scholarly journals Regulation of in vitro and in vivo Hepatic Stellate Cell Activation by the Ayrl Hydrocarbon Receptor

2020 ◽  
Author(s):  
Shivakumar Rayavara Veerabhadraiah
Keyword(s):  
Liver Fibrosis ◽  
Liver Injury ◽  
Cell Activation ◽  
Stellate Cell ◽  
Pathological Condition ◽  
Matrix Material ◽  
Subsequent Development ◽  
Type I

Liver fibrosis is a pathological condition characterized by the excessive deposition of extracellular matrix material by activated hepatic stellate cells (HSCs). We recently reported that activation of the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) increases HSC activation in vitro and in mouse models of experimental liver fibrosis. The goal of this project was to determine the mechanism by which AhR activation impacts HSC activation and the subsequent development of liver fibrosis. It is possible that HSCs are direct cellular targets for TCDD. Alternatively, TCDD could increase HSC activation indirectly by exacerbating hepatocyte damage and inflammation. To investigate this, we generated mice in which the AhR was selectively removed from either hepatocytes or HSCs to determine the ramifications on liver injury, inflammation, and HSC activation in an experimental model of liver fibrosis elicited by chronic administration of TCDD. Results from these studies indicate that TCDD does not directly activate HSCs in the mouse liver to produce fibrosis. Instead, it appears that TCDD-induced changes in hepatocytes, such as the development of steatosis, are what ultimately stimulate HSC activation and produce fibrosis. A second focus of this project was to investigate an endogenous role for AhR signaling in the regulation of HSC activation in the absence of liver injury and inflammation. To this end, I used CRISPR/Cas9 technology to knock down the AhR in the human HSC cell line, LX-2. I discovered that a functional AhR is required for optimal proliferation of activated HSCs. However, other endpoints of HSC activation, such as the production of collagen type I, were not impacted by the removal of AhR signaling. These findings are important because the AhR has been shown to be a druggable target, and there is growing interest in therapeutically modulating AhR activity to prevent or reverse HSC activation. Collectively, results from this project indicate that therapeutically targeting AhR signaling in hepatocytes, instead of AhR signaling in HSCs, might be a preferred approach for limiting HSC activation and preventing or diminishing liver fibrosis.

scholarly journals 3D hESC exosomes enriched with miR-6766-3p ameliorates liver fibrosis by attenuating activated stellate cells through targeting the TGFβRII-SMADS pathway

2021 ◽  
Author(s):  
Ning Wang ◽  
Xiajing Li ◽  
Zhiyong Zhong ◽  
Yaqi Qiu ◽  
Shoupei Liu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Liver Fibrosis ◽  
Liver Injury ◽  
Cell Activation ◽  
Therapeutic Potential ◽  
Stellate Cell ◽  
Embryonic Stem ◽  
Luciferase Reporter ◽  
Fibrotic Liver

Abstract BackgroundExosomes secreted from stem cells exerted salutary effects on the fibrotic liver. Herein, the roles of exosomes derived from human embryonic stem cell (hESC) in anti-fibrosis were extensively investigated. Compared with two-dimensional (2D) culture, the clinical and biological relevance of three-dimensional (3D) cell spheroids were greater because of their higher regeneration potential since they behave more like cells in vivo. In our study, exosomes derived from 3D human embryonic stem cells (hESC) spheroids and the monolayer (2D) hESCs were collected and compared the therapeutic potential for fibrotic liver in vitro and in vivo. ResultsIn vitro, PKH26 labled-hESC-Exosomes were shown to be internalized and integrated into TGFβ-activated-LX2 cells, and reduced the expression of profibrogenic markers, thereby regulating cellular phenotypes. TPEF imaging indicated that PKH26-labled-3D-hESC-Exsomes possessed an enhanced capacity to accumulate in the livers and exhibited more dramatic therapeutic potential in the injured livers of fibrosis mouse model. 3D-hESC-Exosomes decreased profibrogenic markers and liver injury markers, and improved the level of liver functioning proteins, eventually restoring liver function of fibrosis mice. miRNA array revealed a significant enrichment of miR-6766-3p in 3D-hESC-Exosomes, moreover, bioinformatics and dual luciferase reporter assay identified and confirmed the TGFβRII gene as the target of miR-6766-3p. Furthermore, the delivery of miR-6766-3p into activated-LX2 cells decreased cell proliferation, chemotaxis and profibrotic effects, and further investigation demonstrated that the expression of target gene TGFβRII and its downstream SMADs proteins, especially phosphorylated protein p-SMAD2/3 was also notably down-regulated by miR-6766-3p. These findings unveiled that miR-6766-3p in 3D-hESC-Exosomes inactivated SMADs signaling by inhibiting TGFβRII expression, consequently attenuating stellate cell activation and suppressing liver fibrosis. ConclusionsOur results showed that miR-6766-3p in the 3D-hESC-Exosomes inactivates smads signaling by restraining TGFβRII expression, attenuated LX2 cell activation and suppressed liver fibrosis, suggesting that 3D-hESC-Exosome enriched-miR6766-3p is a novel anti-fibrotic therapeutics for treating chronic liver disease. These results also proposed a significant strategy that 3D-Exo could be used as natural nanoparticles to rescue liver injury via delivering antifibrotic miR-6766-3p.

scholarly journals Targeting Follistatin like 1 ameliorates liver fibrosis induced by carbon tetrachloride through TGF-β1-miR29a in mice

2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Xin-Yi Xu ◽  
Yan Du ◽  
Xue Liu ◽  
Yilin Ren ◽  
Yingying Dong ◽  
...  
Keyword(s):  
Liver Fibrosis ◽  
Cell Activation ◽  
Transforming Growth Factor ◽  
Stellate Cell ◽  
Neutralizing Antibody ◽  
Transforming Growth Factor Β1 ◽  
Chemokine Ligand ◽  
Tgf Β1

Abstract Background Hepatic fibrosis is a pathological response of the liver to a variety of chronic stimuli. Hepatic stellate cells (HSCs) are the major source of myofibroblasts in the liver. Follistatin like 1 (Fstl1) is a secreted glycoprotein induced by transforming growth factor-β1 (TGF-β1). However, the precise functions and regulation mechanisms of Fstl1 in liver fibrogenesis remains unclear. Methods Hepatic stellate cell (HSC) line LX-2 stimulated by TGF-β1, primary culture of mouse HSCs and a model of liver fibrosis induced by CCl4 in mice was used to assess the effect of Fstl1 in vitro and in vivo. Results Here, we found that Fstl1 was significantly up regulated in human and mouse fibrotic livers, as well as activated HSCs. Haplodeficiency of Fstl1 or blockage of Fstl1 with a neutralizing antibody 22B6 attenuated CCl4-induced liver fibrosis in vivo. Fstl1 modulates TGF-β1 classic Samd2 and non-classic JNK signaling pathways. Knockdown of Fstl1 in HSCs significantly ameliorated cell activation, cell migration, chemokines C-C Motif Chemokine Ligand 2 (CCL2) and C-X-C Motif Chemokine Ligand 8 (CXCL8) secretion and extracellular matrix (ECM) production, and also modulated microRNA-29a (miR29a) expression. Furthermore, we identified that Fstl1 was a target gene of miR29a. And TGF-β1 induction of Fstl1 expression was partially through down regulation of miR29a in HSCs. Conclusions Our data suggests TGF-β1-miR29a-Fstl1 regulatory circuit plays a key role in regulation the HSC activation and ECM production, and targeting Fstl1 may be a strategy for the treatment of liver fibrosis. Graphical abstract

Snail1 transcription factor is a critical mediator of hepatic stellate cell activation following hepatic injury

2011 ◽  
Vol 300 (2) ◽  
pp. G316-G326 ◽  
Author(s):  
Melania Scarpa ◽  
Alessia R. Grillo ◽  
Paola Brun ◽  
Veronica Macchi ◽  
Annalisa Stefani ◽  
...  
Keyword(s):  
Wound Healing ◽  
Transcription Factor ◽  
Liver Fibrosis ◽  
Liver Injury ◽  
Mrna Expression ◽  
Hepatic Stellate Cell ◽  
Stellate Cell ◽  
Qrt Pcr

Following liver injury, the wound-healing process is characterized by hepatic stellate cell (HSC) activation from the quiescent fat-storing phenotype to a highly proliferative myofibroblast-like phenotype. Snail1 is a transcription factor best known for its ability to trigger epithelial-mesenchymal transition, to influence mesoderm formation during embryonic development, and to favor cell survival. In this study, we evaluated the expression of Snail1 in experimental and human liver fibrosis and analyzed its role in the HSC transdifferentiation process. Liver samples from patients with liver fibrosis and from mice treated by either carbon tetrachloride (CCl4) or thioacetamide (TAA) were evaluated for mRNA expression of Snail1. The transcription factor expression was investigated by immunostaining and real-time quantitative RT-PCR (qRT-PCR) on in vitro and in vivo activated murine HSC. Snail1 knockdown studies on cultured HSC and on CCl4-treated mice were performed by adenoviral delivery of short-hairpin RNA; activation-related genes were quantitated by real-time qRT-PCR and Western blotting. Snail1 mRNA expression resulted upregulated in murine experimental models of liver injury and in human hepatic fibrosis. In vitro studies showed that Snail1 is expressed by HSC and that its transcription is augmented in in vitro and in vivo activated HSC compared with quiescent HSC. At the protein level, we could observe the nuclear translocation of Snail1 in activated HSC. Snail1 knockdown resulted in the downregulation of activation-related genes both in vitro and in vivo. Our data support a role for Snail1 transcription factor in the hepatic wound-healing response and its involvement in the HSC transdifferentiation process.

scholarly journals Dioscin alleviates alcoholic liver fibrosis by attenuating hepatic stellate cell activation via the TLR4/MyD88/NF-κB signaling pathway

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Min Liu ◽  
Youwei Xu ◽  
Xu Han ◽  
Lianhong Yin ◽  
Lina Xu ◽  
...  
Keyword(s):  
Liver Fibrosis ◽  
Signaling Pathway ◽  
Type I Collagen ◽  
Transforming Growth Factor ◽  
Stellate Cell ◽  
Pyrrolidine Dithiocarbamate ◽  
Type I ◽  
Tgf Β1

Abstract The present work aimed to investigate the activities and underlying mechanisms of dioscin against alcoholic liver fibrosis (ALF). In vivo liver fibrosis in mice was induced by an alcoholic liquid diet and in vitro studies were performed on activated HSC-T6 and LX2 cells treated with lipopolysaccharide. Our results showed that dioscin significantly attenuated hepatic stellate cells (HSCs) activation, improved collagen accumulation and attenuated inflammation through down-regulating the levels of myeloid differentiation factor 88 (MyD88), nuclear factor κB (NF-κB), interleukin (IL)-1, IL-6 and tumour necrosis factor-α by decreasing Toll-like receptor (TLR)4 expression both in vivo and in vitro. TLR4 overexpression was also decreased by dioscin, leading to the markedly down-regulated levels of MyD88, NF-κB, transforming growth factor-β1 (TGF-β1), α-smooth muscle actin (α-SMA) and type I collagen (COL1A1) in cultured HSCs. Suppression of cellular MyD88 by ST2825 or abrogation of NF-κB by pyrrolidine dithiocarbamate eliminated the inhibitory effects of dioscin on the levels of TGF-β1, α-SMA and COL1A1. In a word, dioscin exhibited potent effects against ALF via altering TLR4/MyD88/NF-κB signaling pathway, which provided novel insights into the mechanisms of this compound as an antifibrogenic candidate for the treatment of ALF in the future.

scholarly journals Down-Regulation of CXXC5 De-Represses MYCL1 to Promote Hepatic Stellate Cell Activation

2021 ◽  
Vol 9 ◽  
Author(s):  
Xiaoyan Wu ◽  
Wenhui Dong ◽  
Ming Kong ◽  
Haozhen Ren ◽  
Jinglin Wang ◽  
...  
Keyword(s):  
Liver Fibrosis ◽  
Cell Activation ◽  
Zinc Finger Protein ◽  
Stellate Cell ◽  
Underlying Mechanism ◽  
Down Regulation ◽  
Over Expression ◽  
Quiescent Hscs

Liver fibrosis is mediated by myofibroblasts, a specialized cell type involved in wound healing and extracellular matrix production. Hepatic stellate cells (HSC) are the major source of myofibroblasts in the fibrotic livers. In the present study we investigated the involvement of CXXC-type zinc-finger protein 5 (CXXC5) in HSC activation and the underlying mechanism. Down-regulation of CXXC5 was observed in activated HSCs compared to quiescent HSCs both in vivo and in vitro. In accordance, over-expression of CXXC5 suppressed HSC activation. RNA-seq analysis revealed that CXXC5 influenced multiple signaling pathways to regulate HSC activation. The proto-oncogene MYCL1 was identified as a novel target for CXXC5. CXXC5 bound to the proximal MYCL1 promoter to repress MYCL1 transcription in quiescent HSCs. Loss of CXXC5 expression during HSC activation led to the removal of CpG methylation and acquisition of acetylated histone H3K9/H3K27 on the MYCL1 promoter resulting in MYCL1 trans-activation. Finally, MYCL1 knockdown attenuated HSC activation whereas MYCL1 over-expression partially relieved the blockade of HSC activation by CXXC5. In conclusion, our data unveil a novel transcriptional mechanism contributing to HSC activation and liver fibrosis.

scholarly journals The role of let-7b in the inhibition of hepatic stellate cell activation by rSjP40

2021 ◽  
Vol 15 (6) ◽  
pp. e0009472
Author(s):  
Xiaolei Sun ◽  
Li Zhang ◽  
Yuting Jiang ◽  
Aihong Li ◽  
Dandan Zhu ◽  
...  
Keyword(s):  
Liver Fibrosis ◽  
Cell Activation ◽  
Stellate Cell ◽  
Direct Interaction ◽  
Collagen Type I ◽  
Collagen I ◽  
Luciferase Assay ◽  
Type I

Background Hepatic stellate cells (HSCs) are one of the main cell types involved in liver fibrosis induced by many factors, including schistosomes. Previous studies in our lab have shown that recombinant P40 protein from Schistosoma japonicum (rSjP40) can inhibit HSC activation in vitro. Let-7b is a member of the let-7 microRNA family and plays an inhibitory role in a variety of diseases and inflammatory conditions. In this study, we investigated the role of let-7b in the inhibition of HSC activation by rSjP40. Methods Expression of let-7b was detected by quantitative real-time PCR. A dual luciferase assay was used to confirm direct interaction between let-7b and collagen I. We also used western blot to assess protein levels of TGFβRI and collagen type I α1 (COL1A1). Results We found that rSjP40 up-regulates expression of let-7b in HSCs. Let-7b inhibits collagen I expression by directly targeting the 3’UTR region of the collagen I gene. Furthermore, we discovered that let-7b inhibitor partially restores the loss of collagen I expression caused by rSjP40. Conclusion Our research clarifies the role of let-7b in the inhibition of HSC activation by rSjP40 and will provide new insights and ideas for the inhibition of HSC activation and treatment of liver fibrosis.

scholarly journals Nicotinamide Riboside, an NAD + Precursor, Reduces Hepatic Stellate Cell Activation and Attenuates Liver Fibrosis in a Diet-induced Mouse Model of Liver Fibrosis (OR24-06-19)

2019 ◽  
Vol 3 (Supplement_1) ◽  
Author(s):  
Tho Pham ◽  
Minkyung Bae ◽  
Mi-Bo Kim ◽  
Yoojin Lee ◽  
Siqi Hu ◽  
...  
Keyword(s):  
Liver Fibrosis ◽  
Energy Expenditure ◽  
Mouse Model ◽  
Cell Activation ◽  
Stellate Cell ◽  
Body Weight Gain ◽  
Nicotinamide Riboside ◽  
Primary Mouse

Abstract Objectives There is limited pharmacological treatment for liver fibrosis, which can result from chronic liver injury. In this study, we investigated the effect of nicotinamide riboside (NR), a nicotinamide adenine dinucleotide (NAD) precursor, on the development of liver fibrosis in a diet-induced mouse model of liver fibrosis in vivo and in hepatic stellate cells (HSCs) in vitro. Methods Male C57BL/6 J mice were randomly assigned to three groups: a low-fat control (LF; 6% fat by wt), a high-fat/high-sucrose/high-cholesterol control (HF; 35%/34%/2.0% by wt, n = 13) or a HF diet supplemented with NR at 400 mg/kg/day (HF-NR, n = 14) for 20 weeks. Features of liver fibrosis were assessed by molecular, histological, and biochemical analyses to determine the effect of NR. Metabolic rates, energy expenditure and physical activity were measured using indirect calorimetry. Primary mouse and human HSCs, the primary extracellular matrix-producing cell-type in the liver, were used to determine the anti-fibrogenic effects of NR in vitro. Results HF-NR group had reduced body weight gain, which was attributable to increased energy expenditure. NR supplementation did not affect serum alanine aminotransferase levels and markers of steatosis and inflammation in the liver. However, liver trichrome and picrosirius red staining and total collagen quantification showed significant reductions of collagen by NR. Consistently, hepatic collagen 1a1 mRNA and protein were significantly reduced in the HF-NR group. Liver NAD levels were significantly reduced by HF, but was increased by NR supplementation. RNA-Seq analysis of NAD metabolism genes in quiescent and activated HSCs indicated that NAD levels might be reduced in activated HSCs due to repression of NAD salvage pathway, which regenerates NAD from nicotinamide. Indeed, treatment of primary human and mouse HSCs with NR significantly reduced their activation in vitro. Conclusions NR supplementation prevented the development of liver fibrosis in a diet-induced mouse model of liver fibrosis independent of hepatic steatosis and inflammation. The data suggest that NR may directly reduce HSC activation to exert its anti-fibrotic effect. NR may be developed as a potential preventative for human liver fibrosis. Funding Sources The NIH, USDA Multistate Hatch, and USDA Hatch.

scholarly journals Alleviation of Carbon-Tetrachloride-Induced Liver Injury and Fibrosis by Betaine Supplementation in Chickens

2015 ◽  
Vol 2015 ◽  
pp. 1-12 ◽  
Author(s):  
Meng-Tsz Tsai ◽  
Ching-Yi Chen ◽  
Yu-Hui Pan ◽  
Siou-Huei Wang ◽  
Harry J. Mersmann ◽  
...  
Keyword(s):  
Carbon Tetrachloride ◽  
Liver Fibrosis ◽  
Cell Activation ◽  
Transforming Growth Factor ◽  
Stellate Cell ◽  
Food Component ◽  
Metabolic Functions ◽  
Hepatocyte Necrosis

Betaine is a food component with well-reported hepatoprotection effects. However, the effects and mechanisms of betaine on liver fibrosis development are still insufficient. Because metabolic functions of chicken and human liver is similar, we established a chicken model with carbon Tetrachloride- (CCl4-) induced fibrosis for studying antifibrotic effect of betainein vivoandin vitro. Two-week-old male chicks were supplemented with betaine (1%, w/v) in drinking water for 2 weeks prior to the initiation of CCl4treatment (i.p.) until sacrifice. Primary chicken hepatocytes were treated with CCl4and betaine to mimic thein vivosupplementation. The supplementation of betaine significantly alleviated liver fibrosis development along with the inhibition of lipid peroxidation, hepatic inflammation cytokine, and transforming growth factor-β1 expression levels. These inhibitive effects were also accompanied with the attenuation of hepatic stellate cell activation. Furthermore, ourin vitrostudies confirmed that betaine provides antioxidant capacity for attenuating the hepatocyte necrosis by CCl4. Altogether, our results highlight the antioxidant ability of betaine, which alleviates CCl4-induced fibrogenesis process along with the suppression of hepatic stellate cells activation. Since betaine is a natural compound without toxicity, we suggest betaine can be used as a potent nutritional or therapeutic factor for reducing liver fibrosis.

scholarly journals Epiregulin (EREG) and Myocardin Related Transcription Factor A (MRTF-A) Form a Feedforward Loop to Drive Hepatic Stellate Cell Activation

2021 ◽  
Vol 8 ◽  
Author(s):  
Xiaoyan Wu ◽  
Wenhui Dong ◽  
Tianyi Zhang ◽  
Haozhen Ren ◽  
Jinglin Wang ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Liver Fibrosis ◽  
Cell Activation ◽  
Serum Response Factor ◽  
Stellate Cell ◽  
Feedforward Loop ◽  
Related Transcription Factor ◽  
Egf Family

Trans-differentiation of quiescent hepatic stellate cells (HSC) into myofibroblast cells is considered the linchpin of liver fibrosis. A myriad of signaling pathways contribute to HSC activation and consequently liver fibrosis. Epidermal growth factor (EGF) family of cytokines signal through the cognate receptor EGFR to promote HSC activation. In the present study we investigated the transcription regulation of epiregulin (EREG), an EGFR ligand, during HSC activation. We report that EREG expression was significantly up-regulated in activated HSCs compared to quiescent HSCs isolated from mice. In addition, there was an elevation of EREG expression in HSCs undergoing activation in vitro. Of interest, deficiency of myocardin-related transcription factor A (MRTF-A), a well-documented regulator of HSC trans-differentiation, attenuated up-regulation of EREG expression both in vivo and in vitro. Further analysis revealed that MRTF-A interacted with serum response factor (SRF) to bind directly to the EREG promoter and activate EREG transcription. EREG treatment promoted HSC activation in vitro, which was blocked by MRTF-A depletion or inhibition. Mechanistically, EREG stimulated nuclear trans-location of MRTF-A in HSCs. Together, our data portray an EREG-MRTF-A feedforward loop that contributes to HSC activation and suggest that targeting the EREG-MRTF-A axis may yield therapeutic solutions against liver fibrosis.

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