STING-mediated inflammation contributes to Gao binge ethanol feeding model.

Alcohol metabolism causes hepatocytes to release damage-associated molecular patterns (DAMPs). This includes mitochondrial DNA (mtDNA), which is generated and released from damaged hepatocytes and contributes to liver injury by producing proinflammatory cytokines. STING is a pattern recognition receptor of DAMPs known to control the induction of innate immunity in various pathological processes. However, the expression profile and functions of STING in the Gao binge ethanol model remain poorly understood. We demonstrated that STING is upregulated in the Gao binge ethanol model. STING functions as an mtDNA sensor in the Kupffer cells of the liver and induces STING-signaling pathway-dependent inflammation and further aggravates hepatocyte apoptosis in the Gao binge ethanol model. This study provides novel insights into predicting disease progression and developing targeted therapies for alcoholic liver injury.

Alternative activation of macrophages by prostacyclin synthase ameliorates alcohol induced liver injury.

Alcoholic liver disease (ALD) is a major cause of chronic liver disease worldwide. Macrophages exhibit different functional states and are classified as classically activated (M1) and alternatively activated (M2) macrophages. However, the mechanisms that govern M1/M2 polarization in chronic ALD remain to be elucidated. Prostacyclin (PGI2) synthase (PTGIS) is an enzyme of the prostaglandin pathway which catalyzes the conversion of Prostaglandin H2 (PGH2) to PGI2. PTGIS has anti-inflammatory properties. However, the function of PTGIS in ALD has not yet been determined. In this study, we demonstrated that PTGIS was downregulated in ALD and forced PTGIS expression in vivo using recombinant adeno-associated viral vector-packed PTGIS overexpression plasmid, which alleviated the inflammatory response and suppressed the macrophage M1 phenotype in mice. Loss- and gain-of function-experiments demonstrated that forced PTGIS expression inhibited the macrophage switch to the M1 phenotype and promoted M2 polarization. Furthermore, we identified the genes regulated by PTGIS through RNA-sequencing (RNA-seq) analysis. Gene ontology and KEGG pathway analyses showed that PTGIS regulates many genes involved in the immune response and is enriched in the Janus kinase/signal transducers and activators of transcription (JAK/STAT) signal transduction pathway, which plays an important role in regulating macrophage polarization. The proteins interacting with JAKs were predicted using the STRING database. The overlap between the RNA-seq and the STRING database was interleukin-6; this indicated that it was involved in macrophage polarization regulated by JAK/STAT signaling. We further explored the microRNAs that could regulate the expression of PTGIS through TargetScan. The results of luciferase assay illustrated that the expression of PTGIS was regulated by miR-140-3p.1. These results imply that PTGIS plays a pivotal role in ALD, partly by influencing macrophage polarization.

The emerging roles of m6A modification in liver carcinogenesis.

The 'epitranscriptome', a collective term for chemical modifications that influence the structure, metabolism, and functions of RNA, has recently emerged as vitally important for the regulation of gene expression. N6-methyladenosine (m6A), the most prevalent mammalian mRNA internal modification, has been demonstrated to have a pivotal role in almost all vital bioprocesses, such as stem cell self-renewal and differentiation, heat shock or DNA damage response, tissue development, and maternal-to-zygotic transition. Hepatocellular carcinoma (HCC) is prevalent worldwide with high morbidity and mortality because of late diagnosis at an advanced stage and lack of effective treatment strategies. Epigenetic modifications including DNA methylation and histone modification have been demonstrated to be crucial for liver carcinogenesis. However, the role and underlying molecular mechanism of m6A in liver carcinogenesis are mostly unknown. In this review, we summarize recent advances in the m6A region and how these new findings remodel our understanding of m6A regulation of gene expression. We also describe the influence of m6A modification on liver carcinoma and lipid metabolism to instigate further investigations of the role of m6A in liver biological diseases and its potential application in the development of therapeutic strategies.

Circular RNA circFBXW4 suppresses hepatic fibrosis via targeting the miR-18b-3p/FBXW7 axis.

Rationale: Circular RNAs (circRNAs) are a new form of noncoding RNAs that play crucial roles in various pathological processes. However, the expression profile and function of circRNAs in hepatic fibrosis (HF) remain largely unknown. In this study, we show a novel circFBXW4 mediates HF via targeting the miR-18b-3p/FBXW7 axis. Methods: We investigated the expression profile of circRNAs, microRNAs and mRNAs in hepatic stellate cells (HSCs) from HF progression and regression mice by circRNAs-seq and microarray analysis. We found a significantly dysregulated circFBXW4 in HF. Loss-of-function and gain-of-function analysis of circFBXW4 were performed to assess the role of circFBXW4 in HF. Furthermore, we confirmed that circFBXW4 directly binds to miR-18b-3p by luciferase reporter assay, RNA pull down and fluorescence in situ hybridization analysis. Results: We found that circFBXW4 downregulated in liver fibrogenesis. Enforcing the expression of circFBXW4 inhibited HSCs activation, proliferation and induced apoptosis, attenuated mouse liver fibrogenesis injury and showed anti-inflammation effect. Mechanistically, circFBXW4 directly targeted to miR-18b-3p to regulate the expression of FBXW7 in HF. Conclusions: circFBXW4 may act as a suppressor of HSCs activation and HF through the circFBXW4/miR-18b-3p/FBXW7 axis. Our findings identify that circFBXW4 serves as a potential biomarker for HF therapy.

Hesperetin derivative attenuates CCl4-induced hepatic fibrosis and inflammation by Gli-1-dependent mechanisms.

Hepatic fibrosis, a common pathological feature and leading cause of various chronic liver diseases, still lacks effective therapy. Hesperetin derivative (HD) is a derivative of Traditional Chinese Medicine monomer isolated from the fruit peel of Citrusaurantium L. (Rutaceae). In the present study, we revealed the anti-fibrotic effects of HD in CCl4-induced mouse hepatic fibrosis model and in TGF-ß1-activated LX-2 cells, in vivo and in vitro. Results showed that HD prevented CCl4-induced liver injury and histological damage. Consistently, HD inhibited the up-regulation of liver fibrogenesis markers α-SMA, Col1α1, Col3α1 and TIMP-1 in primary hepatic stellate cells (HSCs) and suppressed inflammatory responses in primary liver macrophages from hepatic fibrosis mice. Furthermore, HD promoted the apoptosis of activated HSCs, a key step in the onset of fibrosis regression. Mechanistically, the Hedgehog pathway was involved in HD-treated hepatic fibrosis, and HD specifically contributed to attenuate the aberrant expression of Glioma associated oncogene-1 (Gli-1). Interestingly, blockade of Gli-1 removed the inhibitory effect of HD on activated HSCs, indicating that Gli-1 may play a pivotal role in mediating the anti-fibrotic effect of HD in hepatic fibrosis. Collectively, our results suggest that HD may be a potential anti-fibrotic Traditional Chinese Medicine monomer for the treatment of hepatic fibrosis.

Methylation of RCAN1.4 mediated by DNMT1 and DNMT3b enhances hepatic stellate cell activation and liver fibrogenesis through Calcineurin/NFAT3 signaling.

Background: Liver fibrosis is characterized by extensive deposition of extracellular matrix (ECM) components in the liver. RCAN1 (regulator of calcineurin 1), an endogenous inhibitor of calcineurin (CaN), is required for ECM synthesis during hypertrophy of various organs. However, the functional role of RCAN1 in liver fibrogenesis has not yet been addressed. Methods: We induced experimental liver fibrosis in mice by intraperitoneal injection of 10 % CCl4 twice a week. To investigate the functional role of RCAN1.4 in the progression of liver fibrosis, we specifically over-expressed RCAN1.4 in mice liver using rAAV8-packaged RCAN1.4 over-expression plasmid. Following the establishment of the fibrotic mouse model, primary hepatic stellate cells were isolated. Subsequently, we evaluated the effect of RCAN1.4 on hepatic fibrogenesis, hepatic stellate cell activation, and cell survival. The biological role and signaling events for RCAN1 were analyzed by protein-protein interaction (PPI) network. Bisulfite sequencing PCR (BSP) was used to predict the methylated CpG islands in the RCAN1.4 gene promoter. We used the chromatin immunoprecipitation (ChIP assay) to investigate DNA methyltransferases which induced decreased expression of RCAN1.4 in liver fibrosis. Results: Two isoforms of RCAN1 protein were expressed in CCl4-induced liver fibrosis mouse model and HSC-T6 cells cultured with transforming growth factor-beta 1 (TGF-ß1). RCAN1 isoform 4 (RCAN1.4) was selectively down-regulated in vivo and in vitro. The BSP analysis indicated the presence of two methylated sites in RCAN1.4 promoter and the downregulated RCAN1.4 expression levels could be restored by 5-aza-2'-deoxycytidine (5-azadC) and DNMTs-RNAi transfection in vitro. ChIP assay was used to demonstrate that the decreased RCAN1.4 expression was associated with DNMT1 and DNMT3b. Furthermore, we established a CCl4-induced liver fibrosis mouse model by injecting the recombinant adeno-associated virus-packaged RCAN1.4 (rAAV8-RCAN1.4) over-expression plasmid through the tail vein. Liver- specific-over-expression of RAN1.4 led to liver function recovery and alleviated ECM deposition. The key protein (a member of the NFAT family of proteins) identified on PPI network data was analyzed in vivo and in vitro. Our results demonstrated that RCAN1.4 over-expression alleviates, whereas its knockdown exacerbates, TGF-ß1-induced liver fibrosis in vitro in a CaN/NFAT3 signaling-dependent manner. Conclusions: RCAN1.4 could alleviate liver fibrosis through inhibition of CaN/NFAT3 signaling, and the anti-fibrosis function of RCAN1.4 could be blocked by DNA methylation mediated by DNMT1 and DNMT3b. Thus, RCAN1.4 may serve as a potential therapeutic target in the treatment of liver fibrosis.

SUN2: A potential therapeutic target in cancer.

The incidence of cancer is increasing at an alarming rate despite recent advances in prevention strategies, diagnostics and therapeutics for various types of cancer. The identification of novel biomarkers to aid in prognosis and treatment for cancer is urgently required. Uncontrolled proliferation and dysregulated apoptosis are characteristics exhibited by cancer cells in the initiation of various types of cancer. Notably, aberrant expression of crucial oncogenes or cancer suppressors is a defining event in cancer occurrence. Research has demonstrated that SAD1/UNC84 domain protein-2 (SUN2) serves a suppressive role in breast cancer, atypical teratoid/rhabdoid tumors and lung cancer progression. Furthermore, SUN2 inhibits cancer cell proliferation, migration and promotes apoptosis. Recent reports have also shown that SUN2 serves prominent roles in resistance to the excessive DNA damage that destabilizes the genome and promotes cancer development, and these functions of SUN2 are critical for evading initiation of cancer. Additionally, increasing evidence has demonstrated that SUN2 is involved in maintaining cell nuclear structure and appears to be a central component for organizing the natural nuclear architecture in cancer cells. The focus of the present review is to provide an overview on the pharmacological functions of SUN2 in cancers. These findings suggest that SUN2 may serve as a promising therapeutic target and novel predictive marker in various types of cancer.

Blockade of YAP alleviates hepatic fibrosis through accelerating apoptosis and reversion of activated hepatic stellate cells.

Yes-associated protein (YAP) is a significant downstream protein in the Hippo signaling pathway with important functions in cell proliferation, apoptosis, invasion and migration. YAP also plays a role in the progression and development of various liver diseases. In hepatic fibrosis development and reversion, the proliferation and apoptosis of activated hepatic stellate cells (HSCs) play a critical role. However, the contribution of YAP to hepatic fibrosis progression and reversion and the underlying mechanism have not been investigated. Here we investigated the expression and function of YAP in the proliferation and apoptosis of activated HSCs. We found that YAP expression was increased in liver fibrosis tissues from CCl4-induced model mice and restored to normal level after stopping CCl4 injection and 6 weeks of spontaneously recovery. YAP expression was elevated in HSC-T6 cells treated with TGF-ß1 and recovered after MDI treatment. Silencing of YAP inhibited the activation and proliferation of HSC-T6 cells stimulated by TGF-ß1. In addition, the apoptosis of activated HSC-T6 cells silenced for YAP was slightly enhanced. Furthermore, over-expression of YAP repressed the reversion of activated HSC-T6 cells mediated by MDI reversal. We found that HSC-T6 cells activated by TGF-ß1 showed higher levels of nuclear YAP compared with MDI-treated cells, indicating that YAP was activated in HSC-T6 cells treated by TGF-ß1. We also found that loss of YAP attenuated Wnt/ß-catenin pathway activity in activated HSC-T6 cells. Treatment of VP, an inhibitor of the YAP-TEAD complex, reduced both activation and proliferation of HSC-T6 cells and increased apoptosis. Together these results indicated that reduced expression of YAP contributes to acquisition of the quiescent phenotype in HSCs. Our results suggest that YAP may be a useful target in HSCs activation and reversion.

Suppression of SUN2 by DNA methylation is associated with HSCs activation and hepatic fibrosis.

Hepatic myofibroblasts, activated hepatic stellate cells (HSCs), are the main cell type of extracellular matrix (ECM) deposition during hepatic fibrosis. Aberrant DNA methylation-regulated HSCs activation in liver fibrogenesis has been reported, but the functional roles and mechanisms of DNA methylation in hepatic fibrosis remain to be elucidated. In the present study, reduced representation bisulfite sequencing (RRBS) analysis of primary HSCs revealed hypermethylation patterns in hepatic fibrosis. Interestingly, we found SAD1/UNC84 domain protein-2 (SUN2) gene hypermethylation at CpG sites during liver fibrogenesis in mice with CCl4-induced hepatic fibrosis, which was accompanied by low expression of SUN2. In vivo overexpression of SUN2 following adeno-associated virus-9 (AAV9) administration inhibited CCl4-induced liver injury and reduced fibrogenesis marker expression. Consistently, in vitro experiments showed that enforced expression of SUN2 suppressed HSCs activation and exerted anti-fibrogenesis effects in TGF-ß1-activated HSC-T6 cells. In addition, the signaling mechanisms related to SUN2 expression were investigated in vivo and in vitro. Methyltransferase-3b (DNMT3b) is the principal regulator of SUN2 expression. Mechanistically, inhibition of protein kinase B (AKT) phosphorylation may be a crucial pathway for SUN2-mediated HSCs activation. In conclusion, these findings provide substantial new insights into SUN2 in hepatic fibrosis.

PSTPIP2 connects DNA methylation to macrophage polarization in CCL4-induced mouse model of hepatic fibrosis.

Macrophages play a crucial role in the progression of hepatic fibrosis (HF). In macrophages, epigenetic mechanisms are increasingly being recognized as crucial controllers of their phenotype. However, the functions of macrophage DNA methylation in experimental models of hepatic fibrosis have not been fully addressed. Here, we analyzed isolated hepatic macrophages DNA methylation from CCL4-induced (4 weeks) mice using reduced representation bisulfite sequencing (RRBS). We identified and validated the methylation status of 26 gene promoter regions associated with CpG islands. We further investigated the function of PSTPIP2 in HF by hepatic-adeno-associated virus (AAV9)-PSTPIP2 overexpression. The molecular mechanisms underlying PSTPIPS2-regulated HF were further explored in mice and RAW264.7 cell line. RRBS results show hypermethylation of PSTPIP2 (chr18: 77,843,840-77,843,968) in the 5'-UTR region. PSTPIP2 expression was significantly decreased in isolated hepatic macrophages from CCL4-induced mice. PSTPIP2 hypermethylation is mediated by the methyltransferases DNMT3a and DNMT3b in LPS-induced RAW264.7 cell line. Further investigation indicated that specific overexpression of PSTPIP2 in C57BL/6 mice reduced the inflammatory response and ameliorated liver fibrosis. These data indicated that hypermethylation of PSTPIP2 caused a mixed induction of hepatic classical macrophage (M1) and alternative macrophage (M2) biomarkers in CCL4-induced HF mice. Furthermore, overexpression of PSTPIP2 inhibited the expression of M1 markers by suppressing STAT1 activity, and enhanced the expression of M2 markers by promoting STAT6 activity. In contrast, knockdown of PSTPIP2 promoted M1 polarization and suppressed M2 polarization in vitro. Adding PSTPIP2 expression alleviates liver fibrosis and hepatic inflammation in mice by regulating macrophage polarization.

DNA Methylation of PTGIS Enhances Hepatic Stellate Cells Activation and Liver Fibrogenesis.

The activation of hepatic stellate cells (HSCs) is a central event in the progression of liver fibrosis. Multiple studies proved that DNA methylation might accelerate HSCs activation. However, the specific pathogenesis of liver fibrosis remains not fully addressed. Our laboratory performed Genome methylation screening to find out the methylated gene in mice with liver fibrosis. The pilot experiments showed that the promoter of prostacyclin synthase (PTGIS) gene was hypermethylated in CCl4-induced liver fibrosis mouse model. Moreover, the down-regulated PTGIS expression can be restored by DNMTs-RNAi and 5-aza-2-deoxycytidine (5-azadC), an inhibitor of DNA methyltransferase (DNMTs). Methylation-specific PCR (MSP) showed that the methylation status of PTGIS in HSC-T6 cells cultures with TGF-ß1 (10 ng/mL) was elevated compared with control group. Chromatin immunoprecipitation (ChIP) assay indicated that PTGIS methylation was mainly induced by DNMT1 and DNMT3b. We further investigated the function of PTGIS in liver fibrosis by Recombinant Hepatic-adeno-associated virus (rAAV8)-PTGIS overexpression. The data indicated that overexpression of PTGIS in mouse liver accompanied by elevated apoptosis-related proteins expression in primary HSCs. Conversely, PTGIS silencing mediated by RNAi enhanced the expression of α-SMA and COL1a1 in vitro. Those results illustrated that adding PTGIS expression inhibits the activation of HSCs and alleviates liver fibrosis. Therefore, our study unveils the role of PTGIS in HSCs activation, which may provide a possible explanation for CCl4-mediated liver fibrosis.

SENP2 alleviates CCl4-induced liver fibrosis by promoting activated hepatic stellate cell apoptosis and reversion.

SUMOylation and deSUMOylation, a dynamic process, is proved to be involved in various fibrotic diseases. Here, we found SENP2, one of deSUMOylation protease family member, was decreased in CCl4-induced mice fibrotic liver tissues, primary HSCs and restored after spontaneously recovery. In addition, HSC-T6 cells with TGF-ß1 treatment resulted in a significant reduction of SENP2. Ectopic expression of SENP2 hindered cells activation and proliferation induced by TGF-ß1 while knockdown of SENP2 showed an opposite effect. Importantly, SENP2 promoted apoptosis of HSC-T6 cells activated by TGF-ß1. Furthermore, restoration of SENP2 was observed in inactivated HSCs after adipogenic differentiation mixture (MDI) treatment. Inadequate SENP2 inhibited the reversion of HSC-T6 cells, featured as aberrant expressions of α-SMA and col1a1, two markers of liver fibrosis. It has been reported SENP2 was a suppressant regulator of Wnt/ß-catenin signal pathway. Similarly, we found SENP2 has a negative effect on ß-catenin as well as its downstream genes C-myc and CyclinD1 in liver fibrosis. Collectively, our data indicated SENP2 may be involved in HSCs apoptosis and reversion in liver fibrosis.

Hesperetin derivative-14 alleviates inflammation by activating PPAR-γ in mice with CCl4-induced acute liver injury and LPS-treated RAW264.7 cells.

Hesperetin is a flavanone glycoside compound naturally occurring in the fruit peel of Citrusaurantium L. (Rutaceae). Previous studies revealed that hesperetin possesses various pharmacological effects, including anti-inflammation, anti-tumor, anti-oxidant and neuroprotective properties. Hesperetin derivative-14 (HD-14) is a derivative of hesperetin improved in water solubility and bioavailability. In this study, we indicated that HD-14 (2µM) significantly attenuated inflammation in LPS-treated RAW264.7 cells, besides, HD-14 (100mg/kg) exhibited hepato-protective effects and anti-inflammatory effects on C57BL/6J mice with CCl4-induced acute liver injury. In addition, it was demonstrated that HD-14 dramatically up-regulated the expression of PPAR-γ in vivo and in vitro. Interestingly, over-expression of PPAR-γ had anti-inflammatory effects on the expressions of TNF-α, IL-6, and IL-1ß, whereas, knockdown of PPAR-γ with small interfering RNA had pro-inflammatory effects in LPS-treated RAW264.7 cells. Thus, our findings demonstrated that HD-14 alleviated inflammation by activating PPAR-γ expression at least in part. Further studies founded that HD-14 remarkably inhibited the expression of p-JAK1 and p-STAT1 through up-regulating PPAR-γ. Together, these results suggested that HD-14 served as an activator of PPAR-γ and the JAK1/STAT1 signaling pathway may be involved in the progress of inflammation. Collectively, HD-14 may be utilized as a potential anti-inflammation monomeric compound in the treatment of acute liver injury.

Transmembrane protein 88 attenuates liver fibrosis by promoting apoptosis and reversion of activated hepatic stellate cells.

Transmembrane protein 88 (Tmem88) is a crucial inhibitor for Wnt/ß-catenin pathway in the development of myocardial cells. Due to the important role of ß-catenin in the activation and proliferation of hepatic stellate cells (HSCs), it is necessary to investigate the function of Tmem88 in HSCs. In this study, we found that Tmem88 expression was decreased in the human liver fibrotic tissues, primary HSCs from fibrotic mice and activated HSC-T6 cells. Functionally, Tmem88 could inhibit HSCs activation and proliferation by blocking Wnt/ß-catenin pathway, and promoted the apoptosis of activated HSCs by initiating Bcl-2/Bax/Caspase3 pathway. Moreover, the level of DNA metyltransferase 3a (Dnmt3a) was upregulated in activated HSCs, and siRNA-mediated Dnmt3a silencing led to Tmem88 restoration. These results indicated that Tmem88 played an important role in HSCs activation, proliferation and apoptosis, and Tmem88 expression might be modulated by Dnmt3a.

Nesfatin-1, a potent anorexic agent, decreases exploration and induces anxiety-like behavior in rats without altering learning or memory.

The anorectic neuropeptide nesfatin-1 has recently been characterized as a potential mood regulator, but the accurate effect of nesfatin-1 on anxiety and learning and memory behavior and the possible mechanisms remains unknown. In the present study, to test the hypothesis that nesfatin-1 might affect the anxiety-like and learning and memory behaviors in rats via ERK/CREB/BDNF pathway, nesfatin-1 was administered intraperitoneally to rats with the doses (10, 20, 40µg/kg), and the behavioral performance was tested using the open field task, the Morris water maze (MWM), and the Y maze. Moreover, the protein expression of brain-derived neurotrophic factor (BDNF), total and phosphorylated-ERK in the hippocampus and the prefrontal cortex (PFC) were evaluated. The results showed that chronic administration of nesfatin-1 could decrease the moving distance, the duration in the center, and the frequencies of rearing and grooming in the open field task, decrease the moving distance, frequency, and the preference index of new arm in the Y maze, although there was no significant difference of the performance in the MWM task among groups. Furthermore, 3 weeks' consecutive administration of nesfatin-1 resulted in the decrease of protein expression of BDNF and phosphorylated-ERK in the hippocampus and the PFC. These results provided evidence that exogenous nesfatin-1 could decrease exploration and induce anxiety-like behavior in rats, the mechanism of which might be related to the reduced protein expression of BDNF and phosphorylated-ERK in the hippocampus and the PFC.