Lenvatinib prevents liver fibrosis by inhibiting hepatic stellate cell activation and sinusoidal capillarization in experimental liver fibrosis

Molecular targeted agents are pharmacologically used to treat liver fibrosis and have gained increased attention. The present study examined the preventive effect of lenvatinib on experimental liver fibrosis and sinusoidal capillarization as well as the in vitro phenotypes of hepatic stellate cells. LX-2, a human stellate cell line, was used for in vitro studies. In vivo liver fibrosis was induced in F344 rats using carbon tetrachloride by intraperitoneal injection for 8 weeks, and oral administration of lenvatinib was started two weeks after initial injection of carbon tetrachloride. Lenvatinib restrained proliferation and promoted apoptosis of LX-2 with suppressed phosphorylation of extracellular signal-regulated kinase 1/2 and AKT. It also down-regulated COL1A1, ACTA2 and TGFB1 expressions by inhibiting the transforming growth factor-β1/Smad2/3 pathway. Treatment with lenvatinib also suppressed platelet-derived growth factor-BB-stimulated proliferation, chemotaxis and vascular endothelial growth factor-A production, as well as basic fibroblast growth factor-induced LX-2 proliferation. In vivo study showed that lenvatinib attenuated liver fibrosis development with reduction in activated hepatic stellate cells and mRNA expression of profibrogenic markers. Intrahepatic neovascularization was ameliorated with reduced hepatic expressions of Vegf1, Vegf2 and Vegfa in lenvatinib-treated rats. Collectively, these results suggest the potential use of lenvatinib as a novel therapeutic strategy for liver fibrosis.     

Novel matrine derivative MD-1 attenuates hepatic fibrosis by inhibiting EGFR activation of hepatic stellate cells

Matrine (MT), the effective component of Sophora flavescens Ait, has been shown to have anti-inflammation, immune-suppressive, anti-tumor, and anti-hepatic fibrosis activities. However, the pharmacological effects of MT still need to be strengthened due to its relatively low efficacy and short half-life. In the present study, we report a more effective thio derivative of MT, MD-1, and its inhibitory effects on the activation of hepatic stellate cells (HSCs) in both cell culture and animal models. Cytological experiments showed that MD-1 can inhibit the proliferation of HSC-T6 cells with a half-maximal inhibitory concentration (IC50) of 62 μmol/L. In addition, MD-1 more strongly inhibits the migration of HSC-T6 cells compared to MT and can more effectively induce G0/G1 arrest and apoptosis. Investigating the biological mechanisms underlying anti-hepatic fibrosis in the presence of MD-1, we found that MD-1 can bind the epidermal growth factor receptor (EGFR) on the surface of HSC-T6 cells, which can further inhibit the phosphorylation of EGFR and its downstream protein kinase B (Akt), resulting in decreased expression of cyclin D1 and eventual inhibition of the activation of HSC-T6 cells. Furthermore, in rats with dimethylnitrosamine (DMN)-induced hepatic fibrosis, MD-1 slowed the development and progression of hepatic fibrosis, protecting hepatic parenchymal cells and improving hepatic functions. Therefore, MD-1 is a potential drug for anti-hepatic fibrosis.     

Lipopolysaccharide mediates hepatic stellate cell activation by regulating autophagy and retinoic acid signaling

Bacterial translocation and lipopolysaccharide (LPS) leakage occur at a very early stage of liver fibrosis in animal models. We studied the role of LPS in hepatic stellate cell (HSC) activation and the underlying mechanisms in vitro and in vivo. Herein, we demonstrated that LPS treatment led to a dramatic increase in autophagosome formation and autophagic flux in LX-2 cells and HSCs, which was mediated through the AKT-MTOR and AMPK-ULK1 pathway. LPS significantly decreased the lipid content, including the lipid droplet (LD) number and lipid staining area in HSCs; pretreatment with macroautophagy/autophagy inhibitors or silencing ATG5 attenuated this decrease. Furthermore, lipophagy was induced by LPS through the autophagy-lysosomal pathway in LX-2 cells and HSCs. Additionally, LPS-induced autophagy further reduced retinoic acid (RA) signaling, as demonstrated by a decrease in the intracellular RA level and Rar target genes, resulting in the downregulation of Bambi and promoting the sensitization of the HSC's fibrosis response to TGFB. Compared with CCl₄ injection alone, CCl₄ plus LPS injection exaggerated liver fibrosis in mice, as demonstrated by increased Col1a1 (collagen, type I, α 1), Acta2, Tgfb and Timp1 mRNA expression, ACTA2/α-SMA and COL1A1 protein expression, and Sirius Red staining area, which could be attenuated by injection of an autophagy inhibitor. LPS also reduced lipid content in HSCs in vivo, with this change being attenuated by chloroquine (CQ) administration. In conclusion, LPS-induced autophagy resulted in LD loss, RA signaling dysfunction, and downregulation of the TGFB pseudoreceptor Bambi, thus sensitizing HSCs to TGFB signaling.     

Disruption of myofibroblastic Notch signaling attenuates liver fibrosis by modulating fibrosis progression and regression

The phenotypic transformation of hepatic myofibroblasts (MFs) is involved in the whole process of the progression and regression of liver fibrosis. Notch signaling has been demonstrated to modulate the fibrosis. In this study, we found that Notch signaling in MFs was overactivated and suppressed with the progression and regression of hepatic fibrosis respectively, by detecting Notch signaling readouts in MFs. Moreover, we inactivated Notch signaling specifically in MFs with Sm22α^CreER-RBPj^flox/flox mice (RBPj^MF-KO), and identified that MFs-specific down-regulation of Notch signaling significantly alleviated CCl₄-induced liver fibrosis during the progression and regression. During the progression of liver fibrosis, MFs-specific blockade of Notch signaling inhibited the activation of HSCs to MFs and increases the expression of MMPs to reduce the deposition of ECM. During the regression of fibrosis, blocking Notch signaling in MFs increased the expression of HGF to promote proliferation in hepatocytes and up-regulated the expression of pro-apoptotic factors, Ngfr and Septin4, to induce apoptosis of MFs, thereby accelerating the reversal of fibrosis. Collectively, the MFs-specific disruption of Notch signaling attenuates liver fibrosis by modulating fibrosis progression and regression, which suggests a promising therapeutic strategy for liver fibrosis.     

IRE1α/NOX4 signaling pathway mediates ROS‐dependent activation of hepatic stellate cells in NaAsO2‐induced liver fibrosis

Liver fibrosis is a severe health problem worldwide, and it is characterized by the activation of hepatic stellate cells (HSCs) and excessive deposition of collagen. Prolonged arsenic exposure can induce HSCs activation and liver fibrosis. In the present study, the results showed that chronic NaAsO₂ ingestion could result in liver fibrosis and oxidative stress in Sprague–Dawley rats, along with representative collagen deposition and HSCs activation. In addition, the inositol‐requiring enzyme 1α (IRE1α)–endoplasmic reticulum (ER)‐stress pathway was activated, and the activity of nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) was upregulated in rat livers. Simultaneously, the excessive production of reactive oxygen species (ROS) could induce HSCs activation, and NOX4 played an important role in generating ROS in vitro. Moreover, ER stress occurred with HSCs activation at the same time under NaAsO₂ exposure, and during ER stress, the IRE1α pathway was responsible for NOX4 activation. Therefore, inhibition of IRE1α activation could attenuate the HSCs activation induced by NaAsO₂. In conclusion, the present study manifested that inorganic arsenic exposure could activate HSCs through IRE1α/NOX4‐mediated ROS generation.     

Embryonic liver fodrin involved in hepatic stellate cell activation and formation of regenerative nodule in liver cirrhosis

Transforming growth factor (TGF) β(1) plays a critical role in liver fibrosis. Previous studies demonstrated embryonic liver fodrin (ELF), a β-spectrin was involved in TGF-β/Smad signalling pathway as Smad3/4 adaptor. Here we investigate the role of ELF in pathogenesis of liver cirrhosis. In carbon tetrachloride (CCl(4))-induced mice model of liver cirrhosis, ELF is up-regulated in activated hepatic stellate cells (HSCs), and down-regulated in regenerative hepatocytes of cirrhotic nodules. In activated HSCs in vitro, reduction of ELF expression mediated by siRNA leads to the inhibition of HSC activation and procollagen I expression. BrdU assay demonstrates that down-regulation of ELF expression does not inhibit proliferation of activated HSCs in vitro. Immunostaining of cytokeratin 19 and Ki67 indicates that regenerative hepatocytes in cirrhotic liver are derived from hepatic progenitor cells (HPC). Further study reveals that HPC expansion occurs as an initial phase, before the reduction of ELF expression in regenerative hepatocytes. Regenerative hepatocytes in cirrhotic liver show the change in proliferative activity and expression pattern of proteins involved in G1/S transition, which suggests the deregulation of cell cycle in regenerative hepatocytes. Finally, we find that ELF participates in TGF-β/Smad signal in activated HSCs and hepatocytes through regulating the localization of Smad3/4. These data reveal that ELF is involved in HSC activation and the formation of regenerative nodules derived from HPC in cirrhotic liver.     

CDH11 promotes liver fibrosis via activation of hepatic stellate cells

Liver fibrosis, an important health condition associated with chronic liver injury that provides a permissive environment for cancer development, is characterized by the persistent deposition of extracellular matrix components that are mainly derived from activated hepatic stellate cells (HSCs). CDH11 belongs to a group of transmembrane proteins that are principally located in adherens junctions. CDH11 mediates homophilic cell-to-cell adhesion, which may promote the development of cirrhosis. The goal of this study was to determine whether CDH11 regulates liver fibrosis and to examine its mechanism by focusing on HSC activation. Here we demonstrate that CDH11 expression is elevated in human and mouse fibrotic liver tissues and that CDH11 mediates the profibrotic response in activated HSCs. Our data indicate that CDH11 regulates the TGFβ-induced activation of HSCs. Moreover, cells from CDH11 deficient mice displayed decreased HSC activation in vitro, and CDH11 deficient mice developed liver fibrogenesis in response to chronic damage induced by CCl₄ administration. In addition, CDH11 expression was positively correlated with liver fibrosis in patients with cirrhosis, and could therefore be a prognostic factor in patients with liver fibrosis. Collectively, our findings demonstrate that CDH11 promotes liver fibrosis by activating HSCs and may represent a potential target for anti-fibrotic therapies.     

Mechanistic insights into the effects of SREBP1c on hepatic stellate cell and liver fibrosis

Sterol regulatory element‐binding protein 1c (SREBP1c) plays key roles in maintenance of hepatic stellate cell (HSC) quiescence. The present researches investigated the mechanisms underlying the effects of SREBP1c on HSCs and liver fibrogenesis by HSC‐targeted overexpression of the active SREBP1c using adenovirus in vitro and in vivo. Results demonstrated that SREBP1c exerted inhibitory effects on TAA‐induced liver fibrosis. SREBP1c down‐regulated TGFβ1 level in liver, reduced the receptors for TGFβ1 and PDGFβ, and interrupted the signalling pathways of Smad3 and Akt1/2/3 but not ERK1/2 in HSCs. SREBP1c also led to the decreases in the protein levels of the bromodomain‐containing chromatin‐modifying factor bromodomain protein 4, methionine adenosyltransferase 2B (MAT2B) and TIMP1 in HSCs. In vivo activated HSCs did not express cyclin D1 and cyclin E1 but SREBP1c down‐regulated both cyclins in vitro. SREBP1c elevated PPARγ and MMP1 protein levels in the model of liver fibrosis. The effect of SREBP1c on MAT2B expression was associated with its binding to MAT2B1 promoter. Taken together, the mechanisms underlying the effects of SREBP1c on HSC activation and liver fibrosis were involved in its influences on TGFβ1 level, the receptors for TGFβ1 and PDGFβ and their downstream signalling, and the molecules for epigenetic regulation of genes.     

DHFR silence alleviated the development of liver fibrosis by affecting the crosstalk between hepatic stellate cells and macrophages

Liver fibrogenesis is a dynamic cellular and tissue process which has the potential to progress into cirrhosis of even liver cancer and liver failure. The activation of hepatic stellate cells (HSCs) is the central event underlying liver fibrosis. Besides, hepatic macrophages have been proposed as potential targets in combatting fibrosis. As for the relationship between HSCs and hepatic macrophages in liver fibrosis, it is generally considered that macrophages promoted liver fibrosis via activating HSCs. However, whether activated HSCs could in turn affect macrophage polarization has rarely been studied. In this study, mRNAs with significant differences were explored using exosomal RNA-sequencing of activated Lx-2 cells and normal RNA-sequencing of DHFR loss-of-function Lx-2 cell models. Cell functional experiments in both Lx-2 cells and macrophages animal model experiments were performed. The results basically confirmed exosomes secreted from activated HSCs could promote M1 polarization of macrophages further. Exosome harbouring DHFR played an important role in this process. DHFR silence in HSCs could decrease Lx-2 activation and M1 polarization of M0 macrophages and then alleviate the development of liver fibrosis both in vitro and vivo. Our work brought a new insight that exosomal DHFR derived from HSCs had a crucial role in crosstalk between HSCs activation and macrophage polarization, which may be a potential therapeutic target in liver fibrosis.     

Kaempferol attenuates liver fibrosis by inhibiting activin receptor–like kinase 5

Liver fibrosis is a common public health problem. Patients with liver fibrosis are more likely to develop cirrhosis, or hepatocellular carcinoma (HCC) as a more serious consequence. Numerous therapeutic approaches have emerged, but the final clinical outcome remains unsatisfactory. Here, we discovered a flavonoid natural product kaempferol that could dramatically ameliorate liver fibrosis formation. Our data showed that intraperitoneal injection of kaempferol could significantly decrease the necroinflammatory scores and collagen deposition in the liver tissue. In addition, serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), laminin (LN) and hyaluronic acid (HA) levels were significantly down‐regulated in kaempferol treatment group compared with those in the control group. Our study also demonstrated that kaempferol markedly inhibited the synthesis of collagen and activation of hepatic stellate cells (HSCs) both in vivo and in vitro. Furthermore, the results of Western blotting revealed that kaempferol could down‐regulate Smad2/3 phosphorylation dose‐dependently. These bioactivities of kaempferol may result from its targeted binding to the ATP‐binding pocket of activin receptor–like kinase 5 (ALK5), as suggested by the molecular docking study and LanthaScreen Eu kinase binding assay. Above all, our data indicate that kaempferol may prove to be a novel agent for the treatment of liver fibrosis or other fibroproliferative diseases.     

L-cysteine administration prevents liver fibrosis by suppressing hepatic stellate cell proliferation and activation

Recent studies showed that the function of some amino acids is not only nutritional but also pharmacological. However, the effects of amino acids on liver fibrosis and hepatic stellate cell (HSC) remain unclear. In this research, as a result of screening of amino acids using liver fibrosis induced by DMN administration, L-cysteine was selected as a suppressor of liver fibrosis. Furthermore, the number of activated HSCs, which increased in the fibrotic liver after DMN administration, was decreased in L-cysteine-fed rats. Treatment of freshly isolated HSCs with L-cysteine resulted in inhibition of the increase in smooth muscle alpha-actin (alphaSMA) expression by HSCs and BrdU incorporation into the activated HSCs. These findings suggest that L-cysteine is effective against liver fibrosis. The mechanism of inhibition of fibrosis in the liver is surmized to be direct inhibition of activated HSC proliferation and HSC transformation by L-cysteine.     

Curcumin attenuates angiogenesis in liver fibrosis and inhibits angiogenic properties of hepatic stellate cells

Hepatic fibrosis is concomitant with sinusoidal pathological angiogenesis, which has been highlighted as novel therapeutic targets for the treatment of chronic liver disease. Our prior studies have demonstrated that curcumin has potent antifibrotic activity, but the mechanisms remain to be elucidated. The current work demonstrated that curcumin ameliorated fibrotic injury and sinusoidal angiogenesis in rat liver with fibrosis caused by carbon tetrachloride. Curcumin reduced the expression of a number of angiogenic markers in fibrotic liver. Experiments in vitro showed that the viability and vascularization of rat liver sinusoidal endothelial cells and rat aortic ring angiogenesis were not impaired by curcumin. These results indicated that hepatic stellate cells (HSCs) that are characterized as liver‐specific pericytes could be potential target cells for curcumin. Further investigations showed that curcumin inhibited VEGF expression in HSCs associated with disrupting platelet‐derived growth factor‐β receptor (PDGF‐βR)/ERK and mTOR pathways. HSC motility and vascularization were also suppressed by curcumin associated with blocking PDGF‐βR/focal adhesion kinase/RhoA cascade. Gain‐ or loss‐of‐function analyses revealed that activation of peroxisome proliferator‐activated receptor‐γ (PPAR‐γ) was required for curcumin to inhibit angiogenic properties of HSCs. We concluded that curcumin attenuated sinusoidal angiogenesis in liver fibrosis possibly by targeting HSCs via a PPAR‐γ activation‐dependent mechanism. PPAR‐γ could be a target molecule for reducing pathological angiogenesis during liver fibrosis.     

MiR-542-3p controls hepatic stellate cell activation and fibrosis via targeting BMP-7

There has been an increasing number of studies about microRNAs as key regulators in the development of hepatic fibrosis. Here, we demonstrate that miR-542-3p can promote hepatic fibrosis by downregulating the expression of bone morphogenetic protein 7 (BMP-7), which is known to antagonize transforming growth factor β1 (TGFβ1)-mediated fibrogenesis effect. The expression of miR-542-3p is increased in activated hepatic stellate cells (HSCs). Downregulation of MiR-542-3p by antisense inhibitors can inhibit HSCs activation markers, including α-smooth muscle actin (α-SMA) and collagen as well as TGFβ signaling pathways. MiR-542-3p was significantly upregulated in carbon tetrachloride (CCl₄)-induced hepatic fibrosis in mice, and downregulation of miR-542-3p by lentivirus could prevent the development of hepatic fibrosis. In addition, miR-542-3p can directly bind to the 3′-untranslated region of BMP-7 mRNA, indicating that its profibrotic effect appears to be caused by its inhibition of BMP-7. Our results suggest that downregulation of miR-542-3p prevents liver fibrosis both in vitro and in vivo, highlighting its potential as a novel biomarker or therapeutic target for hepatic fibrosis.     

Deficiency in augmenter of liver regeneration accelerates liver fibrosis by promoting migration of hepatic stellate cell

Background

Augmenter of liver regeneration (ALR) protects liver from various injuries, however, the association of ALR with liver fibrosis, particularly its effect on hepatic stellate cells (HSC), remains unclear. In this study, we investigated the impact of ALR on the activation of HSC, a pivotal event in occurrence of liver fibrosis.

miRNA-130b-5p promotes hepatic stellate cell activation and the development of liver fibrosis by suppressing SIRT4 expression

Liver fibrosis is a progressive disease accompanied by the deposition of extracellular matrix (ECM). Numerous reports have demonstrated that alterations in the expression of microRNAs (miRNAs) are related to liver disease. However, the effect of individual miRNAs on liver fibrosis has not been studied. Hepatic stellate cells (HSCs), being responsible for producing ECM, exert an important influence on liver fibrosis. Then, microarray analysis of non-activated and activated HSCs induced by transforming growth factor β1 (TGF-β1) showed that miR-130b-5p expression was strongly up-regulated during HSC activation. Moreover, the progression of liver fibrosis had a close connection with the expression of miR-130b-5p in different liver fibrosis mouse models. Then, we identified that there were specific binding sites between miR-130b-5p and the 3′ UTR of Sirtuin 4 (SIRT4) via a luciferase reporter assay. Knockdown of miR-130b-5p increased SIRT4 expression and ameliorated liver fibrosis in mice transfected with antagomiR-130b-5p oligos. In general, our results suggested that miR-130b-5p promoted HSC activation by targeting SIRT4, which participates in the AMPK/TGF-β/Smad2/3 signalling pathway. Hence, regulating miR-130b-5p maybe serve as a crucial therapeutic treatment for hepatic fibrosis.