A peculiar role for oxidative stress in non-alcoholic fatty liver disease (NAFLD) and its transition to the inflammatory complication non-alcoholic steatohepatitis (NASH), as well as in its threatening evolution to hepatocellular carcinoma (HCC), is supported by numerous experimental and clinical studies. NADPH oxidases (NOXs) are enzymes producing reactive oxygen species (ROS), whose abundance in liver cells is closely related to inflammation and immune responses. Here, we reviewed recent findings regarding this topic, focusing on the role of NOXs in the different stages of fatty liver disease and describing the current knowledge about their mechanisms of action. We conclude that, although there is a consensus that NOX-produced ROS are toxic in non-neoplastic conditions due to their role in the inflammatory vicious cycle sustaining the transition of NAFLD to NASH, their effect is controversial in the neoplastic transition towards HCC. In this regard, there are indications of a differential effect of NOX isoforms, since NOX1 and NOX2 play a detrimental role, whereas increased NOX4 expression appears to be correlated with better HCC prognosis in some studies. Further studies are needed to fully unravel the mechanisms of action of NOXs and their relationships with the signaling pathways modulating steatosis and liver cancer development.

Liver fibrosis, which is the outcome of wound-healing response to chronic liver damage, represents an unmet clinical need. This study evaluated the anti-fibrotic and anti-inflammatory effects of the polyphenol oleocanthal (OC) extracted from extra virgin olive oil (EVOO) by an in vitro/in vivo approach. The hepatic cell lines LX2 and HepG2 were used as in vitro models. The mRNA expression of pro-fibrogenic markers, namely alpha-smooth muscle actin (α-SMA), collagen type I alpha 1 chain (COL1A1), a panel of metalloproteinases (MMP1, MMP2, MMP3, MMP7, MMP9) and vascular endothelial growth factor A (VEGFA) as well as the pro-oxidant genes NADPH oxidases (NOXs) 1 and 4 were evaluated in TGF-β activated LX2 cells by qRT-PCR. α-SMA and COL1A1 protein expression was assessed by immunofluorescence coupled to confocal microscopy. VEGFA release from LX2 was measured by ELISA. We also evaluated the amount of reactive oxygen species (ROS) produced by H2O2 activated- HepG2 cells. In vivo, OC was administered daily by oral gavage to Balb/C mice with CCl4-induced liver fibrosis. In this model, we measured the mRNA hepatic expression of the three pro-inflammatory interleukins (IL) IL6, IL17, IL23, chemokines such as C-C Motif Chemokine Ligand 2 (CCL2) and C-X-C Motif Chemokine Ligand 12 (CXCL12), and selected miRNAs (miR-181-5p, miR-221-3p, miR-29b-3p and miR-101b-3p) by qRT-PCR. We demonstrated that OC significantly downregulated the gene/protein expression of α-SMA, COL1A1, MMP2, MMP3, MMP7 and VEGF as well as the oxidative enzymes NOX1 and 4 in TGFβ1-activated LX2 cells, and reduced the production of ROS by HepG2. In vivo OC, beside causing a significant reduction of fibrosis at histological assessment, counteracted the CCl4-induced upregulation of pro-fibrotic and inflammatory genes. Moreover, OC upregulated the anti-fibrotic miRNAs (miR-29b-3p and miR-101b-3p) reduced in fibrotic mice, while downregulated the pro-fibrotic miRNAs (miR-221-3p and miR-181-5p), which were dramatically upregulated in fibrotic mice. In conclusion, OC exerts a promising antifibrotic effect via a combined reduction of oxidative stress and inflammation involving putative miRNAs, which in turn reduces hepatic stellate cells activation and liver fibrosis.

Background: Manipulations to decelerate biological aging and extend health span are major challenges for health given the social and healthcare costs of aging population. Excessive oxidative stress and inflammation are primary drivers in biological aging and age-related diseases. Didymin, a natural bioactive flavonoid and the major polyphenol of Monarda didyma L., has showed a crucial anti-inflammatory role. This clinical study aims to investigate the potential benefits of a 12-weeks supplementation of Monarda didyma L. extract on reducing / delaying the biological aging of a susceptible population of aging workers. Methods: This is a randomized, placebo-controlled, double-blind, parallel design and monocentric pilot study. Study population will comprise 80 participants, aged 45-65 years, randomly allocated to both Intervention group (supplementation of Monarda didyma L. extract) and placebo control group (placebo supplementation) for a 12-weeks period of treatment. All study participants, who will be enrolled at the Occupational Medicine Unit – Azienda Ospedale Università of Padova providing a written informed consent, will undergo clinical examination, an interview with structured questionnaires (demographic data, lifestyle, information quality of life and sleeping patterns) and collection of fasting blood for evaluation of indicators of biological aging as primary outcomes (DNA methylation age, telomere length, mitochondrial DNA copy number), as well as secondary outcomes including basic biochemistry and inflammation markers (emocrome, glycemia, insulin, total cholesterol, low and high density lipoproteins, triglycerides, creatinine, telomerase expression, C-reactive protein, interleukin6, aspartate transaminase, alanine transaminase, gamma-glutamyl transferase) and salivary sample for cortisol analysis. Clinical examination and sample collection, to assess primary and secondary outcomes, will be performed at enrollment and at the follow up after of this treatment. Tracking of additional parameters (heart rate, sleep, mobility) will be performed along the study period by using a wearable MiBand 6 watch and a daily diary to determine compliance and record effects on stress, well-being and health status. Discussion: The success of this research could represent a turning point in the healthy aging research, leading to rejuvenation by means of a sustainable and safe intervention, accessible to everybody in order to extend health span. Trial registration: ClinicalTrials.gov PRS (NCT05399966), registration date: May 17, 2022. https://clinicaltrials.gov/ct2/about-studies/learn#WhatIs

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