of collagen synthesis in zinc deficient-hepatic stellate cells and this depletion may be induced by the stimulation of cellular production of H(2)O(2).     

Exogenous expression of caveolin-1 is sufficient for hepatic stellate cell activation

Caveolin-1 (Cav-1) expression is increased in hepatic stellate cells (HSC) upon liver cirrhosis and it functions as an integral membrane protein of lipid rafts and caveolae that regulates and integrates multiple signals as a platform. This study aimed to evaluate the role of Cav-1 in HSC. Thus, the effects of exogenous expression of Cav-1 in GRX cells, a model of activated HSC, were determined. Here, we demonstrated through evaluating well-known HSC activation markers – such as α-smooth muscle actin, collagen I, and glial fibrillary acidic protein – that up regulation of Cav-1 induced GRX to a more activated phenotype. GRX^EGFP-Cav1 presented an increased migration, an altered adhesion pattern, a reorganization f-actin cytoskeleton, an arrested cell cycle, a modified cellular ultrastructure, and a raised endocytic flux. Based on this, GRX ^EGFP-Cav1 represents a new cellular model that can be an important tool for understanding of events related to HSC activation. Furthermore, our results reinforce the role of Cav-1 as a molecular marker of HSC activation.     

FBXO31 modulates activation of hepatic stellate cells and liver fibrogenesis by promoting ubiquitination of Smad7

Liver fibrosis is a critical pathological process in the early stage of many liver diseases, including hepatic cirrhosis and liver cancer. However, the molecular mechanism is not fully revealed. In this study, we investigated the role of F-box protein 31 (FBXO31) in liver fibrosis. We found FBXO31 upregulated in carbon tetrachloride (CCl₄) induced liver fibrosis and in activated hepatic stellate cells, induced by transforming growth factor-β (TGF-β). The enforced expression of FBXO31 caused enhanced proliferation and increased expression of α-smooth muscle actin (α-SMA) and Col-1 in HSC-T6 cells. Conversely, suppression of FBXO31 resulted in inhibition of proliferation and decreased accumulation of α-SMA and Col-1 in HSC-T6 cells. In addition, upregulation of FBXO31 in HSC-T6 cells decreased accumulation of Smad7, the negative regulator of the TGF-β/smad signaling pathway, and suppression of the FBXO31 increased accumulation of Smad7. Immunofluorescence staining showed FBXO31 colocalized with Smad7 in HSC-T6 cells and in liver tissues of BALB/c mice treated with CCl₄. Immunoprecipitation demonstrated FBXO31 interacted with Smad7. Moreover, FBXO31 enhanced ubiquitination of Smad7. In conclusion, FBXO31 modulates activation of HSCs and liver fibrogenesis by promoting ubiquitination of Smad7.