Alcohol Promotes Exosome Biogenesis and Release via Modulating Rabs and miR-192 Expression in Human Hepatocytes

Exosomes are membrane vesicles released by various cell types into the extracellular space under different conditions including alcohol exposure. Exosomes are involved in intercellular communication and as mediators of various diseases. Alcohol use causes oxidative stress that promotes exosome secretion. Here, we elucidated the effects of alcohol on exosome biogenesis and secretion using human hepatocytes. We found that alcohol treatment induces the expression of genes involved in various steps of exosome formation. Expression of Rab proteins such as Rab1a, Rab5c, Rab6, Rab10, Rab11, Rab27a and Rab35 were increased at the mRNA level in primary human hepatocytes after alcohol treatment. Rab5, Rab6 and Rab11 showed significant induction in the livers of patients with alcohol-associated liver disease. Further, alcohol treatment also led to the induction of syntenin, vesicle-associated membrane proteins (VAMPs), and syntaxin that all play various roles in exosome biogenesis and secretion. VAMP3, VAMP5, VAPb, and syntaxin16 mRNA transcripts were increased in alcohol treated cells and in the livers of alcohol-associated liver disease (ALD) patients. Induction in these genes was associated with increases in exosome secretion in alcohol treated hepatocytes. We found that hepatocyte enriched miR-192 and miR-122 levels were significantly decreased in alcohol treated hepatocytes whereas their levels were increased in the cell-free supernatant. The primary transcripts of miR-192 and miR-122 were reduced in alcohol treated hepatocytes, suggesting alcohol partially affects these miRNAs at the transcriptional level. We found that miR-192 has putative binding sites for genes involved in exosome secretion. Inhibition of miR-192 in human hepatoma cells caused a significant increase in Rab27a, Rab35, syntaxin7 and syntaxin16 and a concurrent increase in exosome secretion, suggesting miR-192 regulates exosomes release in hepatocytes. Collectively, our results reveal that alcohol modulates Rabs, VAMPs and syntaxins directly and partly via miR-192 to induce exosome machinery and release.

A Strategy for Screening the Lipid-Lowering Components in Alismatis Rhizoma Decoction Based on Spectrum-Effect Analysis

Alismatis Rhizoma decoction (ARD), comprised of Alisma plantago-aquatica subsp. orientale (Sam.) Sam and Atractylodes macrocephala Koidz. at a ratio of 5 : 2, is a classic traditional Chinese medicine (TCM) formula with successful clinical hypolipidemic effect. This paper aimed to explore the major bioactive compounds and potential mechanism of ARD in the treatment of hyperlipidemia on the basis of spectrum-effect analysis and molecular docking. Nine ARD samples with varying ratios of the constituent herbs were prepared and analyzed by UPLC-Q-TOF/MS to obtain the chemical spectra. Then, the lipid-lowering ability of the nine samples was tested in an oleic acid-induced lipid accumulation model in human hepatoma cells (HepG2). Grey relational analysis and partial least squares regression analysis were then performed to determine the correlation between the chemical spectrums and lipid-lowering efficacies of ARD. The potential mechanisms of the effective compounds were investigated by docking with the farnesoid X receptor (FXR) protein. The results indicated that alisol B 23-acetate, alisol C 23-acetate, and alisol B appeared to be the core effective components on hyperlipidemia in ARD. Molecular docking further demonstrated that all three compounds could bind to FXR and were potential FXR agonists for the treatment of hyperlipidemia. This study elucidated the effective components and potential molecular mechanism of action of ARD for treating hyperlipidemia from a perspective of different compatibility, providing a new and feasible reference for the research of TCM formulas such as ARD.

Protective Effect and Potential Antioxidant Role of Kakadu Plum Extracts on Alcohol-Induced Oxidative Damage in HepG2 Cells

Serial alcohol consumption causes alcoholic liver disease (ALD), which can lead to fatty liver, hepatitis, and cirrhosis. Terminalia ferdinandiana (Kakadu plum) is an indigenous fruit of Australia, which is utilized as a functional food. It is a commercially important antioxidant as it contains a more eloquent level of ascorbic acid than other oranges. In this study, we analyzed the chemical constituents of vitamin C, gallic acid, ellagic acid, and daidzin via High-performance liquid chromatography (HPLC) in the Kakadu plum from two different regions including the Northern Territory (NT) and Western Australia (WA), and compared their biochemical properties. The vitamin C content was much higher (almost 70%) in Kakadu plum (KKD) from the NT than WA. Moreover, ROS generation was inhibited significantly in HepG2 (human hepatoma) cells with the KKD-NT extract treatment when compared to the KKD-WA extract treatment. The cytotoxicity produced by ethanol was significantly suppressed in response to the treatment with both of the samples. In addition, our samples (KKD-NT and KKD-WA) increased the activity of two key enzymes involving alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) that metabolize ethanol. These results show the biochemical confirmation of the mechanism by which KKD exhibits its biological functions including relief from alcohol hangovers as well as protection of the liver cells by the suppression of ROS production and toxic insults.

Nrf1 is not a direct target gene of SREBP1, albeit both are integrated into the rapamycin-responsive regulatory network in human hepatoma cells

It is worth interrogating why no more experimental evidence confirming those findings, since being reported by Manning's group in 2014's Nature (doi: 10.1038/nature13492), has been provided in the hitherto known literature. A key issue arising from their work is of particular concern about whether the mTORC1 signaling to upregulation of Nrf1-targeted proteasomal expression profiles occurs directly by SREBP1. In this study, our experiment evidence revealed that Nrf1 is not a direct target of SREBP1, although both are involved in the rapamycin-responsive regulatory networks. Closely scrutinizing two distinct transcriptomic datasets unraveled no significant changes in transcriptional expression of Nrf1 and almost all proteasomal subunits in siSREBP1 or SREBP1-/- cells, when compared to equivalent controls. However, distinct upstream signaling to Nrf1 dislocation by p97 and its processing by DDI1/2, along with downstream proteasomal expression, may be monitored by mTOR signaling, to various certain extents, depending on distinct experimental settings in different types of cells. Our further evidence has been obtained from DDI1-/- (DDI2insC) cells, demonstrating that putative effects of mTOR on the rapamycin-responsive signaling to Nrf1 and proteasomes may also be executed partially through a DDI1/2-independent mechanism, albeit the detailed regulatory events remain to be determined.

Age-Dependent Decrease in Hepatic Geranylgeranoic Acid Content in C3H/HeN Mice and Its Oral Supplementation Prevents Spontaneous Hepatoma

Geranylgeranoic acid (GGA) has been developed as a preventive agent against second primary hepatoma. Recently, GGA was reported to induce cell death in human hepatoma cells via TLR4-mediated pyroptosis. We have reported that GGA is enzymatically biosynthesized from mevalonic acid in human hepatoma-derived cells and that endogenous GGA is found in most organs of rats. In addition, we found that upregulation of endogenous GGA levels by zaragozic acid A (ZAA) induced cell death in human hepatoma-derived cells. Therefore, we investigated the age-related changes in hepatic GGA and the possibility of suppressing hepatocarcinogenesis by GGA supplementation using male C3H/HeN mice that spontaneously develop hepatoma. We measured endogenous GGA and mRNA of MAOB, a key enzyme of GGA biosynthesis, in the liver of male C3H/HeN mice aged 6–93 weeks. We also tried suppressing spontaneous hepatocarcinogenesis by a single administration of GGA to C3H/HeN mice. Hepatic GGA content and Maob mRNA expression level age-dependently decreased in male C3H/HeN mice; some of which produced spontaneous hepatoma in 2 years. A single oral administration of GGA at 11 months of age significantly prevented hepatoma in terms of the number and weight of tumors per mouse at 24 months. Oral supplementation with GGA or geranylgeraniol significantly increased endogenous hepatic GGA contents dose-dependently; and ZAA dramatically upregulated hepatic GGA. In this study; we found an age-dependent decrease in hepatic endogenous GGA in male C3H/HeN mice and efficient prevention of spontaneous hepatoma by a single administration of GGA at 11 months of age.

Integrative Network Analysis Revealed Genetic Impact of Pyruvate Kinase L/R on Hepatocyte Proliferation and Graft Survival after Liver Transplantation

Background. Pyruvate kinase L/R (PKLR) has been suggested to affect the proliferation of hepatocytes via regulation of the cell cycle and lipid metabolism. However, its impact on the global metabolome and its clinical implications remain unclear. Aims. We aimed to clarify the genetic impact of PKLR on the metabolomic profiles of hepatoma cells and its potential effects on grafts for liver transplantation (LT). Methods. Nontargeted and targeted metabolomic assays were performed in human hepatoma cells transfected with lentiviral vectors causing PKLR overexpression and silencing, respectively. We then constructed a molecular network based on integrative analysis of transcriptomic and metabolomic data. We also assessed the biological functions of PKLR in the global metabolome in LT grafts in patients via a weighted correlation network model. Results. Multiomic analysis revealed that PKLR perturbations significantly affected the pyruvate, citrate, and glycerophospholipid metabolism pathways, as crucial steps in de novo lipogenesis (DNL). We also confirmed the importance of phosphatidylcholines (PC) and its derivative lyso-PC supply on improved survival of LT grafts in patients. Coexpression analysis revealed beneficial effects of PKLR overexpression on posttransplant prognosis by alleviating arachidonic acid metabolism of the grafts, independent of operational risk factors. Conclusion. This systems-level analysis indicated that PKLR affected hepatoma cell viability via impacts on the whole process of DNL, from glycolysis to final PC synthesis. PKLR also improved prognosis after LT, possibly via its impact on the increased genesis of beneficial glycerophospholipids.

Assessing hepatoprotective effects of antioxidants on amiodarone-induced cytotoxicity in human hepatoma HepaRG cell line

Effective therapy of amiodarone-induced hepatotoxicity requires studying the mechanisms of the toxic effects of amiodarone on hepatocytes and assessing the potential impact of hepotoprotective agents. The study was aimed to assess hepatoprotective effects of antioxidants on the amiodarone-induced hepatotoxicity with the use of immortalized human hepatoma cells of the HepaRG cell line. Cell viability was evaluated upon exposure to amiodarone and in the mixture with vitamin Е, N-acetylcysteine and S-adenosylmethionine by impedance measurement; the levels of some hepatotoxicity biomarkers were defined using the Luminex xMAP technology. As a result of the research, the dose-dependent toxic effects of amiodarone were established. The IC50 value of amiodarone in the HepaRG cell line was 3.5 μМ. It is shown that cytotoxic effects decrease and the IC50 value increases in the presence of vitamin Е, N-acetylcysteine and S-adenosylmethionine. Amiodarone reduces the activity of cell cycle regulators: AKT, JNK kinases, and p53 protein. Exposure to amiodarone results in reduced intracellular ATP levels and the release of intracellular enzymes (malate dehydrogenase 1, glutathione S-transferase, sorbitol dehydrogenase, 5'-nucleotidase) into conditioned medium, indicating the necrotic cell death. Thus, vitamin Е, S-adenosylmethionine and N-acetylcysteine reduce amiodarone cytotoxicity in the model of amiodarone-induced damage to hepatocytes and can be considered as hepatoprotective agents in case of the need to protect liver against the hepatotoxic effects of amiodarone.

Anticancer activity study of paeoniflorin Ag(I) complexes against human hepatoma cells 3B cells

A new paeoniflorin Ag(I) complex was synthesized using the hydrothermal method, which was characterized by some spectral techniques like elemental analysis, thermal analysis, 1H nuclear magnetic resonance (1H NMR), Fourier-transform infrared spectroscopy (FTIR), electrospray ionization mass spectrometry (ESI-MS) and geometry optimization. Cell Counting Kit-8 (CCK-8) was used to test the in vitro cytotoxicity of the paeoniflorin Ag(I) complexes in various cells (such as A549, Caco2, HepG2, Hep3B, LO2, H9C2 and PC12 cells). It is shown that the paeoniflorin Ag(I) complexes possessed a remarkable cytotoxicity against the Hep3B cell line, but a low cytotoxicity against the LO2 cell line. Cell cycle progression and membrane potential of Hep3B cells were studied using flow cytometry, and the cell examination of apoptosis was made by Annexin V-FITC/PI staining. Moreover, expressions levels of caspase family proteins and Bcl-2 were also investigated. Our findings demonstrated that the paeoniflorin Ag(I) complexes induced apoptosis of Hep3B cells via the mitochondrial membrane potential access by regulating Bcl-2 and caspase family proteins.

The Phosphatidylserine Receptor TIM-1 Enhances Authentic Chikungunya Virus Cell Entry

Chikungunya virus (CHIKV) is a re-emerging, mosquito-transmitted, enveloped positive stranded RNA virus. Chikungunya fever is characterized by acute and chronic debilitating arthritis. Although multiple host factors have been shown to enhance CHIKV infection, the molecular mechanisms of cell entry and entry factors remain poorly understood. The phosphatidylserine-dependent receptors, T-cell immunoglobulin and mucin domain 1 (TIM-1) and Axl receptor tyrosine kinase (Axl), are transmembrane proteins that can serve as entry factors for enveloped viruses. Previous studies used pseudoviruses to delineate the role of TIM-1 and Axl in CHIKV entry. Conversely, here, we use the authentic CHIKV and cells ectopically expressing TIM-1 or Axl and demonstrate a role for TIM-1 in CHIKV infection. To further characterize TIM-1-dependent CHIKV infection, we generated cells expressing domain mutants of TIM-1. We show that point mutations in the phosphatidylserine binding site of TIM-1 lead to reduced binding, entry, and infection of CHIKV. Ectopic expression of TIM-1 renders immortalized keratinocytes permissive to CHIKV, whereas silencing of endogenously expressed TIM-1 in human hepatoma cells reduces CHIKV infection. Altogether, our findings indicate that, unlike Axl, TIM-1 readily promotes the productive entry of authentic CHIKV into target cells.