Amlexanox attenuates LPS-induced neuroinflammatory responses in microglial cells via inhibition of NF-κB and STAT3 signaling pathways.

Amlexanox is an anti-inflammatory and anti-allergic agent used clinically for the treatment of aphthous ulcers, allergic rhinitis, and asthma. Recent studies have demonstrated that amlexanox, a selective inhibitor of IkB kinase epsilon (IKKε) and TANK-binding kinase 1 (TBK1), suppresses a range of diseases or inflammatory conditions, such as obesity-related metabolic dysfunction and type 2 diabetes. However, the effects of amlexanox on neuroinflammatory responses to amlexanox have not yet been comprehensively studied. In this study, we investigated the novel therapeutic effect of amlexanox on LPS-induced neuroinflammation in vivo, and intraperitoneal injection of amlexanox markedly reduced LPS-induced IKKε levels, proinflammatory cytokines, and microglial activation, as evidenced by ionized calcium-binding adapter molecule 1 (Iba1) immunostaining. Furthermore, amlexanox significantly reduced proinflammatory cytokines and chemokines in LPS-induced bone marrow-derived macrophages (BMDM), murine BV2, and human HMC3 microglial cells. This data provided considerable evidence that amlexanox can be used as a preventive and curative therapy for neuroinflammatory and neurodegenerative diseases. In terms of mechanism aspects, our results demonstrated that the anti-inflammatory action of amlexanox in BV2 microglial cells was through the downregulation of NF-κB and STAT3 signaling pathways. In addition, the combination of amlexanox and SPI (a STAT3 selective inhibitor) showed high efficiency in inhibiting the production of neurotoxic and pro-inflammatory mediators. Overall, our data provide rational insights into the mechanisms of amlexanox as a potential therapeutic strategy for neuroinflammation-related diseases.

Sappanone A Alleviates the Severity of Carbon Tetrachloride-Induced Liver Fibrosis in Mice.

Liver fibrosis is a major challenge to global health because of its various complications, including cirrhosis and hepatocarcinoma, while no effective treatment is available for it. Sappanone A (SA) is a homoisoflavonoid extracted from the heartwood of Caesalpinia sappan Linn. with anti-inflammatory and antioxidant properties. However, the effects of SA on hepatic fibrosis remain unknown. This study aimed to investigate the protective effects of SA on carbon tetrachloride (CCl4)-induced liver fibrosis in mice. To establish a liver fibrosis model, mice were treated intraperitoneally (i.p.) with CCl4 for 4 weeks. SA (25, 50, and 100 mg/kg body weight) was i.p. injected every other day during the same period. Our data indicated that SA decreased liver injury, fibrotic responses, and inflammation due to CCl4 exposure. Consistently, SA reduced oxidative stress and its-mediated hepatocyte death in fibrotic livers. Of note, SA could not directly affect the activation of hepatic stellate cells. Mechanistically, SA treatment lessened oxidative stress-triggered cell death in hepatocytes after CCl4 exposure. SA down-regulated the expression of M1 macrophage polarization markers (CD86 and iNOS) and up-regulated the expression of M2 macrophage polarization markers (CD163, IL-10, and Arg1) in livers and macrophages. Meanwhile, SA induced the activation of peroxisome proliferator-activated receptor gamma (PPARγ). However, decreased inflammatory responses and the trend of M2 macrophage polarization provided by SA were substantially abolished by SR202 (a PPARγ inhibitor) treatment in macrophages. Additionally, SA treatment promoted fibrosis regression. Taken together, our findings revealed that treatment with SA alleviated CCl4-induced fibrotic liver in mice through suppression of oxidative stress-mediated hepatocyte death and promotion of M2 macrophage polarization via PPARγ. Thus, SA might pave the way for a new hepatoprotective agent to treat liver fibrosis.

CXCL5 promotes lipotoxicity of hepatocytes through upregulating NLRP3/Caspase-1/IL-1ß signaling in Kupffer cells and exacerbates nonalcoholic steatohepatitis in mice.

Immune-inflammatory responses play a key role in the development of nonalcoholic steatohepatitis (NASH). Previous studies have demonstrated that CXC motif chemokine ligand 5 (CXCL5) correlates positively with obesity and type 2 diabetes. This study is to explore the functional role of CXCL5 in the pathogenesis of NASH. To establish a NASH model, mice were fed with methionine-and choline-deficient high-fat diet for 6 weeks and anti-CXCL5 mAb was injected during the same period. An in vitro NASH model was established by treating palmitic acid (PA), using a trans-well co-culture system of mouse primary hepatocytes and Kupffer cells (KCs), and recombinant mouse (rm) CXCL5 was treated after PA administration. Our data showed that hepatic CXCL5 levels were highly expressed in the NASH mouse model. CXCL5 neutralization significantly alleviated the severity of NASH livers, demonstrated by pathological analysis, decreased biochemicals, and inflammation. Besides, neutralizing CXCL5 reduced lipid accumulation, cell death, and fibrosis in injured livers. In vitro, rmCXCL5 could not affect the activation of hepatic stellate cells. Also, rmCXCL5 exacerbated PA-induced hepatotoxicity and lipid deposition in hepatocytes co-cultured with KCs rather than in single-cultured hepatocytes. Mechanistically, rmCXCL5 not only promoted NOD-like receptor pyrin domain-containing protein 3 (NLRP3) expression, Cleaved caspase-1 expression, and interleukin 1 beta (IL-1ß) secretion in single-cultured and co-cultured KCs but also increased lipid deposition in co-cultured hepatocytes. In addition, MCC950, an inhibitor of NLRP3, almost abolished the effects of rmCXCL5 on PA-treated co-culture system. Therefore, CXCL5 could exacerbate NASH by promoting lipotoxicity of hepatocytes via upregulating NLRP3/Caspase-1/IL-1ß signaling in KCs.

CXCL5 Promotes Acetaminophen-Induced Hepatotoxicity by Activating Kupffer Cells.

Kupffer cells (KCs) play a key part in the pathological process of acetaminophen (APAP)-induced acute liver injury (ALI), the leading cause of acute liver failure in the world. CXC motif chemokine ligand 5 (CXCL5) exerts proinflammatory effects in acute respiratory distress syndrome and arthritis. In the current study, we aim to reveal the effects of CXCL5 on the activation of KCs and the role of CXCL5 in the pathogenesis of APAP-induced hepatotoxicity. The in vivo study, conducted on mice intraperitoneally injected with APAP (300 mg/kg) to establish the ALI model and then treated with Anti-CXCL5 mAb at 30 min and 12 h after the APAP challenge, showed that CXCL5 expression significantly increased in injured livers, and Anti-CXCL5 mAb mitigated the degree of APAP-evoked ALI in mice which was proven through biochemicals and histological examination. Also, neutralization of CXCL5 had no significant effect on APAP metabolism in the liver but exhibited anti-inflammatory effects and ameliorated hepatocellular death in the injured liver. The in vitro data displayed that recombinant mouse CXCL5 treatment promoted APAP-induced cellular toxicity in primary hepatocytes co-cultured with KCs, compared with single-cultured hepatocytes. Consistent with the result, we found that the Anti-CXCL5 mAb gradient decreased LPS-induced expression of inflammatory cytokines in single-cultured KCs. Therefore, CXCL5 could stimulate KCs to produce inflammatory mediators, therefore damaging hepatocytes from APAP toxicity.

Cyclodextrin-Modified Amphiphilic Microgel with Bifunctional Domains for Infected Wound Healing via Photothermal Antibacterial Therapy and Nitric Oxide Release.

Bacterial infections are one of the major contributing factors to human mortality, which can cause secondary damage to the injured area, such as leading to inflammation, tissue death, and even personal death. Herein, we developed a novel cyclodextrin (CD)-modified amphiphilic microgel with a 3D network nanostructure that encapsulates hydrophilic indocyanine green (ICG) as a trigger for photothermal therapy (PTT) and hydrophobic N,N'-disubstituted-butyl-N,N'-dinitro-1,4-benzenediamine (BNN6) as a heat-sensitive nitric oxide (NO) donor (CD@I-B) to cope with bacteria-infected wound therapy. This biocompatible microgel showed excellent broad-spectrum antibacterial capability under near-infrared (NIR) laser irradiation, while the photothermal conversion process promotes the deswelling of the microgel and release of NO, which synergistically accelerates wound healing. The therapy strategy by synergizing NO delivery with PTT promoted the formation of neovascularization and collagen fiber as well as the elimination of inflammation cells, thus facilitating wound healing. Our study further demonstrates the fantastic opportunities of applying high-performance microgels to provide all-in-one sites for treating wound sterilization and healing.

Nuciferine Effectively Protects Mice against Acetaminophen-Induced Liver Injury.

Acetaminophen (APAP) overdose still poses a major clinical challenge and is a leading cause of acute liver injury (ALI). N-acetylcysteine (NAC) is the only approved antidote to treat APAP toxicity while NAC therapy can trigger side effects including severe vomiting and even shock. Thus, new insights in developing novel therapeutic drugs may pave the way for better treatment of APAP poisoning. Previous research has reported that nuciferine (Nuci) possesses anti-inflammatory and antioxidant properties. Therefore, the objective of this study was proposed to investigate the hepatoprotective effects of Nuci and explore its underlying mechanisms. Mice were intraperitoneally (i.p.) administered with APAP (300 mg/kg) and subsequently injected with Nuci (25, 50, and 100 mg/kg, i.p.) at 30 min after APAP overdose. Then, all mice were sacrificed at 12 h after APAP challenge for further analysis. Nuci-treated mice did not show any side effects and our results revealed that treating Nuci significantly attenuated APAP-induced ALI, as confirmed by histopathological examinations, biochemical analysis, and diminished hepatic oxidative stress and inflammation. The in silico prediction and mRNA-sequencing analysis were performed to explore the underlying mechanisms of Nuci. GO and KEGG enrichment of the predicted target proteins of Nuci includes reactive oxygen species, drug metabolism of cytochrome P450 (CYP450) enzymes, and autophagy. Furthermore, the mRNA-sequencing analyses indicated that Nuci can regulate glutathione metabolic processes and anti-inflammatory responses. Consistently, we found that Nuci increased the hepatic glutathione restoration but decreased APAP protein adducts in damaged livers. Western blot analysis further confirmed that Nuci effectively promoted hepatic autophagy in APAP-treated mice. However, Nuci could not affect the expression levels of the main CYP450 enzymes (CYP1A2, CYP2E1, and CYP3A11). These results demonstrated that Nuci may be a potential therapeutic drug for APAP-induced ALI via amelioration of the inflammatory response and oxidative stress, regulation of APAP metabolism, and activation of autophagy.

Evaluation of transcriptomic molecular classification, biological behavior, and clinicopathological features in hepatocellular carcinoma.

BACKGROUND: Studies in France, Korea, and Singapore found that G1-G6 transcriptomes are involved in hepatocellular carcinoma (HCC) carcinogenesis. However, the suitability of this method in Chinese HCC patients has remained unknown. METHODS: The correlation between the G1-G6 molecular classification and clinicopathological features were analyzed in 107 Chinese HCC patients through the retrospective cohort study. RNA sequencing and bioinformatics analysis were performed to screen related targets and molecular signaling pathways. RESULTS: We found that the G1-G3 subgroups were associated with high serum alpha-fetoprotein (AFP) level, high copy number of hepatitis B virus (HBV) DNA, complex histopathological structure, macrovascular invasion. The G1 subgroup was mainly related to liver cancer stemness, and G3 subgroup showed the worst prognosis. The G5 and G6 subgroups were associated with activation of the Wnt/ß-catenin pathway. Compared with the G4-G6 group, the G1-G3 group showed significantly higher expression levels of regenerating family member 1 beta (REG1B), regenerating family member 3 gamma (REG3G), and inositol 1,4,5-trisphosphate receptor type 1 (ITPR1), and enriched calcium signaling pathway. CONCLUSIONS: This study enhances our understanding of the heterogenicity of China HCC and indicates that the G1-G6 signatures can be used to identify predictive biomarkers against HCC patients in China.

Inhibition of the B7-H3 immune checkpoint limits hepatocellular carcinoma progression by enhancing T lymphocyte-mediated immune cytotoxicity in vitro and in vivo.

PURPOSE: The interaction between tumor cells and immune system in hepatocellular carcinoma (HCC) remains unclear. Great clinical achievements have progressed in HCC patients treated with immune checkpoint inhibitors (ICIs) for programmed death-1 and its ligands. However, response efficacy for these therapies is limited, thereby requiring alternative ICI candidates for HCC treatment. B7 homolog 3 protein (B7-H3), an immunoregulatory protein, plays a significant role in tumor immunity and disease progression. In this study, we evaluated the correlation between B7-H3 expression and prognosis of HCC patients, and investigated the therapeutic potential of B7-H3 targeting in HCC. METHODS: B7-H3 expression was analyzed immunohistochemically in HCC patients, and its relationship with tumor-infiltrating lymphocyte infiltration was assessed. The anti-tumor efficacy of anti-B7-H3 antibody therapy was determined using an in vitro co-culture system and a subcutaneous HCC-bearing murine model. RESULTS: We found that B7-H3 overexpressed in tumor cells and positively correlated with poor prognosis in HCC patients. B7-H3 inhibited the infiltration of CD8+ T cells in tumors. Furthermore, co-culture experiment indicated that inhibiting B7-H3 in tumor cells significantly increased T cells-mediated immune activities and tumor cell killing. Consistently, anti-B7-H3 antibody-treated HCC murine model showed decreased tumor size and enhanced anti-tumor immunity mediated by CD8+ T cells. CONCLUSION: Altogether, our findings suggest that B7-H3 inhibition in tumor cells restores the immune cytotoxicity of T cells, which in turn promotes apoptosis of target cells. Therefore, B7-H3 serves as a key negative regulator in tumor immunity and the promising clinical utility of B7-H3-based immunotherapies for HCC treatment could be developed.

Sappanone A ameliorates acetaminophen-induced acute liver injury in mice.

Sappanone A (SA), a homoisoflavonoid compound extracted from the heartwood of Caesalpinia sappan Linn., exerts anti-inflammatory and antioxidant activities. However, the effects of SA on acetaminophen (APAP) overdose-induced acute liver injury (ALI) have not been determined yet. This study aims to explore the protective effects of SA and the potential mechanisms of action. Mice were pretreated with SA (25, 50, and 100 mg/kg) by intraperitoneal (i.p.) injection for seven days prior to APAP (300 mg/kg, i.p.) administration. At 12 h after APAP injection, serum and liver samples were collected. Primary murine hepatocytes were used to investigate the underlying mechanisms. SA pretreatment dose-dependently attenuated APAP-induced ALI, as validated by reduced serum alanine/aspartate aminotransferase levels, histopathologic lesions, and oxidative stress. Consistently, pretreatment with SA reduced the formation of APAP protein adducts in damaged livers of mice. Mechanistically, SA could facilitate the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and thus promote cellular glutathione (GSH) synthesis. The hepatoprotective outcomes provided by SA were significantly abolished by treatment with ML385, a Nrf2 inhibitor. Besides, anti-inflammatory property of SA reduced inflammatory reaction in injured livers of mice. Of note, posttreatment with SA reveals significant therapeutic influences against APAP-induced ALI in mice. Collectively, our findings demonstrated that pretreated-SA ameliorated APAP-mediated ALI in mice, at least in part, by reducing the generation of APAP protein adducts via Nrf2-enhanced GSH synthesis, and by diminishing hepatic inflammation. Therefore, SA could be a potential hepatoprotective agent for treating ALI.

Metabolic Regulation of Hepatitis B Virus Infection in HBV-Transgenic Mice.

Hepatitis B virus (HBV) infection is a worldwide health burden. Metabolomics analysis has revealed HBV-induced metabolism dysregulation in liver tissues and hepatocytes. However, as an infectious disease, the tissue-specific landscape of metabolic profiles of HBV infection remains unclear. To fill this gap, we applied untargeted nuclear magnetic resonance (NMR) metabolomic analysis of the heart, liver, spleen, lung, kidney, pancreas, and intestine (duodenum, jejunum, ileum) in HBV-transgenic mice and their wild-type littermates. Strikingly, we found systemic metabolic alterations induced by HBV in liver and extrahepatic organs. Significant changes in metabolites have been observed in most tissues of HBV-transgenic mice, except for ileum. The metabolic changes may provide novel therapeutic targets for the treatment of HBV infection. Moreover, tissue-specific metabolic profiles could speed up the study of HBV induced systemic metabolic reprogramming, which could help follow the progression of HBV infection and explain the underlying pathogenesis.

Exogenous activation of toll-like receptor 5 signaling mitigates acetaminophen-induced hepatotoxicity in mice.

Acetaminophen (APAP) poisoning is the most common cause of drug-induced acute liver injury (ALI). Our results showed that toll-like receptor 5 (TLR5) was abundantly expressed in hepatocytes and dramatically downregulated in the toxic mouse livers. Hence, we herein investigated the role of TLR5 signaling after APAP overdose. Mice were intraperitoneally (i.p.) injected with APAP to induce ALI, and then injected with flagellin at one hour after APAP administration. Flagellin attenuated APAP-induced ALI based on decreased histopathologic lesions, serum biochemical, oxidative stress, and inflammation. Furthermore, the protective effects of flagellin were abolished by TH1020 (a TLR5 antagonist) treatment. These results suggest that flagellin exerted protective effects on ALI via TLR5 activation. Mechanistically, flagellin injection promoted the translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) to the nucleus in hepatocytes. Consistent with the in vivo results, flagellin increased the activation of Nrf2 in hepatocytes, resulting in decreased APAP toxicity. ML385, a selective inhibitor of Nrf2, abolished the flagellin-mediated hepatoprotective effects in damaged livers and hepatocytes. Additionally, the flagellin-induced Nrf2 translocation was dependent upon the activation of TLR5-JNK/p38 pathways. These findings suggest that TLR5 signaling-induced Nrf2 activation, at least partially, contributed to the protection against APAP-induced ALI by flagellin treatment.

Orostachys japonicus ameliorates acetaminophen-induced acute liver injury in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Orostachys japonicus A. Berger (O. japonicus), referred to as Wa-song in Korea is a traditional and herbal medicine. Even though it has been traditionally used to treat inflammation- and toxicity-related diseases, the effects of ethanol extract of O. japonicus (OJE) on acetaminophen (N-acetyl-p-aminophenol, APAP) overdose-induced hepatotoxicity have not been determined yet. AIM OF THE STUDY: The present study was aimed to investigate the effects of OJE against APAP-induced acute liver injury (ALI) and explore the underlying mechanisms. MATERIALS AND METHODS: Mice were treated orally with OJE (50, 100, or 200 mg/kg) for seven days before APAP (300 mg/kg) injection. After 12 h of APAP treatment, serum and liver tissues were collected. An in vitro system using primary hepatocytes was also applied in this study. RESULTS: Pretreatment with OJE, especially at a dose of 200 mg/kg, reduced APAP overdose-induced ALI in mice, as evidenced by decreased serum alanine/aspartate aminotransferase levels, histopathological damage, and inflammation. Consistently, OJE pretreatment reduced the gene transcription of cytochrome P450 (CYP) 3A11 and CYP1A2 in livers of mice injected with or without APAP, at least in part, via inactivation of nuclear receptor pregnane X receptor (PXR). Furthermore, the role of PXR in mediating the OJE regulation of CYPs was confirmed in primary hepatocytes, which showed that OJE pretreatment inhibited PXR activity and APAP hepatotoxicity enhanced by pregnenolone 16α-carbonitrile, a mouse agonist of PXR. Besides, the antioxidative activity provided by OJE, involving increases in hepatic glutathione (GSH) content and decreases in malondialdehyde levels, has been shown to exert hepatoprotective effects in normal and injured livers. Moreover, APAP-activated c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) in mice liver were indirectly inhibited by pretreatment with OJE. CONCLUSIONS: Taken together, our findings showed that OJE attenuated APAP-induced ALI by decreasing APAP-metabolizing enzymes via inactivation of PXR and the restoration of hepatic GSH content. Therefore, OJE could be a promising hepatoprotective agent.

Macrophage-Inducible C-Type Lectin Signaling Exacerbates Acetaminophen-Induced Liver Injury by Promoting Kupffer Cell Activation in Mice.

Overdose of acetaminophen (APAP) has become one of the most frequent causes of acute liver failure. Macrophage-inducible C-type lectin (Mincle) acts as a key moderator in immune responses by recognizing spliceosome-associated protein 130 (SAP130), which is an endogenous ligand released by necrotic cells. This study aims to explore the function of Mincle in APAP-induced hepatotoxicity. Wild-type (WT) and Mincle knockout (KO) mice were used to induce acute liver injury by injection of APAP. The hepatic expressions of Mincle, SAP130, and Mincle signaling intermediate (Syk) were markedly upregulated after the APAP challenge. Mincle KO mice showed attenuated injury in the liver, as shown by reduced pathologic lesions, decreased alanine aminotransferase and aspartate aminotransferase levels, downregulated levels of inflammatory cytokines, and decreased neutrophil infiltration. Consistently, inhibition of Syk signaling by GS9973 alleviated APAP hepatotoxicity. Most importantly, Kupffer cells (KCs) were found as the major cellular source of Mincle. The depletion of KCs abolished the detrimental role of Mincle, and the adoptive transfer of WT KC to Mincle KO mice partially reversed the hyporesponsiveness to hepatotoxicity induced by APAP. Furthermore, the expression levels of interleukin (IL)-1ß and neutrophil-attractant CXC chemokines were substantially lower in KCs isolated from APAP-treated Mincle KO mice compared with those from WT mice. Similar results were found in primary Mincle KO KCs treated with a ligand of Mincle (trehalose-6,6-dibehenate) or in conditioned media obtained from APAP-treated hepatocytes. Collectively, Mincle can regulate the inflammatory response of KCs, which is necessary for the complete progression of hepatotoxicity induced by APAP. SIGNIFICANCE STATEMENT: Acetaminophen (APAP) overdose is becoming a main cause of drug-induced acute liver damage in the developed world. This study showed that macrophage-inducible C-type lectin (Mincle) deletion or inhibition of Mincle downstream signaling attenuates APAP hepatotoxicity. Furthermore, Mincle as a modulator of Kupffer cell activation contributes to the full process of hepatotoxicity induced by APAP. This mechanism will offer valuable insights to overcome the limitation of APAP hepatotoxicity treatment.

Asymmetric Total Synthesis of the Highly Strained 4ß-Acetoxyprobotryane-9ß,15α-diol.

The first and asymmetric total synthesis of 4ß-acetoxyprobotryane-9ß,15α-diol, containing a rare and highly strained trans-fused bicyclo[3.3.0]octane ring system, has been achieved. The synthetically challenging [6-5-5] tricyclic ring system in the final product was efficiently and diastereoselectively synthesized via an asymmetric rhodium-catalyzed [4 + 2] cycloaddition reaction, followed by a unique benzilic acid type rearrangement under very mild conditions. The seven contiguous stereocenters were installed efficiently and diastereoselectively.

Dual TBK1/IKKε inhibitor amlexanox mitigates palmitic acid-induced hepatotoxicity and lipoapoptosis in vitro.

The common causes of Non-alcoholic fatty liver disease (NAFLD) are obesity, dyslipidemia, and insulin resistance. Metabolic disorders and lipotoxic hepatocyte damage are hallmarks of NAFLD. Even though amlexanox, a dual inhibitor of TRAF associated nuclear factor κB (NF-κB) activator-binding kinase 1 (TBK1) and IκB kinase epsilon (IKKε), has been reported to effectively improve obesity-related metabolic dysfunctions in mice models, its molecular mechanism has not been fully investigated. This study was designed to investigate the effects of amlexanox on in vitro nonalcoholic steatohepatitis (NASH) model induced by treatment of palmitic acid (PA, 0.4 mM), using a trans-well co-culture system of hepatocytes and Kupffer cells (KCs). Stimulation with PA significantly increased the phosphorylation levels of TBK1 and IKKε in both hepatocytes and KCs, suggesting a potential role of TBK1/IKKε in PA-induced NASH progression. Treatment of amlexanox (50 µM) showed significantly reduced phosphorylation of TBK1 and IKKε and hepatotoxicity as confirmed by decreased levels of lactate dehydrogenase released from hepatocytes. Furthermore, PA-induced inflammation and lipotoxic cell death in hepatocytes were significantly reversed by amlexanox treatment. Intriguingly, amlexanox inhibited the activation of KCs and induced polarization of KCs towards M2 phenotype. Mechanistically, amlexanox treatment decreased the phosphorylation of interferon regulator factor 3 (IRF3) and NF-κB in PA-treated hepatocytes. However, decreased phosphorylation of NF-κB, not IRF3, was found in PA-treated KCs upon amlexanox treatment. Taken together, our findings show that treatment of amlexanox attenuated the severity of PA-induced hepatotoxicity in vitro and lipoapoptosis by the inhibition of TBK1/IKKε-NF-κB and/or IRF3 pathway in hepatocytes and KCs.

AK-1, a Sirt2 inhibitor, alleviates carbon tetrachloride-induced hepatotoxicity in vivo and in vitro.

Background/Aim: Acute liver injury (ALI) is a life-threatening clinical syndrome that is usually caused by toxic chemicals, drugs, or pathogen infections. Sirtuin2 (Sirt2), an NAD+-dependent deacetylase, appears to play detrimental roles in liver injury. Here, we evaluated the therapeutic application targeting Sirt2 in carbon tetrachloride (CCl4)-induced ALI, by using AK-1 (a Sirt2 inhibitor).Methods: For in vivo experiments, a single injection of CCl4 was used to induce ALI. One hour later, mice were intraperitoneally injected with AK-1 and were sacrificed 24 h after CCl4 administration. For in vitro experiments, primary mouse hepatocytes were used to determine the effects of AK-1 on oxidative stress and hepatocellular death induced by CCl4.Results: AK-1 alleviated CCl4-induced ALI as confirmed by histopathologic analysis, and decreased levels of serum biochemicals and inflammatory cytokines. Although it barely affected the expression of hepatic cytochrome P450 enzymes, AK-1 attenuated CCl4-induced oxidative stress and its related cell death. Mechanistically, Sirt2 inhibition significantly increased the nuclear protein level of nuclear factor erythroid 2-related factor 2 (Nrf2), and meanwhile decreased phosphorylation of c-Jun N-terminal kinases (JNK), in normal and injured livers. Similar results were observed in vitro. AK-1 significantly attenuated CCl4-induced cytotoxicity and oxidative stress by up-regulating the activity of Nrf2, and down-regulating JNK signaling in hepatocytes.Conclusions: Our results suggest that AK-1 treatment attenuated oxidative stress and cell death in the ALI model, at least partially, via activating Nrf2 and inhibiting JNK signaling, and that Sirt2 inhibition might be a potential approach to cure ALI.

Toll-Like Receptor 5 Signaling Ameliorates Liver Fibrosis by Inducing Interferon ß-Modulated IL-1 Receptor Antagonist in Mice.

Bacterial flagellin, recognized by cell surface of Toll-like receptor (TLR) 5, is a potent activator of many types of cells, leading to the activation of innate or adaptive immunity, which are pivotal in regulating fibrotic process. However, the exact role of TLR5 signaling in hepatic fibrogenesis remains unclear, and this study aims to elucidate its underlying mechanisms. Flagellin was injected to hepatotoxin- and cholestasis-induced liver fibrosis murine models. Flagellin-induced TLR5 activation significantly decreased the severity of liver fibrosis. Interestingly, the expression levels of IL-1 receptor antagonist (IL1RN) and interferon (IFN)ß markedly increased in fibrotic livers on flagellin treatment. Consistently, in vivo activation of TLR5 signaling markedly increased IFNß and IL1RN expression in the livers. Notably, flagellin injection significantly exacerbated the severity of liver fibrosis in IFN-α/ß receptor 1 (IFNAR1) knockout mice. Furthermore, hepatic expression of IL1RN in the fibrotic livers of IFNAR1 knockout mice was significantly lower than those of wild-type mice. In support of these findings, flagellin-mediated IL1RN production is not sufficient to alleviate the severity of hepatic fibroinflammatory responses in IFNAR1-deficient milieu. Finally, hepatic stellate cells treated with IL1RN had significantly decreased cellular activation and its associated fibrogenic responses. Collectively, manipulation of TLR5 signaling may be a promising therapeutic strategy for the treatment of liver fibrosis.

Dual TBK1/IKKɛ inhibitor amlexanox attenuates the severity of hepatotoxin-induced liver fibrosis and biliary fibrosis in mice.

Although numerous studies have suggested that canonical IκB kinases (IKK) play a key role in the progression of liver fibrosis, the role of non-canonical IKKε and TANK-binding kinase 1 (TBK1) on the development and progression of liver fibrosis remains unclear. To demonstrate such issue, repeated injection of CCl4 was used to induce hepatotoxin-mediated chronic liver injury and biliary fibrosis was induced by 0.1% diethoxycarbonyl-1, 4-dihydrocollidine diet feeding for 4 weeks. Mice were orally administered with amlexanox (25, 50, and 100 mg/kg) during experimental period. Significantly increased levels of TBK1 and IKKε were observed in fibrotic livers or hepatic stellate cells (HSCs) isolated from fibrotic livers. Interestingly, amlexanox treatment significantly inhibited the phosphorylation of TBK1 and IKKε accompanied by reduced liver injury as confirmed by histopathologic analysis, decreased serum biochemical levels and fibro-inflammatory responses. Additionally, treatment of amlexanox promoted the fibrosis resolution. In accordance with these findings, amlexanox treatment suppressed HSC activation and its related fibrogenic responses by partially inhibiting signal transducer and activator of transcription 3. Furthermore, amlexanox decreased the activation and inflammatory responses in Kupffer cells. Collectively, we found that inhibition of the TBK1 and IKKε by amlexanox is a promising therapeutic strategy to cure liver fibrosis.

Ferroptosis Affects the Progression of Nonalcoholic Steatohepatitis via the Modulation of Lipid Peroxidation-Mediated Cell Death in Mice.

Oxidative stress and its associated lipid peroxidation play a key role in nonalcoholic steatohepatitis (NASH). Ferroptosis is a recently recognized type of cell death characterized by an iron-dependent and lipid peroxidation-mediated nonapoptotic cell death. We demonstrate the impact of ferroptosis on the progression of NASH induced by methionine/choline-deficient diet (MCD) feeding for 10 days. RSL-3 (a ferroptosis inducer) treatment showed decreased hepatic expression of glutathione peroxidase 4 (GPX4) and conversely increased 12/15-lipoxygenase, and apoptosis-inducing factor, indicating that ferroptosis plays a key role in NASH-related lipid peroxidation and its associated cell death. Consistently, levels of serum biochemical, hepatic steatosis, inflammation, and apoptosis in MCD-fed mice were exacerbated with RSL-3 treatment. However, MCD-fed mice treated with sodium selenite (a GPX4 activator) showed increase of hepatic GPX4, accompanied by reduced NASH severity. To chelate iron, deferoxamine mesylate salt was used. Administration of deferoxamine mesylate salt significantly reduced NASH severity and abolished the harmful effects of RSL-3 in MCD-fed mice. Finally, treatment with liproxstatin-1 (a ferroptosis inhibitor) repressed hepatic lipid peroxidation and its associated cell death, resulting in decreased NASH severity. Consistent with the in vivo findings, modulation of ferroptosis/GPX4 affected hepatocellular death in palmitic acid-induced in vitro NASH milieu. We conclude that GPX4 and its related ferroptosis might play a major role in the development of NASH.

Cigarette smoking differentially regulates inflammatory responses in a mouse model of nonalcoholic steatohepatitis depending on exposure time point.

Cigarette smoke (CS) is a risk factor for the development of nonalcoholic fatty liver disease. However, the role of mainstream CS (MSCS) in the pathogenesis of nonalcoholic steatohepatitis (NASH) remains unclear. During the first (early exposure) or last (late exposure) three weeks of methionine-choline deficient with high fat diet feeding (6 weeks), each diet group was exposed to MSCS (300 or 600 µg/L). Hepatic or serum biochemical analysis showed that MSCS differentially modulated hepatic injury in NASH milieu, depending on exposure time points. Consistently, NASH-related hepatocellular apoptosis and fibrosis were increased in the early exposure group, but decreased in the late exposure group, except for steatosis. Ex vivo experiments showed that CS extract differentially regulated inflammatory responses in co-cultured hepatocytes and macrophages isolated from steatohepatitic livers after 10 days or 3 weeks of diet feeding. Furthermore, CS differentially up- and down-regulated the expression levels of M1/M2 polarization markers and peroxisome proliferator-activated receptor-gamma (PPARγ) in livers (29% and 38%, respectively) or co-cultured macrophages (2 and 2.5 fold, respectively). Collectively, our findings indicate that opposite effects of MSCS on NASH progression are mediated by differential modulation of PPARγ and its-associated M1/M2 polarization in hepatic macrophages, depending on exposure time points.