In vitro Study of Flavonoids, Fatty Acids, and Steroids on Proliferation of Rat Hepatic Stellate Cells

There is a wealth of evidence that hepatic stellate cells (HSCs) orchestrate most of the important events in liver fibrogenesis. After liver injury, HSCs become activated to a profibrogenic myofibroblastic phenotype and can regulate net deposition of collagens and other matrix proteins in the liver. The proliferation of HSCs is mainly stimulated by the plateletderived growth factor (PDGF). In this study, some compounds from natural resources have been tested for their activity to inhibit PDGF-driven proliferative activity of rat HSCs. Apigenin, quercetin, genistein, daidzin, and biochanin A exhibited > 75% inhibitory activity against HSC-T6. It was found that, γ-linolenic (γ-Ln), eicosapentanoic (EPA) and α- linolenic (α-Ln) acids showed a high inhibitory effect on proliferation of rat HSCs at 50 nmol/l. Cholest-4-ene-3,6-dione and stigmastone-4-en-3,6-dione are the most active steroids with inhibitory activities > 80% and this is most likely due to the presence of the 4-en-3,6-dione moiety in both compounds. These results revealed that the compounds which effectively blocked HSC proliferation may be beneficial in liver fibrosis. Structure-activity relationships (SAR) may provide a basis for rational structure modification.

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Transcriptional factor ATF3 promotes liver fibrosis via activating hepatic stellate cells

Cell Death and Disease ◽

10.1038/s41419-020-03271-6 ◽

2020 ◽

Vol 11 (12) ◽

Author(s):

Zhemin Shi ◽

Kun Zhang ◽

Ting Chen ◽

Yu Zhang ◽

Xiaoxiao Du ◽

...

Keyword(s):

Liver Fibrosis ◽

Hepatic Stellate Cells ◽

In Vitro Study ◽

Stellate Cells ◽

Protective Role ◽

Activating Transcription Factor 3 ◽

Dependent Manner ◽

Liver Fibrogenesis

AbstractThe excessive accumulation of extracellular matrix (ECM) is a key feature of liver fibrosis and the activated hepatic stellate cells (HSCs) are the major producer of ECM proteins. However, the precise mechanisms and target molecules that are involved in liver fibrosis remain unclear. In this study, we reported that activating transcription factor 3 (ATF3) was over-expressed in mice and human fibrotic livers, in activated HSCs and injured hepatocytes (HCs). Both in vivo and in vitro study have revealed that silencing ATF3 reduced the expression of pro-fibrotic genes and inhibited the activation of HSCs, thus alleviating the extent of liver fibrosis, indicating a potential protective role of ATF3 knockdown. However, ATF3 was not involved in either the apoptosis or proliferation of HCs. In addition, our data illustrated that increased nuclear localization of ATF3 promoted the transcription of fibrogenic genes and lnc-SCARNA10, which functioned as a novel positive regulator of TGF-β signaling in liver fibrogenesis by recruiting SMAD3 to the promoter of these genes. Interestingly, further study also demonstrated that lnc-SCARNA10 promoted the expression of ATF3 in a TGF-β/SMAD3-dependent manner, revealing a TGF-β/ATF3/lnc-SCARNA10 axis that contributed to liver fibrosis by activating HSCs. Taken together, our data provide a molecular mechanism implicating induced ATF3 in liver fibrosis, suggesting that ATF3 may represent a useful target in the development of therapeutic strategies for liver fibrosis.

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Gli2-regulated activation of hepatic stellate cells and liver fibrosis by TGF-β signaling

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00310.2020 ◽

2021 ◽

Author(s):

Junyan Yan ◽

Baowei Hu ◽

Wenjie Shi ◽

Xiaoyi Wang ◽

Jiayuan Shen ◽

...

Keyword(s):

Liver Fibrosis ◽

Hepatic Stellate Cells ◽

Stellate Cells ◽

Conditional Knockout ◽

Rna Seq ◽

Expression Levels ◽

Liver Fibrogenesis ◽

Hh Signaling ◽

Signaling Activity

The Hedgehog (Hh) signaling pathway is correlated with hepatic stellate cells (HSCs) activation and liver fibrosis. Gli2 is a key transcription effector of Hh signaling. However, the role of Gli2 in HSC-mediated liver fibrosis progression is largely unknown. In the present study, we investigated the effect of Gli2 on liver fibrogenesis and its possible mechanism using conditional knockout (cKO) Gli2 mice and HSC models. Wild-type (WT) and GFAP-CreERT;Gli2flox/flox male mice were exposed to CCl4 for one month to induce liver fibrosis. Primary HSCs were isolated from mice and the transition of HSCs into a myofibroblastic phenotype was evaluated. Livers from mice underwent histological, immunohistochemical, and immunofluorescence analyses. The expression levels of proteins and genes were evaluated by Western blot (WB) analysis and quantitative real-time polymerase chain reaction (qRT-PCR), respectively. RNA-seq was used to screen differentially expressed genes. Results showed that CCl4 treatment induced liver fibrosis, promoted HSCs activation and proliferation, and up-regulated Hh signaling activity. The cKO of Gli2 in GFAP-CreERT;Gli2flox/flox mice decreased liver fibrosis as well as HSC activation and proliferation. In vitro studies showed that KO of Gli2 in HSCs blocked cell proliferation and activation by decrease of cyclin D1/D2 expression. The RNA-seq results revealed that the expression levels TGF-β1 ligands were down-regulated in Gli2 KO HSCs. Furthermore, overexpression of Gli2 rescued proliferation and activation of HSCs by up-regulation of TGF-β signaling activity. Our data demonstrated that Gli2 regulated HSC activation and liver fibrosis by TGF-β signaling, thus providing support for future Gli2-based investigations of liver fibrosis therapy.

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Inhibitory effect of salvianolic acid B on malondialdehyde-stimulated proliferation of rat hepatic stellate cells in vitro

World Chinese Journal of Digestology ◽

10.11569/wcjd.v14.i5.476 ◽

2006 ◽

Vol 14 (5) ◽

pp. 476

Author(s):

Xiao-Ling Wang ◽

Ping Liu ◽

Yun-Hua Cui ◽

Xu-Dong Hu ◽

Bo-Qin Li ◽

...

Keyword(s):

Hepatic Stellate Cells ◽

Stellate Cells ◽

Inhibitory Effect ◽

Salvianolic Acid B ◽

Salvianolic Acid

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135 Jnk1 IN HEPATIC STELLATE CELLS MODULATES LIVER FIBROGENESIS IN VIVO AND IN VITRO

Journal of Hepatology ◽

10.1016/s0168-8278(13)60137-3 ◽

2013 ◽

Vol 58 ◽

pp. S59-S60

Author(s):

F.J. Cubero ◽

G. Zhao ◽

M. Hatting ◽

Y.A. Nevzorova ◽

F. Schaefer ◽

...

Keyword(s):

Hepatic Stellate Cells ◽

Stellate Cells ◽

Liver Fibrogenesis

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ANTIOXIDATIVE ACTIVITY OF ALPHA-MANGOSTIN IN ACETALDEHYDE-INDUCED HEPATIC STELLATE CELLS: AN IN VITRO STUDY

International Journal of Applied Pharmaceutics ◽

10.22159/ijap.2019.v11s1.16089 ◽

2019 ◽

pp. 164-167

Author(s):

NOVRIANTIKA LESTARI ◽

SAMUEL PRATAMA ◽

KELVIN THEANDRO GOTAMA ◽

VIVIAN SOETIKNO ◽

MELVA LOUISA

Keyword(s):

Liver Fibrosis ◽

Cell Viability ◽

Hepatic Stellate Cells ◽

In Vitro Study ◽

Stellate Cells ◽

Trypan Blue Exclusion ◽

Trypan Blue Exclusion Method ◽

Antioxidative Properties ◽

Causes Of Mortality

Objective: Alcohol accumulation in the liver can cause pathological disorders such as liver fibrosis that can develop into hepatocellular carcinoma,one of the main causes of mortality associated with liver disease. The previous studies have shown that a plant compound, alpha-mangostin, has anantioxidant effect in the inhibition of pancreatic tumor growth in vitro. This study aimed to analyze the antioxidative properties of alpha-mangostin inacetaldehyde-induced liver fibrosis in vitro.Methods: Immortalized hepatic stellate cells (HSCs), of the LX-2 cell line, were incubated with acetaldehyde in the presence or absence of alphamangostin(10 and 20 μM). The cells were then counted and lysed, and LX-2 cell viability was determined with the trypan blue exclusion method. Themalondialdehyde levels, superoxide dismutase activity, and glutathione (GSH) levels were also determined using the cell lysates.Results: Acetaldehyde treatment resulted in a significant increase in HSC cell viability and decreased the production of GSH. Alpha-mangostintreatment resulted in reduced cell viability of the HSCs and prevention of the loss of intracellular GSH.Conclusion: Alpha-mangostin reduced acetaldehyde-induced cell proliferation, and this was affected at least in part by its antioxidative properties

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Interleukin-6 secreted by bipotential murine oval liver stem cells induces apoptosis of activated hepatic stellate cells by activating NF-κB-inducible nitric oxide synthase signaling

Biochemistry and Cell Biology ◽

10.1139/bcb-2016-0011 ◽

2017 ◽

Vol 95 (2) ◽

pp. 263-272 ◽

Cited By ~ 3

Author(s):

Palanivel Gajalakshmi ◽

Syamantak Majumder ◽

Cornelia S. Viebahn ◽

Akila Swaminathan ◽

George C. Yeoh ◽

...

Keyword(s):

Liver Fibrosis ◽

Interleukin 6 ◽

Hepatic Stellate Cells ◽

Critical Role ◽

In Vitro Study ◽

Stellate Cells ◽

Causative Factor ◽

Activated Hepatic Stellate Cells ◽

Induced Apoptosis

Liver fibrosis is now well recognized as the causative factor for increased mortality from complications associated with liver pathologies. Activated hepatic stellate cells (HSCs) play a critical role in the progression of liver fibrosis. Therefore, targeting these activated HSCs to prevent and (or) treat liver disease is a worthwhile approach to explore. In the present in vitro study, we investigated the use of bipotential murine oval liver cells (BMOL) in regulating the functions of activated HSCs to prevent progression of liver fibrosis. We used a conditioned medium-based approach to study the effect of BMOL cells on activated HSC survival and function. Our data showed that BMOL cells block the contraction of activated HSCs by inducing apoptosis of these cells. We demonstrated that BMOL cells secrete soluble factors, such as interleukin-6 (IL-6), which induced apoptosis of activated HSCs. Using both pharmacological and molecular inhibitor approaches, we further identified that IL-6-mediated activation of NF-κB–iNOS–NO–ROS signaling in activated HSCs plays a critical role in BMOL-cell-mediated apoptosis of activated HSCs. Thus, the present study provides an alternative cell-based therapeutic approach to treat liver fibrosis.

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Retinoic acid signaling sensitizes hepatic stellate cells to NK cell killing via upregulation of NK cell activating ligand RAE1

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00212.2007 ◽

2007 ◽

Vol 293 (4) ◽

pp. G809-G816 ◽

Cited By ~ 80

Author(s):

Svetlana Radaeva ◽

Lei Wang ◽

Sergei Radaev ◽

Won-Il Jeong ◽

Ogyi Park ◽

...

Keyword(s):

Retinoic Acid ◽

Hepatic Stellate Cells ◽

Nk Cell ◽

Retinoic Acid Receptor ◽

Neutralizing Antibody ◽

Stellate Cells ◽

Cell Killing ◽

Liver Fibrogenesis ◽

Vitro Model

Hepatic stellate cells (HSCs) store 75% of the body's supply of vitamin A (retinol) and play a key role in liver fibrogenesis. During liver injury, HSCs become activated and susceptible to natural killer (NK) cell killing due to increased expression of the NK cell activating ligand retinoic acid early inducible gene 1 (RAE-1). To study the mechanism by which RAE-1 is upregulated in HSCs during activation, an in vitro model of cultured mouse HSCs was employed. RAE-1 was detected at low levels in quiescent HSCs but upregulated in 4- and 7-day cultured HSCs (early activated HSCs), whereas 21-day cultured HSCs (fully activated HSCs) lost RAE-1 expression. High levels of RAE-1 in 4- and 7-day cultured HSCs correlated with their susceptibility to NK cell killing, which was diminished by treatment with RAE-1 neutralizing antibody. Furthermore, retinoic acid (RA) and retinal dehydrogenase (Raldh) levels were upregulated in early activated HSCs compared with quiescent or fully activated HSCs. Blocking RA synthesis by the Raldh inhibitor or blocking RA signaling by the retinoic acid receptor antagonist abolished upregulation of RAE-1 whereas treatment with RA induced RAE-1 expression in HSCs. In conclusion, during activation, HSCs lose retinol, which is either secreted out or oxidized into RA; the latter stimulates RAE-1 expression and sensitizes early activated HSCs to NK cell killing. In contrast, fully activated HSCs become resistant to NK cell killing because of lack of RAE1 expression, leading to chronic liver fibrosis and disease.

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Pregnenolone-16α-carbonitrile inhibits rodent liver fibrogenesis via PXR (pregnane X receptor)-dependent and PXR-independent mechanisms

Biochemical Journal ◽

10.1042/bj20041598 ◽

2005 ◽

Vol 387 (3) ◽

pp. 601-608 ◽

Cited By ~ 63

Author(s):

Carylyn J. MAREK ◽

Steven J. TUCKER ◽

Dimitrios K. KONSTANTINOU ◽

Lucy J. ELRICK ◽

Dee HAEFNER ◽

...

Keyword(s):

Carbon Tetrachloride ◽

Hepatic Stellate Cells ◽

Stellate Cell ◽

Direct Interaction ◽

Pregnane X Receptor ◽

Stellate Cells ◽

Liver Growth ◽

Liver Fibrogenesis

The effect of liver growth stimulation [using the rodent PXR (pregnane X receptor) activator PCN (pregnenolone-16α-carbonitrile)] in rats chronically treated with carbon tetrachloride to cause repeated hepatocyte necrosis and liver fibrogenesis was examined. PCN did not inhibit the hepatotoxicity of carbon tetrachloride. However, transdifferentiation of hepatic stellate cells and the extent of fibrosis caused by carbon tetrachloride treatment was significantly inhibited by PCN in vivo. In vitro, PCN directly inhibited hepatic stellate cell transdifferentiation to a profibrogenic phenotype, although the cells did not express the PXR (in contrast with hepatocytes), suggesting that PCN acts independently of the PXR. Mice with a functionally disrupted PXR gene (PXR−/−) did not respond to the antifibrogenic effects of PCN, in contrast with wild-type (PXR+/+) mice, demonstrating an antifibrogenic role for the PXR in vivo. However, PCN inhibited the transdifferentiation of PXR−/−-derived mouse hepatic stellate cells in vitro, confirming that there is also a PXR-independent antifibrogenic effect of PCN through a direct interaction with hepatic stellate cells. These data suggest that the PXR is antifibrogenic in rodents in vivo and that a PXR-independent target for PXR activators exists in hepatic stellate cells that also functions to inhibit fibrosis.

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