Attenuated Hepatic Stellate Cells Activation Research Articles

Expression of Cyclin E1 in hepatic stellate cells is critical for the induction and progression of liver fibrosis and hepatocellular carcinoma in mice

Hepatocellular carcinoma (HCC) is one of the most severe malignancies with increasing incidence and limited treatment options. Typically, HCC develops during a multistep process involving chronic liver inflammation and liver fibrosis. The latter is characterized by the accumulation of extracellular matrix produced by Hepatic Stellate Cells (HSCs). This process involves cell cycle re-entry and proliferation of normally quiescent HSCs in an ordered sequence that is highly regulated by cyclins and associated cyclin-dependent kinases (CDKs) such as the Cyclin E1 (CCNE1)/CDK2 kinase complex. In the present study, we examined the role of Cyclin E1 (Ccne1) and Cdk2 genes in HSCs for liver fibrogenesis and hepatocarcinogenesis. To this end, we generated conditional knockout mice lacking Ccne1 or Cdk2 specifically in HSCs (Ccne1∆HSC or Cdk2∆HSC). Ccne1∆HSC mice showed significantly reduced liver fibrosis formation and attenuated HSC activation in the carbon tetrachloride (CCl4) model. In a combined model of fibrosis-driven hepatocarcinogenesis, Ccne1∆HSC mice revealed decreased HSC activation even after long-term observation and substantially reduced tumor load in the liver when compared to wild-type controls. Importantly, the deletion of Cdk2 in HSCs also resulted in attenuated liver fibrosis after chronic CCl4 treatment. Single-cell RNA sequencing revealed that only a small fraction of HSCs expressed Ccne1/Cdk2 at a distinct time point after CCl4 treatment. In summary, we provide evidence that Ccne1 expression in a small population of HSCs is sufficient to trigger extensive liver fibrosis and hepatocarcinogenesis in a Cdk2-dependent manner. Thus, HSC-specific targeting of Ccne1 or Cdk2 in patients with liver fibrosis and high risk for HCC development could be therapeutically beneficial.

Fibroblast Activation Protein Activates Macrophages and Promotes Parenchymal Liver Inflammation and Fibrosis

Fibroblast activation protein (FAP) is expressed on activated fibroblast. Its role in fibrosis and desmoplasia is controversial, and data on pharmacological FAP inhibition are lacking. We aimed to better define the role of FAP in liver fibrosis invivo and invitro. FAP expression was analyzed in mice and patients with fibrotic liver diseases of various etiologies. Fibrotic mice received a specific FAP inhibitor (FAPi) at 2 doses orally for 2 weeks during parenchymal fibrosis progression (6 weeks of carbon tetrachloride) and regression (2 weeks off carbon tetrachloride), and with biliary fibrosis (Mdr2-/-). Recombinant FAP was added to (co-)cultures of hepatic stellate cells (HSC), fibroblasts, and macrophages. Fibrosis- and inflammation-related parameters were determined biochemically, by quantitative immunohistochemistry, polymerase chain reaction, and transcriptomics. FAP+ fibroblasts/HSCs were α-smooth muscle actin (α-SMA)-negative and located at interfaces of fibrotic septa next to macrophages in murine and human livers. In parenchymal fibrosis, FAPi reduced collagen area, liver collagen content, α-SMA+ myofibroblasts, M2-type macrophages, serum alanine transaminase and aspartate aminotransferase, key fibrogenesis-related transcripts, and increased hepatocyte proliferation 10-fold. During regression, FAP was suppressed, and FAPi was ineffective. FAPi less potently inhibited biliary fibrosis. Invitro, FAP small interfering RNA reduced HSC α-SMA expression and collagen production, and FAPi suppressed their activation and proliferation. Compared with untreated macrophages, FAPi regulated macrophage profibrogenic activation and transcriptome, and their conditioned medium attenuated HSC activation, which was increased with addition of recombinant FAP. Pharmacological FAP inhibition attenuates inflammation-predominant liver fibrosis. FAP is expressed on subsets of activated fibroblasts/HSC and promotes both macrophage and HSC profibrogenic activity in liver fibrosis.

Inhibition of protein arginine methyltransferase 1 alleviates liver fibrosis by attenuating the activation of hepatic stellate cells in mice.

Protein arginine methyltransferase 1 (PRMT1) has been reported to be involved in various diseases. The expression of PRMT1 was increased in cirrhotic livers from human patients. However, the role of PRMT1 in hepatic fibrogenesis remains largely unexplored. In this study, we investigated the effect of PRMT1 on hepatic fibrogenesis and its underlying mechanism. We found that PRMT1 expression was significantly higher in fibrotic livers of the mice treated with thioacetamide (TAA) or 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet. Immunofluorescence staining revealed that PRMT1 expression was augmented in both hepatocytes and hepatic stellate cells (HSCs) in the fibrotic livers. Applying a selective inhibitor of PRMT1, PT1001B, significantly suppressed PRMT1 activity and mitigated liver fibrosis in mice. Hepatocyte-specific Prmt1 knockout did not affect liver fibrosis in mice. PRMT1 overexpression promoted the expression of fibrotic genes in the LX-2 cells, whereas knockdown of PRMT1 or treatment with PT1001B exhibited reversal effects, suggesting that PRMT1 plays an important role in HSC activation. Additionally, HSC-specific Prmt1 knockout attenuated HSC activation and liver fibrosis in TAA-induced fibrotic model. RNA-seq analysis revealed that Prmt1 knockout in HSCs significantly suppressed pro-inflammatory NF-κB and pro-fibrotic TGF-β signals, and also downregulated the expression of pro-fibrotic mediators in mouse livers. Moreover, treatment with PT1001B consistently inhibited hepatic inflammatory response in fibrotic model. In conclusion, PRMT1 plays a vital role in HSC activation. Inhibition of PRMT1 mitigates hepatic fibrosis by attenuating HSC activation in mice. Therefore, targeting PRMT1 could be a feasible therapeutic strategy for liver fibrosis.

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

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.

Interleukin-10 Attenuates Liver Fibrosis Exacerbated by Thermoneutrality.

Background: Crosstalk between brown adipose tissue (BAT) and the liver is receiving increasing attention. This study investigated the effect of BAT dysfunction by thermoneutral (TN) housing on liver fibrosis in mice and examined the effect of secreted factors from brown adipocytes on the activation of hepatic stellate cells (HSCs).Methods: The carbon tetrachloride (CCl4)-induced liver fibrosis mouse model was used to evaluate fibrotic changes in the livers of mice housed under standard and TN conditions. The effect of BAT on the activation of HSCs was examined using cultured cells treated with conditioned media from brown adipocytes.Results: Under TN conditions, mice with CCl4-induced liver fibrosis exhibited increased liver injury, collagen deposition, and alpha smooth muscle actin (α-SMA) expression in the liver compared with mice maintained at room temperature. The numbers of liver-infiltrating immune cells and T cells producing IL-17A and IFN-γ were also significantly increased in the livers of mice housed under TN conditions. Treatment of HSCs with conditioned media from brown adipocytes markedly attenuated HSC activation, as shown by down-regulated α-SMA expression at day 4, day 7 and day 10 of culture. At thermoneutrality, with CCl4 administration, IL-10-deficient mice exhibited more severe liver fibrosis than wild-type mice. Interestingly, conditioned media from IL-10-deficient brown adipocytes could up-regulate the expression of α-SMA and induce HSCs activation.Conclusions: BAT inactivation by thermoneutrality contributes to the activation of pro-inflammatory and pro-fibrotic pathways in mice with CCl4-induced liver fibrosis. Normal brown adipocytes secreted factors that impair the activation of HSCs, while this protective effect was lost in IL-10-deficient brown adipocytes. Thus, the BAT–liver axis may serve as a potential therapeutic target for liver fibrosis, and IL-10 may be a key factor regulating the activation of HSCs by BAT.

Attenuation of fibrogenesis by PDE4 inhibitor via decreased cytoskeleton remodeling, activation and contractility of hepatic stellate cells

Liver fibrosis is characterized by accumulation of extracellular matrix (ECM) proteins that occurs due to chronic liver injury. Activated hepatic stellate cells (HSCs) are main source of ECM in the liver. Rho‐associated kinase (Rho‐kinase/ROCK) promotes the assembly of focal adhesions and actin stress fibers by regulating the organization of the cytoskeleton in HSCs. Exchange protein directly regulated by cAMP 1 (EPAC1) attenuated Rho kinase signaling in HSCs and alleviated the development of liver fibrosis. We previously showed that cAMP degrading phosphodiesterase 4 (PDE4) enzymes play a critical role in the pathogenesis of liver fibrosis suggesting that hepatic cAMP signaling is compromised during fibrogenesis. Based on these observations, we hypothesize that preserving cAMP/EPAC1 signaling by inhibiting PDE4 will attenuate HSC activation and fibrotic signaling.C57Bl/6 mice were subjected to repeated carbon tetrachloride (CCl4) injection for 4 weeks to estabilesh liver fibrosis. One group of mice received PDE4 inhibitor, Rolipram (targeted to the liver) twice a week. To examine the direct effect of EPAC1/PDE4 pathway on HSCs, in vitro studies using human HSC cell line (LX2) were also performed. Activation of LX2 HSCs was achieved by using recombinant TGFβ1. Liver tissue proteomic analysis was performed. Pathway enrichment analysis was done using Clarivate analytics software, MetaCore. Sirius Red staining and hydroxyproline (HYP) assay was performed to assess collagen deposition. Real time qPCR and Western blot were performed to validate the findings from proteomic analysis. Statistical analysis was performed using ANOVA.CCl4 administration resulted in a significant accumulation of collagen confirmed by Sirius red staining and hydroxyproline (HYP) content in mice. Importantly, Rolipram prevented HYP accumulation and the expression of genes involved in collagen synthesis (Serpinh1) and crosslinking (Loxl1 and Lox). Additionally, Smad3 (central modulator of canonical TGFβ1 signaling) activation and mRNA levels of Mmp2 and Timp2 were markedly decreased by Rolipram. Pathway enrichment analysis of proteomic data identified several fibrotic pathways impacted by Rolipram, including HSC activation, integrin signaling and cytoskeleton remodeling regulated by Rho‐kinase. Western blot analyses confirmed that Rolipram significantly decreased phosphorylation of myosin light chain (MLC), target of Rho‐associated protein kinase. The effect of PDE4 inhibition on TGFβ1 inducible Smad3 and MLC phosphorylation was recapitulated in LX2 cells by using Rolipram and EPAC1 specific agonist. Moreover, Rolipram decreased Endothelin 1 (ET1) levels both in vivo and in vitro LX2 cells indicating a reduced contractility of HSCs. In LX2 cells TGFβ1 inducible nuclear translocation of Yes‐associated protein (YAP) was also inhibited by Rolipram suggesting that PDE4 inhibition promotes the crosstalk between TGFβ1 and YAP pathway to repress TGFβ1 signaling.These results demonstrate that PDE4 inhibition attenuates liver fibrosis by affecting multiple pathways involved in HSC activation and perpetuation of fibrosis.Support or Funding InformationNIH P20GM113226

Nicotinamide riboside, an NAD+ precursor, attenuates the development of liver fibrosis in a diet-induced mouse model of liver fibrosis

ObjectiveLiver fibrosis is part of the non-alcoholic fatty liver disease (NAFLD) spectrum, which currently has no approved pharmacological treatment. In this study, we investigated whether supplementation of nicotinamide riboside (NR), a nicotinamide adenine dinucleotide (NAD+) precursor, can reduce the development of liver fibrosis in a diet-induced mouse model of liver fibrosis. MethodsMale C57BL/6 J mice were fed a low-fat control (LF), a high-fat/high-sucrose/high-cholesterol control (HF) or a HF diet supplemented with NR at 400 mg/kg/day (HF-NR) for 20 weeks. Features of liver fibrosis were assessed by histological and biochemical analyses. Whole-body energy metabolism was also assessed using indirect calorimetry. Primary mouse and human hepatic stellate cells were used to determine the anti-fibrogenic effects of NR in vitro. ResultsNR supplementation significantly reduced body weight of mice only 7 weeks after mice were on the supplementation, but did not attenuate serum alanine aminotransferase levels, liver steatosis, or liver inflammation. However, NR markedly reduced collagen accumulation in the liver. RNA-Seq analysis suggested that the expression of genes involved in NAD+ metabolism is altered in activated hepatic stellate cells (HSCs) compared to quiescent HSCs. NR inhibited the activation of HSCs in primary mouse and human HSCs. Indirect calorimetry showed that NR increased energy expenditure, likely by upregulation of β-oxidation in skeletal muscle and brown adipose tissue. ConclusionNR attenuated HSC activation, leading to reduced liver fibrosis in a diet-induced mouse model of liver fibrosis. The data suggest that NR may be developed as a potential preventative for human liver fibrosis.

Selective overexpression of cytoglobin in stellate cells attenuates thioacetamide-induced liver fibrosis in mice

Cytoglobin (CYGB), discovered in hepatic stellate cells (HSCs), is known to possess a radical scavenger function, but its pathophysiological roles remain unclear. Here, for the first time, we generated a new transgenic (TG) mouse line in which both Cygb and mCherry reporter gene expression were under the control of the native Cygb gene promoter. We demonstrated that the expression of Cygb-mCherry was related to endogenous Cygb in adult tissues by tracing mCherry fluorescence together with DNA, mRNA, and protein analyses. Administration of a single dose (50 mg/kg) of thioacetamide (TAA) in Cygb-TG mice resulted in lower levels of alanine transaminase and oxidative stress than those in WT mice. After 10 weeks of TAA administration, Cygb-TG livers exhibited reduced neutrophil accumulation, cytokine expression and fibrosis but high levels of quiescent HSCs. Primary HSCs isolated from Cygb-TG mice (HSCCygb-TG) exhibited significantly decreased mRNA levels of α-smooth muscle actin (αSMA), collagen 1α1, and transforming growth factor β-3 after 4 days in culture relative to WT cells. HSCsCygb-TG were resistant to H2O2-induced αSMA expression. Thus, cell-specific overexpression of Cygb attenuates HSC activation and protects mice against TAA-induced liver fibrosis presumably by maintaining HSC quiescence. Cygb is a potential new target for antifibrotic approaches.

PROTECTIVE EFFECT OF DIOSGENIN AGAINST CARBON TETRACHLORIDE AND CISPLATIN INDUCED HEPATOTOXICITY IN RATS

Background: Activation of hepatic stellate cells (HSC) plays central role in the development of liver fibrosis. In HSC activation, the transforming growth factor-1 (TGF-1) is considered to be the main stimuli factor. Diosgenin are the steroidal saponin and found in Trigonella foenum graecum Linn (Fenugreek) and some other species of Dioscorea. Diosgenin attenuates HSC activation by inhibiting transforming growth factor. Aim: In present study an attempt was made to explore the effect of diosgenin on liver fibrosis. Methods: Liver fibrosis was induced in rats by carbon tetrachloride (CCl4) 1 ml/kg intraperitoneally twice a week for 28 days and cisplatin 3mg/kg intraperitoneally at 0, 1, 3 week for 4 weeks. The extent of liver fibrosis was assessed by measuring the weight of liver and levels of total bilirubin (TBL), hydroxyproline (HP) and serum enzymes due to deposition of extracellular matrix (ECM). Results: The administration of diosgenin reduced the liver weight of CCl4 and cisplatin treated animals and reduced the TBL, HP level and serum enzymes significantly and inhibited liver fibrosis induced by CCl4 and cisplatin. Conclusion: The result obtained in the present investigation, Diosgenin treatment exerted significant hepatoprotective effect in animals by inhibiting ECM deposition and HSCs activation.

DNMT1 controls LncRNA H19/ERK signal pathway in hepatic stellate cell activation and fibrosis

Hepatic stellate cells (HSCs) activation is considered as a pivotal event in liver fibrosis. In HSCs activation and fibrosis, epigenetic events are important. Although HSCs activation alters DNA methylation, it is unknown, whether it also affects other epigenetic processes, including LncRNA and its recognition. The aim of this study was to identify the mechanism of DNA methyltransferase 1 (DNMT1) expression and its role in regulating LncRNA H19 during HSCs activation and fibrosis. Expression of DNMT1 and LncRNA H19 were determined in activated HSCs and CCl4-induced rat liver fibrosis tissue. The relationship between the LncRNA H19 and DNMT1 expression was examined in vitro. LncRNA H19 expression was reduced in activated HSCs and rat liver fibrosis tissue, whereas DNMT1 expression and methylation of the LncRNA H19 promoter were increased. Treatment of HSCs of DNMT1-siRNA blocked cell proliferation. Knockdown of DNMT1 elevated H19 expression in activated HSCs, and over-expression of DNMT1 inhibited H19 expression in activated HSCs. Moreover, we investigated the effect of H19 on ERK signal pathway. Treatment HSCs with H19-siRNA increased the expression of p-ERK1/2 in HSCs. Treatment with 5′-aza-2′-deoxycytidine in activated HSCs model reduced fibrosis gene and DNMT1 expression, enhanced H19 expression, and attenuated HSCs activation. These data connect HSCs activation with a DNMT1-LncRNA H19 epigenetic pathway that is important for liver fibrosis.

Insulin signaling as a potential natural killer cell checkpoint in fatty liver disease.

Insulin resistance is a key risk factor in the progression of nonalcoholic fatty liver disease (NAFLD) and may lead to liver fibrosis. Natural killer (NK) cells are thought to exert an antifibrotic effect through their killing of activated hepatic stellate cells (HSCs). Here, we investigated how the interplay between NK cells and HSCs are modified by insulin resistance in NAFLD. Fresh peripheral blood NK cells (clusters of differentiation [CD]56dim, CD16+) were collected from 22 healthy adults and 72 patients with NAFLD not currently taking any medications and without signs of metabolic syndrome. NK cells were assessed for insulin receptor expressions and cytotoxic activity when cultured in medium with HSCs. Fibrosis severities in patients with NAFLD were correlated linearly with elevated serum proinflammatory cytokine expression and insulin resistance severity. At the same time, fibrosis severities inversely correlated with insulin receptor expressions on NK cells as well as with their cytotoxic activities determined by CD107a by flow cytometry. NK cells from donors exhibiting severe fibrosis and insulin resistance exhibited significant mammalian target of rapamycin and extracellular signal‐regulated kinase depletion (through NK cell western blot quantitation), increased apoptosis, and failure to attenuate HSC activation in vitro. While exposure to insulin stimulated the cytotoxic activity of healthy NK cells, rapamycin prevented this effect and reduced NK insulin receptor expressions. Conclusion: Elevated insulin levels in F1 and F2 fibrosis enhances NK cell cytotoxic activity toward HSCs and prevents fibrosis progression by insulin receptors and downstream mammalian target of rapamycin and extracellular signal‐regulated kinase pathways. At more advanced stages of insulin resistance (F3 and F4 fibrosis), impaired NK cell activity rooted in low insulin receptor expression and or low serum insulin levels could further deteriorate fibrosis and may likely lead to cirrhosis development. (Hepatology Communications 2018;2:285‐298)

Hepatic stellate cell-specific deletion of SIRT1 exacerbates liver fibrosis in mice

Liver fibrosis is widely perceived as a host defense mechanism that aids tissue repair following liver injury. Excessive fibrogenesis, however, serves to disrupt normal liver structure and precedes such irrevocable human pathologies as cirrhosis and hepatocellular carcinoma. Activation of hepatic stellate cells (HSCs) is a hallmark event during liver fibrosis. In the present study we investigated the mechanism by which the lysine deacetylase SIRT1 regulates HSC activation. We report here that SIRT1 levels were decreased in the liver in different mouse models and in cultured HSCs undergoing activation. SIRT1 down-regulation paralleled HDAC4 up-regulation. HDAC4 was recruited to the SIRT1 promoter during HSC activation and removed acetylated histones H3 and H4 from the SIRT1 promoter leading to SIRT1 trans-repression. HDAC4 silencing restored SIRT1 expression and attenuated HSC activation in SIRT1-dependent manner. More important, selective deletion of SIRT1 in HSCs exacerbated CCl4-induced liver fibrosis in mice. Mechanistically, SIRT1 deacetylated PPARγ to block HSC activation. Together, our data reveal an HDAC4-SIRT1-PPARγ axis that contributes to the regulation of HSC activation and liver fibrosis.

Lysosome-mediated degradation of a distinct pool of lipid droplets during hepatic stellate cell activation

Activation of hepatic stellate cells (HSCs) is a critical step in the development of liver fibrosis. During activation, HSCs lose their lipid droplets (LDs) containing triacylglycerols (TAGs), cholesteryl esters, and retinyl esters (REs). We previously provided evidence for the presence of two distinct LD pools, a preexisting and a dynamic LD pool. Here we investigate the mechanisms of neutral lipid metabolism in the preexisting LD pool. To investigate the involvement of lysosomal degradation of neutral lipids, we studied the effect of lalistat, a specific lysosomal acid lipase (LAL/Lipa) inhibitor on LD degradation in HSCs during activation in vitro The LAL inhibitor increased the levels of TAG, cholesteryl ester, and RE in both rat and mouse HSCs. Lalistat was less potent in inhibiting the degradation of newly synthesized TAG species as compared with a more general lipase inhibitor orlistat. Lalistat also induced the presence of RE-containing LDs in an acidic compartment. However, targeted deletion of the Lipa gene in mice decreased the liver levels of RE, most likely as the result of a gradual disappearance of HSCs in livers of Lipa-/- mice. Lalistat partially inhibited the induction of activation marker α-smooth muscle actin (α-SMA) in rat and mouse HSCs. Our data suggest that LAL/Lipa is involved in the degradation of a specific preexisting pool of LDs and that inhibition of this pathway attenuates HSC activation.

Wnt2b attenuates HSCs activation and liver fibrosis through negative regulating TLR4 signaling

The Wingless-type MMTV integration site family member 2b (Wnt2b) has been found to be a principal mediator of liver development and regeneration. However, the significance of Wnt2b in the pathogenesis of fibrosis-related liver diseases remains undefined. Here, we report that Wnt2b was highly expressed in the fibrotic liver tissues, exhibiting protective effects against activation of hepatic stellate cells (HSCs) and fibrosis progression. We identified a negative regulation of Wnt2b on the toll-like receptor 4 (TLR4) activation-mediated pro-fibrogenic effects. Wnt2b was shown not only to directly suppress LPS-induced HSCs activation, but also to inhibit TLR4-enhanced the sensitivity of HSCs to transforming growth factor beta (TGF-β). Mechanistic study showed that Wnt2b suppresses TLR4 signaling through inhibiting the expression of TLR4 as well as the activation of NF-κB and MAPKs. These findings provided new insights into the pathophysiology of liver fibrosis by characterizing Wnt2b as a novel endogenous suppressor of TLR4 signaling, maintaining tissue homeostasis during the early stage of hepatic fibrosis-associated liver diseases.

Knockdown of Fstl1 attenuates hepatic stellate cell activation through the TGF‑β1/Smad3 signaling pathway.

Follistatin‑like 1 (Fstl1) is a secreted glycoprotein that belongs to the follistatin and SPARC (secreted protein, acidic and rich in cysteine) families and was identified to serve a critical role in lung fibrosis. However, the role of Fstl1 in liver fibrosis remains undefined. Therefore, the aim of the present study was to investigate the role of Fstl1 in liver fibrosis. The results indicated that Fstl1 was highly expressed in human hepatic fibrosis tissues and activated hepatic stellate cells (HSCs). Furthermore, knockdown of Fstl1effectively suppressed HSC proliferation and the protein expression levels of α‑SMA and collagen I in transforming growth factor (TGF)‑β1‑treated HSCs. Mechanistically, knockdown of Fstl1 remarkably decreased the phosphorylation level of Smad3 in TGF‑β1‑induced HSCs. Taken together, the present study demonstrated that Fstl1serves an important role in liver fibrosis and target deletion of Fstl1 attenuated HSCs activation through suppressing TGF‑β1/Smad3 signaling pathway. Therefore, Fstl1 may be a potential therapeutic target for the treatment of liver fibrosis.

Sirtuin 3 (SIRT3) Regulates α-Smooth Muscle Actin (α-SMA) Production through the Succinate Dehydrogenase-G Protein-coupled Receptor 91 (GPR91) Pathway in Hepatic Stellate Cells

Sirtuin 3 (SIRT3) is an NAD(+)-dependent protein deacetylase. Recent studies have shown that SIRT3 expression is decreased in nonalcoholic fatty liver disease (NAFLD). Moreover, SIRT3 is a key regulator of succinate dehydrogenase (SDH), which catalyzes the oxidation of succinate to fumarate. Increased succinate concentrations and the specific G protein-coupled receptor 91 (GPR91) are involved in the activation of hepatic stellate cells (HSCs). In this study, we aimed to establish whether SIRT3 regulated the SDH activity, succinate, and GPR91 expression in HSCs and an animal model of NAFLD. Our goal was also to determine whether succinate released from hepatocytes regulated HSC activation. Inhibiting SIRT3 using SIRT3 siRNA exacerbated HSC activation via the SDH-succinate-GPR91 pathway, and SIRT3 overexpression or honokiol treatment attenuated HSC activation in vitro In isolated liver and HSCs from methionine- and choline-deficient (MCD) diet-induced NAFLD, the expression of SIRT3 and SDH activity was decreased, and the succinate concentrations and GPR91 expression were increased. Moreover, we found that GPR91 knockdown or resveratrol treatment improved the steatosis in MCD diet-fed mice. This investigation revealed a novel mechanism of the SIRT3-SDH-GPR91 cascade in MCD diet-induced HSC activation in NAFLD. These findings highlight the biological significance of novel strategies aimed at targeting SIRT3 and GPR91 in HSCs with the goal of improving NAFLD treatment.

Effectiveness of Losartan-Loaded Hyaluronic Acid (HA) Micelles for the Reduction of Advanced Hepatic Fibrosis in C3H/HeN Mice Model.

Advanced hepatic fibrosis therapy using drug-delivering nanoparticles is a relatively unexplored area. Angiotensin type 1 (AT1) receptor blockers such as losartan can be delivered to hepatic stellate cells (HSC), blocking their activation and thereby reducing fibrosis progression in the liver. In our study, we analyzed the possibility of utilizing drug-loaded vehicles such as hyaluronic acid (HA) micelles carrying losartan to attenuate HSC activation. Losartan, which exhibits inherent lipophilicity, was loaded into the hydrophobic core of HA micelles with a 19.5% drug loading efficiency. An advanced liver fibrosis model was developed using C3H/HeN mice subjected to 20 weeks of prolonged TAA/ethanol weight-adapted treatment. The cytocompatibility and cell uptake profile of losartan-HA micelles were studied in murine fibroblast cells (NIH3T3), human hepatic stellate cells (hHSC) and FL83B cells (hepatocyte cell line). The ability of these nanoparticles to attenuate HSC activation was studied in activated HSC cells based on alpha smooth muscle actin (α-sma) expression. Mice treated with oral losartan or losartan-HA micelles were analyzed for serum enzyme levels (ALT/AST, CK and LDH) and collagen deposition (hydroxyproline levels) in the liver. The accumulation of HA micelles was observed in fibrotic livers, which suggests increased delivery of losartan compared to normal livers and specific uptake by HSC. Active reduction of α-sma was observed in hHSC and the liver sections of losartan-HA micelle-treated mice. The serum enzyme levels and collagen deposition of losartan-HA micelle-treated mice was reduced significantly compared to the oral losartan group. Losartan-HA micelles demonstrated significant attenuation of hepatic fibrosis via an HSC-targeting mechanism in our in vitro and in vivo studies. These nanoparticles can be considered as an alternative therapy for liver fibrosis.

Schistosoma japonicum protein SjP40 inhibits TGF-β1-induced activation of hepatic stellate cells.

SjP40 is a major egg antigen of Schistosoma japonicum. In the present study, the authors investigated the effect of SjP40 in vitro on transforming growth factor-β1 (TGF-β1)- stimulated hepatic stellate cells (HSCs). LX-2, an immortalized human HSC line, was treated with purified recombinant SjP40 (rSjP40) in the presence or absence of TGF-β1. Quantitative real-time polymerase chain reaction and western blot analysis were performed to determine messenger ribonucleic acid and protein of fibrogenic genes and TGF-β signaling pathway. The results showed that expression of fibrogenic genes was significantly reduced by rSjP40. Furthermore, rSjP40 also suppressed the TGF-β1-induced upregulation of Smads and ERK proteins. We also found that the effect of rSjP40 on HSCs was similar to SB431542, an inhibitor of type I TGF-β receptor. In conclusion, the data suggest that SjP40 attenuates HSC activation, which might be, at least in part, mediated by inhibiting the TGF-β and ERK signaling pathways.

Mycophenolic acid induces apoptosis of hepatic stellate cells in an in vitro model of HCV

Introduction. Hepatitis C virus (HCV) infection is an important risk factor for the development of liver fibrosis and progression to cirrhosis. Liver transplantation as terminal treatment option for liver disease requires life-long immunosuppression. However, immunomodulatory therapy may promote reinfection and renewed fibrogenesis. Immunosupressive agents may also affect the life cycle of hepatic stellate cells (HSC), the main source of extracellular matrix. We thus aimed to characterize the effects of three common immunosuppressive agents on HSC apoptosis with or without engulfment of HCV infected apoptotic bodies.Material and methods. LX2 cells were incubated with three different immunosuppressants (rapamycine, mycophenolic acid or cyclosporine A) and co-incubated for 24 and 48 h with apoptotic bodies (AB), produced from Huh7 cells or from Con1 cells (Huh7-cells containing a subgenomic HCV replicon). The engulfment of AB was confirmed by immunofluorescence staining. HSC viability, apoptosis rate and expression of profibrogenic and proapoptotic genes were quantified.Results. In LX2 cells that engulfed Conl AB, the treatment with mycophenolic acid induced HSC apoptosis and reduced collagen lalpha 1 expression compared to cylosporine A or rapamycine treatment. In conclusion mycophenolic acid is a potent inducer of HSC apoptosis and attenuates HSC activation and consecutively fibrogenesis in HCV infection. Translational studies will need to confirm these mono-culture results in vivo.

Interleukin-22 ameliorates liver fibrogenesis by attenuating hepatic stellate cell activation and downregulating the levels of inflammatory cytokines.

To investigate the effect of interleukin (IL)-22 on hepatic fibrosis in mice and the possible mechanism involved. Liver fibrosis was induced in male BALB/c mice by CCl₄. Recombinant IL-22 (rmIL-22) was administered intraperitoneally in CCl₄-treated mice. Fibrosis was assessed by histology and Masson staining. The activation of hepatic stellate cells (HSCs) was investigated by analysis of α-smooth muscle actin expression. The frequencies of T helper (Th) 22 cells, Th17 cells and Th1 cells, the expression of inflammatory cytokines [IL-22, IL-17A, interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), IL-6, IL-1β] and transcription factors [aryl hydrocarbon receptor (AHR), RAR-related orphan receptor (RORγt), T-bet] mRNA in the liver were investigated. In addition, the plasma levels of IL-22, IL-17A, IFN-γ, TNF-α, IL-6 and IL-1β were evaluated. Significant elevations in circulating Th22 cells, Th17 cells, Th1 cells, IL-22, IL-17A, and IFN-γ were observed in the hepatic fibrosis group compared with the control group (P < 0.01). Treatment with rmIL-22 in mice with hepatic fibrosis ameliorated the severity of hepatic fibrosis, which was confirmed by lower hepatic fibrosis pathological scores (P < 0.01). RmIL-22 decreased the frequencies of Th22 cells (6.71% ± 0.97% vs 8.09% ± 0.74%, P < 0.01), Th17 cells (4.34% ± 0.37% vs 5.71% ± 0.24%, P < 0.01), Th1 cells (3.09% ± 0.49% vs 4.91% ± 0.73%, P < 0.01), and the levels of IL-22 (56.23 ± 3.08 vs 70.29 ± 3.01, P < 0.01), IL-17A (30.74 ± 2.77 vs 45.68 ± 2.71, P < 0.01), and IFN-γ (74.78 ± 2.61 vs 124.89 ± 2.82, P < 0.01). Down-regulation of IL-22, IL-17A, IFN-γ, TNF-α, IL-6, IL-1β, AHR RORγt, and T-bet gene expression in the liver was observed in the rmIL-22 group (P < 0.01). The frequencies of Th22, Th17 and Th1 cells are elevated in hepatic fibrosis. RmIL-22 can attenuate HSC activation and down-regulate the levels of inflammatory cytokines, thereby ameliorating liver fibrogenesis.