Ferroptosis contributes to ethanol-induced hepatic cell death via labile iron accumulation and GPx4 inactivation.

Alcohol abuse is a significant cause of global morbidity and mortality, with alcoholic liver disease (ALD) being a common consequence. The pathogenesis of ALD involves various cellular processes, including oxidative stress, inflammation, and hepatic cell death. Recently, ferroptosis, an iron-dependent form of programmed cell death, has emerged as a potential mechanism in many diseases. However, the specific involvement and regulatory mechanisms of ferroptosis in ALD remain poorly understood. Here we aimed to investigate the presence and mechanism of alcohol-induced ferroptosis and the involvement of miRNAs in regulating ferroptosis sensitivity. Our findings revealed that long-term ethanol feeding induced ferroptosis in male mice, as evidenced by increased expression of ferroptosis-related genes, lipid peroxidation, and labile iron accumulation in the liver. Furthermore, we identified dysregulation of the methionine cycle and transsulfuration pathway, leading to severe glutathione (GSH) exhaustion and indirect deactivation of glutathione peroxidase 4 (GPx4), a critical enzyme in preventing ferroptosis. Additionally, we identified miR-214 as a ferroptosis regulator in ALD, enhancing hepatocyte ferroptosis by transcriptionally activating the expression of ferroptosis-driver genes. Our study provides novel insights into the involvement and regulatory mechanisms of ferroptosis in ALD, highlighting the potential therapeutic implications of targeting ferroptosis and miRNAs in ALD management.

miR-29c-3p promotes alcohol dehydrogenase gene cluster expression by activating an ADH6 enhancer.

Alcohol dehydrogenases (ADHs) play vital roles in alcohol metabolism and alcohol toxicity, yet little is known about microRNA-mediated regulation of the ADH gene cluster. Here, we showed that miR-29c activated ADH gene cluster transcription by targeting an enhancer element within the ADH6 gene. miR-29c is differentially expressed in alcoholic liver disease. Following biochemical and molecular evidence demonstrated that miR-29c increased ADH6 mRNA and protein levels without affecting the stability of the ADH6 transcript. Further evidence showed that exogenous miR-29c translocated into the nucleus and then unconventionally bound an enhancer element within the ADH6 gene. Luciferase reporter assay and chromatin immunoprecipitation data indicated that miR-29c activated the enhancer and increased the enrichment of RNA polymerase II at the promoter regions of ADH1A, ADH1B, ADH1C, ADH4, and ADH6. Finally, exogenous miR-29c transfection promoted the expression of ADH1A, ADH1B, ADH1C, and ADH4 pre-mRNA and mRNA transcripts from the ADH gene cluster. In conclusion, our data suggest that miR-29c might be a novel epigenetic regulator involved in ADH gene cluster activation.

A novel epigenetic mechanism unravels hsa-miR-148a-3p-mediated CYP2B6 downregulation in alcoholic hepatitis disease.

Cytochrome P450 (CYP) enzymes play critical roles in drug transformation, and the total CYPs are markedly decreased in alcoholic hepatitis (AH), a fatal alcoholic liver disease. miRNAs are endogenous small noncoding RNAs that regulate many essential biological processes. Knowledge concerning miRNA regulation of CYPs in AH disease is limited. Here we presented the changes of key CYPs in liver samples of AH patients retrieved from GEO database, performed in silico prediction of miRNAs potentially targeting the dysregulated CYP transcripts, and deciphered a novel mechanism underlying miRNA mediated CYPs expression in liver cells. Nine miRNAs were predicted to regulate CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2J2, and CYP3A4, among which hsa-miR-148a-3p was selected as a case study. Biochemical and molecular evidences demonstrated that miR-148a promoted CYP2B6 expression by increasing mRNA stability via directly binding to the 3'UTR sequence, and that this positive posttranscriptional regulation was AGO1/2-dependent. Further, luciferase reporter gene assay and RNA secondary structure analysis illustrated that the seedless target site, not the seed target site, controlled miR-148a-mediated CYP2B6 upregulation. Moreover, we identified HNF4A as a liver-specific transcription factor of MIR-148A through EMSA and chromatin immunoprecipitation experiments. In conclusion, ethanol downregulated miR-148a in hepatocytes through HNF4A regulation, which eventually decreased CYP2B6 expression. Our finding will benefit the understanding of dysregulated drug metabolism in AH patients and highlight an unconventional mechanism for epigenetic regulation of CYP gene expression.

A novel mechanism underlying alcohol dehydrogenase expression: hsa-miR-148a-3p promotes ADH4 expression via an AGO1-dependent manner in control and ethanol-exposed hepatic cells.

The alcohol dehydrogenases (ADHs) and aldehyde dehydrogenases (ALDHs) play critical roles in alcoholism development and alcohol toxicology; however, few studies have focused on the miRNA-mediated mechanisms underlying the expressions of alcohol-metabolizing enzymes. In the present study, we showed the expression changes of each alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) in the liver samples of alcoholic hepatitis (AH) patients, and predicted the miRNAs targeting the dysregulated alcohol-metabolizing genes by a systematic in silico analysis. 13 miRNAs were predicted to regulate the expressions of ADH1A, ADH4, and ALDH2, respectively, with hsa-miR-148a-3p (miR-148a) showing the most significant down-regulation in AH patients. Following experimental evidence using HepG2 cells proved that miR-148a promoted ADH4 expression by directly binding to the coding sequence of ADH4 and increasing the mRNA stability via an AGO1-dependent manner. Additional assays showed that secondary structure of ADH4 transcript affected the target accessibility and binding of miR-148a-3p. In sum, our results suggest that the expressions of key alcohol-metabolizing enzymes are repressed in AH patients, and the non-canonical positive regulation of miR-148a on ADH4 reveals a new regulationary mechanism for ADH genes.

A toxicity pathway-oriented approach to develop adverse outcome pathway: AHR activation as a case study.

With numerous new chemicals introduced into the environment everyday, identification of their potential hazards to the environment and human health is a considerable challenge. Developing adverse outcome pathway (AOP) framework is promising in helping to achieve this goal as it can bring In Vitro testing into toxicity measurement and understanding. To explore the toxic mechanism underlying environmental chemicals via the AOP approach, an integration of adequate experimental data with systems biology understanding is preferred. Here, we describe a novel method to develop reliable and sensible AOPs that relies on chemical-gene interactions, toxicity pathways, molecular regulations, phenotypes, and outcomes information obtained from comparative toxicogenomics database (CTD) and Ingenuity Pathway Analysis (IPA). Using Benzo(a)pyrene (BaP), a highly studied chemical as a stressor, we identified the pivotal IPA toxicity pathways, the molecular initiating event (MIE), and candidate key events (KEs) to structure AOPs in the liver and lung, respectively. Further, we used the corresponding CTD information of multiple typical AHR-ligands, including 2,3,7,8-tetrachlorodibenzoparadioxin (TCDD), valproic acid, quercetin, and particulate matter, to validate our AOP networks. Our approach is likely to speed up AOP development as providing a time- and cost-efficient way to collect all fragmented bioinformation in published studies. It also facilitates a better understanding of the toxic mechanism of environmental chemicals, and potentially brings new insights into the screening of critical paths in the AOP network.

CYC31, A Natural Bromophenol PTP1B Inhibitor, Activates Insulin Signaling and Improves Long Chain-Fatty Acid Oxidation in C2C12 Myotubes.

3-bromo-4,5-Bis(2,3-dibromo-4,5-dihydroxybenzyl)-1,2-benzenediol (CYC31) is a bromophenol protein tyrosine phosphatase 1B (PTP1B) inhibitor isolated from the red alga Rhodomela confervoides. Here, the effect of CYC31 on the insulin signaling and fatty-acid-induced disorders in C2C12 myotubes was investigated. Molecular docking assay showed that CYC31 was embedded into the catalytic pocket of PTP1B. A cellular study found that CYC31 increased the activity of insulin signaling and promoted 2-NBDG uptake through GLUT4 translocation in C2C12 myotubes. Further studies showed that CYC31 ameliorated palmitate-induced insulin resistance in C2C12 myotubes. Moreover, CYC31 treatment significantly increased the mRNA expression of carnitine palmitoyltransferase 1B (CPT-1B) and fatty acid binding protein 3 (FABP3), which was tightly linked with fatty acid oxidation. These findings suggested that CYC31 could prevent palmitate-induce insulin resistance and could improve fatty acid oxidation through PTP1B inhibition.