MG53's non-physiologic interaction with insulin receptor: lack of effect on insulin-stimulated Akt phosphorylation in muscle, heart and liver tissues.

Rationale: MG53's known function in facilitating tissue repair and anti-inflammation has broad applications to regenerative medicine. There is controversy regarding MG53's role in the development of type 2 diabetes mellitus. Objective: This study aims to address this controversy - whether MG53's myokine function contributes to inhibition of insulin signaling in muscle, heart, and liver tissues. Study design: We determined the binding affinity of the recombinant human MG53 (rhMG53) to the insulin receptor extracellular domain (IR-ECD) and found low affinity of interaction with Kd (>480 nM). Using cultured C2C12 myotubes and HepG2 cells, we found no effect of rhMG53 on insulin-stimulated Akt phosphorylation (p-Akt). We performed in vivo assay with C57BL/6J mice subjected to insulin stimulation (1 U/kg, intraperitoneal injection) and observed no effect of rhMG53 on insulin-stimulated p-Akt in muscle, heart and liver tissues. Conclusion: Overall, our data suggest that rhMG53 can bind to the IR-ECD, however has a low likelihood of a physiologic role, as the Kd for binding is ~10,000 higher than the physiologic level of MG53 present in the serum of rodents and humans (~10 pM). Our findings question the notion proposed by Xiao and colleagues - whether targeting circulating MG53 opens a new therapeutic avenue for type 2 diabetes mellitus and its complications.

Structural characterization, antioxidant and immunomodulatory activities of a polysaccharide from a traditional Chinese rice wine, Guangdong Hakka Huangjiu.

Hakka Huangjiu, a traditional Chinese rice wine, boasts a rich history and is known for its immunomodulatory, antibacterial, anti-aging and anti-fatigue effects. However, there is limited research on the primary active components and molecular mechanism of the bioactivity of Hakka Huangjiu. To address this gap, this study assessed the structural characteristics, antioxidant, and immunomodulatory activities of the polysaccharide-1 of Guangdong Hakka Huangjiu (HP1). Structural analysis revealed that HP1 had a low molecular weight polysaccharide of 5550 Da, primarily consisting of glucose (93.2 %), with smaller amounts of xylose, mannuronic acid and galactose. Methylation and NMR analysis suggested that the main glycosidic linkages present in HP1 are α-D-Glcp-(1→, →4)-α-D-Glcp-(1 â†’ and →6) -α-D-Glcp-(1→. Furthermore, HP1 exhibited dose-dependent DPPH·, ABTS+ and OH· scavenging activity. HP1 exhibited significant protection of HepG2 cells from H2O2 damage. Additionally, HP1 induced the release of NO, TNF-α, IL-6 and iNOS in RAW264.7 cells. HP1 treatment significantly increased mRNA expression of TNF-α, IL-6, iNOS, COX-2, IL-1ß and TGF-ß1. These results suggested that polysaccharides HP1 may have potential as a novel natural antioxidant and immunomodulatory product for use in nutraceuticals and functional foods.

Targeting insulin resistance: myricetin and isorhamnetin from Hardwickia binata, and luteolin from Hedysarum alpinum enhance glucose uptake and AMPK signaling in HepG2 cells.

This study examined five plants (Xylopia aethiopica, Agave sisalana, Hardwickia binata, Hedysarum alpinum, and Toxicodendron vernicifluum) for their potential to address insulin resistance in type 2 diabetes. In-vitro assays showed that H. binata leaves and H. alpinum flowers inhibited α-glucosidase and α-amylase while enhancing glucose uptake in normal and insulin-resistant HepG2 cells. Phytochemical screening and SPE purification identified the key constituents responsible for the effects. The chromatographic and spectral analysis confirmed flavonoids in H. binata (myricetin, isorhamnetin, quercetin, kaempferol, and catechin) and H. alpinum (luteolin, quercetin, kaempferol, and apigenin). Myricetin, isorhamnetin, and luteolin significantly increased glucose uptake, enhanced hexokinase and pyruvate kinase activities, and promoted IRec and IRS-1 phosphorylation, modulating insulin signalling. They activated AMPK and Akt, with molecular docking confirming strong AMPK binding. These findings suggest that H. binata, H. alpinum, and their flavonoids are promising candidates for managing insulin resistance and type 2 diabetes, warranting further research.

Isolation, Characterization, Moisturization and Anti-HepG2 Cell Activities of a Novel Polysaccharide from Cyanobacterium aponinum.

Polysaccharides from cyanobacteria are extensively reported for their complex structures, good biocompatibility, and diverse bioactivities, but only a few cyanobacterial species have been exploited for the biotechnological production of polysaccharides. According to our previous study, the newly isolated marine cyanobacterium Cyanobacterium aponinum SCSIO-45682 was a good candidate for polysaccharide production. This work provided a systematic study of the extraction optimization, isolation, structural characterization, and bioactivity evaluation of polysaccharides from C. aponinum SCSIO-45682. Results showed that the crude polysaccharide yield of C. aponinum reached 17.02% by hot water extraction. The crude polysaccharides showed a porous and fibrous structure, as well as good moisture absorption and retention capacities comparable to that of sodium alginate. A homogeneous polysaccharide (Cyanobacterium aponinum polysaccharide, CAP) was obtained after cellulose DEAE-52 column and Sephadex G-100 column purification. CAP possessed a high molecular weight of 4596.64 kDa. It was mainly composed of fucose, galactose, and galacturonic acid, with a molar ratio of 15.27:11.39:8.64. The uronic acid content and sulfate content of CAP was 12.96% and 18.06%, respectively. Furthermore, CAP showed an in vitro growth inhibition effect on human hepatocellular carcinoma (HepG2) cells. The above results indicated the potential of polysaccharides from the marine cyanobacterium C. aponinum SCSIO-45682 as a moisturizer and anticancer addictive applied in cosmetical and pharmaceutical industries.

Oxidative Stress and Annexin A2 Differential Expression in Free Fatty Acids-Induced Non-Alcoholic Fatty Liver Disease in HepG2 Cells.

Non-alcoholic fatty liver disease (NAFLD) is a rising global burden, affecting one in four adults. Despite the increasing prevalence of NAFLD, the exact cellular and molecular mechanisms remain unclear, and effective therapeutic strategies are still limited. In vitro models of NAFLD are critical to understanding the pathogenesis and searching for effective therapies; thus, we evaluated the effects of free fatty acids (FFAs) on NAFLD hallmarks and their association with the modulation of Annexin A2 (ANXA2) and Keratin 17 (KRT17) in HepG2 cells. Our results show that oleic and palmitic acids can differentially induce intracellular lipid accumulation, cell death, and promote oxidative stress by increasing lipid peroxidation, protein carbonylation, and antioxidant defense depletion. Moreover, a markedly increased expression of inflammatory cytokines demonstrated the activation of inflammation pathways associated with lipotoxicity and oxidative stress. ANXA2 overexpression and KRT17 nuclear translocation were also observed, supporting the role of both molecules in the progression of liver disease. Taken together, these data provide insights into the interplay between ANXA2 and KRT17 in NAFLD, paving the way for understanding molecular mechanisms involved with the disease and developing new therapeutic strategies.

Hepatoprotective effect of flavonoid rich fraction of Sesbania grandiflora: Results of In vivo, in vitro, and molecular docking studies.

BACKGROUND: Phytochemicals and their derivatives are promising target drugs for various ailments and have served as therapeutic agents for several decades. Using in vivo and in vitro models and molecular docking, this study investigated the pharmacological potential of a flavonoid-rich fraction of the ethanolic extract of Sesbania grandiflora (SG). OBJECTIVES: This research aimed to determine whether flavonoid-rich whole-plant extracts of SGs have any cytoprotective or in vivo hepatoprotective effects. Additionally, the study was intended to elucidate the molecular connections between the discovered flavonoid flavonols and PPARα target proteins linked to liver problems, for which an in silico molecular docking investigation was performed. MATERIALS AND METHODS: To separate the flavonoid components, the entire Sesbania grandiflora plant was first extracted using ethanol as a solvent by soxhlet extraction. The resulting ethanolic extract was then fractionated. The cytoprotective and hepatoprotective properties were evaluated via in vitro and in vivo experiments. SGOT, SGPT, triglyceride, bilirubin, and total protein levels were used to evaluate hepatotoxicity in animal models. In vitro studies on Hepatocellular Carcinoma G2 (HepG2) cell lines have examined their cytotoxic effects and antioxidant activity. The most promising flavonoid-flavanol compounds were identified by conducting molecular docking studies against PPARα target protein (PDB ID: 3VI8) using MOE software. RESULTS: In vivo, the serum levels of SGOT, SGPT, total triglyceride and total bilirubin were measured in experimental animals treated with the flavonoid-rich ethanolic extract of SG. Significant reductions in the levels of these hepatic injury markers were observed, indicating the hepatoprotective potential of the extract. Elevated levels of liver biomarkers in the untreated group indicated liver injury or dysfunction. The treated groups showed significant restoration of these biomarkers, suggesting the hepatoprotective potential of SG. The IC50 value for the total flavonoid content of SG was 190.28 µg/ml, indicating its safety in inhibiting HepG2 cell growth. Flavonoid treatment decreased cell viability but did not affect antioxidant parameters in hepatocytes. In addition, SG restored the damaged hepatocyte architecture. Molecular docking studies revealed the binding affinities of flavonoids for PPARα. These findings suggest that a promising lead candidate for the development of therapeutic medicines against anti-TB drug-induced hepatotoxicity has been identified. CONCLUSION: Our findings demonstrate the hepatoprotective potential of the flavonoid-rich fraction of Sesbania grandiflora both in vivo and in vitro. This study provides valuable insights into its mechanism of action, highlighting its promising therapeutic application in the management of liver disorders. This study highlights the hepatoprotective and cytoprotective potential of the total flavonoid-rich fraction of SG.

Collagen I Increases Palmitate-Induced Lipotoxicity in HepG2 Cells via Integrin-Mediated Death.

Various strategies have been employed to improve the reliability of 2D, 3D, and co-culture in vitro models of nonalcoholic fatty liver disease, including using extracellular matrix proteins such as collagen I to promote cell adhesion. While studies have demonstrated the significant benefits of culturing cells on collagen I, its effects on the HepG2 cell line after exposure to palmitate (PA) have not been investigated. Therefore, this study aimed to assess the effects of PA-induced lipotoxicity in HepG2 cultured in the absence or presence of collagen I. HepG2 cultured in the absence or presence of collagen I was exposed to PA, followed by analyses that assessed cell proliferation, viability, adhesion, cell death, mitochondrial respiration, reactive oxygen species production, gene and protein expression, and triacylglycerol accumulation. Culturing HepG2 on collagen I was associated with increased cell proliferation, adhesion, and expression of integrin receptors, and improved cellular spreading compared to culturing them in the absence of collagen I. However, PA-induced lipotoxicity was greater in collagen I-cultured HepG2 than in those cultured in the absence of collagen I and was associated with increased α2ß1 receptors. In summary, the present study demonstrated for the first time that collagen I-cultured HepG2 exhibited exacerbated cell death following exposure to PA through integrin-mediated death. The findings from this study may serve as a caution to those using 2D models or 3D scaffold-based models of HepG2 in the presence of collagen I.

Hepatoprotective effect of date fruit extract against ethanol-induced apoptosis in human hepatoma (HepG2) cells.

Ethanol is a well-known hepatotoxic agent and date fruits have been associated with their biological actions. In current study, we have investigated the hepatoprotective potential of DFE on ethanol-induced cellular damages in human hepatoma (HepG2) cells. The hepatoprotective potential was assessed by exposing the HepG2 cells to non-toxic concentrations (15, 30, and 60 µg/mL) of DFE for 24 h; then toxic concentration (500 µM) of ethanol. Our results demonstrated that pretreatment with DFE significantly prohibited ethanol-induced hepatotoxicity in HepG2 cells. We observed that DFE treatment increased cell viability, reduced LDH leakage, restored cellular morphology, and inhibited caspase-3 enzyme activity in a dose dependent way, induced by ethanol. Further DFE was also effective in restoring the LPO, GSH, and catalase levels towards normal altered by ethanol. Our results also revealed that ethanol-induced ROS generation was significantly inhibited by DFE. The ethanol-induced mRNA expression of apoptotic related genes (p53, caspase-3, caspase-7, Bax, and Bcl-2) were also normalized by pretreatment with DFE. The findings from this study indicated that DFE can significantly protect HepG2 cells against ethanol-induced hepatotoxicity. Our study also provides scientific validation for the traditional use of DFE, aiming to understand its hepatoprotective potential. Altogether, to the best of our knowledge, this is the first study demonstrated that ethanol-induced hepatotoxicity can be prohibited by the DFE. Thus, DFE has a potential application in nutraceuticals as a therapeutic agent to prevent liver diseases.

Chlorpyrifos induces cytotoxicity via oxidative stress and mitochondrial dysfunction in HepG2 cells.

Chlorpyrifos (CPF), a widely used broad-spectrum organophosphate pesticide, has been associated with various adverse health effects in animals and humans. While its primary mechanism of action involves the irreversible inhibition of acetylcholinesterase, secondary mechanisms have also been suggested. The aim of the present study was to explore the secondary mechanisms of action involved in CPF-induced acute cytotoxicity using human hepatocarcinoma HepG2 cells. In particular, we investigated oxidative stress and mitochondrial function by assessing reactive oxygen species (ROS) generation, lipid peroxidation (LPO) and mitochondrial membrane potential (ΔΨm) alteration. Results showed that 24-h exposure to CPF (78.125-2500 µM) decreased cell viability in a concentration-dependent manner (IC50 = 280.87 ± 26.63 µM). Sub-toxic CPF concentrations (17.5, 35 and 70 µM) induced increases in ROS generation (by 83%), mitochondrial superoxide (by 7.1%), LPO (by 11%), and decreased ΔΨm (by 20%). CPF also upregulated Nrf2 protein expression, indicating the role of the latter in modulating the cellular response to oxidative insults. Overall, our findings suggest that CPF caused hepatotoxicity through oxidative stress and mitochondrial dysfunction. Given the re-emerging use of CPF, this study emphasizes the need for comprehensive analysis to elucidate its toxicity on non-target organs and associated mechanisms.

Cytotoxicity evaluation of organophosphorus flame retardants using electrochemical biosensors and elucidation of associated toxic mechanisms.

In recent years, organophosphorus flame retardants (OPFRs) have been widely used as substitutes for brominated flame retardants with excellent properties, and their initial toxicological effects on the water ecosystem and human health have gradually emerged. However, to date, research on the cytotoxicity and health risks of OPFRs is still limited. Therefore, this study aims to systematically explore the cytotoxic effects and toxic mechanisms of OPFRs on cells. Human liver cancer (HepG2) cells were adopted as an ideal model for toxicity evaluation due to their rapid growth and metabolism. This study proposes a sensitive electrochemical cell-based sensor constructed on a graphitized multi-walled carbon nanotube/ionic liquid/gold nanoparticle-modified electrode. The sensor was used to detect the cytotoxicity of tri(2-butylxyethyl) phosphate (TBEP), tributyl phosphate (TnBP), triphenyl phosphate (TPhP), tri(1,3-dichloro-2-propyl) phosphate (TDCIPP), tri(2-chloropropyl) phosphate (TCPP) and tri(2-chloroethyl) phosphate (TCEP) in the liquid medium, providing insight into their toxicity in water environments. The half-maximal inhibitory concentration (IC50) of TBEP, TnBP, TPhP, TDCIPP, TCPP and TCEP on HepG2 cells were 179.4, 194.9, 219.8, 339.4, 511.8 and 859.0 µM, respectively. Additionally, the cytotoxic mechanism of six OPFRs was discussed from the perspective of oxidative stress and apoptosis, and four indexes were correlated with toxicity. Furthermore, transcriptome sequencing was conducted, followed by a thorough analysis of the obtained sequencing results. This analysis demonstrated a significant enrichment of the p53 and PPAR pathways, both of which are closely associated with oxidative stress and apoptosis. This study presents a simplified and efficient technique for conducting in vitro toxicity studies on organophosphorus flame retardants in a water environment. Moreover, it establishes a scientific foundation for further investigation into the mechanisms of cytotoxicity associated with these compounds.

Structural characterization, physicochemical properties and hypoglycemic activity of soluble dietary fibers from salt stressed mung bean sprouts.

Low-salt stress germination is an effective way to improve the nutritional composition of food crops. A novel soluble dietary fiber (MS-SDF) was isolated from low-salt stress mung bean sprouts that were exposed to low-salt stress using anion exchange and gel permeation techniques. Structural analysis revealed that MS-SDF was a homogeneous heteropolysaccharide with an average molecular weight of 164.997 KDa. It featured a loose structure and contained the characteristic functional groups typical of polysaccharides. MS-SDF was composed of arabinose, galactose, glucose, and mannose with a molar ratio of 3.95:3.86:82.69:9.02. The structure was mainly composed of →6)-α-D-Glcp-(1→, →5)-α-L-Araf-(1→, and →3,6)-α-D-Glcp-(1→ as the main chain. Branched at O-3 position with single ß-D-Manp-(1→ as major the side chain. Furthermore, in vitro hypoglycemic assays indicate that MS-SDF exhibits α-glucosidase inhibitory activity, significantly enhancing glucose uptake, glycogen synthesis, and pyruvate kinase activity in insulin-resistant HepG2 cells. Overall, MS-SDF could be used as a promising source of functional hypoglycemic foods.

Improvement of glucolipid metabolism and oxidative stress via modulating PI3K/Akt pathway in insulin resistance HepG2 cells by chickpea flavonoids.

Chickpea (Cicer arietinum L.) is a significant dietary source of flavonoids and the hypoglycemic activity were investigated in this study. Firstly, total twenty nine chickpea flavonoids were identified by UPLC-MS/MS with ononin, cyanidin-3-O-glucoside, astragalin, cynaroside, kaempferol-3-O-rutinoside, biochanin A, and daidzin being the most abundant among them. Our results demonstrated that chickpea flavonoids regulated glucose metabolism and lipid metabolism, and reduced oxidative stress in insulin resistance HepG2 cells. Furthermore, insulin resistance was ameliorated by chickpea flavonoids through the activation of insulin receptor substrate1 (IRS1), phosphoinositide 3-kinase (PI3K), and phosphorylated protein kinase B (Akt) in HepG2 cells. More importantly, key differential metabolites include L-tryptophan, L-tyrosine, l-glutamine and linoleic acid were reserved by chickpea flavonoids and correlated with glucolipid metabolism and oxidative stress in IR-HepG2 cells. In conclusion, these results indicated that chickpea flavonoids might act as potential natural products regulating insulin resistance in HepG2 cells.

α-amanitin induce inflammatory response by activating ROS/NF-κB-NLRP3 signaling pathway in human hepatoma HepG2 cells.

α-amanitin (AMA) is a hepatotoxic mushroom toxin responsible for over 90% of mushroom poisoning fatalities worldwide, seriously endangering human life and health. Few evidences have indicated that AMA leads to inflammatory responses and inflammatory infiltration in vitro and in vivo. However, the molecular mechanism remains unknown. In this study, human hepatocellular carcinomas cells (HepG2) were exposed to AMA at various concentrations for short period of times. Results revealed that AMA increased ROS production and elevated the releases of malondialdehyde (MDA) and lactate dehydrogenase (LDH), resulting in oxidative damage in HepG2 cells. Also, AMA exposure significantly increased the secreted levels of inflammatory cytokines and activated the NLRP3 inflammasome. The inflammatory responses were reversed by NLRP3 inhibitor MCC950 and NF-κB inhibitor Bay11-7082. Additionally, N-acetylcysteine (NAC) blocked the upregulation of the NF-κB/NLRP3 signaling pathway and remarkably alleviated the inflammatory response. These results demonstrated that AMA could induce inflammation through activating the NLRP3 inflammasome triggered by ROS/NF-κB signaling pathway. Our research provides new insights into the molecular mechanism of AMA-induced inflammation damage and may contribute to establish new prevention strategies for AMA hepatotoxicity.

Lactucopicrin promotes fatty acid ß-oxidation and attenuates lipid accumulation through adenosine monophosphate-activated protein kinase activation in free fatty acid-induced human hepatoblastoma cancer cells.

With its annually increasing prevalence, non-alcoholic fatty liver disease (NAFLD) has become a serious threat to people's life and health. After a preliminary research, we found that Lactucopicrin has pharmacological effects, such as lowering blood lipids and protecting the liver. Further research showed its significant activation for fatty acid ß-oxidase hydroxyacyl-coenzyme A (CoA) dehydrogenase trifunctional multienzyme complex subunit alpha (HADHA), so we hypothesized that Lactucopicrin could ameliorate lipid accumulation in hepatocytes by promoting fatty acid ß-oxidation. In this study, free fatty acid (FFA)-induced human hepatoblastoma cancer cells (HepG2) were used to establish an in vitro NAFLD model to investigate the molecular basis of Lactucopicrin in regulating lipid metabolism. Staining with Oil red O and measurements of triglyceride (TG) content, fatty acid ß-oxidase (FaßO) activity, reactive oxygen species (ROS) content, mitochondrial membrane potential, and adenosine triphosphate (ATP) content were used to assess the extent to which Lactucopicrin ameliorates lipid accumulation and promotes fatty acid ß-oxidation. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot methods were used to explore the regulatory effects of Lactucopicrin on factors related to fatty acid ß-oxidation. Results showed that Lactucopicrin downregulated phosphorylated mammalian target of rapamycin (P-mTOR) by activating the adenosine monophosphate-activated protein kinase (AMPK) pathway and upregulated the messenger RNA (mRNA) and protein expression levels of coactivators (peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α)), transcription factors (peroxisome proliferator-activated receptor α (PPARα) and peroxisome proliferator-activated receptor γ (PPARγ)), and oxidative factors (carnitine palmitoyltransferase 1A (CPT1A) and HADHA). This phenomenon resulted in a significant increase in FaßO activity, ATP content, and JC-1 and a significant decrease in ROS level, TG content, and intracellular lipid droplets. With the addition of Dorsomorphin, all the effects of Lactucopicrin intervention were suppressed. In summary, Lactucopicrin promotes fatty acid ß-oxidation by activating the AMPK pathway, thereby ameliorating FFA-induced intracellular lipid accumulation in HepG2 cells.

Exosomal delivery of miR-200b-3p suppresses the growth of hepatocellular carcinoma cells by targeting ERG- and VEGF-mediated angiogenesis.

Hepatocellular carcinoma (HCC) remains a lethal malignancy with limited treatment options. Recent discoveries have highlighted the pivotal role of miRNAs in HCC progression. We previously reported that the expression of miR-200b-3p was decreased in HCC cells and exosomal miR-200b-3p from hepatocytes inhibited angiogenesis by suppressing the expression of the endothelial transcription factor ERG (erythroblast transformation-specific (ETS)-related gene), leading to the hypothesis that the delivery of this miRNA may inhibit angiogenesis and suppress HCC growth in vivo. Here, we tested this hypothesis by using human HCC inoculation models. First, we transfected the human HepG2 HCC cells and established a stable cell line that overexpressed a high level of miR-200b-3p. When miR-200b-3p-overexpressing cells were injected into severe combined immunedeficiency (SCID)-beige mice, tumor growth was significantly reduced compared to tumors of control cells, with a reduction in the expression of ERG and vascular endothelial growth factor (VEGF) and subsequent angiogenesis. Intra-tumoral injection of exosomes containing high levels of miR-200b-3p also reduced the growth of parental HepG2 tumors with reduced ERG and VEGF expression and angiogenesis. These results validate the inhibitory role of miR-200b-3p in tumor angiogenesis, thereby suppressing HCC tumor growth, and provide a novel insight into its potential therapeutic application.

Effects of multi-walled carbon nanotubes on gene and microRNA expression in human hepatocarcinoma HepG2 cells.

The usage of multi-walled carbon nanotubes (MWCNT) has increased exponentially in the past years, but, potential toxicity mechanisms are not clear. We studied the transcriptomic alterations induced by one multi-walled carbon nanotube (MWCNT) and its -OH and -COOH functionalized derivatives in human HepG2 cells. We showed that all three MWCNT treatments induced alterations in stress-related signaling pathways, inflammation-related signaling pathways, cholesterol synthesis pathways, proliferation-related pathways, senescence-related pathways and cancer-related pathways. In stress-related pathways, the acute phase response was induced in all three MWCNTs and all doses treated and ranked high. Other stress-related pathways were also related to the oxidative-induced signaling pathways, such as NRF-2 mediated oxidative stress response, hepatic fibrosis/Stella cell activation, iNOS signaling, and Hif1α signaling. Many inflammation-related pathways were altered, such as IL-8, IL-6, TNFR1, TNFR2, and NF-κB signaling pathways. These results were consistent with our previous results with exposures to the same three multi-walled carbon nanotubes in human lung BEAS-2B and also with results in mice and rats. From the microRNA target filter analysis, TXNIP & miR-128-3p interaction was present in all three MWCNT treatments, and maybe important for the induction of oxidative stress. CXCL-8 & miR-146-5p and Wee1 & miR-128-3p were only present in the cells treated with the parent and the OH-functionalized MWCNTs. These mRNA-miRNA interactions were involved in oxidative stress, inflammation, cell cycle, cholesterol biosynthesis and cancer related pathways. Target filter analysis also showed altered liver hyperplasia/hyperproliferation and hepatic cancer pathways. In short, target filter analysis complemented the transcriptomic analysis and pointed to specific gene/microRNA interactions that can help inform mechanism of action. Moreover, our study showed that the signaling pathways altered in HepG2 cells correlated well with the toxicity and carcinogenicity observed in vivo, indicating that HepG2 may be a good in vitro predictive model for MWCNT toxicity studies.

One-Step Formation Method of Plasmid DNA-Loaded, Extracellular Vesicles-Mimicking Lipid Nanoparticles Based on Nucleic Acids Dilution-Induced Assembly.

We propose a nucleic acids dilution-induced assembly (NADIA) method for the preparation of lipid nanoparticles. In the conventional method, water-soluble polymers such as nucleic acids and proteins are mixed in the aqueous phase. In contrast, the NADIA method, in which self-assembly is triggered upon dilution, requires dispersion in an alcohol phase without precipitation. We then investigated several alcohols and discovered that propylene glycol combined with sodium chloride enabled the dispersion of plasmid DNA and protamine sulfate in the alcohol phase. The streamlined characteristics of the NADIA method enable the preparation of extracellular vesicles-mimicking lipid nanoparticles (ELNPs). Among the mixing methods using a micropipette, a syringe pump, and a microfluidic device, the lattermost was the best for decreasing batch-to-batch differences in size, polydispersity index, and transfection efficiency in HepG2 cells. Although ELNPs possessed negative ζ-potentials and did not have surface antigens, their transfection efficiency was comparable to that of cationic lipoplexes. We observed that lipid raft-mediated endocytosis and macropinocytosis contributed to the transfection of ELNPs. Our strategy may overcome the hurdles linked to supply and quality owing to the low abundance and heterogeneity in cell-based extracellular vesicles production, making it a reliable and scalable method for the pharmaceutical manufacture of such complex formulations.

Metabolomic and sphingolipidomic profiling of human hepatoma cells exposed to widely used pharmaceuticals.

Pharmaceutical compounds have become one of the main contaminants of emerging concern (CECs) due to their high usage and increased release into the environment. This study aims to assess the effects caused by three widely consumed hepatotoxic pharmaceutical compounds: an antibiotic (amoxicillin), an antiepileptic (carbamazepine), and an antidepressant (trazodone), on human health when indirectly exposed to toxicologically relevant concentrations (30, 15, and 7.5 µM for amoxicillin and carbamazepine, and 4, 2, and 1 µM for trazodone). A combination of semi-targeted metabolomic and targeted sphingolipid analyses was chosen to unravel the metabolic alterations in human hepatic cells exposed to these CECs at three concentrations for 24 h. HepG2 hepatoma cells were encapsulated in sodium alginate spheroids to improve the physiological relevance of this in vitro approach. Statistical analysis was used to identify the most affected metabolites and sphingolipids for each drug exposure. The results revealed small but significant changes in response to carbamazepine and trazodone exposures, affecting sphingolipid, glycerophospholipid precursors, and amino acid metabolism. Under both drug treatments, a decrease in various ceramide species (related to cell signaling) was observed, along with reduced taurine levels (related to the biosynthesis of bile acid conjugates) and carnitine levels (suggesting an impact on energy production). These and other drug-specific changes indicate that cellular functions in liver cells might be altered under low doses of these CECs, potentially affecting the health of other organs.

Schisandra chinensis Bee Pollen Extract Inhibits Proliferation and Migration of Hepatocellular Carcinoma HepG2 Cells via Ferroptosis-, Wnt-, and Focal Adhesion-Signaling Pathways.

Purpose: Bee pollen possesses favorable anticancer activities. As a medicinal plant source, Schisandra chinensis bee pollen (SCBP) possesses potential pharmacological properties, such as reducing cisplatin-induced liver injury, but its anti-liver cancer effect is still rarely reported. This paper aims to investigate the effect and mechanism of SCBP extract (SCBPE) on hepatocellular carcinoma HepG2 cells. Methods: The effect of SCBPE on cell proliferation and migration of HepG2 cells was evaluated based on MTT assay, morphology observation, or scratching assay. Furthermore, tandem mass tag-based quantitative proteomics was used to study the effect mechanisms. The mRNA expression levels of identified proteins were verified by RT-qPCR. Results: Tandem mass tag-based quantitative proteomics showed that 61 differentially expressed proteins were obtained in the SCBPE group compared with the negative-control group: 18 significantly downregulated and 43 significantly upregulated proteins. Bioinformatic analysis showed the significantly enriched KEGG pathways were predominantly ferroptosis-, Wnt-, and hepatocellular carcinoma-signaling ones. Protein-protein interaction network analysis and RT-qPCR validation revealed SCBPE also downregulated the focal adhesion-signaling pathway, which is abrogated by PF-562271, a well-known inhibitor of FAK. Conclusion: This study confirmed SCBPE suppressed the cell proliferation and migration of hepatocellular carcinoma HepG2 cells, mainly through modulation of ferroptosis-, Wnt-, hepatocellular carcinoma-, and focal adhesion-signaling pathways, providing scientific data supporting adjuvant treatment of hepatocellular carcinoma using SCBP.

Stearidonic acid improves eicosapentaenoic acid status: studies in humans and cultured hepatocytes.

Background: Ahiflower oil from the seeds of Buglossoides arvensis is rich in α-linolenic acid (ALA) and stearidonic acid (SDA). ALA and SDA are potential precursor fatty acids for the endogenous synthesis of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which are n3-long chain polyunsaturated fatty acids (n3-LC-PUFAS), in humans. Since taurine, an amino sulfonic acid, is often associated with tissues rich in n3-LC-PUFAS (e.g., in fatty fish, human retina), taurine may play a role in EPA- and DHA-metabolism. Objective: To examine the capacity of the plant-derived precursor fatty acids (ALA and SDA) and of the potential fatty acid metabolism modulator taurine to increase n3-LC-PUFAS and their respective oxylipins in human plasma and cultivated hepatocytes (HepG2 cells). Methods: In a monocentric, randomized crossover study 29 healthy male volunteers received three sequential interventions, namely ahiflower oil (9 g/day), taurine (1.5 g/day) and ahiflower oil (9 g/day) + taurine (1.5 g/day) for 20 days. In addition, cultivated HepG2 cells were treated with isolated fatty acids ALA, SDA, EPA, DHA as well as taurine alone or together with SDA. Results: Oral ahiflower oil intake significantly improved plasma EPA levels (0.2 vs. 0.6% of total fatty acid methyl esters (FAMES)) in humans, whereas DHA levels were unaffected by treatments. EPA-levels in SDA-treated HepG2 cells were 65% higher (5.1 vs. 3.0% of total FAMES) than those in ALA-treated cells. Taurine did not affect fatty acid profiles in human plasma in vivo or in HepG2 cells in vitro. SDA-rich ahiflower oil and isolated SDA led to an increase in EPA-derived oxylipins in humans and in HepG2 cells, respectively. Conclusion: The consumption of ahiflower oil improves the circulating levels of EPA and EPA-derived oxylipins in humans. In cultivated hepatocytes, EPA and EPA-derived oxylipins are more effectively increased by SDA than ALA.