Canonical Wnt signaling promotes HSC glycolysis and liver fibrosis through an LDH-A/HIF-1α transcriptional complex.

BACKGROUND AND AIMS: Aerobic glycolysis reprogramming occurs during HSC activation, but how it is initiated and sustained remains unknown. We investigated the mechanisms by which canonical Wnt signaling regulated HSC glycolysis and the therapeutic implication for liver fibrosis. APPROACH AND RESULTS: Glycolysis was examined in HSC-LX2 cells upon manipulation of Wnt/ß-catenin signaling. Nuclear translocation of lactate dehydrogenase A (LDH-A) and its interaction with hypoxia-inducible factor-1α (HIF-1α) were investigated using molecular simulation and site-directed mutation assays. The pharmacological relevance of molecular discoveries was intensified in primary cultures, rodent models, and human samples. HSC glycolysis was enhanced by Wnt3a but reduced by ß-catenin inhibitor or small interfering RNA (siRNA). Wnt3a-induced rapid transactivation and high expression of LDH-A dependent on TCF4. Wnt/ß-catenin signaling also stimulated LDH-A nuclear translocation through importin ß2 interplay with a noncanonical nuclear location signal of LDH-A. Mechanically, LDH-A bound to HIF-1α and enhanced its stability by obstructing hydroxylation-mediated proteasome degradation, leading to increased transactivation of glycolytic genes. The Gly28 residue of LDH-A was identified to be responsible for the formation of the LDH-A/HIF-1α transcription complex and stabilization of HIF-1α. Furthermore, LDH-A-mediated glycolysis was required for HSC activation in the presence of Wnt3a. Results in vivo showed that HSC activation and liver fibrosis were alleviated by HSC-specific knockdown of LDH-A in mice. ß-catenin inhibitor XAV-939 mitigated HSC activation and liver fibrosis, which were abrogated by HSC-specific LDH-A overexpression in mice with fibrosis. CONCLUSIONS: Inhibition of HSC glycolysis by targeting Wnt/ß-catenin signaling and LDH-A had therapeutic promise for liver fibrosis.

New horizons for the role of RNA N6-methyladenosine modification in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common malignancy, presenting a formidable challenge to the medical community owing to its intricate pathogenic mechanisms. Although current prevention, surveillance, early detection, diagnosis, and treatment have achieved some success in preventing HCC and controlling overall disease mortality, the imperative to explore novel treatment modalities for HCC remains increasingly urgent. Epigenetic modification has emerged as pivotal factors in the etiology of cancer. Among these, RNA N6-methyladenosine (m6A) modification stands out as one of the most prevalent, abundant, and evolutionarily conserved post-transcriptional alterations in eukaryotes. The literature underscores that the dynamic and reversible nature of m6A modifications orchestrates the intricate regulation of gene expression, thereby exerting a profound influence on cell destinies. Increasing evidence has substantiated conspicuous fluctuations in m6A modification levels throughout the progression of HCC. The deliberate modulation of m6A modification levels through molecular biology and pharmacological interventions has been demonstrated to exert a discernible impact on the pathogenesis of HCC. In this review, we elucidate the multifaceted biological functions of m6A modifications in HCC, and concurrently advancing novel therapeutic strategies for the management of this malignancy.

Induction of DNMT1-dependent demethylation of SHP-1 by the natural flavonoid compound Baicalein overcame Imatinib-resistance in CML CD34+ cells.

BACKGROUND: The most significant cause of treatment failure in chronic myeloid leukemia (CML) is a persistent population of minimal residual cells. Emerging evidences showed that methylation of SHP-1 contributed to Imatinib (IM) resistance. Baicalein was reported to have an effect on reversal of chemotherapeutic agents resistance. However, the molecular mechanism of Baicalein on JAK2/STAT5 signaling inhibition against drug resistance in bone marrow (BM) microenvironment that had not been clearly revealed. METHODS: We co-cultured hBMSCs and CML CD34+ cells as a model of SFM-DR. Further researches were performed to clarify the reverse mechanisms of Baicalein on SFM-DR model and engraftment model. The apoptosis, cytotoxicity, proliferation, GM-CSF secretion, JAK2/STAT5 activity, the expression of SHP-1 and DNMT1 were analyzed. To validate the role of SHP-1 on the reversal effect of Baicalein, the SHP-1 gene was over-expressed by pCMV6-entry shp-1 and silenced by SHP-1 shRNA, respectively. Meanwhile, the DNMT1 inhibitor decitabine was used. The methylation extent of SHP-1 was evaluated using MSP and BSP. The molecular docking was replenished to further explore the binding possibility of Baicalein and DNMT1. RESULTS: BCR/ABL-independent activation of JAK2/STAT5 signaling was involved in IM resistance in CML CD34+ subpopulation. Baicalein significantly reversed BM microenvironment-induced IM resistance not through reducing GM-CSF secretion, but interfering DNMT1 expression and activity. Baicalein induced DNMT1-mediated demethylation of the SHP-1 promoter region, and subsequently activated SHP-1 re-expression, which resulted in an inhibition of JAK2/STAT5 signaling in resistant CML CD34+ cells. Molecular docking model indicated that DNMT1 and Baicalein had binding pockets in 3D structures, which further supported Baicalein might be a small-molecule inhibitor targeting DNMT1. CONCLUSIONS: The mechanism of Baicalein on improving the sensitivity of CD34+ cells to IM might be correlated with SHP-1 demethylation by inhibition of DNMT1 expression. These findings suggested that Baicalein could be a promising candidate by targeting DNMT1 to eradicate minimal residual disease in CML patients. Video Abstract.

m6A methylation-induced NR1D1 ablation disrupts the HSC circadian clock and promotes hepatic fibrosis.

The roles of nuclear receptor subfamily 1 group d member 1 (NR1D1) and the circadian clock in liver fibrosis remain unclear. Here, we showed that liver clock genes, especially NR1D1, were dysregulated in mice with carbon tetrachloride (CCl4)-induced liver fibrosis. In turn, disruption of the circadian clock exacerbated experimental liver fibrosis. NR1D1-deficient mice were more sensitive to CCl4-induced liver fibrosis, supporting a critical role of NR1D1 in liver fibrosis development. Validation at the tissue and cellular levels showed that NR1D1 was primarily degraded by N6-methyladenosine (m6A) methylation in a CCl4-induced liver fibrosis model, and this result was also validated in rhythm-disordered mouse models. In addition, the degradation of NR1D1 further inhibited the phosphorylation of dynein-related protein 1-serine site 616 (DRP1S616), resulting in weakened mitochondrial fission function and increased mitochondrial DNA (mtDNA) release in hepatic stellate cell (HSC), which in turn activated the cGMP-AMP synthase (cGAS) pathway. Activation of the cGAS pathway induced a local inflammatory microenvironment that further stimulated liver fibrosis progression. Interestingly, in the NR1D1 overexpression model, we observed that DRP1S616 phosphorylation was restored, and cGAS pathway was also inhibited in HSCs, resulting in improved liver fibrosis. Taken together, our results suggest that targeting NR1D1 may be an effective approach to liver fibrosis prevention and management.

Oroxylin A regulates cGAS DNA hypermethylation induced by methionine metabolism to promote HSC senescence.

Relevant studies have recognized the important role of hepatic stellate cell (HSC) senescence in anti-liver fibrosis. Cellular senescence is believed to be regulated by the cGAS-STING signaling pathway. However, underlying exact mechanisms of cGAS-STING pathway in hepatic stellate cell senescence are still unclear. Here, we found that Oroxylin A could promote senescence in HSC by activating the cGAS-STING pathway. Moreover, activation of the cGAS-STING pathway was dependent on DNMT3A downregulation, which suppressed cGAS gene DNA methylation. Interestingly, the attenuation of DNMT activity relied on the reduction of methyl donor SAM level. Noteworthy, the downregulation of SAM levels implied the imbalance of methionine cycle metabolism, and MAT2A was considered to be an important regulatory enzyme in metabolic processes. In vivo experiments also indicated that Oroxylin A induced senescence of HSCs in mice with liver fibrosis, and DNMT3A overexpression partly offset this effect. In conclusion, we discovered that Oroxylin A prevented the methylation of the cGAS gene by preventing the production of methionine metabolites, which promoted the senescence of HSCs. This finding offers a fresh hypothesis for further research into the anti-liver fibrosis mechanism of natural medicines.

COVID-19-associated liver injury: Adding fuel to the flame.

COVID-19 is mainly characterized by respiratory disorders and progresses to multiple organ involvement in severe cases. With expansion of COVID-19 and SARS-CoV-2 research, correlative liver injury has been revealed. It is speculated that COVID-19 patients exhibited abnormal liver function, as previously observed in the SARS and MERS pandemics. Furthermore, patients with underlying diseases such as chronic liver disease are more susceptible to SARS-CoV-2 and indicate a poor prognosis accompanied by respiratory symptoms, systemic inflammation, or metabolic diseases. Therefore, COVID-19 has the potential to impair liver function, while individuals with preexisting liver disease suffer from much worse infected conditions. COVID-19 related liver injury may be owing to direct cytopathic effect, immune dysfunction, gut-liver axis interaction, and inappropriate medication use. However, discussions on these issues are infancy. Expanding research have revealed that angiotensin converting enzyme 2 (ACE2) expression mediated the combination of virus and target cells, iron metabolism participated in the virus life cycle and the fate of target cells, and amino acid metabolism regulated immune response in the host cells, which are all closely related to liver health. Further exploration holds great significance in elucidating the pathogenesis, facilitating drug development, and advancing clinical treatment of COVID-19-related liver injury. This article provides a review of the clinical and laboratory hepatic characteristics in COVID-19 patients, describes the etiology and impact of liver injury, and discusses potential pathophysiological mechanisms.

ß-Elemene induces apoptosis by activating the P53 pathway in human hypertrophic scar fibroblasts.

Hypertrophic scar (HS) is a condition characterized by excessive synthesis and deposition of collagen. There are many clinical methods to alleviate HS, but most of them are accompanied by many complications. To investigate the effects of ß-Elemene, extracted from the ginger family plant Wenyujin, on human hypertrophic scar fibroblast (hHSFs). Cultured hHSFs and human normal fibroblasts, observed the effect of ß-Elemene on apoptosis, extracellular matrix, and endoplasmic reticulum stress (ERS) by western blot, Real Time-Polymerase Chain Reaction (RT-PCR), and flow cytometry. Based on our findings, it is clear that ß-Elemene could inhibit the expression of α-smooth muscle actin (α-SMA), collagen I, and fibronectin, reduced collagen deposition. Further studies had found that ß-Elemene could increase the expression of ERS-related proteins CHOP and Calnexin in a dose-dependent manner, thereby promoting the aggregation of cleaved-caspase-3 and inducing hHSFs to undergo poptosis. This process may depend on the regulation of P53. The results of our study indicates that ß-Elemene induced hHSFs to undergo apoptosis though ERS pathway in a P53-dependent manner, which means that our research provided a new strategy for the development of drugs for the treatment of HS.

Modification of lysine deacetylation regulates curcumol-induced necroptosis through autophagy in hepatic stellate cells.

The excessive deposition of extracellular matrix (ECM) is the main characteristic of liver fibrosis, and hepatic stellate cells (HSCs) are the main source of ECM. The removal of activated HSCs has a reversal effect on liver fibrosis. Western blot and MTT analysis indicated that curcumol could relieve hepatic fibrosis by promoting HSCs receptor-interacting protein kinase 1/3 (RIP1/RIP3)-dependent necroptosis. Importantly, autophagy flow was monitored by constructing the mRFP-GFP-LC3 plasmid, and it was found that curcumol cleared activated HSCs in a necroptosis manner that was dependent on autophagy. Our study suggested that the activation of necrosome formed by RIP1 and RIP3 depended on Atg5, and that autophagosomes were also necessary for curcumol-induced necroptosis. Furthermore, microscale thermophoresis and co-immunoprecipitation assay results proved that curcumol could target Sirt1 to regulate autophagy by reducing the acetylation level of Atg5. The HSCs-specific silencing of Sirt1 exacerbated CCl4 -induced liver fibrosis in mice. The deacetylation of Atg5 not only accelerated the accumulation of autophagosomes but also enhanced the interaction between Atg5 and RIP1/RIP3 to induce necroptosis. Overall, our study indicated that curcumol could activate Sirt1 to promote Atg5 deacetylation and enhanced its protein-protein interaction function, thereby inducing autophagy and promoting the necroptosis of HSCs to reduce liver fibrosis.

Puerarin attenuates intracerebral hemorrhage-induced early brain injury possibly by PI3K/Akt signal activation-mediated suppression of NF-κB pathway.

Intracerebral hemorrhage (ICH) can induce intensively oxidative stress, neuroinflammation, and brain cell apoptosis. However, currently, there is no highly effective treatment available. Puerarin (PUE) possesses excellent neuroprotective effects by suppressing the NF-κB pathway and activating the PI3K/Akt signal, but its role and related mechanisms in ICH-induced early brain injury (EBI) remain unclear. In this study, we intended to observe the effects of PUE and molecular mechanisms on ICH-induced EBI. ICH was induced in rats by collagenase IV injection. PUE was intraperitoneally administrated alone or with simultaneously intracerebroventricular injection of LY294002 (a specific inhibitor of the PI3K/Akt signal). Neurological deficiency, histological impairment, brain edema, hematoma volume, blood-brain barrier destruction, and brain cell apoptosis were evaluated. Western blot, immunohistochemistry staining, reactive oxygen species (ROS) measurement, and enzyme-linked immunosorbent assay were performed. PUE administration at 50 mg/kg and 100 mg/kg could significantly reduce ICH-induced neurological deficits and EBI. Moreover, PUE could notably restrain ICH-induced upregulation of the NF-κB pathway, pro-inflammatory cytokines, ROS level, and apoptotic pathway and activate the PI3K/Akt signal. However, LY294002 delivery could efficaciously weaken these neuroprotective effects of PUE. Overall, PUE could attenuate ICH-induced behavioral defects and EBI possibly by PI3K/Akt signal stimulation-mediated inhibition of the NF-κB pathway, and this made PUE a potential candidate as a promising therapeutic option for ICH-induced EBI.

The mechanism research on the anti-liver fibrosis of emodin based on network pharmacology.

AIMS: This study was designated to illustrate the underlying mechanisms of emodin anti-liver fibrosis via network pharmacology and experiment. METHODS: The TSMCP and Genecards database were applied to screen the relevant targets of emodin or liver fibrosis. The essential target was selected by using Cytoscape to analyze the topological network of potential targets. Furthermore, we constructed a preliminary molecule docking study to explore the binding site by Surflex-Dock suite SYBYL X 2.0. The DAVID database was selected for gene functional annotations and KEGG enrichment analysis. Moreover, we demonstrated the ameliorating effect of emodin on carbon tetrachloride (CCl4 )-induced liver injury in mice. We also verified the network predictions in vitro via various techniques. RESULTS: The collected results showed that 35 targets were related to emodin, and 6,198 targets were associated with liver fibrosis. The Venn analysis revealed that 17 intersection targets were correlated with emodin anti-liver fibrosis. The topological network analysis suggested that the p53 was the remarkable crucial target. Besides, the molecule docking results showed that emodin could directly interact with p53 by binding the active site residues ASN345, GLN331, and TYR347. Finally, KEGG pathway enrichment results indicated that essential genes were mainly enriched in mitogen-activated protein kinase (MAPK) signaling pathways. Moreover, our study confirmed that emodin alleviated CCl4 -induced liver injury in mice, inducing hepatic stellate cells (HSCs) apoptosis via regulating the p53/ERK/p38 axis. CONCLUSIONS: This study partially verified the network pharmacological prediction of emodin inducing HSCs cell apoptosis through the p53/ERK/p38 axis.

A novel lncRNA PLK4 up-regulated by talazoparib represses hepatocellular carcinoma progression by promoting YAP-mediated cell senescence.

A growing number of studies recognize that long non-coding RNAs (lncRNAs) are essential to mediate multiple tumorigenic processes, including hepatic tumorigenesis. However, the pathological mechanism of lncRNA-regulated liver cancer cell growth remains poorly understood. In this study, we identified a novel function lncRNA, named polo-like kinase 4 associated lncRNA (lncRNA PLK4, GenBank Accession No. RP11-50D9.3), whose expression was dramatically down-regulated in hepatocellular carcinoma (HCC) tissues and cells. Interestingly, talazoparib, a novel and highly potent poly-ADP-ribose polymerase 1/2 (PARP1/2) inhibitor, could increase lncRNA PLK4 expression in HepG2 cells. Importantly, we showed that talazoparib-induced lncRNA PLK4 could function as a tumour suppressor gene by Yes-associated protein (YAP) inactivation and induction of cellular senescence to inhibit liver cancer cell viability and growth. In summary, our findings reveal the molecular mechanism of talazoparib-induced anti-tumor effect, and suggest a potential clinical use of talazoparib-targeted lncRNA PLK4/YAP-dependent cellular senescence for the treatment of HCC.

Novel mitochondrion-targeting copper(II) complex induces HK2 malfunction and inhibits glycolysis via Drp1-mediating mitophagy in HCC.

[Cu(ttpy-tpp)Br2 ]Br (abbreviated as CTB) is a novel mitochondrion-targeting copper(II) complex synthesized by our research group, which contains tri-phenyl-phosphonium (TPP) groups as its lipophilic property. In this study, we explored how CTB affects mitochondrial functions and exerts its anti-tumour activity. Multiple functional and molecular analyses including Seahorse XF Bioanalyzer Platform, Western blot, immunofluorescence analysis, co-immunoprecipitation and transmission electron microscopy were used to elucidate the underlying mechanisms. Human hepatoma cells were subcutaneously injected into right armpit of male nude mice for evaluating the effects of CTB in vivo. We discovered that CTB inhibited aerobic glycolysis and cell acidification by impairing the activity of HK2 in hepatoma cells, accompanied by dissociation of HK2 from mitochondria. The modification of HK2 not only led to the complete dissipation of mitochondrial membrane potential (MMP) but also promoted the opening of mitochondrial permeability transition pore (mPTP), contributing to the activation of mitophagy. In addition, CTB co-ordinately promoted dynamin-related protein 1 (Drp1) recruitment in mitochondria to induce mitochondrial fission. Our findings established a previously unrecognized role for copper complex in aerobic glycolysis of tumour cells, revealing the interaction between mitochondrial HK2-mediated mitophagy and Drp1-regulated mitochondrial fission.

Endoplasmic reticulum stress and protein degradation in chronic liver disease.

Endoplasmic reticulum (ER) stress is easily observed in chronic liver disease, which often causes accumulation of unfolded or misfolded proteins in the ER, leading to unfolded protein response (UPR). Regulating protein degradation is an integral part of UPR to relieve ER stress. The major protein degradation system includes the ubiquitin-proteasome system (UPS) and autophagy. All three arms of UPR triggered in response to ER stress can regulate UPS and autophagy. Accumulated misfolded proteins could activate these arms, and then generate various transcription factors to regulate the expression of UPS-related and autophagy-related genes. The protein degradation process regulated by UPR has great significance in many chronic liver diseases, including non-alcoholic fatty liver disease (NAFLD), alcoholic liver disease (ALD), viral hepatitis, liver fibrosis, and hepatocellular carcinoma(HCC). In most instances, the degradation of excessive proteins protects cells with ER stress survival from apoptosis. According to the specific functions of protein degradation in chronic liver disease, choosing to promote or inhibit this process is promising as a potential method for treating chronic liver disease.

Oroxylin A induces apoptosis of activated hepatic stellate cells through endoplasmic reticulum stress.

Hepatic stellate cell (HSC) activation plays an indispensable role in hepatic fibrosis. Inducing apoptosis of activated HSCs can attenuate or reverse fibrogenesis. In this study, we initially found that oroxylin A (OA) protected CCl4-induced liver injury accompanied by endoplasmic reticulum stress (ERS) activation of HSCs in mice. In vitro, OA treatment markedly reduced fibrogenesis by modulating extracellular matrix synthesis and degradation. OA inhibited cell proliferation and induced cell cycle arrest of HSCs at S phase. Further, OA was observed to induce HSC apoptosis, as indicated by caspase activation. Using the eIF2α dephosphorylation inhibitor salubrinal, we found that ERS pathway activation was required for OA to induce HSC apoptosis. ERS-related proteins were significantly upregulated by OA treatment, and salubrinal abrogated the effects of OA on HSCs. Thus, we inferred that OA attenuated HSC activation by promoting ERS. In vivo, inhibition of ERS by salubrinal partly abrogated the hepatoprotective effect of OA in CCl4-treated mice. In conclusion, our findings suggest a role for ERS in the mechanism underlying amelioration of hepatic fibrosis by OA.

Dihydroartemisinin attenuates alcoholic fatty liver through regulation of lipin-1 signaling.

Alcoholic liver disease (ALD) is generated from excessive alcohol consumption, characterized by hepatic steatosis. Mechanistically, excessive hepatic lipid accumulation was attributed to the aberrant lipin-1 signaling during the development of alcoholic steatosis in rodent species and human. Dihydroartemisinin (DHA) has been recently identified to relieve hepatocytes necrosis and prevent from hepatic steatosis in alcohol-induced liver diseases; however, the role of DHA in ALD has not been elucidated completely. Therefore, this study was aimed to further identify the potential mechanisms of pharmacological effects of DHA on ALD. Results demonstrated that DHA regulated the expression and nucleocytoplasmic shuttling of lipin-1 in mice with chronic ethanol exposure. Results confirmed that the disruption of lipin-1 signaling abolished the suppression of DHA on alcohol-induced hepatic steatosis. Interestingly, DHA also significantly improved liver injury, and inflammation mediated by lipin-1 signaling in chronic alcohol-fed mice. in vivo experiments further consolidated the concept that DHA protected against hepatocyte lipoapoptosis dependent on the regulation of nucleocytoplasmic shuttling of lipin-1 signaling, resulting in attenuated ratio of Lpin1 ß/α. Obvious increases in cell apoptosis were observed in alcohol-treated lipin1ß-overexpressed mice. Although DHA attenuated cell apoptosis, overexpression of lipin-1ß neutralized DHA action. DHA ameliorated activation of endoplasmic reticulum stress through inhibiting activation of JNK and CHOP, which was abrogated by overexpression of lipin-1ß. In summary, DHA significantly improved liver injury, steatosis and hepatocyte lipoapoptosis in chronic alcohol-fed mice via regulation of lipin-1 signaling.

P53-dependent induction of ferroptosis is required for artemether to alleviate carbon tetrachloride-induced liver fibrosis and hepatic stellate cell activation.

Ferroptosis is recently reported as a new mode of regulated cell death. Its essential characteristics are disturbed redox homeostasis, overloaded iron, and increased lipid peroxidation. However, the role of ferroptosis in liver fibrosis remains poorly understood. In this study, we attempted to investigate the effect of artemether (ART) on ferroptosis in hepatic fibrosis and to further clarify the possible mechanisms. Our data showed that ART treatment markedly attenuated liver injury and reduced fibrotic scar formation in the mouse model of liver fibrosis. Moreover, experiments in vitro also confirmed that ART treatment significantly decreased expression of hepatic stellate cell (HSC) activation markers. Interestingly, HSCs treated by ART presented morphological features of ferroptosis. Furthermore, ART remarkably triggered ferroptosis by promoting the accumulation of iron and lipid peroxides, whereas inhibition of ferroptosis by specific inhibitor ferrostatin-1 (Fer-1) completely abolished ART-induced antifibrosis effect. More importantly, our discovery determined that tumor suppressor P53 was an upstream molecule in the facilitation of ART-induced HSC ferroptosis. Conversely, knockdown of P53 by siRNA evidently blocked ART-induced HSC ferroptosis in turn exacerbated liver fibrosis. Overall, our findings revealed that P53-dependent induction of ferroptosis is necessary for ART to ameliorate CCl4 -induced hepatic fibrosis and inhibit HSC activation. © 2018 IUBMB Life, 71(1):45-56, 2019.

TPP-related mitochondrial targeting copper (II) complex induces p53-dependent apoptosis in hepatoma cells through ROS-mediated activation of Drp1.

BACKGROUND: In recent years, copper complexes have gradually become the focus of potential anticancer drugs due to their available redox properties and low toxicity. In this study, a novel mitochondrion-targeting copper (II) complex, [Cu (ttpy-tpp)Br2] Br (simplified as CTB), is first synthesized by our group. CTB with tri-phenyl-phosphine (TPP), a targeting and lipophilic group, can cross the cytoplasmic and mitochondrial membranes of tumor cells. The present study aims to investigate how CTB affects mitochondrial functions and exerts its anti-tumor activity in hepatoma cells. METHODS: Multiple molecular experiments including Flow cytometry, Western blot, Immunofluorescence, Tracker staining, Transmission Electron Microscopy and Molecular docking simulation were used to elucidate the underlying mechanisms. Human hepatoma cells were subcutaneously injected into right armpit of male nude mice for evaluating the effects of CTB in vivo. RESULTS: CTB induced apoptosis via collapse of mitochondrial membrane potential (MMP), ROS production, Bax mitochondrial aggregation as well as cytochrome c release, indicating that CTB-induced apoptosis was associated with mitochondrial pathway in human hepatoma cells. Mechanistic study revealed that ROS-related mitochondrial translocation of p53 was involved in CTB-mediated apoptosis. Simultaneously, elevated mitochondrial Drp1 levels were also observed, and interruption of Drp1 activation played critical role in p53-dependent apoptosis. CTB also strongly suppressed the growth of liver cancer xenografts in vivo. CONCLUSION: In human hepatoma cells, CTB primarily induces mitochondrial dysfunction and promotes accumulation of ROS, leading to activation of Drp1. These stimulation signals accelerate mitochondrial accumulation of p53 and lead to the eventual apoptosis. Our research shows that CTB merits further evaluation as a chemotherapeutic agent for the treatment of Hepatocellular carcinoma (HCC).

Blockade of glycolysis-dependent contraction by oroxylin a via inhibition of lactate dehydrogenase-a in hepatic stellate cells.

BACKGROUND: Contraction of hepatic stellate cells (HSCs) plays an important role in the pathogenesis of liver fibrosis by regulating sinusoidal blood flow and extracellular matrix remodeling. Here, we investigated how HSC contraction was affected by the natural compound oroxylin A, and elucidated the underlying mechanism. METHODS: Cell contraction and glycolysis were examined in cultured human HSCs and mouse liver fibrosis model upon oroxylin A intervention using diversified cellular and molecular assays, as well as genetic approaches. RESULTS: Oroxylin A limited HSC contraction associated with inhibiting myosin light chain 2 phosphorylation. Oroxylin A blocked aerobic glycolysis in HSCs evidenced by reduction in glucose uptake and consumption and lactate production. Oroxylin A also decreased extracellular acidification rate and inhibited the expression and activity of glycolysis rate-limiting enzymes (hexose kinase 2, phosphofructokinase 1 and pyruvate kinas type M2) in HSCs. Then, we identified that oroxylin A blockade of aerobic glycolysis contributed to inhibition of HSC contraction. Furthermore, oroxylin A inhibited the expression and activity of lactate dehydrogenase-A (LDH-A) in HSCs, which was required for oroxylin A blockade of glycolysis and suppression of contraction. Oral administration of oroxylin A at 40 mg/kg reduced liver injury and fibrosis, and inhibited HSC glycolysis and contraction in mice with carbon tetrachloride-induced hepatic fibrosis. However, adenovirus-mediated overexpression of LDH-A significantly counteracted the oroxylin A's effects in fibrotic mice. CONCLUSIONS: Blockade of aerobic glycolysis by oroxylin A via inhibition of LDH-A reduced HSC contraction and attenuated liver fibrosis, suggesting LDH-A as a promising target for intervention of hepatic fibrosis.

Perilipin 5 and liver fatty acid binding protein function to restore quiescence in mouse hepatic stellate cells.

Hepatic stellate cell (HSC) activation occurs along with decreased Perilipin5 (Plin5) and liver fatty acid-binding protein (L-Fabp) expression and coincident lipid droplet (LD) depletion. Conversely, the activated phenotype is reversible in WT HSCs upon forced expression of Plin5. Here, we asked if L-Fabp expression is required for Plin5-mediated rescue of the quiescent phenotype. Lentiviral Plin5 transduction of passaged L-Fabp-/- HSCs failed to reverse activation markers or restore lipogenic gene expression and LD formation. However, adenoviral L-Fabp infection of lentiviral Plin5 transduced L-Fabp-/- HSCs restored both the quiescent phenotype and LD formation, an effect also mediated by adenoviral intestine-Fabp or adipocyte-Fabp. Expression of exogenous Plin5 in activated WT HSCs induced a transcriptional program of lipogenic gene expression including endogenous L-Fabp, but none of the other FABPs. We further demonstrated that selective, small molecule inhibition of endogenous L-Fabp also eliminated the ability of exogenous Plin5 to rescue LD formation and reverse activation of WT HSCs. This functional coordination of L-Fabp with Plin5 was 5'-AMP-activated protein kinase (AMPK)-dependent and was eliminated by AMPK inhibition. Taken together, our results indicate that L-Fabp is required for Plin5 to activate a transcriptional program that restores LD formation and reverses HSC activation.

Oroxylin A prevents angiogenesis of LSECs in liver fibrosis via inhibition of YAP/HIF-1α signaling.

Angiogenesis of liver sinusoidal endothelial cells (LSECs) accompanies with hypoxia in liver fibrosis and they are of mutual promotion, which has raised wide concern. Here we established murine model of liver fibrosis and found that oroxylin A (40 mg/kg) could ameliorate angiogenesis in liver fibrosis may related to hypoxia inducible factor 1α (HIF-1α). The underlying mechanism was further investigated by isolating and culturing murine primary LSECs. Hypoxia induced vascular endothelial growth factor A (VEGF-A), angiopoietin 2 (Ang-2), and platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) elevated in LSECs were reduced by oroxylin A or acriflavine (ACF, an HIF-1α inhibitor), indicating HIF-1α involved the angiogenesis of LSECs. Additionally, interference with Yes-associated protein (YAP) significant downregulated the protein expression of HIF-1α and VEGF-A, while YAP plasmid exhibited an opposite effect. We next found that oroxylin A inhibited hypoxia-induced nuclear translocation of YAP, which may influence the accumulation of HIF-1α and subsequently decrease transcription of downstream target gene including VEGF-A and Ang-2, thereby exerting an anti-angiogenic activity.