Andrea Ferrigno

Background: Metabolic-dysfunction-associated steatotic liver disease (MASLD) is a multifactorial liver disease in which mitochondrial dysfunction, oxidative stress, and inflammation play key roles in driving the progression toward metabolic dysfunction-associated steatohepatitis (MASH) and hepatocellular carcinoma (HCC). Dysfunctional mitochondria generate excess reactive oxygen species (ROS), impair antioxidant defenses, activate pro-inflammatory pathways and hepatic stellate cells, and perpetuate liver injury. Mitochondrial Complex I is a major ROS source, particularly under conditions of dysregulated energy metabolism. Since Complex I inhibition by metformin was shown to reduce ROS and activate the adenosine monophosphate-activated protein kinase (AMPK), this study aimed to evaluate whether a novel Complex I Modulator (CIM, BI4500) could attenuate oxidative stress, inflammation, and consequently reduce lipid accumulation and fibrosis in a methionine- and choline-deficient diet (MCD)-fed rat model of MASH. Methods: Rats were fed an MCD or an isocaloric control diet for six weeks. From week four, animals received daily oral treatment with CIM (10 mg/kg) or vehicle (Natrosol). At the endpoint, liver tissue was collected for histological, biochemical, and molecular analyses. Lipid droplet area, inflammatory infiltration, and collagen deposition were evaluated on tissue sections; total lipid content and oxidative stress markers were assessed in homogenates and isolated mitochondria. Molecular pathways related to oxidative stress, lipid metabolism, and fibrosis were assessed at protein and mRNA levels. Results: CIM treatment significantly reduced oxidative stress (ROS, lipid peroxidation, nitrogen species), promoting AMPK activation and metabolic reprogramming. This included increased expression of peroxisome proliferator-activated receptor alpha (PPAR-α) and its target genes, and decreased sterol regulatory element binding protein-1c (SREBP-1c)-driven lipogenesis. These changes halted fibrosis progression, as confirmed by Picro-Sirius Red staining and fibrosis markers. Conclusions: these findings indicate that Complex I modulation may represent a promising strategy to counteract MASLD progression toward MASH.

This study evaluated the protective role of farnesoid-X-receptor (FXR) agonist INT-787 in the control of mitochondrial changes using a metabolic dysfunction-associated steatohepatitis (MASH) model. Lep-ob/ob mice were fed a control diet (CD) for 21 weeks (wks), or a high-fat diet (HFD) for 9 or 21 wks; in the 21 wk HFD groups, INT-787 (30 mg/kg/day) dosed via HFD admixture was added. The hepatic ATP, ROS, GSH and MIC19, which stabilizes the structure of inner mitochondrial membrane (IMM), were quantified. Transmission electron microscopy (TEM) analysis was also performed. INT-787 increased hepatic ATP, which was downregulated after HFD 9 and 21 wks. Hepatic ROS increased and GSH decreased after 21 wks and were recovered by INT-787. MIC19 mRNA level decreased after HFD 21 wks, and it was completely restored after INT-787 administration. TEM analysis showed that INT-787 reverted the mitochondrial alterations as documented by restored mitochondrial length, number of mitochondrial cristae junctions (CJs), and distance between endoplasmic reticulum (ER) and outer mitochondrial membrane (OMM) when compared with HFD groups. These results underline the involvement of the FXR pathway in the control of mitochondrial damage, thus revealing a previously undiscovered mechanism mediated by FXR activation: the upregulation of IMM protein MIC19, which is essential for maintaining cristae integrity and mitochondrial function.

Venetoclax is a Bcl-2 homology domain 3 (BH3) mimetic currently approved for the treatment of chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML) that has proven to be highly effective in reinstating apoptosis in leukemic cells through the highly selective inhibition of the anti-apoptotic protein B-cell lymphoma-2 (Bcl-2). Clinically, venetoclax has provided lasting remissions through the inhibition of CLL and AML blasts. However, this activity has often come at the cost of grade III/IV neutropenia due to hematopoietic cells’ dependence on Bcl-2 for survival. As life-threatening infections are an important complication in these patients, an effective management of neutropenia is indispensable to maximize patient outcomes. While there is general consensus over dose reduction and scheduling modifications to minimize the risk of neutropenia, the impact of these modifications on survival is uncertain. Moreover, guidelines do not yet adequately account for patient-specific and disease-specific risk factors that may predict toxicity, or the role combination treatment plays in exacerbating neutropenia. The objective of this review is to discuss the venetoclax-induced mechanism of hematological toxicity, the potential predictive risk factors that affect patient vulnerability to neutropenia, and the current consensus on practices for management of neutropenia.

The aim of this Special Issue is to provide an update on the diagnosis and treatment of nonalcoholic fatty liver disease (NAFLD), which is the most prevalent liver disease worldwide; however, there are still no specific treatment agents

The reversion-inducing cysteine-rich protein with Kazal motifs (RECK), a membrane-anchored glycoprotein, negatively regulates various membrane proteins involved in the tissue governing extracellular matrix (ECM) remodeling such as metalloproteases (MMPs) and the sheddases ADAM10 and ADAM17. The significance of the present review is to summarize the current understanding of the pathophysiological role of RECK, a newly discovered signaling pathway associated with different liver injuries. Specifically, this review analyzes published data on the downregulation of RECK expression in hepatic ischemia/reperfusion (I/R) injury, liver-related cancers, including hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), as well as in the progression of nonalcoholic fatty liver disease (NAFLD) to non-alcoholic steatohepatitis (NASH). In addition, this review discusses the regulation of RECK by inducers, such as FXR agonists. The RECK protein has also been suggested as a potential diagnostic and prognostic marker for liver injury or as a biomarker with predictive value for drug treatment efficacy.

Background and Aims: We have previously shown in a model of hepatic ischaemia/reperfusion injury that the farnesoid X receptor (FXR) agonist obeticholic acid (OCA) restores reversion‐inducing‐cysteine‐rich protein with Kazal motifs (RECK), an inverse modulator of metalloproteases (MMPs) and inhibitor of the sheddases ADAM10 and ADAM17 involved in inflammation and fibrogenesis. Here, the effects of FXR agonists OCA and INT‐787 on hepatic levels of RECK, MMPs, ADAM10 and ADAM17 were compared in a diet‐induced ob/ob mouse model of non‐alcoholic steatohepatitis (NASH).Methods: Lep ob/ob NASH mice fed a high‐fat diet (HFD) or control diet (CD) for 9 weeks (wks) were treated with OCA or INT‐787 0.05% dosed via HFD admixture (30 mg/kg/day) or HFD for further 12 wks. Serum alanine transaminase (ALT) and inflammatory cytokines, liver RECK, MMP‐2 and MMP‐9 activity as well as ADAM10, ADAM17, collagen deposition (Sirius red), hepatic stellate cell activation (α‐SMA) and pCK⁺ reactive biliary cells were quantified.Results: Only INT‐787 significantly reduced serum ALT, IL‐1β and TGF‐β. A downregulation of RECK expression and protein levels observed in HFD groups (at 9 and 21 wks) was counteracted by both OCA and INT‐787. HFD induced a significant increase in liver MMP‐2 and MMP‐9; OCA administration reduced both MMP‐2 and MMP‐9 while INT‐787 markedly reduced MMP‐2 expression. OCA and INT‐787 reduced both ADAM10 and ADAM17 expression and number of pCK⁺ cells. INT‐787 was superior to OCA in decreasing collagen deposition and α‐SMA levels.Conclusion: INT‐787 is superior to OCA in controlling specific cell types and clinically relevant anti‐inflammatory and antifibrotic molecular mechanisms in NASH.

The endogenous antioxidant defense plays a big part in the pathogenesis of non-alcoholic fatty liver disease (NAFLD), a common metabolic disorder that can lead to serious complications such as cirrhosis and cancer. HuR, an RNA-binding protein of the ELAV family, controls, among others, the stability of MnSOD and HO-1 mRNA. These two enzymes protect the liver cells from oxidative damage caused by excessive fat accumulation. Our aim was to investigate the expression of HuR and its targets in a methionine-choline deficient (MCD) model of NAFLD. To this aim, we fed male Wistar rats with an MCD diet for 3 and 6 weeks to induce NAFLD; then, we evaluated the expression of HuR, MnSOD, and HO-1. The MCD diet induced fat accumulation, hepatic injury, oxidative stress, and mitochondrial dysfunction. A HuR downregulation was also observed in association with a reduced expression of MnSOD and HO-1. Moreover, the changes in the expression of HuR and its targets were significantly correlated with oxidative stress and mitochondrial injury. Since HuR plays a protective role against oxidative stress, targeting this protein could be a therapeutic strategy to both prevent and counteract NAFLD.

The autofluorescence of specific fatty acids, retinoids, and bilirubin in crude serum can reflect changes in liver functional engagement in maintaining systemic metabolic homeostasis. The role of these fluorophores as intrinsic biomarkers of pharmacological actions has been investigated here in rats administered with obeticholic acid (OCA), a Farnesoid-X Receptor (FXR) agonist, proven to counteract the increase of serum bilirubin in hepatic ischemia/reperfusion (I/R) injury. Fluorescence spectroscopy has been applied to an assay serum collected from rats submitted to liver I/R (60/60 min ± OCA administration). The I/R group showed changes in the amplitude and profiles of emission spectra excited at 310 or 366 nm, indicating remarkable alterations in the retinoid and fluorescing fatty acid balance, with a particular increase in arachidonic acid. The I/R group also showed an increase in bilirubin AF, detected in the excitation spectra recorded at 570 nm. OCA greatly reversed the effects observed in the I/R group, confirmed by the biochemical analysis of bilirubin and fatty acids. These results are consistent with a relationship between OCA anti-inflammatory effects and the acknowledged roles of fatty acids as precursors of signaling agents mediating damaging responses to harmful stimuli, supporting serum autofluorescence analysis as a possible direct, real-time, cost-effective tool for pharmacological investigations.

The blockade of metabotropic glutamate receptor type 5 (mGluR5) was previously found to reduce fat accumulation in HEPG2 cells. Here, we evaluated the effects of mGluR5 blockade in a mouse model of steatosis. Male ob/ob mice fed a high-fat diet were treated with MPEP or vehicle. After 7 weeks, liver biopsies were collected, and nuclei were isolated from fresh tissue. Lipid droplet area and collagen deposition were evaluated on tissue slices; total lipids, lipid peroxidation, and ROS were evaluated on tissue homogenates; PPARα, SREBP-1, mTOR, and NF-κB were assayed on isolated nuclei by Western Blot. Target genes of the above-mentioned factors were assayed by RT-PCR. Reduced steatosis and hepatocyte ballooning were observed in the MPEP group with respect to the vehicle group. Concomitantly, increased nuclear PPARα and reduced nuclear SREBP-1 levels were observed in the MPEP group. Similar trends were obtained in target genes of PPARα and SREBP-1, Acox1 and Acc1, respectively. MPEP administration also reduced oxidative stress and NF-κB activation, probably via NF-κB inhibition. Levels of common markers of inflammation (Il-6, Il1β and Tnf-α) and oxidative stress (Nrf2) were significantly reduced. mTOR, as well as collagen deposition, were unchanged. Concluding, MPEP, a selective mGluR5 negative allosteric modulator, reduces both fat accumulation and oxidative stress in a 7-week murine model of steatosis. Although underlying mechanisms need to be further investigated, this is the first in vivo study showing the beneficial effects of MPEP in a murine model of steatosis.