A TLR4/MD2 fusion protein inhibits LPS-induced pro-inflammatory signaling in hepatic stellate cells

Activated hepatic stellate cells (HSCs) play a key role in hepatic fibrogenesis. In injured liver they are the main extracellular matrix protein producing cell type and further perpetuate hepatic injury by secretion of pro-inflammatory mediators. Since LPS-mediated signaling through toll-like receptor 4 (TLR4) has been identified as key fibrogenic signal in HSCs we aimed to test TLR4 as potential target of therapy via ligand-binding soluble receptors. Incubation of human HSCs with a fusion protein between the extracellular domain of TLR4 and MD2 which binds LPS inhibited LPS-induced NFκB and JNK activation. TLR4/MD2 abolished LPS-induced secretion of IL-6, IL-8, MCP1, and RANTES in HSCs. In addition, TLR4/MD2 fused to human IgG-Fc neutralized LPS activity. Since TLR4 mutant mice are resistant to liver fibrosis, the TLR4/MD2 soluble receptor might represent a new therapeutic molecule for liver fibrogenesis in vivo.     

MicroRNA-326 attenuates hepatic stellate cell activation and liver fibrosis by inhibiting TLR4 signaling

Hepatic fibrosis is a chronic inflammatory and reversible repair reaction of the liver under the continuous action of virus or various injuries. In this study, we aimed at identifying the role of miR-326 in the hepatic stellate cell (HSC) activation and liver fibrosis and its potential mechanism. In this study, the liver fibrosis mouse model was developed by injecting CCl₄. Liver tissue morphology was observed and the expression level of α-smooth muscle actin, collagen1α1 and miR-326 was measured. Target gene identification was performed by loss-of-function and gain-of-function. The effect of miR-326 on the expression level of the cytokines associated with the TLR4/MyD88/nuclear factor-κB (NF-κB) pathway was assessed in vitro and in vivo. We show that miR-326 was downregulated in CCl₄-induced fibrotic mice and activated HSCs. The target gene of miR-326 is TLR4. Moreover, miR-326 inhibited the activation of HSCs in vitro through TLR4/MyD88/NF-κB signaling. miR-326 attenuated hepatic fibrosis and inflammation of CCl₄-induced mice in vivo. Our results demonstrate for the first time that miR-326 inhibits HSC activation through TLR4/MyD88/NF-κB signaling. Furthermore, miR-326 plays critical roles in attenuating liver fibrosis and inflammation, suggesting the therapeutic potential of miRNAs.     

HGF and Direct Mesenchymal Stem Cells Contact Synergize to Inhibit Hepatic Stellate Cells Activation through TLR4/NF-kB Pathway

Aims

Bone marrow-derived mesenchymal stem cells (BMSCs) can reduce liver fibrosis. Apart from the paracrine mechanism by which the antifibrotic effects of BMSCs inhibit activated hepatic stellate cells (HSCs), the effects of direct interplay and juxtacrine signaling between the two cell types are poorly understood. The purpose of this study was to explore the underlying mechanisms by which BMSCs modulate the function of activated HSCs.

Methods

We used BMSCs directly and indirectly co-culture system with HSCs to evaluate the anti-fibrosis effect of BMSCs. Cell proliferation and activation were examined in the presence of BMSCs and HGF. c-met was knockdown in HSCs to evaluate the effect of HGF secreted by BMSCs. The TLR4 and Myeloid differentiation primary response gene 88(MyD88) mRNA levels and the NF-kB pathway activation were determined by real-time PCR and western blotting analyses. The effect of BMSCs on HSCs activation was investigated in vitro in either MyD88 silencing or overexpression in HSCs. Liver fibrosis in rats fed CCl₄ with and without BMSCs supplementation was compared. Histopathological examinations and serum biochemical tests were compared between the two groups.

Results

BMSCs remarkably inhibited the proliferation and activation of HSCs by interfering with LPS-TLR4 pathway through a cell–cell contact mode that was partially mediated by HGF secretion. The NF-kB pathway is involved in HSCs activation inhibition by BMSCs. MyD88 over expression reduced the BMSC inhibition of NF-kB luciferase activation. BMSCs protected liver fibrosis in vivo.

Conclusion

BMSCs modulate HSCs in vitro via TLR4/MyD88/NF-kB signaling pathway through cell–cell contact and secreting HGF. BMSCs have therapeutic effects on cirrhosis rats. Our results provide new insights into the treatment of hepatic fibrosis with BMSCs.     

MicroRNA-145 induces the senescence of activated hepatic stellate cells through the activation of p53 pathway by ZEB2

Activation of quiescent hepatic stellate cells (HSCs) is the major event in liver fibrosis, along with enhancement of cell proliferation and overproduction of extracellular matrix. Recent findings suggest that senescence of activated HSCs might limit the development of liver fibrosis. The p53, a guardian of the genome is associated with liver fibrosis, has been shown to regulate HSCs senescence. In this study, we report that microRNA-145 (miR-145) and p53 were downregulated in vivo and in vitro, concomitant with the enhanced expression of zinc finger E-box binding homeobox 2 (ZEB2). In addition, overexpression of miR-145 and p53 led to upregulation of the number of senescence-associated β-galactosidase-positive HSCs and the expression of senescence markers p16 and p21, along with the reduced abundance of HSC activation markers α-smooth muscle actin and type I collagen in activated HSCs. Furthermore, silencing of ZEB2 promoted senescence of activated HSCs. Moreover, we also demonstrated that miR-145 specifically targeted the 3′-untranslated regions of ZEB2. In vitro promoter regulation studies show that ZEB2 could bind to the E-box of the p53 promoter as well as inhibit its promoter activity and thus suppress the expression of p53, which in turn repressed activated HSCs senescence. Taken together, our results describe a novel miR-145-ZEB2-p53 regulatory line might participate in the senescence of activated HSCs and might carry potential therapeutic targets for restraining liver fibrosis.     

MyD88 in hepatic stellate cells enhances liver fibrosis via promoting macrophage M1 polarization

During liver fibrosis, quiescent HSCs (qHSCs) are activated to become activated HSCs (aHSCs)/myofibroblasts. The signal adapter MyD88, an essential component of TLR signaling, plays an important role in liver fibrosis. However, far less is known about the specific effects of MyD88 signaling in both qHSCs and aHSCs in the progress of liver fibrosis. Here, we used a CCl₄-induced mouse fibrosis model in which MyD88 was selectively depleted in qHSCs (GFAP^MyD88−/− mice) or aHSCs (α-SMA^MyD88−/− mice). MyD88 deficiency in qHSCs or aHSCs attenuated liver fibrosis in mice and inhibited α-SMA-positive cell activation. Inhibition of MyD88 in HSCs decreased α-SMA and collagen I levels, inflammatory cell infiltration, and pro-inflammatory gene expression. Furthermore, MyD88 signaling in HSCs increased the secretion of CXCL10, which promoted macrophage M1 polarization through CXCR3, leading to activation of the JAK/STAT1 pathway. Inhibition of CXCL10 attenuated macrophage M1 polarization and reduced liver fibrosis. Thus, MyD88 signaling in HSCs crucially contributes to liver fibrosis and provides a promising therapeutic target for the prevention and treatment of liver fibrosis.Subject terms: Mechanisms of disease, Kupffer cells     

Resolution of liver cirrhosis using vitamin A-coupled liposomes to deliver siRNA against a collagen-specific chaperone

There are currently no approved antifibrotic therapies for liver cirrhosis. We used vitamin A-coupled liposomes to deliver small interfering RNA (siRNA) against gp46, the rat homolog of human heat shock protein 47, to hepatic stellate cells. Our approach exploits the key roles of these cells in both fibrogenesis as well as uptake and storage of vitamin A. Five treatments with the siRNA-bearing vitamin A-coupled liposomes almost completely resolved liver fibrosis and prolonged survival in rats with otherwise lethal dimethylnitrosamine-induced liver cirrhosis in a dose- and duration-dependent manner. Rescue was not related to off-target effects or associated with recruitment of innate immunity. Receptor-specific siRNA delivery was similarly effective in suppressing collagen secretion and treating fibrosis induced by CCl(4) or bile duct ligation. The efficacy of the approach using both acute and chronic models of liver fibrosis suggests its therapeutic potential for reversing human liver cirrhosis.     

Augmenter of liver regeneration (ALR) gene therapy attenuates CCl₄-induced liver injury and fibrosis in rats

Liver fibrosis represents a process of healing and scarring in response to chronic liver injury. Augmenter of liver regeneration (ALR) has been shown to protect hepatocytes from various toxins. The aim of this study was to investigate the effects of ALR gene therapy on liver injury and fibrosis induced by CCl(4) in rats and further explore the underlying mechanisms. Human ALR expression plasmid was delivered via the tail vein. ALR gene therapy might protect the liver from CCl(4)-induced injury and fibrogenesis by attenuating the mitochondrial dysfunction, suppressing oxidative stress, and inhibiting activation of HSCs. This report demonstrated that ALR gene therapy protected against the ATP loss, increased the activity of ATPase, decreased intrahepatic reactive oxygen species level, and down-regulated transforming growth factor-β1, platelet-derived growth factor-BB, and α-smooth muscle actin expression. Following gene transfer liver function tests were significantly improved. In brief, ALR gene therapy might be an effective therapeutic reagent for liver fibrosis with potential clinical applications.     

Augmenter of liver regeneration (ALR) gene therapy attenuates CCl4-induced liver injury and fibrosis in rats

Liver fibrosis represents a process of healing and scarring in response to chronic liver injury. Augmenter of liver regeneration (ALR) has been shown to protect hepatocytes from various toxins. The aim of this study was to investigate the effects of ALR gene therapy on liver injury and fibrosis induced by CCl₄ in rats and further explore the underlying mechanisms. Human ALR expression plasmid was delivered via the tail vein. ALR gene therapy might protect the liver from CCl₄-induced injury and fibrogenesis by attenuating the mitochondrial dysfunction, suppressing oxidative stress, and inhibiting activation of HSCs. This report demonstrated that ALR gene therapy protected against the ATP loss, increased the activity of ATPase, decreased intrahepatic reactive oxygen species level, and down-regulated transforming growth factor-β1, platelet-derived growth factor-BB, and α-smooth muscle actin expression. Following gene transfer liver function tests were significantly improved. In brief, ALR gene therapy might be an effective therapeutic reagent for liver fibrosis with potential clinical applications.     

Targeted delivery of siRNA to hepatocytes and hepatic stellate cells by bioconjugation

Previously, we successfully conjugated galactosylated poly(ethylene glycol) (Gal-PEG) to oligonucleotides (ODNs) via an acid labile ester linker (Zhu et al., Bioconjugate Chem. 2008, 19, 290-8). In this study, sense strands of siRNA were conjugated to Gal-PEG and mannose 6-phosphate poly(ethylene glycol) (M6P-PEG) for targeted delivery of siRNAs to hepatocytes and hepatic stellate cells (HSCs), respectively. These siRNA conjugates were purified by ion exchange chromatography and verified by gel retardation assay. To evaluate their RNAi functions, the validated siRNA duplexes targeting firefly luciferase and transforming growth factor beta 1 (TGF-β1) mRNA were conjugated to Gal-PEG and M6P-PEG, and their gene silencing efficiencies were determined after transfection into HepG2 and HSC-T6 cells. The disulfide bond between PEG and siRNA was cleaved by dithiothreitol, leading to the release of intact siRNA. Both Gal-PEG-siRNA and M6P-PEG-siRNA conjugates could silence luciferase gene expression by about 40% without any transfection reagents, while the gene silencing effects reached more than 98% with the help of cationic liposomes at the same dose. Conjugation of TGF-β1 siRNA with Gal-PEG and M6P-PEG could silence endogenous TGF-β1 gene expression as well. In conclusion, these siRNA conjugates have the potential for targeted delivery of siRNAs to hepatocytes and hepatic stellate cells for efficient gene silencing in vivo.     

1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)-formulated, immune-stimulatory vascular endothelial growth factor a small interfering RNA (siRNA) increases antitumoral efficacy in murine orthotopic hepatocellular carcinoma with liver fibrosis

Most experimental therapy studies are performed in mice that bear subcutaneous or orthotopic hepatoma but are otherwise healthy and nonfibrotic. The majority of hepatocellular carcinoma (HCC), however, develops in patients suffering from preexisting liver fibrosis. We investigated the efficacy of a standard experimental therapeutic approach to interrupt the vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR) cascade via VEGF-A silencing, with or without 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP; cationic lipid) formulation, in HCC mice with preexisting liver fibrosis. The data show that intraperitoneal treatment with naked VEGF-A small interfering RNA (siRNA; 200 microg/kg) was inefficient to treat HCC implanted into fibrotic livers. VEGF-A siRNA containing an immunostimulatory motif in combination with DOTAP formulation significantly reduced hepatic VEGF-A expression and additionally activated the innate and adapted immune system as shown by an increased intrahepatic interferon type 1 response (68-fold increased beta-interferon expression). DOTAP-formulated VEGF-A siRNA markedly improved VEGF-A siRNA uptake and enhanced the antitumor response. This study shows for the first time the therapeutic feasibility of using synergistic effects (gene silencing and activation of the immune system) united in one siRNA sequence to reduce HCC growth and metastasis in mice with preexisting liver fibrosis. We expect that these results will help to direct and improve future experimental gene-silencing approaches and establish more efficient antitumoral therapies against HCC.     

PLK1 regulates hepatic stellate cell activation and liver fibrosis through Wnt/β‐catenin signalling pathway

As an outcome of chronic liver disease, liver fibrosis involves the activation of hepatic stellate cells (HSCs) caused by a variety of chronic liver injuries. It is important to explore approaches to inhibit the activation and proliferation of HSCs for the treatment of liver fibrosis. PLK1 is overexpressed in many human tumour cells and has become a popular drug target in tumour therapy. Therefore, further study of the function of PLK1 in the cell cycle is valid. In the present study, we found that PLK1 expression was elevated in primary HSCs isolated from CCl₄‐induced liver fibrosis mice and LX‐2 cells stimulated with TGF‐β1. Knockdown of PLK1 inhibited α‐SMA and Col1α1 expression and reduced the activation of HSCs in CCl₄‐induced liver fibrosis mice and LX‐2 cells stimulated with TGF‐β1. We further showed that inhibiting the expression of PLK1 reduced the proliferation of HSCs and promoted HSCs apoptosis in vivo and in vitro. Furthermore, we found that the Wnt/β‐catenin signalling pathway may be essential for PLK1‐mediated HSCs activation. Together, blocking PLK1 effectively suppressed liver fibrosis by inhibiting HSC activation, which may provide a new treatment strategy for liver fibrosis.     

Retinol binding protein-albumin domain III fusion protein deactivates hepatic stellate cells

Liver fibrosis is characterized by accumulation of extracellular matrix, and activated hepatic stellate cells (HSCs) are the primary source of the fibrotic neomatrix and considered as therapeutic target cells. We previously showed that albumin in pancreatic stellate cells (PSCs), the key cell type for pancreatic fibrogenesis, is directly involved in the formation of vitamin A-containing lipid droplets, inhibiting PSC activation. In this study, we evaluated the anti-fibrotic activity of both albumin and retinol binding protein-albumin domain III fusion protein (R-III), designed for stellate cell-targeted delivery of albumin III, in rat primary HSCs and investigated the underlying mechanism. Forced expression of albumin or R-III in HSCs after passage 2 (activated HSCs) induced lipid droplet formation and deactivated HSCs, whereas point mutations in high-affinity fatty acid binding sites of albumin domain III abolished their activities. Exogenous R-III, but not albumin, was successfully internalized into and deactivated HSC-P2. When HSCs at day 3 after plating (pre-activated HSCs) were cultured in the presence of purified R-III, spontaneous activation of HSCs was inhibited even after passage 2, suggestive of a potential for preventive effect. Furthermore, treatment of HSCs-P2 with R-III led to a significant reduction in both cytoplasmic levels of all-trans retinoic acid and the subsequent retinoic acid signaling. Therefore, our data suggest that albumin deactivates HSCs with reduced retinoic acid levels and that R-III may have therapeutic and preventive potentials on liver fibrosis.     

LncRNA Meg8 suppresses activation of hepatic stellate cells and epithelial-mesenchymal transition of hepatocytes via the Notch pathway

Long non-coding RNAs (lncRNAs) play an important role in various physiological and pathological processes. However, the biological role of lncRNA Meg8 in liver fibrosis is largely unknown. In this study, we found that Meg8 was over-expressed in activated hepatic stellate cells (HSCs), injured hepatocytes (HCs) and fibrotic livers. Furthermore, we revealed that Meg8 suppressed the expression of the pro-fibrogenic and proliferation genes in activated HSCs. In addition, silencing Meg8 significantly inhibited the expression of the epithelial markers, while noticeably promoted the expression of the mesenchymal markers in primary HCs and AML12 cells. Mechanistically, we demonstrated that Meg8 suppressed HSCs activation and epithelial-mesenchymal transition (EMT) of HCs through inhibiting the Notch pathway. In conclusion, our findings indicate that Meg8 may serve as a novel protective molecule and a potential therapeutic target of liver fibrosis.     

Selective activation of Toll-like receptor 7 in activated hepatic stellate cells may modulate their profibrogenic phenotype

Cholestatic liver injury may activate HSCs (hepatic stellate cells) to a profibrogenic phenotype, contributing to liver fibrogenesis. We have previously demonstrated the involvement of TLR (Toll-like receptor) 7?in the pathogenesis of biliary atresia. In the present study we investigated the ability of TLR7 to modulate the profibrogenic phenotype in HSCs. Obstructive jaundice was associated with significant down-regulation of TLR7. Primary HSCs isolated from BDL (bile duct ligation) rats with obstructive jaundice exhibited reduced expression of TLR7 and increased expression of α-SMA (α-smooth muscle actin) and collagen-α1 compared with sham rats, reflecting HSC-mediated changes. Treatment of primary activated rat HSCs and rat T6 cells with CL075, a TLR7 and TLR8 ligand, significantly decreased expression of MCP-1 (monocyte chemotactic protein-1), TGF-β1 (transforming growth factor-β1), collagen-α1 and MMP-2 (matrix metalloproteinase-2), and inhibited cell proliferation and migration. In contrast, silencing TLR7 expression with shRNA (short hairpin RNA) in T6 cells effectively blocked the effects of CL075 stimulation, reversing the changes in MCP-1, TGF-β1 and collagen-α1 expression and accelerating cell migration. Our results indicate that obstructive jaundice is associated with down-regulation of TLR7 and up-regulation of profibrogenic gene expression in HSCs. Selective activation of TLR7 may modulate the profibrogenic phenotype in activated HSCs associated with cholestatic liver injury.     

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.     

HucMSC-derived exosomes delivered BECN1 induces ferroptosis of hepatic stellate cells via regulating the xCT/GPX4 axis

Activated hepatic stellate cells (HSCs) are significant in liver fibrosis. Our past investigations have shown that human umbilical cord mesenchymal stem cells (hucMSCs) and their secreted exosomes (MSC-ex) could alleviate liver fibrosis via restraining HSCs activation. However, the mechanisms underlying the efficacy were not clear. Ferroptosis is a regulatory cell death caused by excessive lipid peroxidation, and it plays a vital role in the occurrence and development of liver fibrosis. In the present study, we aimed to study the proferroptosis effect and mechanism of MSC-ex in HSCs. MSC-ex were collected and purified from human umbilical cord MSCs. Proferroptosis effect of MSC-ex was examined in HSCs line LX-2 and CCl4 induced liver fibrosis in mice. Gene knockdown or overexpression approaches were used to investigate the biofactors in MSC-ex-mediated ferroptosis regulation. Results: MSC-ex could trigger HSCs ferroptosis by promoting ferroptosis-like cell death, ROS formation, mitochondrial dysfunction, Fe²⁺ release, and lipid peroxidation in human HSCs line LX-2. Glutathione peroxidase 4 (GPX4) is a crucial regulator of ferroptosis. We found that intravenous injection of MSC-ex significantly decreased glutathione peroxidase 4 (GPX4) expression in activated HSCs and collagen deposition in experimental mouse fibrotic livers. Mechanistically, MSC-ex derived BECN1 promoted HSCs ferroptosis by suppressing xCT-driven GPX4 expression. In addition, ferritinophagy and necroptosis might also play a role in MSC-ex-promoted LX-2 cell death. Knockdown of BECN1 in MSC diminished proferroptosis and anti-fibrosis effects of MSC-ex in LX-2 and fibrotic livers. MSC-ex may promote xCT/GPX4 mediated HSCs ferroptosis through the delivery of BECN1 and highlights BECN1 as a potential biofactor for alleviating liver fibrosis.Subject terms: Translational research, Stem-cell research     

肝纤维化过程中Toll样受体4在肝脏的表达分布及意义

目的动态观察大鼠肝纤维化过程中Toll样受体4（Toll-like receptor4,TLR4）蛋白在肝脏的表达,探讨TLR4与肝纤维化发生发展的关系。方法以四氯化碳皮下注射复制大鼠肝纤维化模型,设立正常对照组和模型1周组、2周组、4周组、6周组。常规HE染色和天狼猩红胶原染色观察肝脏病变;检测肝组织羟脯氨酸和血浆内毒素含量;免疫组化和Western blot检测TLR4在肝组织中的表达,检测α-SMA观察活化的肝星状细胞（HSCs）。结果与正常对照组比较,CCl4作用2周时,肝组织羟脯氨酸含量开始明显增多（P〈0.01）;模型组各组血浆内毒素含量呈梯度上升（P〈0.01）,且与肝组织羟脯氨酸含量呈显著正相关关系（P〈0.01）;CCl4作用1周后肝组织TLR4的表达即明显增强（P〈0.01）,4周时和6周时有所下降（与2周组相比,P〈0.05）,但仍高于正常对照组（P〈0.01）。TLR4阳性细胞包括枯否细胞、活化的HSCs及少量的肝细胞和内皮细胞。结论内毒素及其受体TLR4的改变可能在肝纤维化中起重要作用。