Effect of High Concentrations of Fructose on the Expression of TGF-β and α-SMA Genes in Human Hepatic Stellate Cells

Background: Liver fibrosis is a reversible response to wound-healing that occurs in most forms of chronic liver damage, beginning with the activation of hepatic stellate cells (HSCs). The increased expression of genes, such as beta-converting growth factor (TGF-β) and actin-alpha smooth muscle (α-SMA) indicates the activation of HSCs. During liver damage, HSCs are activated and converted to myofibroblasts. As a result, the expression of TGF-β and αSMA genes in HSCs increases and leads to liver fibrosis. High fructose intake is known to have harmful effects on human health. Due to the persistent increase in high fructose intake via many beverages and foods in industrialized countries, much concern has been raised about the effect of fructose on liver damage, but its role in activating human HSCs has not been studied. Objectives: We aimed to investigate the effect of high fructose concentration on human HSCs activation by measuring the level of mRNA expression of TGF-β and α-SMA genes involved in liver fibrosis. Methods: Human HSCs were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) plus 10% Fetal Bovine Serum (FBS) at 37°C in 5% CO2. Cells were incubated in media containing 25 and 30 mM fructose for 48 h. The control group was incubated in DMEM without fructose. The cells were serum-starved for 24 h before treatment. Then, the total RNA was extracted, reversely transcribed into cDNA, and underwent Quantitative Real-time PCR (qRT-PCR). Results: The results indicated that the mRNA expression of TGF-β and αSMA genes significantly increased by treating with 25 and 30 mM fructose in HSCs when compared to the control group (P < 0.05). Conclusions: The increase in the mRNA of TGF-β and αSMA genes is used as a standard marker for HSC activation, leading to liver fibrosis. The results demonstrated that high fructose concentration could activate HSCs and increase the levels of TGF-β and αSMA in these cells. Thus, controlling fructose consumption and identifying the mechanism of fructose action is important to treat and reduce liver injury.

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Effect of Fibroblast Growth Factor 21 on the Expression of TLR4 and BAMBI Genes in Cholesterol-Treated Human Hepatic Stellate Cells

Jentashapir Journal of Cellular and Molecular Biology ◽

10.5812/jjcmb.113672 ◽

2021 ◽

Vol 12 (3) ◽

Author(s):

Elham Shakerian ◽

Narges Mohammad Taghvaei ◽

Zohre Askari ◽

Reza Afarin

Keyword(s):

Growth Factor ◽

Mrna Expression ◽

Fibroblast Growth Factor ◽

Hepatic Stellate Cells ◽

Stellate Cells ◽

Fibroblast Growth Factor 21 ◽

Control Group ◽

Type I ◽

Fibroblast Growth ◽

Human Hscs

Background: Activated hepatic stellate cells (HSCs) are the primary mediators in the progression of hepatic fibrosis. The activation of toll-like receptor 4 (TLR4) signaling leads to the downregulation of the transmembrane inhibitory transforming growth factor-beta (TGF-β) pseudoreceptor BMP and activin membrane-bound inhibitor (BAMBI) on HSCs. Fibroblast growth factor 21 (FGF21) is a natural secretory protein in the body with effects, such as the reduction of fat accumulation and oxidation of lipids; however; no study has investigated FGF21 ability to prevent the progression of liver fibrosis. Objectives: This study aimed to examine the beneficial effects of FGF21 to reduce cholesterol-activated human HSCs. Methods: The human HSCs were incubated in media containing different concentrations of cholesterol, including 25, 50, 75, 100, 125, and 150 μM, for 24 h and then incubated with FGF21 for 24 h. Total ribonucleic acids were extracted and reversely transcribed into complementary deoxyribonucleic acid. A quantitative real-time polymerase chain reaction was performed in this study. Results: The results showed that the messenger ribonucleic acid (mRNA) expression of TGF-β, collagen, type I, alpha 1 (collagen1α), and TLR4 genes increased significantly in the presence of cholesterol (75 and 100 μM), compared to that of the control group (* P < 0.05, ** P < 0.01, and *** P < 0.001); nevertheless, the mRNA expression of the BAMBI gene significantly reduced, compared to that of the control group (* P < 0.05). The FGF21 significantly reduced the mRNA expression of TGF-β, collagen1α, and TLR4 genes (# P < 0.05). The mRNA expression of the BAMBI gene significantly increased with FGF21 (# P < 0.05). Conclusions: It was concluded that the treatment with FGF21 reduces the cholesterol-activated HSCs by decreasing the mRNA expression of the TLR4, TGF-β, and collagen1α genes and increasing the mRNA expression of the BAMBI gene.

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Astaxanthin Attenuates the Changes in the Expression of miRNAs Involved in the Activation of Hepatic Stellate Cells (P02-011-19)

Current Developments in Nutrition ◽

10.1093/cdn/nzz029.p02-011-19 ◽

2019 ◽

Vol 3 (Supplement_1) ◽

Author(s):

Minkyung Bae ◽

Ji-Young Lee

Keyword(s):

Liver Fibrosis ◽

Mirna Expression ◽

Hepatic Stellate Cells ◽

Expression Profiles ◽

Stellate Cells ◽

Expression Levels ◽

Candidate Mirnas ◽

Primary Mouse ◽

Mirna Expression Profiles ◽

Human Hscs

Abstract Objectives MicroRNAs (miRNAs) are known to be associated with human diseases, including liver fibrosis. We previously demonstrated that astaxanthin (ASTX), a xanthophyll carotenoid, has anti-fibrogenic effects in hepatic stellate cells (HSCs). HSCs are the major cell type responsible for the accumulation of extracellular matrix during the development of liver fibrosis once they are activated. The objective of this study was to compare miRNA expression profiles in activated HSCs (aHSCs) with those of quiescent HSCs (qHSCs) to identify miRNAs that may play crucial roles in the activation of HSCs. We also determined the effect of ASTX on the changes in miRNAs during HSC activation. Methods Primary mouse HSCs were cultured on uncoated plastic dishes for activation. The cells cultured for 1 day and 7 days after isolation served as qHSCs and aHSCs, respectively. qHSCs were treated with/without 25 µM ASTX during the activation for 7 days. miRNA expression profiles were determined using a miScript miRNA PCR array for mouse fibrosis. miRNAs whose expression were altered by more than 2-folds during HSC activation and by ASTX were selected. Their expression levels were further confirmed by quantitative real-time PCR in primary mouse and human HSCs and LX-2 cells, a human HSC cell line. Results Compared with qHSCs, the expression levels of 14 miRNAs and 23 miRNAs were increased and decreased by more than 2-folds, respectively, in aHSCs. Among 14 miRNAs increased in aHSCs, the expression of miR-192–5p, miR-382–5p, and miR-874–3p was reduced by ASTX. In addition, ASTX increased the expression of miR-19a-3p, miR-19b-3p, and miR-101a-3p which were among the 23 miRNAs that were decreased in aHSCs. Of the selected 6 miRNAs, miR-382–5p was chosen for further analysis based on its high expression in HSCs and the magnitude of differences between groups. Unlike in primary mouse HSCs, the expression of miR-382–5p was not altered by transforming growth factor β1, a fibrogenic cytokine, or by ASTX in primary human HSCs and LX-2 cells, which are cells somewhat activated. Conclusions We identified candidate miRNAs that may be important for the activation of HSCs from qHSCs, which were also sensitive to ASTX. Of the candidate miRNAs, miR-382–5p is likely involved in the early stage of HSC activation, i.e., transdifferentiation of qHSCs to aHSCs. Funding Sources NIH.

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Coagulation, Microenvironment and Liver Fibrosis

Cells ◽

10.3390/cells7080085 ◽

2018 ◽

Vol 7 (8) ◽

pp. 85 ◽

Cited By ~ 13

Author(s):

Niccolò Bitto ◽

Eleonora Liguori ◽

Vincenzo Mura

Keyword(s):

Liver Damage ◽

Hepatic Stellate Cells ◽

Thrombin Generation ◽

Tissue Injury ◽

Stellate Cells ◽

Coagulation Cascade ◽

Protease Activated Receptors ◽

Chronic Liver Damage ◽

Liver Fibrogenesis ◽

The Impact

Fibrosis is the main consequence of any kind of chronic liver damage. Coagulation and thrombin generation are crucial in the physiological response to tissue injury; however, the inappropriate and uncontrolled activation of coagulation cascade may lead to fibrosis development due to the involvement of several cellular types and biochemical pathways in response to thrombin generation. In the liver, hepatic stellate cells and sinusoidal endothelial cells orchestrate fibrogenic response to chronic damage. Thrombin interacts with these cytotypes mainly through protease-activated receptors (PARs), which are expressed by endothelium, platelets and hepatic stellate cells. This review focuses on the impact of coagulation in liver fibrogenesis, describes receptors and pathways involved and explores the potential antifibrotic properties of drugs active in hemostasis in studies with cells, animal models of liver damage and humans.

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S-allyl-glutathione improves experimental liver fibrosis by regulating Kupffer cell activation in rats

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00023.2017 ◽

2018 ◽

Vol 314 (2) ◽

pp. G150-G163 ◽

Cited By ~ 7

Author(s):

Shigekazu Takemura ◽

Hideki Azuma ◽

Mayuko Osada-Oka ◽

Shoji Kubo ◽

Toshihiko Shibata ◽

...

Keyword(s):

Heat Shock ◽

Liver Fibrosis ◽

Heat Shock Protein ◽

Mrna Expression ◽

Primary Culture ◽

Kupffer Cell ◽

Hepatic Stellate Cells ◽

Stellate Cells ◽

Diallyl Sulfide ◽

M2 Phenotype

S-allyl-glutathione (SAG) is one of the metabolites of diallyl sulfide (DAS), a component of garlic. DAS has shown preventative effects on carcinogenesis in animal models. However, whether synthetic SAG can improve liver fibrosis has not been investigated. We examined the potential preventive effects of SAG on acute and chronic models of liver fibrosis by chronic carbon tetrachloride (CCl4) administration. SAG inhibited liver fibrogenesis induced by CCl4 in a dose-dependent manner and reduced heat shock protein-47 (HSP47), a collagen-specific chaperone, and other fibrosis markers. In fibrosis regression models, after administration of either CCl4 for 9 wk or dimethyl nitrosamine (DMN) for 6 wk, SAG markedly accelerated fibrolysis in both models. In the regression stage of DMN-treated liver, SAG normalized the ratio of M2 phenotype (expression of mannose receptor) in Kupffer cells (KCs). Consistent with these results, the culture supernatants of SAG-treated M2-phenotype KCs inhibited collagen-α1(I) chain (COL1A1) mRNA expression in primary culture-activated rat hepatic stellate cells (HSCs). However, SAG did not directly inhibit HSC activation. In an acute model of CCl4 single injection, SAG inhibited hepatic injury dose dependently consistent with the inhibited the elevation of the bilirubin and ALT levels. These findings suggest that SAG could improve the fibrogenic and fibrolysis cascade via the regulation of excess activated and polarized KCs. SAG may also serve as a preventive and therapeutic agent in fibrosis of other organs for which current clinical therapy is unavailable. NEW & NOTEWORTHY S-allyl-glutathione (SAG) is a metabolite of diallyl sulfide, a component of garlic. SAG increased hepatic glutathione levels and GSH-to-GSSG ratio in normal rats. SAG treatment before or after liver fibrosis from chronic CCl4 administration improved liver fibrosis and regression. SAG decreased heat shock protein-47 (HSP47), a collagen-specific chaperone, and other fibrosis markers in CCl4-treated livers. SAG-treated Kupffer cell conditioned medium also inhibited collagen-α1(I) chain (COL1A1) mRNA expression and other markers in primary culture hepatic stellate cells.

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Hepatic stellate cells activation and liver fibrosis after chronic administration of ethanol

Current Issues in Pharmacy and Medical Sciences ◽

10.1515/cipms-2016-0014 ◽

2016 ◽

Vol 29 (2) ◽

pp. 66-70

Author(s):

Katarzyna Kot-Bakiera ◽

Ewelina Wawryk-Gawda ◽

Beata Cichacz-Kwiatkowska ◽

Barbara Jodlowska-Jedrych

Keyword(s):

Alcohol Consumption ◽

Liver Fibrosis ◽

Kupffer Cells ◽

Hepatic Stellate Cells ◽

Smooth Muscle Actin ◽

Stellate Cells ◽

Control Group ◽

Chronic Alcohol ◽

Chronic Alcohol Consumption ◽

Group A

Abstract Hepatic stellate cells (HSC) are a nonparenchymal population of liver cells. In normal conditions, they store vitamin A, control the turnover of the extracellular matrix, and regulate the contractility of the sinusoids. Acute and chronic damage such as that brought about by alcohol activates the stellate cells and they are then responsible for the liver's inflammatory fibrotic response. Hence, alcohol consumption leads to hepatitis, steatosis, fibrosis and cirrhosis of liver by way of different mechanisms depending on effect upon the nonparenchymal cells of the liver. The aim of our study was to assess the histological changes in the liver of rats after chronic alcohol consumption. In our work, we evaluated the intensity of liver fibrosis and the number of Kupffer cells and active hepatic stellate cells present within a test population. In the experiment, we used 10 Wistar rats of 250 gram weight. The animals were placed within one of two groups: A (experimental) and C (control). Group A received alcohol for 4 weeks, while group C received just water. The rats of both groups were decapitated 24 hours after the end of the experiment. The samples of liver were then evaluated after H&E, Masson’s trichrome staining and an immunohistochemical reaction to desmin (a marker of quiescent HSC) and α-smooth muscle actin (marker of active HSC) antibody. In our work, we observed intensive fibrosis in the portal spaces and perivenular areas in group A samples. Moreover, Kupffer cells and stellate cells with positive α-SMA expression were more numerous in group A than in the group C, and these correlate with the area of intensive fibrosis. The expression of desmin in the HSC was seen in both groups to a similar level. Conclusion: Chronic alcohol consumption activates the transdifferentiation of hepatic stellate cells into the positive α-SMA myofibroblast-like cells which are responsible for fibrogenesis.

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Integrin αVβ1 Regulates Procollagen I Production through a Non-canonical Transforming Growth Factor β Signaling Pathway in Human Hepatic Stellate Cells

Biochemical Journal ◽

10.1042/bcj20200749 ◽

2021 ◽

Author(s):

Zuoning Han ◽

Yanling Ma ◽

Gary Cao ◽

Zhengping Ma ◽

Ruihua Chen ◽

...

Keyword(s):

Growth Factor ◽

Liver Fibrosis ◽

Hepatic Stellate Cells ◽

Transforming Growth Factor ◽

Specific Inhibitor ◽

Stellate Cells ◽

Transforming Growth Factor Β ◽

Current Data ◽

Human Hscs ◽

Tgf Β1

Hepatic stellate cells (HSCs) are thought to play key roles in the development of liver fibrosis. Extensive evidence has established the concept that αV integrins are involved in the activation of latent transforming growth factor β (TGF-β), a master regulator of the fibrotic signaling cascade. Based on mRNA and protein expression profiling data, we found that αVβ1 integrin is the most abundant member of the αV integrin family in either quiescent or TGF-β1-activated primary human HSCs. Unexpectedly, either a selective αVβ1 inhibitor, Compound 8 (C8), or a pan-αV integrin inhibitor, GSK3008348, decreased TGF-β1-activated procollagen I production in primary human HSCs, in which the role of β1 integrin was confirmed by ITGB1 siRNA. In contrast to an Activin receptor-like kinase 5 (Alk5) inhibitor, C8 and GSK3008348 failed to inhibit TGF-β1 induced SMAD3 and SMAD2 phosphorylation, but inhibited TGF-β-induced phosphorylation of ERK1/2 and STAT3, suggesting that αVβ1 integrin is involved in non-canonical TGF-β signaling pathways. Consistently, ITGB1 siRNA significantly decreased phosphorylation of ERK1/2. Furthermore, a selective inhibitor of MEK1/2 blocked TGF-β1 induced phosphorylation of ERK1/2 and decreased TGF-β1 induced procollagen I production, while a specific inhibitor of STAT3 had no effect on TGF-β1 induced procollagen I production. Taken together, current data indicate that αVβ1 integrin can regulate TGF-β signaling independent of its reported role in activating latent TGF-β. Our data further support that αVβ1 inhibition is a promising therapeutic target for the treatment of liver fibrosis.

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Increased production of ADAMTS13 in hepatic stellate cells contributes to enhanced plasma ADAMTS13 activity in rat models of cholestasis and steatohepatitis

Thrombosis and Haemostasis ◽

10.1160/th08-11-0732 ◽

2009 ◽

Vol 102 (08) ◽

pp. 389-396 ◽

Cited By ~ 14

Author(s):

Naoko Watanabe ◽

Yukio Kume ◽

Yumiko Satoh ◽

Makoto Kaneko ◽

Daiya Takai ◽

...

Keyword(s):

Smooth Muscle ◽

Bile Duct ◽

Liver Fibrosis ◽

Mrna Expression ◽

Hepatic Stellate Cells ◽

Stellate Cells ◽

Potential Contribution ◽

Adamts13 Activity ◽

Rat Models ◽

Duct Ligation

SummaryAlthough hepatic stellate cells, endothelial cells, glomerular podocytes and plateles were reported to be a source of ADAMTS13, it is not clarified which source is involved in the regulation of plasma ADAMTS13 activity. It was demonstrated previously that selective hepatic stellate cell damage in rats caused decreased plasma ADAMTS13 activity.To further elucidate the potential contribution of hepatic stellate cells to the regulation of plasma ADAMTS13 activity, this study examined plasma ADAMTS13 activity when hepatic stellate cells proliferate during the process of liver fibrosis by employing rat models of liver fibrosis due to cholestasis, bile duct ligation, and steatohepatitis, a choline-deficient L-amino acid-defined-diet. ADAMTS13 expression was increased with co-localisation with smooth muscle α-actin, a marker of hepatic stellate cells, in bile duct-ligated livers up to four weeks, in which a close correlation between ADAMTS13 and smooth muscle α-actin mRNA expressions was determined. Plasma ADAMTS13 activity, measured by a sandwich ELISA involving a specific substrate to ADAMTS13, was increased in bile duct-ligated rats with a significant correlation with ADAMTS13 mRNA expression levels in the liver. Furthermore, ADAMTS13 mRNA expression was increased with enhanced mRNA expression in smooth muscle α-actin in the livers of rats fed a choline-deficient L-amino aciddefined-diet for 16 weeks, in which increased plasma ADAMTS13 activity was determined. Thus, increased plasma ADAMTS13 activity in cholestasis and steatohepatitis in rats may be due, at least in part, to enhanced ADAMTS13 production in the liver, suggesting a significant role of hepatic stellate cells in the regulation of plasma ADAMTS13 activity.

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Long-Term Exposure to Low-Dose Di-(2-ethylhexyl) Phthalate Impairs Cholesterol Metabolism in Hepatic Stellate Cells and Exacerbates Liver Fibrosis

International Journal of Environmental Research and Public Health ◽

10.3390/ijerph17113802 ◽

2020 ◽

Vol 17 (11) ◽

pp. 3802

Author(s):

Chun-Ya Lee ◽

Fat-Moon Suk ◽

Yuh-Ching Twu ◽

Yi-Jen Liao

Keyword(s):

Liver Fibrosis ◽

Liver Damage ◽

Hepatic Stellate Cells ◽

Low Dose ◽

Cholesterol Metabolism ◽

Mapk Pathway ◽

Stellate Cells ◽

Major Incident ◽

Ethylhexyl Phthalate

Phthalates are often added to plastic products to increase their flexibility. Di-(2-ethylhexyl) phthalate (DEHP) is one of the most common plasticizers. Previously, a major incident involving phthalate-contaminated foodstuffs occurred, where phthalates were deliberately added to foodstuffs as a substitute for emulsifiers, resulting in a threat to public health. DEHP exposure can cause liver damage and further lead to cancer; however, the effects of long-term exposure to low-dose DEHP on hepatic stellate cells (HSCs) and on liver fibrosis are still unclear. In this study, we showed that chronic exposure to low-dose DEHP results in an accumulation of cholesterol in HSCs by disturbing the cholesterol metabolism and enhancing endogenous cholesterol synthesis. In addition, long-term exposure to low-dose DEHP reduces the sensitivity of HSCs to platelet-derived growth factor BB (PDGF-BB)-induced proliferation by blocking the MAPK pathway. Dysfunction of mitochondrial respiration and induction of caspase 3/PARP-dependent apoptosis were observed in HSCs following chronic, low-dose exposure. The carbon tetrachloride (CCl4)-induced liver fibrosis mouse model showed that long-term administration of DEHP significantly promoted liver damage, inflammatory infiltration, cholesterol accumulation, and deposition of hepatic collagen. In conclusion, long-term exposure to low-dose DEHP may perturb the cholesterol metabolism in HSCs and accelerate liver damage and fibrosis.

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