FXR agonists INT-787 and OCA increase RECK and inhibit liver steatosis and inflammation in diet-induced ob/ob mouse model of NASH.

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-1ß and TGF-ß. 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.

MPEP Attenuates Intrahepatic Fat Accumulation in Obese Mice.

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, Il1ß 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.

MCD Diet Rat Model Induces Alterations in Zinc and Iron during NAFLD Progression from Steatosis to Steatohepatitis.

We evaluate the effects of the methionine-choline-deficient (MCD) diet on serum and hepatic zinc (Zn) and iron (Fe) and their relationships with matrix metalloproteinases (MMPs) and their modulators (TIMPs and RECK) as well as hepatic fatty acids using male Wistar rats fed 2-, 4- and 8-week MCD diets. Serum and hepatic Zn decrease after an 8-week MCD diet. Serum Fe increases after an 8-week MCD diet and the same occurs for hepatic Fe. An increase in hepatic MMP activity, associated with a decrease in RECK and TIMPs, is found in the MCD 8-week group. Liver Fe shows a positive correlation versus MMPs and RECK, and an inverse correlation versus TIMPs. A positive correlation is found comparing liver Zn with stearic, vaccenic and arachidonic acids, and an inverse correlation is found with linolenic and docosatetraenoic acids. An opposite trend is found between liver Fe versus these fatty acids. During NAFLD progression from steatosis to steatohepatitis, MCD rats exhibit an increase in Zn and a decrease in Fe levels both in serum and tissue associated with alterations in hepatic MMPs and their inhibitors, and fatty acids. The correlations detected between Zn and Fe versus extracellular matrix modulators and fatty acids support their potential role as therapeutic targets.

Long-term cold storage preservation does not affect fatty livers from rats fed with a methionine and choline deficient diet.

BACKGROUND: Waiting lists that continue to grow and the lack of organs available for transplantation necessitate the use of marginal livers, such as fatty livers. Since steatotic livers are more susceptible to damage from ischemia and reperfusion, it was investigated whether fatty livers with different lipidomic profiles show a different outcome when subjected to long-term cold storage preservation. METHODS: Eight-week-old male Wistar rats fed for 2 weeks by a methionine-choline-deficient (MCD) diet or control diet were employed in this study. Livers were preserved in a University of Wisconsin (UW) solution at 4 °C for 6, 12 or 24 h and, after washout, reperfused for 2 h with a Krebs-Henseleit buffer at 37 °C. Hepatic enzyme release, bile production, O2-uptake, and portal venous pressure (PVP) were evaluated. The liver fatty acid profile was evaluated by a gas chromatography-mass spectrometry (GC/MS). RESULTS: MCD rats showed higher LDH and AST levels with respect to the control group. When comparing MCD livers preserved for 6, 12 or 24 h, no differences in enzyme release were found during both the washout or the reperfusion period. The same trend occurred for O2-uptake, PVP, and bile flow. A general decrease in SFA and MUFA, except for oleic acid, and a decrease in PUFA, except for arachidonic, eicosadienoic, and docosahexanaeoic acids, were found in MCD rats when compared with control rats. Moreover, the ratio between SFA and the various types of unsaturated fatty acids (UFA) was significantly lower in MCD rats. CONCLUSIONS: Although prolonged cold ischemia negatively affects the graft outcome, our data suggest that the quality of lipid constituents could influence liver injury during cold storage: the lack of an increased hepatic injury in MCD may be justified by low SFA, which likely reduces the deleterious tendency toward lipid crystallization occurring under cold ischemia.

Metabotropic Glutamate Receptor Blockade Reduces Preservation Damage in Livers from Donors after Cardiac Death.

We previously demonstrated that the blockade of mGluR5 by 2-methyl-6(phenylethynyl)pyridine (MPEP) reduces both cold and warm ischemia/reperfusion injury. Here we evaluated whether MPEP reduces the hepatic preservation injury in rat livers from cardiac-death-donors (DCDs). Livers from DCD rats were isolated after an in situ warm ischemia (30 min) and preserved for 22 h at 4 °C with UW solution. Next, 10 mg/Kg MPEP or vehicle were administered 30 min before the portal clamping and added to the UW solution (3 µM). LDH released during washout was quantified. Liver samples were collected for iNOS, eNOS, NO, TNF-α, ICAM-1, caspase-3 and caspase-9 protein expression and nuclear factor-erythroid-2-related factor-2 (Nrf2) gene analysis. Lower LDH levels were detected in control grafts versus DCD groups. An increase in eNOS and NO content occurred after MPEP treatment; iNOS and TNF-α content was unchanged. ICAM-1 expression was reduced in the MPEP-treated livers as well as the levels of caspase-3 and caspase-9. Nrf2, oxidative stress-sensitive gene, was recovered to control value by MPEP. These results suggest that MPEP can be used to reclaim DCD livers subjected to an additional period of cold ischemia during hypothermic storage. MPEP protects against apoptosis and increased eNOS, whose overexpression has been previously demonstrated to be protective in hepatic ischemia/reperfusion damage.

Nonalcoholic Fatty Liver Disease and Non-Alcoholic Steatohepatitis: Current Issues and Future Perspectives in Preclinical and Clinical Research.

Nonalcoholic fatty liver disease (NAFLD) is a continuum of liver abnormalities often starting as simple steatosis and to potentially progress into nonalcoholic steatohepatitis (NASH), fibrosis, cirrhosis and hepatocellular carcinoma. Because of its increasing prevalence, NAFLD is becoming a major public health concern, in parallel with a worldwide increase in the recurrence rate of diabetes and metabolic syndrome. It has been estimated that NASH cirrhosis may surpass viral hepatitis C and become the leading indication for liver transplantation in the next decades. The broadening of the knowledge about NASH pathogenesis and progression is of pivotal importance for the discovery of new targeted and more effective therapies; aim of this review is to offer a comprehensive and updated overview on NAFLD and NASH pathogenesis, the most recommended treatments, drugs under development and new drug targets. The most relevant in vitro and in vivo models of NAFLD and NASH will be also reviewed, as well as the main molecular pathways involved in NAFLD and NASH development.

Detailed Molecular Mechanisms Involved in Drug-Induced Non-Alcoholic Fatty Liver Disease and Non-Alcoholic Steatohepatitis: An Update.

Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are some of the biggest public health challenges due to their spread and increasing incidence around the world. NAFLD is characterized by intrahepatic lipid deposition, accompanied by dyslipidemia, hypertension, and insulin resistance, leading to more serious complications. Among the various causes, drug administration for the treatment of numerous kinds of diseases, such as antiarrhythmic and antihypertensive drugs, promotes the onset and progression of steatosis, causing drug-induced hepatic steatosis (DIHS). Here, we reviewed in detail the major classes of drugs that cause DIHS and the specific molecular mechanisms involved in these processes. Eight classes of drugs, among the most used for the treatment of common pathologies, were considered. The most diffused mechanism whereby drugs can induce NAFLD/NASH is interfering with mitochondrial activity, inhibiting fatty acid oxidation, but other pathways involved in lipid homeostasis are also affected. PubMed research was performed to obtain significant papers published up to November 2021. The key words included the class of drugs, or the specific compound, combined with steatosis, nonalcoholic steatohepatitis, fibrosis, fatty liver and hepatic lipid deposition. Additional information was found in the citations listed in other papers, when they were not displayed in the original search.

Obeticholic Acid Reduces Kidney Matrix Metalloproteinase Activation Following Partial Hepatic Ischemia/Reperfusion Injury in Rats.

We have previously demonstrated that the farnesoid X receptor (FXR) agonist obeticholic acid (OCA) protects the liver via downregulation of hepatic matrix metalloproteinases (MMPs) after ischemia/reperfusion (I/R), which can lead to multiorgan dysfunction. The present study investigated the capacity of OCA to modulate MMPs in distant organs such as the kidney. Male Wistar rats were dosed orally with 10 mg/kg/day of OCA (5 days) and were subjected to 60-min partial hepatic ischemia. After 120-min reperfusion, kidney biopsies (cortex and medulla) and blood samples were collected. Serum creatinine, kidney MMP-2, and MMP-9-dimer, tissue inhibitors of MMPs (TIMP-1, TIMP-2), RECK, TNF-alpha, and IL-6 were monitored. MMP-9-dimer activity in the kidney cortex and medulla increased after hepatic I/R and a reduction was detected in OCA-treated I/R rats. Although not significantly, MMP-2 activity decreased in the cortex of OCA-treated I/R rats. TIMPs and RECK levels showed no significant differences among all groups considered. Serum creatinine increased after I/R and a reduction was detected in OCA-treated I/R rats. The same trend occurred for tissue TNF-alpha and IL-6. Although the underlying mechanisms need further investigation, this is the first study showing, in the kidney, beneficial effects of OCA by reducing TNF-alpha-mediated expression of MMPs after liver I/R.

SerpinB3 administration protects liver against ischemia-reperfusion injury.

We have investigated the change in SerpinB3 during hepatic ischemia and the potential role of its antiprotease activity in cell protection by the administration of wild-type SerpinB3 (SerpinB3-WT) or active loop-deleted recombinant SerpinB3 protein (SerpinB3-D) in a rat model of ischemia (60 min)/reperfusion (60 min) (I/R). A time-dependent increase of SerpinB3, both at transcription and protein level, was found in ischemic livers after 60, 120 and 180 min. SerpinB3-WT decreased polymorphonuclear cell infiltration and serum enzymes and increased ATP when compared with I/R group. These events were not obtained using SerpinB3-D. No significant changes in both liver SerpinB3 mRNA and protein were found in all I/R groups considered. The present data show that the administration of SerpinB3-WT reduced the I/R injury and this effect appears to be dependent on its anti-protease activity.

Changes in Glutathione Content in Liver Diseases: An Update.

Glutathione (GSH), a tripeptide particularly concentrated in the liver, is the most important thiol reducing agent involved in the modulation of redox processes. It has also been demonstrated that GSH cannot be considered only as a mere free radical scavenger but that it takes part in the network governing the choice between survival, necrosis and apoptosis as well as in altering the function of signal transduction and transcription factor molecules. The purpose of the present review is to provide an overview on the molecular biology of the GSH system; therefore, GSH synthesis, metabolism and regulation will be reviewed. The multiple GSH functions will be described, as well as the importance of GSH compartmentalization into distinct subcellular pools and inter-organ transfer. Furthermore, we will highlight the close relationship existing between GSH content and the pathogenesis of liver disease, such as non-alcoholic fatty liver disease (NAFLD), alcoholic liver disease (ALD), chronic cholestatic injury, ischemia/reperfusion damage, hepatitis C virus (HCV), hepatitis B virus (HBV) and hepatocellular carcinoma. Finally, the potential therapeutic benefits of GSH and GSH-related medications, will be described for each liver disorder taken into account.

The selective blockade of metabotropic glutamate receptor-5 attenuates fat accumulation in an in vitro model of benign steatosis.

It has been previously found that the blockade of metabotropic glutamate receptor type 5 (mGluR5) protects against hepatic ischemia/reperfusion injury and acetaminophen toxicity. The role of mGluR5 in NAFLD has not yet been elucidated. Here, we evaluated the effects of mGluR5 blockade in an in vitro model of steatosis. HepG2 cells were pre-incubated for 12 h with an mGluR5 agonist, a negative allosteric modulator (DHPG and MPEP, respectively) or vehicle, then treated with 1.5 mM oleate/palmitate (O/P) for another 12 h. Cell viability was evaluated with the MTT assay; fat accumulation was measured using the fluorescent dye nile red; SREBP-1, PPAR-α, iNOS and Caspase-3 protein expression were evaluated by Western blot; NFkB activity was evaluated as pNFkB/NFkB ratio. mGluR5 modulation did not alter cell viability in O/P-incubated cells; MPEP prevented intracellular lipid accumulation in O/P treated cells; MPEP administration was also associated with a reversion of O/P-induced changes in SREBP-1 and PPAR-α expression, involved in free fatty acid (FFA) metabolism and uptake. No changes were observed in iNOS and Caspase-3 expression, or in NFkB activity. In conclusion, mGluR5 pharmacological blockade reduced fat accumulation in HepG2 cells incubated with O/P, probably by modulating the expression of SREBP-1 and PPAR-α.