Evaluation of capillary miR-122 as a prognostic biomarker of paracetamol-induced liver toxicity.

INTRODUCTION: Paracetamol (acetaminophen) overdose is a leading cause of acute liver failure in many Western countries. Diagnostic tools for this poisoning may be suboptimal in some cases and new biomarkers have been investigated. We investigated the role of capillary microRNA-122 (miR-122) as a prognostic biomarker of liver injury in the clinical management of patients with paracetamol overdose. METHODS: In a paracetamol overdose patient cohort, miR-122 was measured by quantitative polymerase chain reaction in a blood drop obtained by a finger prick at the end of an antidote cycle treatment with N-acetylcysteine treatment (12 h). Liver injury was defined as serum alanine aminotransferase (ALT) activity > 100 IU/L collected at 10 or 20 h after the start of treatment. Pearson's correlation analyses were performed. RESULTS: In patients with paracetamol overdose, capillary miR-122 was positively correlated with ALT measured at 10 h and at 20 h (r = 0.83, P < 0.0001; r = 0.96, P < 0.0001, respectively). CONCLUSION: This work supports the potential use of capillary miR-122 as a prognostic biomarker of liver injury throughout clinical management of patients with paracetamol overdose. Capillary miR-122 can be measured in a blood drop collected by a finger prick, a minimally invasive diagnostic test for patient stratification.

METTL3-mediated m6A mRNA modification was involved in cadmium-induced liver injury.

Cadmium is an environmental pollutant that has extensive deleterious effects. However, the mechanisms underlying the hepatotoxicity induced by long-term exposure to cadmium remained undefined. In the present study, we explored the role of m6A methylation in the development of cadmium-induced liver disease. We showed a dynamic change of RNA methylation in liver tissue from mice administrated with cadmium chloride (CdCl2) for 3, 6 and 9 months, respectively. Particularly, the METTL3 expression was declined in a time-dependent manner, associated with the degree of liver injury, indicating the involvement of METTL3 in hepatotoxicity induced by CdCl2. Moreover, we established a mouse model with liver-specific over-expression of Mettl3 and administrated these mice with CdCl2 for 6 months. Notably, METTL3 highly expressed in hepatocytes attenuated CdCl2-induced steatosis and liver fibrosis in mice. In vitro assay also showed METTL3 overexpression ameliorated the CdCl2-induced cytotoxicity and activation of primary hepatic stellate cells. Furthermore, transcriptome analysis identified 268 differentially expressed genes both in mice liver tissue treated with CdCl2 for 3 months and 9 months. Among them, 115 genes were predicted to be regulated by METTL3 determined by m6A2Target database. Further analysis revealed the perturbation of metabolic pathway, glycerophospholipid metabolism, ErbB signaling pathway, Hippo signaling pathway, and choline metabolism in cancer, and circadian rhythm, led to hepatotoxicity induced by CdCl2. Collectively, our findings reveal new insight into the crucial role of epigenetic modifications in hepatic diseases caused by long-term exposure to cadmium.

Contribution of HIF-1α/BNIP3-mediated autophagy to lipid accumulation during irinotecan-induced liver injury.

Irinotecan is a topoisomerase I inhibitor which has been widely used to combat several solid tumors, whereas irinotecan therapy can induce liver injury. Liver injury generally leads to tissue hypoxia, and hypoxia-inducible factor-1α (HIF-1α), a pivotal transcription factor, mediates adaptive pathophysiological responses to lower oxygen condition. Previous studies have reported a relationship between HIF-1α and autophagy, and autophagy impairment is a common characteristic in a variety of diseases. Here, irinotecan (50 mg/kg) was employed on mice, and HepG2 and L-02 cells were cultured with irinotecan (10, 20 and 40 µM). In vivo study, we found that irinotecan treatment increased final liver index, serum aminotransferase level and hepatic lipid accumulation. Impaired autophagic flux and activation of HIF-1α/BNIP3 pathway were also demonstrated in the liver of irinotecan-treated mice. Moreover, irinotecan treatment significantly deteriorated hepatic oxidative stress, evidenced by increased MDA and ROS contents, as well as decreased GSH-Px, SOD and CAT contents. Interestingly, protein levels of NLRP3, cleaved-caspase 1 and IL-1ß were enhanced in the liver of mice injected with irinotecan. In vitro study, irinotecan-treated HepG2 and L-02 cells also showed impaired autophagic flux, while HIF-1α inhibition efficaciously removed the accumulated autophagosomes induced by irinotecan. Additionally, irinotecan treatment aggravated lipid accumulation in HepG2 and L-02 cells, and HIF-1α inhibition reversed the effect of irinotecan. Furthermore, HIF-1α inhibition weakened irinotecan-induced NLRP3 inflammasome activation in HepG2 cells. Taken together, our results suggest that irinotecan induces liver injury by orchestrating autophagy via HIF-1α/BNIP3 pathway, and HIF-1α inhibition could alleviate irinotecan-induced lipid accumulation in HepG2 and L-02 cells, which will provide a new clue and direction for the prevention of side effects of clinical chemotherapy drugs.

Lycopene Alleviates Chronic Stress-Induced Liver Injury by Inhibiting Oxidative Stress-Mediated Endoplasmic Reticulum Stress Pathway Apoptosis in Rats.

The liver is the major organ of metabolism and is extremely vulnerable to chronic stress. Lycopene (LYC) is a natural carotenoid with potent antioxidant and chronic disease potential. However, whether LYC protects against chronic restraint stress (CRS)-induced liver injury and the underlying mechanisms remain unclear. In this study, rats were restrained for 21 days for 6 h per day, with or without gavage of LYC (10 mg/kg). Serum ALT (85.99 ± 4.07 U/L) and AST (181.78 ± 7.35 U/L) and scores of liver injury were significantly increased in the CRS group. LYC significantly promoted the nuclear translocation of Nrf2, elevated the expression of antioxidant genes, and attenuated reactive oxygen radicals (ROS) levels within the liver. Cellular thermal shift assay (CETSA) and molecular docking results indicated that LYC competitively binds to Keap1 with the lowest molecule affinity of -9.0 kcal/mol. Moreover, LYC significantly relieved the hepatic endoplasmic reticulum swelling and decreased the expression of endoplasmic reticulum stress (ERS) hallmarks like GRP78, CHOP, and cleaved caspase-12. Meanwhile, LYC also mitigated CRS-induced hepatocyte apoptosis. Interestingly, every other day, the intraperitoneal injection of the Nrf2 inhibitor brusatol (0.4 mg/kg) significantly counteracted the protective effect of LYC. In conclusion, LYC protects against CRS-induced liver injury by activating the Nrf2 signaling pathway, scavenging ROS, and further attenuating ERS-associated apoptosis pathways.

Hepatic CYP2B10 is highly induced by binge ethanol and contributes to acute-on-chronic alcohol-induced liver injury.

BACKGROUND: The chronic-plus-binge model of ethanol consumption, where chronically (8-week) ethanol-fed mice are gavaged a single dose of ethanol (E8G1), is known to induce steatohepatitis in mice. However, how chronically ethanol-fed mice respond to multiple binges of ethanol remains unknown. METHODS: We extended the E8G1 model to three gavages of ethanol (E8G3) spaced 24 h apart, sacrificed each group 9 h after the final gavage, analyzed liver injury, and examined gene expression changes using microarray analyses in each group to identify mechanisms contributing to liver responses to binge ethanol. RESULTS: Surprisingly, E8G3 treatment induced lower levels of liver injury, steatosis, inflammation, and fibrosis as compared to mice after E8G1 treatment. Microarray analyses identified several pathways that may contribute to the reduced liver injury after E8G3 treatment compared to E8G1 treatment. The gene encoding cytochrome P450 2B10 (Cyp2b10) was one of the top upregulated genes in the E8G1 group and was further upregulated in the E8G3 group, but only moderately induced after chronic ethanol consumption, as confirmed by RT-qPCR and western blot analyses. Genetic disruption of Cyp2b10 worsened liver injury in E8G1 and E8G3 mice with higher blood ethanol levels compared to wild-type control mice, while in vitro experiments revealed that CYP2b10 did not directly promote ethanol metabolism. Metabolomic analyses revealed significant differences in hepatic metabolites from E8G1-treated Cyp2b10 knockout and WT mice, and these metabolic alterations may contribute to the reduced liver injury in Cyp2b10 knockout mice. CONCLUSION: Hepatic Cyp2b10 expression is highly induced after ethanol binge, and such upregulation reduces acute-on-chronic ethanol-induced liver injury via the indirect modification of ethanol metabolism.

Upregulated miR-322-5p regulates cell cycle and promotes cell proliferation and apoptosis by directly targeting Wee1 in mice liver injury.

Liver injury from any number of causes (e.g. chemical material, drugs and diet, viral infection) is a global health problem, and its mechanism is not clearly understood. MicroRNAs (miRNAs) expression profiling is gaining popularity because miRNAs, as key regulators in gene expression networks, can influence many biological processes and have also shown promise as biomarkers for disease. Previous studies reported the regulation effects of miRNAs in liver injury, whereas function and molecular mechanisms of miR-322-5p were still unclear. Therefore, our study focused on the biological role of miR-322-5p in carbon tetrachloride (CCl4)-induced liver injury proliferation, apoptosis, and cell cycle. A mouse model of CCl4-induced liver injury was established, and the transcriptomes and miRNAs transcriptomes of 2d and 5d liver tissues after injury were sequenced. The expression of miR-322-5p and the cell cycle genes were detected in liver tissues and Hepa1-6 cell line by miRNA RT-PCR, qRT-PCR. The effects of miR-322-5p on liver cell proliferation, cell cycle and apoptosis were evaluated using MTS assays and flow cytometry analysis. The relationship between miR-322-5p and Wee1 was predicted and confirmed by bioinformatics analysis and a dual luciferase reporter assay. Functional experiments, including an MTS assay and flow cytometric analysis, were performed to study the effects of Wee1. MiR-322-5p was upregulated in injury liver tissues, and downregulated miR-322-5p was proved to inhibit proliferation, apoptosis and arrest cell cycle at G2/M in vitro. The dual-luciferase reporter assay results indicated that miR-322-5p has a binding site at position 285 in the Wee1 3´UTR. The effects of miR-322-5p in proliferation and cell cycle regulation can be abolished by Wee1 through rescue experiments. By directly targeting Wee1 influenced the expression of several cell cycle factors, including Cyclin dependent kinase 1 (Cdk1), cyclin B1 (Ccnb1) and Cell division cyclin 25C (Cdc25C). MiR-322-5p may function as a suppressive factor by negatively controlling Wee1, thus, highlighting the potential role of miR-322-5p as a therapeutic target for liver injury.Abbreviations: ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; GSH: Glutathione, γ-glutamyl cysteinel + glycine; CCl4: Carbon tetrachloride; HE: Haematoxylin and eosin; KEGG: Kyoto Encyclopedia of Genes and Genomes.

DNMT3a-mediated methylation of PSTPIP2 enhances inflammation in alcohol-induced liver injury via regulating STAT1 and NF-κB pathway.

Alcohol-induced liver injury (ALI) is associated with inflammatory responses regulated by macrophages. Activation of macrophages plays a crucial role in ALI while DNA methylation-regulated gene silencing is associated with inflammation processes in macrophages. Proline-Serine-Threonine Phosphatase Interacting Protein 2 (PSTPIP2), which belongs to the Fes/CIP4 homology-Bin/Amphiphysin/Rvs domain family of proteins and plays a role in macrophages. Previous studies have shown that Pstpip2 can be methylated. Herein, its expression was found to be significantly downregulated in primary liver macrophages isolated from EtOH-fed mice and EtOH-induced RAW264.7 cells. Overexpression of PSTPIP2 using liver-specific recombinant AAV serotype 9 (rAAV9)-PSTPIP2 in EtOH-fed mice dramatically alleviated liver injury and inflammatory responses. In addition, silencing of PSTPIP2 aggravated the alcohol-induced inflammatory response in vitro. Mechanistically, PSTPIP2 might affect macrophage-induced inflammatory responses by regulating the STAT1 and NF-κB signaling pathways. The downregulation of PSTPIP2 in ALI may be associated with DNA methylation. Methylation-specific PCR and western blotting analyses showed that EtOH induced abnormal DNA methylation patterns and increased the protein expression levels of DNMT1, DNMT3a, and DNMT3b. The chromatin immunoprecipitation assay showed that DNMT3a could directly bind to the Pstpip2 promoter and act as a principal regulator of PSTPIP2 expression. Moreover, silencing of DNMT3a significantly restored the EtOH-induced low expression of PSTPIP2 and inhibited EtOH-induced inflammation. Overall, these findings provide a detailed understanding of the possible functions and mechanisms of PSTPIP2 in ALI, thus providing new substantive research to elucidate the pathogenesis of ALI and investigate potential targeted treatment strategies.

Orphan Nuclear Receptor RORγ Modulates the Genome-Wide Binding of the Cholesterol Metabolic Genes during Mycotoxin-Induced Liver Injury.

Maintaining lipid homeostasis is crucial to liver function, the key organ that governs the whole-body energy metabolism. In contrast, lipid dysregulation has been implicated in mycotoxin-induced liver injury, by which the pathophysiological regulation and the molecular components involved remain elusive. Here we focused on the potential roles of orphan nuclear receptor (NR) RORγ in lipid programming, and aimed to explore its action on cholesterol regulation in the liver of mycotoxin-exposed piglets. We found that liver tissues were damaged in the mycotoxin-exposed piglets compared to the healthy controls, revealed by histological analysis, elevated seral ALT, AST and ALP levels, and increased caspase 3/7 activities. Consistent with the transcriptomic finding of down-regulated cholesterol metabolism, we demonstrated that both cholesterol contents and cholesterol biosynthesis/transformation gene expressions in the mycotoxin-exposed livers were reduced, including HMGCS1, FDPS, SQLE, EBP, FDFT1 and VLDLR. Furthermore, we reported that RORγ binds to the cholesterol metabolic genes in porcine hepatocytes using a genome-wide ChIP-seq analysis, whereas mycotoxin decreased the RORγ binding occupancies genome-wide, especially at the cholesterol metabolic pathway. In addition, we revealed the enrichment of co-factors p300 and SRC, the histone marks H3K27ac and H3K4me2, together with RNA Polymerase II (Pol-II) at the locus of HMGCS1 in hepatocytes, which were reduced by mycotoxin-exposure. Our results provide a deep insight into the cholesterol metabolism regulation during mycotoxin-induced liver injury, and propose NRs as therapeutic targets for anti-mycotoxin treatments.

Hypoxia Preconditioning Promotes the Proliferation and Migration of Human Urine-Derived Stem Cells in Chronically Injured Liver of Mice by Upregulating CXCR4.

Our previous studies reported that urine-derived stem cells (USCs) possess a strong self-renewal ability and multidirectional differentiation potential and thus are an ideal candidate cell source for hepatocellular transplantation. USC transplantation may repair the pathological changes of chronic liver injury to a certain extent, and hypoxia pretreatment may improve the recovery efficiency of USCs. Therefore, the present study aimed to investigate the possible mechanism of the improved recovery efficiency of hypoxia-pretreated USCs. A chronic liver injury model was established by intraperitoneal injection of carbon tetrachloride into nude mice. USCs were transplanted via caudal vein injection. Hematoxylin and eosin staining and Masson's staining were performed to determine the pathology of the liver. Immunofluorescence and frozen section biopsy were performed to determine differentiation and cell fusion in vivo. Cell coculture was used to detect cell fusion in vitro. The proliferative ability of USCs was evaluated using cell viability and colony formation assays, and the migratory functions of USCs were evaluated using wound healing and transwell assays. The degeneration of hepatocytes and the level of fibrosis in the hypoxia transplantation group were improved compared with the normoxia transplantation group. It was found that exogenous USCs may be differentiated into functional hepatocytes or fused with hepatocytes in vivo. C-X-C motif chemokine (CXC) ligand 12 (CXCL12) expression levels in liver tissue of the chronic liver injury model were upregulated compared with those in the control group. The expression of CXC receptor 4 (CXCR4) in hypoxia-pretreated USCs was also significantly upregulated. The results suggested that USCs fused with different types of liver cells and that hypoxia treatment promoted the fusion rate in vitro by upregulating CXCR4 signaling. Furthermore, hypoxia pretreatment promoted cell proliferation, migration, and cell fusion by inducing CXCR4 signaling, leading to USC-elicited liver tissue recovery following injury in vivo.

Dietary iso-α-acids prevent acetaldehyde-induced liver injury through Nrf2-mediated gene expression.

Acetaldehyde is the major toxic metabolite of alcohol (ethanol) and enhances fibrosis of the liver through hepatic stellate cells. Additionally, alcohol administration causes the accumulation of reactive oxygen species (ROS), which induce hepatocyte injury-mediated lipid peroxidation. Iso-α-acids, called isohumulones, are bitter acids in beer. The purpose of this study was to investigate the protective effects of iso-α-acids against alcoholic liver injury in hepatocytes in mice. C57BL/6N mice were fed diets containing isomerized hop extract, which mainly consists of iso-α-acids. After 7 days of feeding, acetaldehyde was administered by a single intraperitoneal injection. The acetaldehyde-induced increases in serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were suppressed by iso-α-acids intake. Hepatic gene expression analyses showed the upregulation of detoxifying enzyme genes, glutathione-S-transferase (GST) and aldehyde dehydrogenase (ALDH). In vitro, iso-α-acids upregulated the enzymatic activities of GST and ALDH and induced the nuclear translocation of nuclear factor-erythroid-2-related factor 2 (Nfe2l2; Nrf2), a master regulator of antioxidant and detoxifying systems. These results suggest that iso-α-acid intake prevents acetaldehyde-induced liver injury by reducing oxidative stress via Nrf2-mediated gene expression.

De Novo Profiling of Long Non-Coding RNAs Involved in MC-LR-Induced Liver Injury in Whitefish: Discovery and Perspectives.

Microcystin-LR (MC-LR) is a potent hepatotoxin for which a substantial gap in knowledge persists regarding the underlying molecular mechanisms of liver toxicity and injury. Although long non-coding RNAs (lncRNAs) have been extensively studied in model organisms, our knowledge concerning the role of lncRNAs in liver injury is limited. Given that lncRNAs show low levels of sequence conservation, their role becomes even more unclear in non-model organisms without an annotated genome, like whitefish (Coregonus lavaretus). The objective of this study was to discover and profile aberrantly expressed polyadenylated lncRNAs that are involved in MC-LR-induced liver injury in whitefish. Using RNA sequencing (RNA-Seq) data, we de novo assembled a high-quality whitefish liver transcriptome. This enabled us to find 94 differentially expressed (DE) putative evolutionary conserved lncRNAs, such as MALAT1, HOTTIP, HOTAIR or HULC, and 4429 DE putative novel whitefish lncRNAs, which differed from annotated protein-coding transcripts (PCTs) in terms of minimum free energy, guanine-cytosine (GC) base-pair content and length. Additionally, we identified DE non-coding transcripts that might be 3' autonomous untranslated regions (3'UTRs) of mRNAs. We found both evolutionary conserved lncRNAs as well as novel whitefish lncRNAs that could serve as biomarkers of liver injury.

Inhibition of EZH2 ameliorates bacteria-induced liver injury by repressing RUNX1 in dendritic cells.

Fulminant hepatic failure (FHF) is a clinical syndrome characterized by a sudden and severe impairment in liver function. However, the precise mechanism of immune dysregulation that is significant to FHF pathogenesis remains unclear. Enhancer of zeste homolog 2 (EZH2) has been implicated in inflammation as a regulator of immune cell function. In this study, we investigated the role of EZH2 in an animal model of human FHF induced by Propionibacterium acnes (P. acnes) and lipopolysaccharide (LPS). We demonstrated that EZH2 depletion in dendritic cells (DCs) and pharmacological inhibition of EZH2 using GSK126 both significantly ameliorated liver injury and improved the survival rates of mice with P. acnes plus LPS-induced FHF, which could be attributed to the decreased infiltration and activation of CD4+ T cells in the liver, inhibition of T helper 1 cells and induction of regulatory T cells. The expression of EZH2 in DCs was increased after P. acnes administration, and EZH2 deficiency in DCs suppressed DC maturation and prevented DCs from efficiently stimulating CD4+ T-cell proliferation. Further mechanistic analyses indicated that EZH2 deficiency directly increased the expression of the transcription factor RUNX1 and thereby suppressed the immune functions of DCs. The functional dependence of EZH2 on RUNX1 was further illustrated in DC-specific Ezh2-deficient mice. Taken together, our findings establish that EZH2 exhibits anti-inflammatory effects through inhibition of RUNX1 to regulate DC functions and that inhibition of EZH2 alleviates P. acnes plus LPS-induced FHF, probably by inhibiting DC-induced adaptive immune responses. These results highlight the effect of EZH2 on DCs, serving as a guide for the development of a promising immunotherapeutic strategy for FHF.

[Mechanism of Taurohyodeoxycholate-induced Biliary Phospholipid Efflux -Understanding the Function of the ABCB4 Enhancer for Developing Therapeutic Agents against Bile Salt-induced Liver Injury].

Biliary lipids primarily consist of bile salts, phospholipids, and cholesterol. Bile salts have potent detergent properties and deleterious effects on the cell membrane and are cytotoxic to hepatocytes. We have previously reported that phosphatidylcholine (PC), the predominant bile phospholipid, protects hepatocytes from the cytotoxicity of bile salts, whereas cholesterol reverses the cytoprotective effects of PC against bile salts. ABCB4, a member of the ATP-binding cassette transporter family, secretes biliary phospholipids, especially PC, from the hepatocytes into the bile. Using Abcb4 knockout mice and HEK293 cells that stably expressed ABCB4, we examined the effects of taurine- or glycine-conjugated cholate, ursodeoxycholate, and hyodeoxycholate on the ABCB4-mediated efflux of PC. We observed that the biliary secretion of PC in wild-type mice significantly increased following infusion of all the tested bile salts, especially taurohyodeoxycholate. On the other hand, the biliary secretion of PC in Abcb4 knockout mice was not affected by the bile salt infusions. The results also demonstrated that the efflux of PC from ABCB4-expressing HEK293 cells was significantly stimulated by taurohyodeoxycholate, which has a strong potential to form mixed micelles with PC. Furthermore, the results of our study emphasized the possibility that the specific interactions of bile salts with ABCB4 are necessary for the release of PC molecules from the binding pocket of ABCB4 into the aqueous environment. Further understanding of this mechanism will aid in the development of novel therapeutic agents for cholestatic liver diseases.

Pharmacological evidence for the involvement of ryanodine receptors in halothane-induced liver injury in mice.

Drug-induced liver injury (DILI) is a major safety concern in drug development. Halothane (HAL), an inhaled anesthetic, induces severe and idiosyncratic liver injury. Ryanodine receptors (RyR) are major intracellular calcium release channels found on the plasma membrane of the endoplasmic reticulum (ER). It has been reported that disordered hepatic calcium homeostasis is a feature of HAL-induced liver injury (HILI) in guinea pigs. However, there are no reports on whether RyR could mediate the pathogenesis of HILI. The aim of the present study was to investigate the effect of RyR on HILI. Ryanodine (RYA, RyR agonist, 50 µg/kg, i.p.) was administered to BALB/c female mice 1 h before HAL administration (15 mmol/kg, i.p.), which significantly elevated plasma transaminase levels and induced severe hepatic inflammation and necrosis. In contrast, dantrolene sodium (DAN, RyR antagonist) treatment significantly suppressed HILI in a dose- and time-dependent manner and alleviated liver damage. The number of infiltrated neutrophils in the liver were higher in the group treated with HAL + RYA than in the group treated with HAL alone, while DAN treatment decreased neutrophil infiltration in HILI. The hepatic mRNA levels of proinflammatory cytokines; chemokines; and factors related to danger signals, neutrophils, oxidative and ER stress, pro-apoptosis, and RyR were significantly increased with RYA pretreatment, whereas these levels were decreased with DAN treatment. These results suggest that RYA exacerbates HILI, and DAN exerts a protective effect against HILI. Hence, our study provides a novel insight regarding the effect of RyR in the mechanism underlying HILI.

Role of Mesencephalic Astrocyte-Derived Neurotrophic Factor in Alcohol-Induced Liver Injury.

Consumption of alcohol in immoderate quantity induces endoplasmic reticulum (ER) stress response (alcohol-induced ER stress). Mesencephalic astrocyte-derived neurotrophic factor (MANF), an ER stress-inducible protein, works as an evolutionarily conserved regulator of systemic and liver metabolic homeostasis. In this study, the effects of MANF on alcohol-induced liver injury were explored by using hepatocyte-specific MANF-knockout mice (MANF ΔHep) in a chronic-plus-binge alcohol feeding model. We found that alcohol feeding upregulated MANF expression and MANF ΔHep mice exhibited more severe liver injury with extra activated ER stress after alcohol feeding. In addition, we found that MANF deficiency activated iNOS and p65 and increased the production of NO and anti-inflammatory cytokines, which was further enhanced after alcohol treatment. Meanwhile, MANF deletion upregulated the levels of CYP2E1, 4-HNE, and MDA and downregulated the levels of GSH and SOD. These results indicate that MANF has potential protection on alcohol-induced liver injury, and the underlying mechanisms may be associated with meliorating the overactivated ER stress triggered by inflammation and oxidative stress via inhibiting and reducing NO/NF-κB and CYP2E1/ROS, respectively. Therefore, MANF might be a negative regulator in alcohol-induced ER stress and participate in the crosstalk between the NF-κB pathway and oxidative stress in the liver. Conclusions. This study identifies a specific role of MANF in alcohol-induced liver injury, which may provide a new approach for the treatment of ALI.

Dendrobine attenuates isoniazid- and rifampicin-induced liver injury by inhibiting miR-295-5p.

The present study aims to investigate the protective effects of Dendrobine and its underlying mechanisms on liver injury induced by isoniazid (INH) and rifampicin (RIF). A mouse model of liver injury was induced by intragastrically administration of 100 mg/kg INH and 100 mg/kg RIF for 14 days. The mice were intragastrically administrated with Dendrobine (50, 100, and 200 mg/kg) before the administration of INH and RIF. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined. Oxidative stress markers including glutathione, superoxide dismutase, and malondialdehyde in the liver were measured and liver histopathological examinations were performed. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot were applied to determine the mRNA and protein expressions, respectively. Luciferase reporter assay was used to evaluate the interactions between miR-295-5p and CYP1A2. Dendrobine significantly decreased serum ALT and AST and inhibited the liver index and ameliorated the liver histological changes induced by INH and RIF. Besides, Dendrobine also regulated oxidative stress status in the liver by the regulation of CYP1A2. Moreover, mmu-miR-295-5p was identified to target CYP1A2 and to regulate the expression of CYP1A2. In summary, Dendrobine ameliorated INH and RIF induced mouse liver injury by miR-295-5p-mediated CYP1A2 expression.

Inflammation-associated suppression of metabolic gene networks in acute and chronic liver disease.

Inflammation has been recognized as essential for restorative regeneration. Here, we analyzed the sequential processes during onset of liver injury and subsequent regeneration based on time-resolved transcriptional regulatory networks (TRNs) to understand the relationship between inflammation, mature organ function, and regeneration. Genome-wide expression and TRN analysis were performed time dependently in mouse liver after acute injury by CCl4 (2 h, 8 h, 1, 2, 4, 6, 8, 16 days), as well as lipopolysaccharide (LPS, 24 h) and compared to publicly available data after tunicamycin exposure (mouse, 6 h), hepatocellular carcinoma (HCC, mouse), and human chronic liver disease (non-alcoholic fatty liver, HBV infection and HCC). Spatiotemporal investigation differentiated lobular zones for signaling and transcription factor expression. Acute CCl4 intoxication induced expression of gene clusters enriched for inflammation and stress signaling that peaked between 2 and 24 h, accompanied by a decrease of mature liver functions, particularly metabolic genes. Metabolism decreased not only in pericentral hepatocytes that underwent CCl4-induced necrosis, but extended to the surviving periportal hepatocytes. Proliferation and tissue restorative TRNs occurred only later reaching a maximum at 48 h. The same upstream regulators (e.g. inhibited RXR function) were implicated in increased inflammation and suppressed metabolism. The concomitant inflammation/metabolism TRN occurred similarly after acute LPS and tunicamycin challenges, in chronic mouse models and also in human liver diseases. Downregulation of metabolic genes occurs concomitantly to induce inflammation-associated genes as an early response and appears to be initiated by similar upstream regulators in acute and chronic liver diseases in humans and mice. In the acute setting, proliferation and restorative regeneration associated TRNs peak only later when metabolism is already suppressed.

Association of CYP2C19 and UGT1A4 polymorphisms with voriconazole-induced liver injury.

Aim: This study investigated the association between voriconazole-induced liver injury and gene polymorphisms of CYP2C19 and UGT1A4. Materials & methods: Thirty-eight adult patients who received voriconazole therapy were included in the study. Genotype of CYP2C19 was detected using gene chip hybrid analysis. The UGT1A4 142T>G was genotyped using PCR-RFLP analysis. Results: Ten patients (26.3%) had voriconazole-induced liver injury and were considered as the case group There was no significant difference between the two groups in genotype and allele frequencies of CYP2C19*2 and UGT1A4 142T>G (p > 0.05), however, the GA frequency of CYP2C19 *3 in the drug-induced liver injury case group was higher than that in the control group (p < 0.05). Compared with patients carrying *1/*1 or *1/*2, there was no significant difference in voriconazole trough concentration of the patients with *1/*3 (p > 0.05). Conclusion: There was no significant correlation between voriconazole-induced liver injury and gene polymorphisms of CYP2C19 and UGT1A4.

Tanshinone IIA prevents rifampicin-induced liver injury by regulating BSEP/NTCP expression via epigenetic activation of NRF2.

BACKGROUND & AIMS: Rifampicin (RFP)-induced cholestatic liver injury is characterized by impaired hepatic bile acid (BA) transport. Bile salt efflux pump (BSEP) and Na+/taurocholate cotransporter (NTCP) are the major BA transporters. However, little is known about the mechanisms underlying these transporters. METHODS: The role of tanshinone IIA (TAN IIA) in preventing RFP-induced liver injury was evaluated in vitro and in vivo, based on the regulatory mechanism of nuclear factor erythroid 2-related factor 2 (NRF2)-BSEP/NTCP signalling. The epigenetic induction of NRF2 by TAN IIA was investigated as well as the influence on BSEP and NTCP transcriptional activation and NRF2 DNA-binding ability. RESULTS: TAN IIA strongly induced BSEP and NTCP expression in hepatocytes. NRF2 knockdown abrogated the induction. We found two NRF2 binding sites on the human BSEP promoter, called musculoaponeurotic fibrosarcoma recognition elements (MAREs), and one MARE on the NTCP promoter. Human BSEP and NTCP promoter luciferase reporter gene plasmids were stimulated by NRF2. Mutations of the predicted MAREs abolished NRF2 transcriptional activation. TAN IIA induced the expression of ten-eleven translocation 2 (TET2) to mediate the demethylation of NRF2, which promoted NRF2 DNA-binding on the BSEP and NTCP promoters and their transcriptional activation. Finally, in vivo, Nrf2 played an important role in RFP-induced liver injury (more serious liver injury in Nrf2-/- mice), and TAN IIA prevented it. CONCLUSIONS: These results indicate that NRF2 regulates the target transporters BSEP and NTCP, depending on the DNA demethylation by TET2. Pharmacological activation of NRF2 by TAN IIA may be beneficial for RFP-induced liver injury.

Long non-coding RNAs play regulatory roles in acetaminophen-induced liver injury.

OBJECTIVE: To explore the expression profile and role of hepatic long non-coding RNA (lncRNA) in acetaminophen-induced liver injury mouse model by analyzing lncRNA-mRNA co-expression. METHODS: Serum aminotransferase, liver pathology and inflammatory cells were analyzed in mice model at different time points after treated with acetaminophen 300 mg/kg. High-throughput RNA sequencing was performed to investigate hepatic expression profiles of messenger RNA (mRNA) and lncRNA. The relationship between the lncRNA and mRNA was delineated by the co-expression network using Cytoscape software. Differential mRNAs co-expressed with lncRNAs were analyzed using Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes enrichment. Differential mRNAs and lncRNAs were selected for quantitative reverse transcription polymerase chain reaction validation, and the conservation of lncRNA between human and mouse was analyzed. RESULTS: Liver injury was more severe at 24 hours than at 6 hours. There was a substantial infiltration of monocytes instead of neutrophil and Kupffer cells at 24 hours compared with 6 hours. The mRNAs co-expressed with the differential lncRNAs at 24 vs 6 hours were mainly enriched in protein processing in endoplasmic reticulum, MAPK and PPAR signaling pathways. The co-expression network delineated with four lncRNAs and 94 mRNAs presented the core position of lncRNA in the network. A conservation analysis indicated that four differential mouse lncRNAs (NONMMUT023651.2, NONMMUT029382.2, NONMMUT029383.2 and NONMMUT102053.1) could all be mapped to the relevant human lncRNAs. CONCLUSION: Four lncRNAs may play regulatory roles through metabolic and apoptosis-related pathways during hepatic homeostasis maintenance and repair progress.