Erdi Sozen

Objective: Nuclear factor erythroid 2-related factor 2 (NRF2), a key transcription factor involved in redox homeostasis, plays a critical role in preventing lipid peroxidation against various hepatic disorders, including metabolic dysfunction-associated fatty liver disease (MAFLD). Growing body of evidence suggests that excessive fructose consumption contributes to hepatic lipid accumulation. Methods: Lipid accumulation in hepatocytes was induced by fructose treatment, while small interfering RNA (siRNA) transfection was used to suppress sterol regulatory element-binding protein-1c (SREBP-1c) expression. The size and the number of lipid droplets (LDs), as well as nuclear translocation of NRF2, were evaluated using microscopic analysis. Protein and mRNA expression levels were assessed by western blotting and qRT-PCR, respectively. Oxidative stress was analyzed using flow cytometry. Results: Fructose administration significantly increased LD accumulation and oxidative stress, accompanied by NRF2 activation and SREBP-1c expression. Silencing of SREBP-1c in fructose-treated hepatocytes markedly reduced LD accumulation and attenuated activation of the NRF2 signaling pathway. In contrast, inhibition of heme oxygenase-1 (HO-1) partially reversed these effects. Conclusion: The NRF2/HO-1 axis is involved in cellular adaptive response to fructose-induced oxidative stress and excessive LD accumulation. Significance statement: Elucidating the involvement of the NRF2/HO-1 axis between fructose metabolism and LD accumulation may provide valuable insights for the development of therapeutic strategies targeting fatty liver disorders.

Metabolic dysfunction‐associated fatty liver disease (MAFLD), defined by fat accumulation in more than 5% of hepatocytes, is a common metabolic syndrome worldwide. However, 30%–40% of MAFLD cases progress to metabolic dysfunction‐associated steatohepatitis (MASH), increasing the importance of the disease. MicroRNAs (miRNAs), non‐coding RNA molecules approximately 21 nucleotides long, are used as biomarkers in many diseases and play a crucial role in regulating cellular processes by affecting gene expression. It is also known that miRNAs are effective in the progression of MASH and its profile depends on the stage of the disease. Therefore, we determined the relationship between MASH and miRNA profiles in an in vivo trial using an established model of cholesterol‐induced MASH in rabbits. We also evaluated the impact of α‐tocopherol, which is known to have a protective effect in MAFLD/MASH transition, on miRNA profiles. Regarding the limited information using rabbits, we first performed miRNA screening and identified miRNAs that are already described in rabbits or other organisms as well as the putative ones. Among those, two putative miRNAs (miR‐230 and miR‐1146) determined by sequencing may be important in the diagnosis and treatment of the disease. Furthermore, levels of five miRNAs (miR‐122‐5p, miR‐199‐5p, miR‐145‐5p, miR‐27b‐3p, miR‐34a‐5p) and their relevance in the pathogenesis of MASH were determined by RT‐PCR and target gene prediction, respectively. In conclusion, the present study provides novel information regarding dysregulated miRNAs in high‐cholesterol diet‐induced MASH and the impact of α‐tocopherol.

Phagocytic engulfment of apoptotic cells, particularly neutrophils by macrophages, known as efferocytosis, is crucial in preventing secondary necrosis and promoting tissue repair. 17‐Oxo‐DHA, an electrophilic metabolite of docosahexaenoic acid (DHA), is generated in macrophages and has been reported to contribute to inflammation resolution by enhancing efferocytosis. However, many gaps remain in our understanding of the pro‐resolving effects of 17‐oxo‐DHA. Our results reveal that 17‐oxo‐DHA augments the efferocytic activity of bone marrow‐derived macrophages (BMDMs) by stimulating the biosynthesis of resolvin D2 (RvD2), one of the prototypic pro‐resolving mediators (SPMs), while reducing the expressions of IL‐6 and TNF‐α. Mechanistically, either gene silencing of Nrf2 or pharmacological inhibition of its target protein HO‐1 suppresses 17‐oxo‐DHA‐induced efferocytosis, decreasing the levels of 15‐LOX, COX‐2, and various SPMs. Notably, treatment of macrophages with SPMs was able to restore 17‐oxo‐DHA‐induced efferocytosis even when HO‐1 activity was suppressed. Thus, our study suggests critical roles of SPMs and the Nrf2/HO‐1 axis in mediating 17‐oxo‐DHA‐induced efferocytosis, which are novel candidate therapeutic targets in non‐resolving inflammatory diseases.

Objective: Sterol regulatory element binding protein 1c (SREBP-1c), a transcription factor involved in the biosynthesis of fatty acids, is critical in metabolic dysfunction-associated fatty liver disease (MAFLD) by promoting lipid accumulation and metabolic dysregulation that leads to hepatic pathologies. Fructose, becoming increasingly common in diets, activates SREBP-1c by increasing acetyl-CoA production. Present study aimed to sought the effect of SREBP-1c in fructose induced lipid accumulation. Methods: A fructose-induced lipid accumulation model was developed in mouse hepatocyte cells (AML12), where SREBP-1c expression was inhibited through siRNA transfection. Following different fructose concentrations, viability was determined by MTT assay, and the protein expression of SREBP-1c protein was determined by western blotting. The number of lipid droplets (LDs) was quantified microscopically, and lipogenic mRNA expressions of FASN, SCD1, GPAM, ACLY, ACSL1 and ACACA were detected by qRT-PCR. Results: Western blotting and microscopic analysis indicated that 25 mM for 72 hours of fructose increased total LDs, together with SREBP-1c levels, without affecting cell viability. The mRNA expression of SREBP-1c decreased in the presence of siRNA, confirming siRNA efficacy. SREBP-1c silencing reduced the number of fructose-induced total LDs. As lipogenic mRNA expressions, SREBP-1c silencing reduced SCD1 and ACLY, while other genes were unaffected. Conclusion: Silencing of SREBP-1c in hepatocytes demonstrated its beneficial effect by reducing fructose-induced LD accumulation.

Naturally occurring vitamin E is a lipophilic plant-derived molecule corresponding to the 2R forms of alpha-tocopherol. A series of natural analogs or tocochromanols are present in nature, including β-, γ- and δ-tocopherol (βT, γT, δT), the corresponding tocotrienols (αTE, βTE, γTE, δTE) and tocomonoenols. Differences between these analogs as lipophilic antioxidants and modulators of molecular processes suggest specific therapeutic properties against various disorders associated with acute and chronic inflammation. However, hepatic metabolism of these compounds via cytochrome P450-initiated side chain ω-oxidation involves the production of long-chain metabolites (LCMs) followed by intermediate (ICMs) and short-chain metabolites (SCMs), respectively. Despite the initial studies indicating these metabolites as catabolic-end products, recent findings identify their importance in providing biological functions. In this scope, LCMs, especially 13'-carboxychromanols (13'-COOHs), have been reported to hold stronger anti-inflammatory capacity than their unmetabolized precursors due to their ability to inhibit 5-lipoxygenase and cyclooxygenase-catalyzed eicosanoid formation, as well as their modulation of the pro-inflammatory transcriptional protein nuclear factor κB (NF-κB). Also, these LCMs have been reported to enhance detoxification and lipid metabolism pathways associated with cellular inflammation by modulating the nuclear receptors peroxisome proliferator-activated receptor-γ (PPARγ) and pregnane x receptor (PXR). These properties of LCMs will be described in this narrative review article focusing on recent information regarding their bioavailability, anti-inflammatory effects, and mechanisms of action in acute and chronic inflammatory disorders.

Despite limited number of studies, oxysterols are known to contribute to the progression of nonalcoholic steatohepatitis (NASH) by affecting lipid/cholesterol metabolism and elevating proinflammatory and profibrotic processes. Accordingly, we used a high cholesterol-mediated in vivo NASH model and aimed to determine alterations in fatty acid content and oxysterol levels together with their effects on cholesterol/lipid metabolism during the progression of the disease. We further investigated the beneficial role of alpha-tocopherol. To this end, in our hypercholesterolemic rabbit model, we determined fatty acid profile by GC-MS while 25-, 27-, 4 beta-, 7 alpha, and 24(S)-Hydroxycholesterol levels by means of LC-MS/MS. Additionally, lipid (SREBP-1c, PPAR alpha, PPAR gamma) and cholesterol metabolism-related proteins (LXR alpha, SREBP2 and ABCA1) were determined by immunoblotting. In conclusion, the present findings provide a complete analysis of the hepatic alterations in lipid and oxysterol profiles mediated by a high-cholesterol diet. In addition, this study explains the protective effect of alpha-tocopherol on lipogenesis and oxysterol production in hypercholesterolemia-induced NASH. We believe that present study will guide to novel theories in the progression and therapeutic targeting of fatty liver diseases.

Inflammation and apoptosis signaling are crucial steps in the progression from nonalcoholic fatty liver disease (NAFLD) to nonalcoholic steatohepatitis (NASH). Alpha‐tocopherol, the most active form of vitamin E, is an important modulator of signaling mechanisms, but its involvement to cholesterol‐induced NASH pathogenesis remains poorly defined. Herein we have reported a novel effect of α‐tocopherol in the transition from hepatic steatosis to NASH. High cholesterol diet alone (without α‐tocopherol) in rabbits elevated NASH development as indicated by increased inflammatory response, apoptotic activity and liver fibrosis. Such elevation results from induction of signaling mechanisms since the expressions of IL1β, phospho c‐Jun/c‐Jun ratio, JNK, caspase 9, CHOP and Bax were increased, and recruitment of macrophage, α‐smooth muscle actin (α‐SMA) and COL1A1 into the liver tissue were induced. Alpha‐tocopherol supplementation inhibited inflammatory response, apoptosis and fibrosis development without affecting lipid accumulation in high cholesterol‐induced NASH. Specifically, α‐tocopherol lowered the inflammatory level as observed by reduced macrophage infiltration and JNK/c‐Jun signaling. Lower inflammatory status co‐occurred with the reduction of CHOP and Bax expressions as well as fibrosis‐related COL1A1 and α‐SMA levels. Taken together, α‐tocopherol supplementation inhibits cholesterol‐induced NASH development by lowering JNK/c‐Jun/inflammation axis in addition to JNK/CHOP/apoptosis signaling, which might contribute to resistance against NAFLD/NASH transition.

Aging has been characterized with the accumulation of oxidized proteins, as a consequence of progressive decline in proteostasis capacity. Among others, proteasomal system is an efficient protein turnover complex to avoid aggregation of oxidized proteins. Heat shock protein 70 (HSP70) is another critical player that is involved in some key processes including the correct folding of misfolded proteins and targeting aggregated proteins to the proteasome for rapid degradation. The aim of this study was to determine the role of proteasomal system and heat shock proteins to maintain proteome balance during replicative senescence in mild hyperthermia conditions. Our results demonstrated that HSP40/70 machinery is induced by mild hyperthermia conditions independent from senescence conditions. Since HSP70 is largely responsible for the rapidly inducible cell protection following hyperthermia, the activation of “heat shock response” resulted in the elevation of HSP40/70 expressions as well as the proteasome activity. Interestingly, when HSP70 expression was inhibited, increased proteasomal activation was shown to be responsive to mild hyperthermia. Since HSP70 is involved in various stress-related pathways such as oxidative and endoplasmic reticulum stress, depletion of HSP70 expression may induce proteasomal degradation to maintain proteome balance of the cell. Thus, our data suggests that in mild heat stress conditions, molecular chaperone HSP70 plays an important role to avoid protein oxidation and aggregation; however, activities of proteasomal system are induced when HSP70 expression is depleted.