METTL3 facilitates hepatic fibrosis progression via m6A-YTHDF2 dependent silencing of GPR161

Hepatic fibrosis (HF) is a very common condition seen in millions of patients with various liver diseases. N6-methyladenosine (m6A) plays critical roles in various biological and pathological processes. However, the role of m6A and its main methyltransferase METTL3 in HF remains obscure. Here, we reported that METTL3 expression was elevated in HSCs from CCl4 induced fibrotic liver. METTL3 knockdown in HSCs mediated by recombinant adeno-associated-virus serotype 9 packed short hairpin RNA against METTL3 alleviated liver injury and fibrosis compared to empty carrier group. Mechanistically, the decreased liver fibrosis in CCl4-treated HSC-specific METTL3 knockdown mice was due to the increased GPR161 that is a suppressor of Hedgehog pathway, a well-known pathway to activate in liver injury and regeneration. As expect, GPR161 transferred into HSCs alleviated liver fibrosis and HSC activation. Forced GPR161 expression inhibited Gli3 activated form nuclear accumulation and subsequently suppressed fibrosis-associate gene expression. Conclusion, HSC-specific deletion of METTL3 inhibits liver fibrosis via elevated GPR161 expression, which subsequently suppressed Hedgehog pathway activation and fibrosis-associated genes expression, providing novel therapeutic targets for HF therapy.

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MiR-200c-3p targets SESN1 and represses the IL-6/AKT loop to prevent cholangiocyte activation and cholestatic liver fibrosis

Laboratory Investigation ◽

10.1038/s41374-021-00710-6 ◽

2021 ◽

Author(s):

Yongfeng Song ◽

Melanie Tran ◽

Li Wang ◽

Dong-Ju Shin ◽

Jianguo Wu

Keyword(s):

Liver Fibrosis ◽

Liver Injury ◽

Feedback Loop ◽

Ductular Reaction ◽

Adeno Associated Virus ◽

Matrix Deposition ◽

Duct Ligation ◽

Virus Serotype ◽

Cholestatic Liver Injury

AbstractCholestasis causes ductular reaction in the liver where the reactive cholangiocytes not only proliferate but also gain a neuroendocrine-like phenotype, leading to inflammatory cell infiltration and extracellular matrix deposition and contributing to the development and progression of cholestatic liver fibrosis. This study aims to elucidate the role of miR-200c in cholestasis-induced biliary liver fibrosis and cholangiocyte activation. We found that miR-200c was extremely abundant in cholangiocytes but was reduced by cholestasis in a bile duct ligation (BDL) mouse model; miR-200c was also decreased by bile acids in vitro. Phenotypically, loss of miR-200c exacerbated cholestatic liver injury, including periductular fibrosis, intrahepatic inflammation, and biliary hyperplasia in both the BDL model and the 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) model. We identified sestrin 1 (SESN1) as a target of miR-200c. Sesn1−/−-BDL mice showed mitigation of cholestatic liver injury. On a molecular level, the pro-proliferative IL-6/AKT feedback loop was activated in Mir200c−/− livers but was inhibited in Sesn1−/− livers upon cholestasis in mice. Furthermore, rescuing expression of miR-200c by the adeno-associated virus serotype 8 ameliorated BDL-induced liver injury in Mir200c−/− mice. Taken together, this study demonstrates that miR-200c restrains the proliferative and neuroendocrine-like activation of cholangiocytes by targeting SESN1 and inhibiting the IL-6/AKT feedback loop to protect against cholestatic liver fibrosis. Our findings provide mechanistic insights regarding biliary liver fibrosis, which may help to reveal novel therapeutic targets for the treatment of cholestatic liver injury and liver fibrosis.

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Hepatic Stellate Cell–Macrophage Crosstalk in Liver Fibrosis and Carcinogenesis

Seminars in Liver Disease ◽

10.1055/s-0040-1708876 ◽

2020 ◽

Vol 40 (03) ◽

pp. 307-320

Author(s):

Michitaka Matsuda ◽

Ekihiro Seki

Keyword(s):

Liver Fibrosis ◽

Liver Injury ◽

Cell Activation ◽

Stellate Cell ◽

Tumor Development ◽

Mesenchymal Cell ◽

Chronic Liver ◽

Health Concern ◽

Chronic Liver Injury ◽

Fibrotic Liver

AbstractChronic liver injury due to viral hepatitis, alcohol abuse, and metabolic disorders is a worldwide health concern. Insufficient treatment of chronic liver injury leads to fibrosis, causing liver dysfunction and carcinogenesis. Most cases of hepatocellular carcinoma (HCC) develop in the fibrotic liver. Pathological features of liver fibrosis include extracellular matrix (ECM) accumulation, mesenchymal cell activation, immune deregulation, and angiogenesis, all of which contribute to the precancerous environment, supporting tumor development. Among liver cells, hepatic stellate cells (HSCs) and macrophages play critical roles in fibrosis and HCC. These two cell types interplay and remodel the ECM and immune microenvironment in the fibrotic liver. Once HCC develops, HCC-derived factors influence HSCs and macrophages to switch to protumorigenic cell populations, cancer-associated fibroblasts and tumor-associated macrophages, respectively. This review aims to summarize currently available data on the roles of HSCs and macrophages in liver fibrosis and HCC, with a focus on their interaction.

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

10.21203/rs.3.rs-736691/v1 ◽

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.

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Adeno-Associated Virus Serotype 2 Vector–Mediated Reintroduction of microRNA-19b Attenuates Hepatic Fibrosis

Human Gene Therapy ◽

10.1089/hum.2017.035 ◽

2018 ◽

Vol 29 (6) ◽

pp. 674-686 ◽

Cited By ~ 5

Author(s):

Elizabeth Brandon-Warner ◽

Jennifer H. Benbow ◽

Jacob H. Swet ◽

Nicole A. Feilen ◽

Catherine R. Culberson ◽

...

Keyword(s):

Hepatic Fibrosis ◽

Adeno Associated Virus ◽

Virus Serotype

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Raf Kinase Inhibitory Protein Down-Expression Exacerbates Hepatic Fibrosis In Vivo and In Vitro

Cellular Physiology and Biochemistry ◽

10.1159/000452524 ◽

2016 ◽

Vol 40 (1-2) ◽

pp. 49-61 ◽

Cited By ~ 12

Author(s):

Quanfang Huang ◽

Chunhong Liang ◽

Ling Wei ◽

Jinlan Nie ◽

Shengjuan Lu ◽

...

Keyword(s):

Liver Fibrosis ◽

Liver Injury ◽

Hepatic Fibrosis ◽

Chronic Liver ◽

Chronic Liver Injury ◽

Inhibitory Protein ◽

Raf Kinase ◽

Raf Kinase Inhibitory Protein ◽

Liver Tissues

Background/Aims: Raf kinase inhibitory protein (RKIP) is closely associated with numerous tumors and participates in their development through regulating the growth, apoptosis, invasion and metastasis of tumor cells. However, the role of RKIP in chronic liver injury and particularly in liver fibrosis is still unclear. Methods: In the present study, hepatic fibrosis was induced by porcine serum (PS) in rats and primary hepatic stellate cells (HSCs) were isolated from rat livers. Moreover, locostatin was used to interfere with RKIP expression. Results: RKIP expression was significantly inhibited by locostatin in both liver tissues of rats and primary HSCs. Down-regulating RKIP expression resulted in serious liver injury, extensive accumulation of collagen, and significant increase in the levels of ALT, AST and TNF-α during liver fibrosis in rats. Moreover, down-regulating RKIP significantly promoted HSCs proliferation and colony formation in vitro. Reduced RKIP significantly increased the production of collagen and the level of α-SMA as well as the expression of MMP-1 and MMP-2 in both liver tissues and primary HSCs. Furthermore, down-regulating RKIP promoted the activation of the ERK and TLR4 signaling pathways. Conclusion: Our findings clearly indicate an inverse correlation between RKIP level and the degree of the liver injury and fibrosis. The decrease in RKIP expression may exacerbate chronic liver injury and liver fibrosis.

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Inhibition of Mastermind-like 1 alleviates liver fibrosis induced by carbon tetrachloride in rats

Experimental Biology and Medicine ◽

10.1177/1535370218810892 ◽

2018 ◽

Vol 243 (14) ◽

pp. 1099-1108

Author(s):

Shaoping Zheng ◽

Yixiong Chen ◽

Shaojiang Zheng ◽

Zhihui He ◽

Zhihong Weng

Keyword(s):

Carbon Tetrachloride ◽

Liver Fibrosis ◽

Hepatic Fibrosis ◽

Hepatic Stellate Cells ◽

Stellate Cells ◽

Underlying Mechanism ◽

Signal Pathways ◽

Fibrotic Liver ◽

Hepatic Fibrogenesis ◽

Signal Transductions

Mastermind-like 1 (MAML1) functions in critical transcriptional coactivation in Notch and Wnt/β-catenin signal pathways, which participate in hepatic fibrosis. This study is aimed to reveal the potential role of MAML1 in liver fibrosis and identify its underlying mechanism. In present research, the enhanced expression of MAML1 was found in the fibrotic liver tissues in carbon tetrachloride (CCl4)-induced hepatic fibrosis in rats, and MAML1 expression increased gradually during the activation of hepatic stellate cells (HSCs) isolated from the normal rat. Further studies showed that blocking MAML1 expression efﬁciently decreased the expression of α-SMA and collagen I (Col1a1) in HSCs. Interestingly, MAML1 may modulate HSCs activation via interrupting both Notch and Wnt/β-catenin signal transductions, and the inhibition of MAML1 by a recombinant adeno-associated virus type 1 vector carrying shRNA targeting MAML1 alleviated CCl4-induced hepatic fibrosis in rats. These findings suggest that the selective regulation of MAML1 expression may be a feasible therapeutic approach to reverse liver fibrosis. Impact statement Liver fibrosis is a common wound-healing response to all kinds of liver injuries. Hepatic stellate cells (HSCs) activation is the key event during liver fibrogenesis. Thus, the elucidation of mechanisms for regulating HSCs activation is helpful for identifying novel anti-fibrotic targets and strategies. MAML1, an important component of Notch signal, functions in critical transcriptional coactivation in the Notch and Wnt/β-catenin signal pathways. In the present study, we investigated the potential function of MAML1 during hepatic fibrogenesis in rats. Our results demonstrated that MAML1 participates in liver fibrosis through modulating HSCs activation via interrupting both the Notch and Wnt/β-catenin signal transductions. Additionally, the inhibition of MAML1 markedly attenuated CCl4-induced hepatic fibrogenesis in rats. Our results shed a light for the exploitation of a new therapeutic strategy for hepatic fibrosis via targeting MAML1.

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LPS signaling enhances hepatic fibrogenesis caused by experimental cholestasis in mice

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00405.2005 ◽

2006 ◽

Vol 290 (6) ◽

pp. G1318-G1328 ◽

Cited By ~ 83

Author(s):

Fuyumi Isayama ◽

Ian N. Hines ◽

Michael Kremer ◽

Richard J. Milton ◽

Christy L. Byrd ◽

...

Keyword(s):

Bile Duct ◽

Liver Fibrosis ◽

Liver Injury ◽

Hepatic Fibrosis ◽

Bile Duct Ligation ◽

Wild Type ◽

Hepatic Macrophages ◽

Duct Ligation ◽

Lps Signaling ◽

Experimental Cholestasis

Although it is clear that bile acid accumulation is the major initiator of fibrosis caused by cholestatic liver disease, endotoxemia is a common side effect. However, the depletion of hepatic macrophages with gadolinium chloride blunts hepatic fibrosis. Because endotoxin is a key activator of hepatic macrophages, this study was designed to test the hypothesis that LPS signaling through CD14 contributes to hepatic fibrosis caused by experimental cholestasis. Wild-type mice and CD14 knockout mice (CD14−/−) underwent sham operation or bile duct ligation and were killed 3 wk later. Measures of liver injury, such as focal necrosis, biliary cell proliferation, and inflammatory cell influx, were not significantly different among the strains 3 wk after bile duct ligation. Markers of liver fibrosis such as Sirius red staining, liver hydroxyproline, and α-smooth muscle actin expression were blunted in CD14−/− mice compared with wild-type mice after bile duct ligation. Despite no difference in lymphocyte infiltration, the macrophage/monocyte activation marker OX42 (CD11b) and the oxidative stress/lipid peroxidation marker 4-hydroxynonenal were significantly upregulated in wild-type mice after bile duct ligation but not in CD14−/− mice. Increased profibrogenic cytokine mRNA expression in the liver after bile duct ligation was significantly blunted in CD14−/− mice compared with the wild type. The hypothesis that LPS was involved in experimental cholestatic liver fibrosis was tested using mice deficient in LPS-binding protein (LBP−/−). LBP−/− mice had less liver injury and fibrosis (Siruis red staining and hydroxyproline content) compared with wild-type mice after bile duct ligation. In conclusion, these data demonstrate that endotoxin in a CD14-dependent manner exacerbates hepatic fibrogenesis and macrophage activation to produce oxidants and cytokines after bile duct ligation.

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Plumbagin Ameliorates CCl4-Induced Hepatic Fibrosis in Rats via the Epidermal Growth Factor Receptor Signaling Pathway

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2015/645727 ◽

2015 ◽

Vol 2015 ◽

pp. 1-12 ◽

Cited By ~ 5

Author(s):

Si Chen ◽

Yi Chen ◽

Bi Chen ◽

Yi-jing Cai ◽

Zhuo-lin Zou ◽

...

Keyword(s):

Growth Factor ◽

Liver Fibrosis ◽

Epidermal Growth Factor ◽

Hepatic Fibrosis ◽

Hepatic Stellate Cells ◽

Growth Factor Receptor ◽

Stellate Cells ◽

Heparin Binding ◽

Fibrotic Liver ◽

Epidermal Growth

Epidermal growth factor (EGF) and its signaling molecules, EGFreceptor (EGFR) and signal transducer and activator of transcription factor 3 (STAT3), have been considered to play a role in liver fibrosis and cirrhosis. Plumbagin (PL) is an extracted component from the plant and has been used to treat different kinds of cancer. However, its role in regulation of EGFR and STAT3 during liver fibrosis has not been investigated. In this study, the effects of PL on the regulation of EGFR and STAT3 were investigated in carbon tetrachloride (CCl4) induced liver fibrosis and hepatic stellate cells (HSC-T6). PL significantly attenuated liver injury and fibrosis in CCl4treated rats. At concentrations of 2 to 6 μM, PL did not induce significant cytotoxicity of HSC-T6 cells. Moreover, PL reduced phosphorylation of EGFR and STAT3 in both fibrotic liver and heparin-binding EGF-like growth factor (HB-EGF) treated HSC-T6 cells. Furthermore, PL reduced the expression ofα-SMA, EGFR, and STAT3 in both fibrotic liver and HB-EGF treated HSC-T6 cells. In conclusion, plumbagin could ameliorate the development of hepatic fibrosis through its downregulation of EGFR and STAT3 in the liver, especially in hepatic stellate cells.

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