scholarly journals Effect of Exogenous Fetuin-A on TGF-β/Smad Signaling in Hepatic Stellate Cells

2016 ◽  
Vol 2016 ◽  
pp. 1-6 ◽  
Author(s):  
Yulai Zhou ◽  
Shuang Yang ◽  
Pan Zhang
Keyword(s):  
Western Blot ◽  
Hepatic Stellate Cells ◽  
Stellate Cells ◽  
Relative Content ◽  
Negative Regulation ◽  
Rt Pcr ◽  
Fetuin A ◽  
Smad3 Protein ◽  
Smad7 Protein ◽  
Excessive Activation

Objective. To explore the effects of low concentration of exogenous fetuin-A intervention on TGF-β1 induced LX2 cells through detection of the expression of mRNA and protein of Smad2, Smad3, and Smad7. Methods. MTT assay was used to detect the LX2 cells proliferation and the regression equation calculating software was applied to determine IC50 of fetuin-A. RT-PCR was used to determine the relative content of Smad2, Smad3, and Smad7 mRNA in LX2 cells. Western blot was used to detect the LX2 cells relative content of Smad2, Smad3, Smad7 protein expression, respectively. Results. The analysis from RT-PCR and western blot showed that when compared with the other groups TGF-β1 + fetuin-A group increased the expression of Smad2 and Smad3 while decreased the expression of Smad7 (P<0.05). Conclusion. Fetuin-A may improve the excessive activation of hepatic stellate cells which is caused by an enhanced positive regulation of Smad2 and Smad3 protein and the deficiency in negative regulation of Smad7 protein. This is through inhibiting the expression of Smad2 and Smad3 gene and promoting the expression of Smad7 gene. As a result, the development of liver fibrosis will be reduced.

scholarly journals MiR-571 affects the development and progression of liver fibrosis by regulating the Notch3 pathway

2021 ◽  
Author(s):  
Shuo Cong ◽  
Yongmei Liu ◽  
Yi Li ◽  
Yu Chen ◽  
Rui Chen ◽  
...  
Keyword(s):  
Liver Fibrosis ◽  
Western Blot ◽  
Signaling Pathway ◽  
Hepatic Stellate Cells ◽  
Stellate Cells ◽  
Rt Pcr ◽  
Promote Cell Proliferation ◽  
Notch 3 ◽  
And Migration ◽  
Set Up

Abstract Exploring the expression of miR-571 in patients with liver fibrosis and its role in the progression of liver fibrosis. A total of 74 patients with chronic hepatitis and cirrhosis accompanied by liver fibrosis in our institution from September to December 2018 were collected for study, and the expression of miR-571 in patients with different progressions of liver fibrosis was determined by RT-PCR and Western blot analysis. Set up Notch3 up group and Notch3 down regulated group, RT-PCR and Western blot were used to determine the effect of Notch signaling on the expression of fibrogenic α-SMA, collagen I. CCK-8, cell scratch assays, Transwell assays, flow cytometry were used to determine the effect of miR-571 on LX-2 proliferation, migration, apoptosis in human stem stellate cells, and RT-PCR, Western blot assays were performed to determine the effect of miR-571 on the Notch3 signaling pathway and the expression of profibrogenic factors. miR-571 is up-regulated in patients with liver fibrosis and is associated with the progression of liver fibrosis. Notch3 signaling pathway can promote the expression of fibroblast in human hepatic stellate cells; miR-571 can inhibit the apoptosis of human hepatic stellate cells, promote cell proliferation and migration; up regulation of miR-571 can promote the expression of Notch3 and Jagged 1; up regulation of miR-571 can also promote the expression of fibroblast. miR-571 can promote the activation of human stem stellate cells and the expression of fibroblasts through Notch 3 signaling pathway.

scholarly journals MiR-571 affects the development and progression of liver fibrosis by regulating the Notch3 pathway

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Shuo Cong ◽  
Yongmei Liu ◽  
Yi Li ◽  
Yu Chen ◽  
Rui Chen ◽  
...  
Keyword(s):  
Liver Fibrosis ◽  
Western Blot ◽  
Signaling Pathway ◽  
Hepatic Stellate Cells ◽  
Stellate Cells ◽  
Rt Pcr ◽  
Related Factors ◽  
Promote Cell Proliferation ◽  
And Migration ◽  
Set Up

AbstractExploring the expression of miR-571 in patients with liver fibrosis and its role in the progression of liver fibrosis. A total of 74 patients with liver fibrosis in our institution from September to December 2018 were collected for study, and the expression of miR-571, Notch3 and Jagged1 in patients with different progressions of liver fibrosis was determined by RT-PCR and Western blot analysis. Set up Notch3 up group and Notch3 down regulated group, RT-PCR and Western blot were used to determine the effect of Notch signaling on the expression of fibrogenic factors. CCK-8, cell scratch assays, Transwell assays, flow cytometry were used to determine the effect of miR-571 on LX-2 proliferation, migration, apoptosis in human stem stellate cells, and RT-PCR, Western blot assays were performed to determine the effect of miR-571 on the Notch3 signaling pathway and the expression of profibrogenic factors. miR-571, Notch3 and Jagged1 are up-regulated in patients with liver fibrosis and is associated with the progression of liver fibrosis. Notch3 signaling pathway can promote the expression of fibroblast in human hepatic stellate cells; miR-571 can inhibit the apoptosis of human hepatic stellate cells, promote cell proliferation and migration; up regulation of miR-571 can promote the expression of Notch3 and Jagged1, and up-regulation of miR-571 also promoted the expression of related fibroblasts. MiR-571 can promote the activation of human stem cell stellate cells and the expression of fibroblast related factors through Notch3 signaling pathway.

Exposure to bacterial cell wall products triggers an inflammatory phenotype in hepatic stellate cells

2005 ◽  
Vol 289 (3) ◽  
pp. G571-G578 ◽  
Author(s):  
Paola Brun ◽  
Ignazio Castagliuolo ◽  
Massimo Pinzani ◽  
Giorgio Palù ◽  
Diego Martines
Keyword(s):  
Cell Wall ◽  
Liver Damage ◽  
Hepatic Stellate Cells ◽  
Bacterial Cell ◽  
Stellate Cells ◽  
Bacterial Cell Wall ◽  
Rt Pcr ◽  
Systemic Complications ◽  
Bacterial Endotoxins ◽  
Extracellular Matrix Components

Activated hepatic stellate cells (HSCs) secrete extracellular matrix components during hepatic fibrosis, but recent studies have shown that HSCs can also release a variety of proinflammatory cytokines. Moreover, bacterial endotoxemia is not only associated with systemic complications in the late stages of liver failure but is also a direct cause of liver damage, activating resident inflammatory cells. In this study, we investigated whether HSCs can respond directly to bacterial cell wall products acquiring a new phenotype. RT-PCR and immunocytochemistry assays were used to show that murine HSCs expressed specific mRNA transcripts and proteins for LPS and lipoteichoic acid (LTA) receptor systems and peptidoglycan recognition proteins. Exposing HSCs to bacterial endotoxins led to phosphorylation of mitogen-activated protein kinase ERK1 and the development of a proinflammatory phenotype. After exposure to LPS, LTA, or N-acetyl muramyl peptide, transforming growth factor-β1, IL-6, and monocyte chemoattractant protein-1 (MCP-1) mRNA specific transcripts and proteins increased significantly in HSCs, as assayed by quantitative real-time RT-PCR and ELISA. These LPS-mediated effects in HSCs were receptor dependent, because LPS-induced ERK1 phosphorylation, IL-6, and MCP-1 mRNA and protein level upregulation were significantly less pronounced in HSCs isolated from C3H/HeJ mice lacking Toll-like receptor 4. In conclusion, our results show that murine HSCs express functional receptors for bacterial endotoxins, and HSCs exposed to bacterial products develop a strong proinflammatory phenotype. We speculate that high levels of bacterial endotoxins in the portal vein may directly induce a proinflammatory phenotype in HSCs that contributes to liver damage.

scholarly journals Expression of P2Y nucleotide receptors and ectonucleotidases in quiescent and activated rat hepatic stellate cells

2004 ◽  
Vol 287 (2) ◽  
pp. G417-G424 ◽  
Author(s):  
Jonathan A. Dranoff ◽  
Mika Ogawa ◽  
Emma A. Kruglov ◽  
Marianna D. A. Gaça ◽  
Jean Sévigny ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Hepatic Stellate Cells ◽  
Stellate Cells ◽  
P2y Receptors ◽  
Extracellular Nucleotides ◽  
Nucleoside Triphosphate ◽  
P2y Receptor ◽  
Rt Pcr ◽  
Nucleoside Triphosphate Diphosphohydrolase ◽  
Receptor Inhibitor

Extracellular nucleotides regulate a variety of cellular activities, including proliferation of fibrogenic cells outside of the liver. However, the expression of receptors for extracellular nucleotides in hepatic stellate cells (HSC) is unknown. Thus our aims were to investigate the expression of mediators of nucleotide signaling in HSC and to determine whether extracellular nucleotides regulate HSC function. Confocal video microscopy was used to observe nucleotide-induced changes in cytosolic Ca2+ (Cai2+) in live HSC. P2Y receptor subtype expression and ectonucleotidase expression in quiescent and activated HSC were determined using RT-PCR, Northern blot, immunoblot, and confocal immunofluorescence. Functional ectonucleotidase activity was assessed using a colorimetric method. Nucleotide-sensitive procollagen-1 mRNA expression in activated HSC was assessed using real-time RT-PCR. Extracellular ATP increased Cai2+ in HSC; this was inhibited by the P2 receptor inhibitor suramin. Quiescent HSC expressed the P2Y subtypes P2Y2 and P2Y4 and were activated by ATP and UTP, whereas activated HSC expressed the P2Y subtype P2Y6 and were activated by UDP and ATP. Activated but not quiescent HSC expressed the ectonucleotidase nucleoside triphosphate diphosphohydrolase 2, extracellular UDP tripled procollagen-1 mRNA expression in activated HSC, and this was inhibited by the P2Y receptor inhibitor suramin. HSC express functional P2Y receptors and switch the expression of P2Y receptor subtypes on activation. Moreover, HSC differentially regulate nucleoside triphosphate diphosphohydrolase expression after activation. Because activation of P2Y receptors in activated HSC regulates procollagen-1 transcription, P2Y receptors may be an attractive target to prevent or treat liver fibrosis.

Fluorofenidone attenuates hepatic fibrosis by suppressing the proliferation and activation of hepatic stellate cells

2014 ◽  
Vol 306 (3) ◽  
pp. G253-G263 ◽  
Author(s):  
Yu Peng ◽  
Huixiang Yang ◽  
Nasui Wang ◽  
Yan Ouyang ◽  
Yanrong Yi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Liver Fibrosis ◽  
Western Blot ◽  
Hepatic Fibrosis ◽  
Hepatic Stellate Cells ◽  
Cyclin E ◽  
Stellate Cells ◽  
Collagen I ◽  
Phase Cell ◽  
Cyclin D

Fluorofenidone (AKF-PD) is a novel pyridone agent. The purpose of this study is to investigate the inhibitory effects of AKF-PD on liver fibrosis in rats and the involved molecular mechanism related to hepatic stellate cells (HSCs). Rats treated with dimethylnitrosamine or CCl4 were randomly divided into normal, model, AKF-PD treatment, and pirfenidone (PFD) treatment groups. The isolated primary rat HSCs were treated with AKF-PD and PFD respectively. Cell proliferation and cell cycle distribution were analyzed by bromodeoxyuridine and flow cytometry, respectively. The expression of collagen I and α-smooth muscle actin (α-SMA) were determined by Western blot, immunohistochemical staining, and real-time RT-PCR. The expression of cyclin D1, cyclin E, and p27kip1 and phosphorylation of MEK, ERK, Akt, and 70-kDa ribosomal S6 kinase (p70S6K) were detected by Western blot. AKF-PD significantly inhibited PDGF-BB-induced HSC proliferation and activation by attenuating the expression of collagen I and α-SMA, causing G0/G1 phase cell cycle arrest, reducing expression of cyclin D1 and cyclin E, and promoting expression of p27kip1. AKF-PD also downregulated PDGF-BB-induced MEK, ERK, Akt, and p70S6K phosphorylation in HSCs. In rat liver fibrosis, AKF-PD alleviated hepatic fibrosis by decreasing necroinflammatory score and semiquantitative score, and reducing expression of collagen I and α-SMA. AKF-PD attenuated the progression of hepatic fibrosis by suppressing HSCs proliferation and activation via the ERK/MAPK and PI3K/Akt signaling pathways. AKF-PD may be used as a potential novel therapeutic agent against liver fibrosis.

scholarly journals Nonmuscle myosin II shRNA inhibit migration and contraction in rat hepatic stellate cells through regulating AKT/mTOR/S6K/4EBP1 signaling pathway

2018 ◽  
Author(s):  
Zhenghong Li ◽  
Yun Feng ◽  
Ruling Zhang ◽  
Peiwen Wang ◽  
Lungen Lu ◽  
...  
Keyword(s):  
Western Blot ◽  
Real Time ◽  
Signaling Pathway ◽  
Hepatic Stellate Cells ◽  
Real Time Pcr ◽  
Stellate Cell ◽  
Myosin Ii ◽  
Stellate Cells ◽  
Nonmuscle Myosin ◽  
Nonmuscle Myosin Ii

AbstractMigration and contraction of activated hepatic stellate cell (HSC) are essential factors for cirrhosis formation and development. It has been demonstrated that blebbistatin, a nonmuscle myosin II (NMMII) inhibitor, can inhibit the migration and contraction of HSC, whereas the main cell signaling pathway is still unknown. Mammalian target of rapamycin (mTOR) signaling pathway may be involved in many cells migration and contraction, whether NMMII and mTOR have any crosslinks draw our attention. In the currently study, we used LV-RNAi to specifically attenuate mTOR and NMMII in rat HSC. We aimed to examine the effect of mTOR LV-RNAi on the migration and contraction of HSC and explore the crosslink between mTOR cell signal and NMMII. Using real-time PCR and western blot, we found that mTOR and the downstream factors including S6K and 4EBP1 all up-regulated with the activation of HSC, mTOR and NMMII LV-RNAi was transfected into activated HSC using lipofectamine 2000. The levels of mRNA and proteins were also examined using real-time PCR and western blot respectively. The expression of mTOR can be down-regulated by NMMII LV-RNAi significantly, as well as the expression of S6K, 4EBP1, α-SMA and collagen I, but the level of AKT was up-regulated. Then we used Transwell system and collagen lattices to examine the NMMII and mTOR LV-RNAi efficiency on HSC migration and contraction, as we hypothesized, both of the LV-RNAi could inhibit HSC migration and contraction significantly. These results indicated that nonmuscle myosin II shRNA inhibit migration and contraction in rat hepatic stellate cells through the regulation of mTOR/S6K/4EBP1 signaling pathway

scholarly journals Thunbergia laurifolia Exhibits Antifibrotic Effects in Human Hepatic Stellate Cells

2017 ◽  
Vol 2017 ◽  
pp. 1-9 ◽  
Author(s):  
Ratchadaporn Namsen ◽  
Noppamas Rojanasthien ◽  
Seewaboon Sireeratawong ◽  
Piyanuch Rojsanga ◽  
Wutigri Nimlamool ◽  
...  
Keyword(s):  
Western Blot ◽  
Transforming Growth Factor Beta ◽  
Blot Analysis ◽  
Hepatic Stellate Cells ◽  
Western Blot Analysis ◽  
Transforming Growth Factor ◽  
Stellate Cells ◽  
Convincing Evidence ◽  
High Concentrations ◽  
Thunbergia Laurifolia

Leaves of Thunbergia laurifolia (TL) have been reported to have antioxidation, anti-inflammatory, detoxifying, and hepatoprotective effects. However, studies relating to antifibrotic activity have not been reported. Currently, there is no standard treatment for hepatic fibrosis. This study aimed to investigate the antifibrotic activity of TL in human hepatic stellate LX-2 cells. Results from cell viability and cell death assays showed that the extract at high concentrations was toxic to LX-2 cells. TL extract reversed the transformation of LX-2 cells to myofibroblast-like characteristics in response to stimulation by transforming growth factor-beta 1. This action may be associated with the effect of TL in suppressing α-SMA and collagen-I production observed by immunofluorescence study and western blot analysis. Additionally, TL extract significantly decreased MMP-9 activity which is consistent with the reduction of MMP-9, MMP-2, and TIMP-1 gene expression. The effect of TL in suppressing fibrosis may be associated with its ability to inhibit the activation of p38 MAPK and Erk1/2 kinases as examined by western blot analysis. Our study provides convincing evidence that TL possesses antifibrotic activity which may be through the suppression of TGF-β1-mediated production of MMPs, collagen-1, and α-SMA in hepatic stellate cells.

scholarly journals De novo expression of glutamine synthetase during transformation of hepatic stellate cells into myofibroblast-like cells

1998 ◽  
Vol 335 (3) ◽  
pp. 697-700 ◽  
Author(s):  
Johannes G. BODE ◽  
Thorsten PETERS-REGEHR ◽  
Axel M. GRESSNER ◽  
Dieter HÄUSSINGER
Keyword(s):  
Enzyme Activity ◽  
Smooth Muscle ◽  
Glutamine Synthetase ◽  
Western Blot ◽  
Hepatic Stellate Cells ◽  
De Novo ◽  
Smooth Muscle Actin ◽  
Stellate Cells ◽  
Cell Protein ◽  
Muscle Actin

The expression of glutamine synthetase (GS) was studied in cultured quiescent hepatic stellate cells (HSC) and during their transformation into myofibroblast-like cells. GS mRNA was detectable in quiescent HSC (1-day culture); however, the enzyme protein was not expressed, as assessed by Western blot analysis, immunocytochemistry and the absence of detectable enzyme activity. Similar findings were obtained after 2 days of culture; in addition, the mRNA levels had dropped by about 70%, but they increased again thereafter during the process of HSC transformation in culture, as indicated by the expression of α-smooth-muscle actin. In parallel with the accumulation of α-smooth-muscle actin, GS was expressed, as shown by Western blot analysis and immunocytochemistry, and enzyme activity increased from undetectable levels in quiescent cells to 0.13±0.01 µmol/h per mg of cell protein within 7–14 days. This value compares with GS activity in liver parenchymal cells of 0.57±0.03 µmol/h per mg of cell protein. The findings suggest that activation of HSC results in the de novo expression of GS protein and activity, and this may serve as another marker of HSC transformation.

scholarly journals 3,5-Diethoxy-3′-Hydroxyresveratrol (DEHR) Ameliorates Liver Fibrosis via Caveolin-1 Activation in Hepatic Stellate Cells and in a Mouse Model of Bile Duct Ligation Injury

Molecules ◽  
2018 ◽  
Vol 23 (11) ◽  
pp. 2833 ◽  
Author(s):  
Phil Lee ◽  
Hye-Jin Park ◽  
Namki Cho ◽  
Hong Kim
Keyword(s):  
Bile Duct ◽  
Liver Fibrosis ◽  
Western Blot ◽  
Hepatic Stellate Cells ◽  
Bile Duct Ligation ◽  
B Cell Lymphoma ◽  
Stellate Cells ◽  
Transfected Cells ◽  
Caveolin 1 ◽  
Duct Ligation

Hepatic stellate cells (HSCs) are involved in the pathogenesis of liver fibrosis. Resveratrol, 3,5,4′-trihydroxystilbene, is a dietary polyphenol found in natural food products. Here, we evaluated the anti-proliferative effects of a synthetic resveratrol derivative, 3,5-diethoxy-3′-hydroxyresveratrol (DEHR), on HSCs. Flow cytometry and Western blot analyses showed that DEHR induces apoptosis through the upregulation of cleaved caspase-3 and poly (ADP-ribose) polymerase expression and reduction in the level of an anti-apoptotic protein B-cell lymphoma 2 (Bcl2). As caveolin-1 (CAV1), a competitive inhibitor of heme oxygenase 1 (HO-1), is related to apoptotic proteins in hepatic cells, we focused on the role of CAV1 in DEHR-induced apoptosis in HSCs through Western blot analyses. Our results showed that the inhibitory effect of DEHR on cell viability was stronger in HO-1 siRNA-transfected cells but weakened in CAV1 siRNA-transfected cells. Collagen concentration was significantly reduced, whereas CAV1 expression increased after treatment of a bile duct ligation injury-induced liver fibrosis model with DEHR for four weeks. We confirmed that DEHR treatment significantly reduced fibrous hyperplasia around the central veins, using hematoxylin and eosin and Sirius red staining. DEHR ameliorates liver fibrosis in vitro and in vivo, possibly through a mechanism involving CAV1.

scholarly journals Forsythiaside A Regulates Activation of Hepatic Stellate Cells by Inhibiting NOX4-Dependent ROS

2022 ◽  
Vol 2022 ◽  
pp. 1-17
Author(s):  
Mengting Zhou ◽  
Xingtao Zhao ◽  
Li Liao ◽  
Ying Deng ◽  
Meichen Liu ◽  
...  
Keyword(s):  
Liver Fibrosis ◽  
Western Blot ◽  
Hepatic Fibrosis ◽  
Hepatic Stellate Cells ◽  
Antioxidant Activities ◽  
Stellate Cells ◽  
Tnf Α ◽  
And Migration ◽  
Tgf Β1 ◽  
Proliferation And Migration

Hepatic stellate cells (HSCs) activation is an important step in the process of hepatic fibrosis. NOX4 and reactive oxygen species expressed in HSCs play an important role in liver fibrosis. Forsythiaside A (FA), a phenylethanoid glycoside extracted and isolated from Forsythiae Fructus, has significant antioxidant activities. However, it is not clear whether FA can play a role in inhibiting the HSCs activation through regulating NOX4/ROS pathway. Therefore, our purpose is to explore the effect and mechanism of FA on HSCs activation to alleviate liver fibrosis. LX2 cells were activated by TGF-β1 in vitro. MTT assay and Wound Healing assay were used to investigate the effect of FA on TGF-β1-induced LX2 cell proliferation and migration. Elisa kit was used to measure the expression of MMP-1 and TIMP-1. Western blot and RT-qPCR were used to investigate the expression of fibrosis-related COLI, α-SMA, MMP-1 and TIMP-1, and inflammation-related TNF-α, IL-6 and IL-1β. The hydroxyproline content was characterized using a biochemical kit. The mechanism of FA to inhibit HSCs activation and apoptosis was detected by DCF-DA probe, RT-qPCR, western blot and flow cytometry. NOX4 siRNA was used to futher verify the effect of FA on NOX4/ROS pathway. The results showed that FA inhibited the proliferation and migration of LX2 cells and adjusted the expression of MMP-1, TIMP-1, COLI, α-SMA, TNF-α, IL-6 and IL-1β as well as promoted collagen metabolism to show potential in anti-hepatic fibrosis. Mechanically, FA down-regulated NOX4/ROS signaling pathway to improve oxidation imbalances, and subsequently inhibited PI3K/Akt pathway to suppress proliferation. FA also promoted the apoptosis of LX2 cells by Bax/Bcl2 pathway. Furthermore, the effects of FA on TGF-β1-induced increased ROS levels and α-SMA and COLI expression were weaken by silencing NOX4. In conclusion, FA had potential in anti-hepatic fibrosis at least in part by remolding of extracellular matrix and improving oxidation imbalances to inhibit the activation of HSCs and promote HSCs apoptosis.

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