The effects of cGAS-STING inhibition in liver disease, kidney disease, and cellular senescence.

The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling pathway is one of the fundamental mechanisms of the body's defense, which responds to the abnormal presence of double-stranded DNA in the cytoplasm to establish an effective natural immune response. In addition to detecting microbial infections, the cGAS pathway may be triggered by any cytoplasmic DNA, which is absent from the normal cytoplasm, and only conditions such as senescence and mitochondrial stress can lead to its leakage and cause sterile inflammation. A growing body of research has shown that the cGAS-STING pathway is strongly associated with sterile inflammation. In this study, we reviewed the regulatory mechanisms and biological functions of the cGAS-STING pathway through its involvement in aseptic inflammation in liver disease, kidney disease, and cellular senescence.

Pyroptosis: shedding light on the mechanisms and links with cancers.

Pyroptosis, a novel form of programmed cell death (PCD) discovered after apoptosis and necrosis, is characterized by cell swelling, cytomembrane perforation and lysis, chromatin DNA fragmentation, and the release of intracellular proinflammatory contents, such as Interleukin (IL) 8, IL-1ß, ATP, IL-1α, and high mobility group box 1 (HMGB1). Our understanding of pyroptosis has increased over time with an increase in research on the subject: gasdermin-mediated lytic PCD usually, but not always, requires cleavage by caspases. Moreover, new evidence suggests that pyroptosis induction in tumor cells results in a strong inflammatory response and significant cancer regression, which has stimulated great interest among scientists for its potential application in clinical cancer therapy. It's worth noting that the side effects of chemotherapy and radiotherapy can be triggered by pyroptosis. Thus, the intelligent use of pyroptosis, the double-edged sword for tumors, will enable us to understand the genesis and development of cancers and provide potential methods to develop novel anticancer drugs based on pyroptosis. Hence, in this review, we systematically summarize the molecular mechanisms of pyroptosis and provide the latest available evidence supporting the antitumor properties of pyroptosis, and provide a summary of the various antitumor medicines targeting pyroptosis signaling pathways.

Ferroptosis: Shedding Light on Mechanisms and Therapeutic Opportunities in Liver Diseases.

Cell death is a vital physiological or pathological phenomenon in the development process of the organism. Ferroptosis is a kind of newly-discovered regulated cell death (RCD), which is different from other RCD patterns, such as apoptosis, necrosis and autophagy at the morphological, biochemical and genetic levels. It is a kind of iron-dependent mode of death mediated by lipid peroxides and lipid reactive oxygen species aggregation. Noteworthily, the number of studies focused on ferroptosis has been increasing exponentially since ferroptosis was first found in 2012. The liver is the organ that stores the most iron in the human body. Recently, it was frequently found that there are different degrees of iron metabolism disorder and lipid peroxidation and other ferroptosis characteristics in various liver diseases. Numerous investigators have discovered that the progression of various liver diseases can be affected via the regulation of ferroptosis, which may provide a potential therapeutic strategy for clinical hepatic diseases. This review aims to summarize the mechanism and update research progress of ferroptosis, so as to provide novel promising directions for the treatment of liver diseases.

MicroRNA-195-3p promotes hepatic stellate cell activation and liver fibrosis by suppressing PTEN expression.

Liver fibrosis is a reversible wound healing reaction characterized by abnormal accumulation of extracellular matrix (ECM) in response to liver injury. Recent studies have shown that it can be epigenetically regulated, especially by microRNAs (miRNAs). It has been acknowledged that activation of hepatic stellate cells (HSCs) is a pivotal step in the initiation and progression of liver fibrosis. Notably, our results showed that miR-195-3p was increased in HSCs isolated from CCl4-treated mice and that the increase was more pronounced as the degree of liver fibrosis increased. Moreover, treatment of LX-2 cells, a human immortalized hepatic stellate cell line, with TGF-ß1 resulted remarkable upregulation of miR-195-3p. Gain-of-function and loss-of-function experiments have suggested that the increased levels of miR-195-3p inhibit the expression of phosphatase and tension homolog deleted on chromosome 10 (PTEN), a negative regulator of the PI3K/Akt/mTOR signaling pathway in liver fibrosis, thereby contributing to HSC activation and proliferation and promoting the expression of profibrotic genes, such as α-SMA and collagen I, in LX-2 cells, which accelerates the accumulation of fibrous extracellular matrix deposition in the liver, while knockdown of miR-195-3p induced the opposite effect. Taken together, these results provide evidence for the harmful role of miR-195-3p in CCl4-treated mouse liver fibrosis.

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.

Interleukin-9 attenuates inflammatory response and hepatocyte apoptosis in alcoholic liver injury.

Alcoholic liver injury is a liver cell dysfunction disease caused by long-term or excessive alcohol consumption. Inhibiting the production of inflammatory factors is an important way to alleviate liver injury. Interleukin-9 (IL-9) is one of the members of IL-2Rγc family. It has multiple biological functions. Previous studies have shown that IL-9 is a cytokine that is closely related to inflammatory disease, allergic diseases, autoimmune diseases, and parasitic infections. However, no systematic studies have been performed to address the role of IL-9 in ALI. This project aims to investigate the effects of IL-9 on macrophage-related inflammatory response and hepatocyte apoptosis in alcohol-induced liver injury by injecting adeno-associated virus (AAV9) into tail vein. In the ALI model group, western blot and ELISA assays demonstrated that the expression of IL-9 was reduced. Overexpression of IL-9 relieved the injury and reduced the serum levels of IL-6, TNF-α in EtOH-induced ALI mouse model. Moreover, by using western blot, it was indicated that IL-9 can inhibit the expression of pro-apoptotic protein, such as cleaved caspase 3 and Bax. In vitro, mouse recombinant protein IL-9 inhibited the expression of IL-6, TNF-α in EtOH-induced RAW264.7 cells. Moreover, flow cytometry and western blot results displayed that macrophage-derived IL-9 inhibited hepatocyte apoptosis. After silencing STAT3 in AML-12 cells, the anti-apoptotic effect of macrophage-derived IL-9 was further enhanced. These results indicate that IL-9 reduces the production of pro-inflammatory factors in ALI. Furthermore, macrophage-derived IL-9 can reduce hepatocyte apoptosis by inhibiting the activation of the STAT3 pathway.

The miR-455-3p/HDAC2 axis plays a pivotal role in the progression and reversal of liver fibrosis and is regulated by epigenetics.

Histone deacetylases (HDACs), especially HDAC2, play a role in alleviating liver fibrosis; however, the specific upstream regulation mechanism is unknown. Herein, TargetScan was used to predict the potential upstream targets of HDAC2, and the role of miR-455-3p was explored. The dual luciferase reporter assay showed that miR-455-3p binds to the 3' UTR of HDAC2 mRNA. Additionally, miR-455-3p was downregulated in the liver tissues of patients with cirrhosis and mice with liver fibrosis, as well as in primary HSCs isolated from fibrotic mouse livers and TGF-ß-treated LX-2 cells. In contrast, it is highly expressed in the reversal stage of hepatic fibrosis and MDI-cultured LX-2 cells. Our functional analyses showed that miR-455-3p overexpression facilitated apoptosis and reduced the expression of pro-fibrotic markers and the proliferation of activated LX-2 cells. On the contrary, miR-455-3p inhibition converted inactivated LX-2 cells into activated, proliferative, fibrogenic cells. Interestingly, restoration of HDAC2 expression partially blocked the function of miR-455-3p. Downregulated miR-455-3p expression can be restored by DNA methyltransferases in activated LX-2 cells. Methylation-specific PCR, bisulfite sequencing PCR, and chromatin immunoprecipitation assays indicated that the methylation level of miR-455-3p promoter CpG islands was elevated in TGF-ß-treated LX-2 cells and that miR-455-3p was downregulated in activated LX-2 cells by DNA hypermethylation, which is mediated by DNMT3b and DNMT1. In conclusion, miR-455-3p acts as a liver fibrosis suppressor by targeting HDAC2, and its deficiency further aggravates the reversal phase of fibrosis. Thus, the epigenetics mediated miR-455-3p/HDAC2 axis may serve as a novel potential therapeutic target for clinical treatment of hepatic fibrosis.

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.

Circular RNA circPSD3 alleviates hepatic fibrogenesis by regulating the miR-92b-3p/Smad7 axis.

Recently, circular RNAs (circRNAs) have been frequently reported to be involved in hepatocellular carcinoma (HCC) development and progression. However, the role of circRNAs in hepatic fibrosis (HF) is still unclear. Our previous high-throughput screen revealed changes in many circRNAs in mice with carbon tetrachloride (CCl4)-induced HF. For instance, the expression of circPSD3, a circRNA derived from the Pleckstrin and Sec7 domain-containing 3 (PSD3) gene, was considerably downregulated in primary hepatic stellate cells (HSCs) and liver tissues of mice with CCl4-induced HF compared to those in the vehicle group. In vivo overexpression of circPSD3 using AAV8-circPSD3 arrested the deterioration of CCl4-induced HF as indicated by reduced serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) content, liver hydroxyproline level, collagen deposition, and pro-fibrogenic gene and pro-inflammatory cytokine levels. Moreover, in vitro loss-of-function and gain-of-function analyses suggested that circPSD3 inhibited the activation and proliferation of HSCs. Mechanistically, circPSD3 served as a sponge for miR-92b-3p, subsequently promoting the expression of Smad7. In conclusion, our present findings reveal a novel mechanism by which circPSD3 alleviates hepatic fibrogenesis by targeting the miR-92b-3p/Smad7 axis, and they also indicate that circPSD3 may serve as a potential biomarker for HF.

NLRP12 negatively regulates EtOH-induced liver macrophage activation via NF-κB pathway and mediates hepatocyte apoptosis in alcoholic liver injury.

Alcohol-induced liver injury is characterized by abnormal liver dysfunction and excessive inflammation response. Recent years a wealth of data have been yielded indicating that EtOH (ethyl alcohol)-induced macrophage activation along with liver inflammation plays a dominating role in the progression of alcohol-induced liver injury. Here we found high expression of NLRP12 (Nucleotide-binding oligomerization domain protein 12, which is generally considered to be a negative regulator of inflammatory response) in EtOH-fed mouse liver tissue, primary Kupffer cells and EtOH-induced RAW264.7 cells. Additionally, overexpression of NLRP12 following Ad (adenovirus)-NLRP12-EGFP contributed to the attenuation of steatosis and inflammation in EtOH-fed mice model and EtOH-primed RAW264.7 cells. In parallel, Knockdown of NLRP12 aggravated the inflammatory response in RAW264.7 cells triggered by EtOH. Meanwhile, after administration of overexpression or inhibition of NLRP12 expression in vitro, the expression of phosphorylated protein of NF-kB signaling pathway was significantly affected. After increasing or decreasing the expression of NLRP12 in RAW264.7 cells, AML-12 cells were cultured with the supernatant of RAW264.7 cells stimulated by EtOH, and the percent of apoptosis ratio of AML-12 cells was remarkably altered. The study suggested that reduced inflammatory response induced by NLRP12-mediated inhibition of NF-kB pathway participated in the decrease of hepatocyte apoptosis in alcohol-induced liver injury. Collectively, these findings suggested the significance of NLRP12-mediated macrophage activation in alcohol-induced liver injury.

LncRNA NEAT1: Shedding light on mechanisms and opportunities in liver diseases.

With advances in genome and transcriptome research technology, the function and mechanism of lncRNAs in physiological and pathological states have been gradually revealed. Nuclear Enriched Abundant Transcript 1 (NEAT1, a long non-coding RNA), a vital component of paraspeckles, plays an indispensable role in the formation and integrity of paraspeckles. Throughout the research history, NEAT1 is mostly aberrantly upregulated in various cancers, and high expression of NEAT1 often contributes to poor prognosis of patients. Notably, the role and mechanism of NEAT1 in liver diseases have been increasingly reported. NEAT1 accelerates the progression of non-alcoholic fatty liver disease (NAFLD), liver fibrosis and hepatocellular carcinoma, while exerting a protective role in the pathogenesis of acute-on-chronic liver failure by inhibiting the inflammatory response. In this review, we will elaborate on relevant studies on the different casting of NEAT1 in liver diseases, especially focusing on its regulatory mechanisms and new opportunities for alcoholic liver disease.

Circular RNA expression profile of liver tissues in an EtOH-induced mouse model of alcoholic hepatitis.

Alcoholic hepatitis (AH) is a widely prevalent liver-related disease that results from long-term alcohol consumption. However, there is still a lack of effective treatment. Previous studies have reported that circular RNAs (circRNAs) are related to the development of various diseases. However, the function of circRNAs and their roles in AH are largely unknown. Therefore, we used bioinformatics analysis to investigate changes in circRNA expression and predict their functions in AH. An AH model was established in C57BL/6J mouse treated by Gao-binge modeling method, and then the circRNA profile of liver tissues was screened by Next Generation Sequencing. By comparing circRNA expression in liver tissues in AH model groups and normal controls, we identified that circRNAs were differently expressed during AH pathogenesis, and then differential expression levels of selected circRNAs were validated by qRT-PCR. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were employed to predict the functions of these circRNAs. A total of 20 circRNAs were found to be differentially expressed in AH model groups (p ≤ 0.05) compared with the expression of the normal controls respectively. Among them, 9 circRNAs were significantly up-regulated, and the other 11 were down-regulated. Ten circRNAs were randomly selected to verify the reliability of these profiles by qRT-PCR. After obtaining the parental genes of the differentially expressed circRNAs, the top 30 enrichment GO entries and KEGG pathways were annotated. Then, we selected significantly differentially expressed circRNA (mm9_ circ_018725) for further analysis in vitro. Although the exact mechanisms and biological functions of these circRNAs in the development of AH need further exploration, our findings do suggest that knockdown of mm9_ circ_018725 could inhibit hepatocyte apoptosis induced by EtOH in vitro. In addition, suppression of mm9_ circ_018725 reduced the release of pro-inflammatory cytokines from EtOH-stimulated Raw264.7 cells. Thus, our study brings us closer to understanding the pathogenic mechanisms and finding new molecular targets for the clinical treatment of alcoholic hepatitis.

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.

4-Methylcoumarin-[5,6-g]-hesperetin attenuates inflammatory responses in alcoholic hepatitis through PPAR-γ activation.

4-Methylcoumarin-[5,6-g]-hesperetin (4-MCH) is a hesperidin derivative produced by the structural modification of hesperetin. Alcoholic hepatitis (AH) is the origin of many serious liver diseases that are accompanied by hepatic inflammation. In this study, we detected the anti-inflammatory activity of 4-MCH in EtOH fed mice and examined the potential molecular mechanism of this activity. We found that 4-MCH suppressed the release of inflammatory cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in primary liver macrophages isolated from mice and in EtOH-treated RAW264.7 cells. In addition, we showed that the expression of peroxisome proliferator-activated receptor-γ (PPAR-γ) was down-regulated in vivo and in vitro in AH. Furthermore, 4-MCH acted as an activator of PPAR-γ, which could therefore ameliorate the inhibitory effects of EtOH on the expression of PPAR-γ. The impairment of PPAR-γ function (T0070907 or PPAR-γ siRNA treatment) resulted in greater inflammation than that in the control group. Conversely, over-expression of PPAR-γ further reduced the release of inflammatory cytokines from EtOH-stimulated RAW264.7 cells. Additional investigations showed that 4-MCH significantly inhibited the phosphorylation of p65. Collectively, these results indicate that 4-MCH alleviated the inflammatory reaction through PPAR-γ activation via the NF-κB-p65 signaling pathway, which regulates the expression of IL-6 and TNF-α in AH.