Drug-Drug Interaction Potential, Cytotoxicity, and Reactive Oxygen Species Production of Salix Cortex Extracts Using Human Hepatocyte-Like HepaRG Cells

Herbal preparations of willow bark (Salix cortex) are available in many countries as non-prescription medicines for pain and inflammation, and also as dietary supplements. Currently only little information on toxicity and drug interaction potential of the extracts is available. This study now evaluated the effects of two Salix cortex extracts on human hepatocyte-like HepaRG cells, in view of clinically relevant CYP450 enzyme activity modulation, cytotoxicity and production of reactive oxygen species (ROS). Drug metabolism via the CYP450 enzyme system is considered an important parameter for the occurrence of drug-drug interactions, which can lead to toxicity, decreased pharmacological activity, and adverse drug reactions. We evaluated two different bark extracts standardized to 10 mg/ml phenolic content. Herein, extract S6 (S. pentandra, containing 8.15 mg/ml total salicylates and 0.08 mg/ml salicin) and extract B (industrial reference, containing 5.35 mg/ml total salicylates and 2.26 mg/ml salicin) were tested. Both Salix cortex extracts showed no relevant reduction in cell viability or increase in ROS production in hepatocyte-like HepaRG cells. However, they reduced CYP1A2 and CYP3A4 enzyme activity after 48 h at ≥25 μg/ml, this was statistically significant only for S6. CYP2C19 activity inhibition (0.5 h) was also observed at ≥25 μg/ml, mRNA expression inhibition by 48 h treatment with S6 at 25 μg/ml. In conclusion, at higher concentrations, the tested Salix cortex extracts showed a drug interaction potential, but with different potency. Given the high prevalence of polypharmacy, particularly in the elderly with chronic pain, further systematic studies of Salix species of medical interest should be conducted in the future to more accurately determine the risk of potential drug interactions.

Primary Human Hepatocyte Spheroids as Tools to Study the Hepatotoxic Potential of Non-Pharmaceutical Chemicals

Drug-induced liver injury, including cholestasis, is an important clinical issue and economic burden for pharmaceutical industry and healthcare systems. However, human-relevant in vitro information on the ability of other types of chemicals to induce cholestatic hepatotoxicity is lacking. This work aimed at investigating the cholestatic potential of non-pharmaceutical chemicals using primary human hepatocytes cultured in 3D spheroids. Spheroid cultures were repeatedly (co-) exposed to drugs (cyclosporine-A, bosentan, macitentan) or non-pharmaceutical chemicals (paraquat, tartrazine, triclosan) and a concentrated mixture of bile acids for 4 weeks. Cell viability (adenosine triphosphate content) was checked every week and used to calculate the cholestatic index, an indicator of cholestatic liability. Microarray analysis was performed at specific time-points to verify the deregulation of genes related to cholestasis, steatosis and fibrosis. Despite the evident inter-donor variability, shorter exposures to cyclosporine-A consistently produced cholestatic index values below 0.80 with transcriptomic data partially supporting its cholestatic burden. Bosentan confirmed to be hepatotoxic, while macitentan was not toxic in the tested concentrations. Prolonged exposure to paraquat suggested fibrotic potential, while triclosan markedly deregulated genes involved in different types of hepatotoxicity. These results support the applicability of primary human hepatocyte spheroids to study hepatotoxicity of non-pharmaceutical chemicals in vitro.

Exposure to Perfluoro-Octanoic Acid Associated With Upstream Uncoupling of the Insulin Signaling in Human Hepatocyte Cell Line

Perfluoro–alkyl substances (PFAS) are chemical pollutants with prevalent stability and environmental persistence. Exposure to PFAS, particularly perfluoro-octanoic acid (PFOA), has been associated with increased diabetes-related cardiovascular mortality in subjects residing areas of high environmental contamination, however the exact pathogenic mechanism remains elusive. Here we used HepG2 cells, an in vitro model of human hepatocyte, to investigate the possible role of PFOA exposure in the alteration of hepatic glucose metabolism. HepG2 cells were exposed for 24 hours to PFOA at increasing concentration from 0 to 1000 ng/mL and then stimulated with 100 nm Insulin (Ins). The consequent effect on glycogen synthesis, glucose uptake and Glut-4 glucose transporter translocation was then evaluated by, respectively, Periodic Acid Schiff (PAS) staining, 2-deoxyglucose (2-DG) uptake assay and immunofluorescence. Exposure to PFOA was associated with reduced glycogen synthesis and glucose uptake, at concentration equal or greater than, respectively, 0,1 ng/mL and 10 ng/mL, with parallel impaired membrane translocation of Glut-4 upon Ins stimulation. Western blot analysis showed early uncoupling of Insulin Receptor (InsR) activation from the downstream Akt and GSK3 phosphorylation. Computational docking analysis disclosed the possible stabilizing effect of PFOA on the complex between InsR and GM3 ganglioside, previously shown to be associated with the low grade chronic inflammation-related insulin resistance. Consistently, long term treatment with glucosyl-ceramide synthase inhibitor PDMP was able to largely restore glycogen synthesis, glucose uptake and Glut-4 translocation upon Ins stimulation in HepG2 exposed to PFOA. Our data support a novel pathogenic mechanism linking exposure to PFOA to derangement of hepatocyte cell metabolism.

Proteomic Profiling of Exosomes From Hemorrhagic Moyamoya Disease and Dysfunction of Mitochondria in Endothelial Cells

Background and Purpose: Moyamoya disease (MMD) is a rare steno-occlusive and slowly progressing cerebrovascular disorder. The detailed mechanism of the underlying pathogenesis is still blurry. Methods: Tandem Mass Tag-labeled quantitative proteomics was performed on serum-derived exosomes (SDEs) extracted from adult patients diagnosed with pure ischemic MMD or hemorrhagic MMD and healthy controls. Then mouse brain vascular endothelial cell (EC), human umbilical vein EC, neuroblastoma cell, and human hepatocyte cell were treated with exosomes, and changes of the protein expression in mouse brain vascular EC cells were identified. Results: Proteomics analysis results showed that 859 shared proteins were detected in SDEs from ischemic and hemorrhagic MMD patients with 231 differently expressed compared with healthy controls. Bioinformatic analysis revealed dysregulated cell growth and maintenance and indicated disturbed actin dynamics in MMD, with CFL1 (Cofilin-1) and ACTR2/3 (actin-related protein 2/3; also known as ARP2/3) downregulated in ischemic and hemorrhagic patients’ SDEs. We also found immunity dysfunction in hemorrhagic MMD. Following treatment with MMD SDEs, mouse brain vascular EC cells showed significantly higher levels of proliferation and more ethynyl-2-deoxyuridine-positive cells compared with the healthy control group, while there were no obvious changes in the human umbilical vein EC and human hepatocyte cell. Interestingly, we also found that SDEs from ischemic MMD promoted neuroblastoma cell proliferation. Proteomic analysis of mouse brain vascular EC cells suggested that SDEs from hemorrhagic MMD patients induced dysfunction of the mitochondria in cerebrovascular ECs. Conclusions: This study highlighted potential molecular mechanisms underlying the pathogenesis of MMD patients, thereby providing new therapeutic strategies for MMD.