Indoleamine 2,3-dioxygenase 1-mediated iron metabolism in macrophages contributes to lipid deposition in nonalcoholic steatohepatitis.

BACKGROUND: Non-alcoholic steatohepatitis (NASH) is a rapidly progressing chronic liver disease of global significance. However, the underlying mechanisms responsible for NASH remain unknown. Indoleamine 2,3-dioxygenase 1 (IDO1) has been recognized as essential factor in immune response and metabolic regulation. Here we aimed to investigate the functions and mechanisms of the IDO1 in macrophages on hepatic lipid deposition and iron metabolism in NASH. METHODS: The effect of IDO1 in NASH was evaluated by WT and IDO1-/- mice model fed with methionine/choline-deficient (MCD) diet in vivo. Macrophages scavenger clodronate liposomes (CL) and overexpressing of IDO1 in macrophages by virus were employed as well. Lipid deposition was assessed through pathological examination and lipid droplet staining, while iron levels were measured using an iron assay kit and western blotting. Primary hepatocytes and bone marrow-derived macrophages were treated with oleic acid/palmitic acid (OA/PA) to assess IDO1 expression via Oil Red O staining and immunofluorescence staining in vitro. RESULTS: Pathological images demonstrated that the increase of IDO1 exacerbated lipid accumulation in the livers of mice with MCD diet, while reduction of iron accumulation was observed in the liver and the serum of MCD-fed mice. Scavenging of macrophages effectively mitigated both lipid and iron accumulation. In addition, the deficiency of IDO1 in macrophages significantly mitigated lipid accumulation and iron overload in hepatic parenchymal cells. Finally, lentivirus-mediated overexpression of IDO1 in liver macrophages exacerbated hepatic steatosis and iron deposition in NASH. CONCLUSIONS: Our results demonstrated that effective inhibition of IDO1 expression in macrophages in NASH alleviated hepatic parenchymal cell lipid accumulation and iron deposition, which provided new insights for the future treatment of NASH.

STING modulates iron metabolism to promote liver injury and inflammation in acute immune hepatitis.

The pathogenesis of Autoimmune Hepatitis (AIH) is closely associated with perturbations in iron ion metabolism, during which Stimulator of Interferon Genes (STING) plays an important role. However, the precise regulatory mechanism remains elusive. In this study, we investigated the relationship between iron dysregulation and STING activation in Concanavalin A (ConA)-induced AIH liver injury. STING knockout (STING-/-) mice and AAV (Adeno-Associated virus)-Sting1-RNAi-treated mice were involved and subjected in AIH. We observed that increased iron dysregulation was linked with STING activation, but this effect was effectively reversed by the administration of iron chelating agent Desferoxamine (DFO) and the antioxidant Ferrostatin-1 (Fer-1). Notably, the iron transport protein Transferrin (TF) and Transferrin Receptor (TfR) exhibited significant accumulation in AIH along with upregulated expression of ferritin protein. Additionally, the deficiency of STING reduced hepatic iron accumulation, mitigated oxidative stress, and attenuated macrophage activation during ConA treatment. Furthermore, liver-specific knockdown of STING using AAV-Sting1-RNAi significantly ameliorated liver iron dysregulation and oxidative stress response induced by Kupffer cells (KCs). KC-derived STING exacerbates liver damage severity in AIH through promoting disturbances in hepatic iron ion metabolism as well as oxidative stress response. These findings provide valuable insights into the pathogenesis of AIH and may pave the way for potential therapeutic strategies targeting STING and iron metabolism in the future.

Erchen decoction alleviates the progression of NAFLD by inhibiting lipid accumulation and iron overload through Caveolin-1 signaling.

ETHNOPHARMACOLOGICAL RELEVANCE: A combination of 6 different Chinese herbs known as Erchen decoction (ECD) has been traditionally used to treat digestive tract diseases and found to have a protective effect against nonalcoholic fatty liver disease (NAFLD). Despite its efficacy in treating NAFLD, the precise molecular mechanism by which Erchen Decoction regulated iron ion metabolism to prevent disease progression remained poorly understood. AIM OF STUDY: Our study attempted to confirm the specific mechanism of ECD in reducing lipid and iron in NAFLD from the perspective of regulating the expression of Caveolin-1 (Cav-1). STUDY DESIGN: In our study, the protective effect of ECD was investigated in Palmitic Acid + Oleic Acid-induced hepatocyte NAFLD model and high-fat diet-induced mice NAFLD model. To investigate the impact of Erchen Decoction (ECD) on lipid metabolism and iron metabolism via mediating Cav-1 in vitro, Cav-1 knockdown cell lines were established using lentivirus-mediated transfection techniques. MATERIALS AND METHODS: We constructed NAFLD model by feeding with high-fat diet for 12 weeks in vivo and Palmitic Acid + Oleic Acid treatment for 24 h in vitro. The regulation of Lipid and iron metabolism results by ECD were detected by serological diagnosis, immunofluorescent and immunohistochemical staining, and western blotting. The binding ability of 6 small molecules of ECD to Cav-1 was analyzed by molecular docking. RESULTS: We demonstrated that ECD alleviated the progression of NAFLD by inhibiting lipid accumulation, nitrogen oxygen stress, and iron accumulation in vivo and in vitro experiments. Furthermore, ECD inhibited lipid and iron accumulation in liver by up-regulating the expression of Cav-1, which indicated that Cav-1 was an important target for ECD to exert its curative effect. CONCLUSIONS: In summary, our study demonstrated that ECD alleviated the accumulation of lipid and iron in NAFLD through promoting the expression of Cav-1, and ECD might serve as a novel Cav-1 agonist to treat NAFLD.

Caveolin-1 is critical for hepatic iron storage capacity in the development of nonalcoholic fatty liver disease.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is associated with disordered lipid and iron metabolism. Our previous study has substantiated the pivotal role of Caveolin-1 (Cav-1) in protecting hepatocytes and mediating iron metabolism in the liver. This study aimed to explore the specific mechanisms underlying the regulation of iron metabolism by Cav-1 in NAFLD. METHODS: Hepatocyte-specific Cav-1 overexpression mice and knockout mice were used in this study. Cav-1-knockdown of RAW264.7 cells and mouse primary hepatocytes were performed to verify the changes in vitro. Moreover, a high-fat diet and palmitic acid plus oleic acid treatment were utilized to construct a NAFLD model in vivo and in vitro, respectively, while a high-iron diet was used to construct an in vivo iron overload model. Besides, iron concentration, the expression of Cav-1 and iron metabolism-related proteins in liver tissue or serum were detected using iron assay kit, Prussian blue staining, Western blotting, immunofluorescence staining, immunohistochemical staining and ELISA. The related indicators of lipid metabolism and oxidative stress were evaluated by the corresponding reagent kit and staining. RESULTS: Significant disorder of lipid and iron metabolism occurred in NAFLD. The expression of Cav-1 was decreased in NAFLD hepatocytes (P < 0.05), accompanied by iron metabolism disorder. Cav-1 enhanced the iron storage capacity of hepatocytes by activating the ferritin light chain/ferritin heavy chain pathway in NAFLD, subsequently alleviating the oxidative stress induced by excess ferrous ions in the liver. Further, CD68+CD163+ macrophages expressing Cav-1 were found to accelerate iron accumulation in the liver, which was contrary to the effect of Cav-1 in hepatocytes. Positive correlations were also observed between the serum Cav-1 concentration and the serum iron-related protein levels in NAFLD patients and healthy volunteers (P < 0.05). CONCLUSIONS: These findings confirm that Cav-1 is an essential target protein that regulates iron and lipid metabolic homeostasis. It is a pivotal molecule for predicting and protecting against the development of NAFLD.

Naringin ameliorates liver fibrosis in zebrafish by modulating IDO1-mediated lipid metabolism and inflammatory infiltration.

Liver fibrosis (LF) is an important reparative process in response to acute or chronic hepatic injury, which has the potential to advance towards cirrhosis and hepatocellular carcinoma. Dietary naringin consumption contributes to protection against LF in animal studies, while the exact protective mechanism of naringin remains unclear. This study aimed to investigate the molecular mechanisms behind the potential protective effect of naringin against TAA-induced LF in zebrafish. In this study, we utilized zebrafish to create the LF model and investigate the therapeutic mechanism of naringin. Firstly, we evaluated the changes in hepatic fibrosis and lipid accumulation in the liver following naringin treatment with oil red O, Nile red, and Sirius red and immunohistochemistry. In addition, we employed an ROS probe to directly measure oxidative stress and monitor inflammatory cell migration in a zebrafish transgenic line. Morpholino was used in the knockdown of IDO1 in order to verify its vital role in LF. Our findings demonstrated that naringin exhibited anti-inflammatory and anti-fibrotic action in conjunction with a reversal in lipid accumulation, oxidative stress and suppression of macrophage infiltration and activation of hepatic stellate cells. Furthermore, the results showed that the antifibrotic effect of naringin was removed upon IDO1 knockdown, proving that naringin exerts a protective effect by regulating IDO1. Naringin demonstrates remarkable protective effects against LF, effectively counteracting inflammation and hepatic steatosis in zebrafish liver. These findings suggest that naringin may function as an effective IDO1 inhibitor, holding the potential for clinical translation as a therapeutic agent for the treatment of LF.

Ginsenosides Rg1 regulate lipid metabolism and temperature adaptation in Caenorhabditis elegans.

Background: Obesity is a risk factor for aging and many diseases, and the disorder of lipid metabolism makes it prominent. This study aims to investigate the effect of ginsenoside Rg1 on aging, lipid metabolism and stress resistance. Methods: Rg1 was administered to Caenorhabditis elegans (C. elegans) cultured in NGM or GNGM. The lifespan, locomotory activity, lipid accumulation, cold and heat stress resistance and related mRNA expression of the worms were examined. Gene knockout mutants were used to clarify the effect on lipid metabolism of Rg1. GFP-binding mutants were used to observe the changes in protein expression. Results: We reported that Rg1 reduced lipid accumulation and improved stress resistance in C. elegans. Rg1 significantly reduced the expression of fatty acid synthesis-related genes and lipid metabolism-related genes in C. elegans. However, Rg1 did not affect the fat storage in fat-5/fat-6 double mutant or nhr-49 mutant. Combined with network pharmacology, we clarified the possible pathways and targets of Rg1 in lipid metabolism. In addition, Rg1-treated C. elegans showed a higher expression of anti-oxidative genes and heat shock proteins, which might contribute to stress resistance. Conclusion: Rg1 reduced fat accumulation by regulating lipid metabolism via nhr-49 and enhanced stress resistance by its antioxidant effect in C. elegans.

Exocarpium Citri Grandis alleviates the aggravation of NAFLD by mitigating lipid accumulation and iron metabolism disorders.

ETHNOPHARMACOLOGICAL RELEVANCE: Exocarpium Citri grandis (ECG, Huajuhong in Chinese), the epicarp of C. grandis 'Tomentosa', has been used for hundreds of years as an anti-inflammatory, expectorant, hypoglycemic, and lipid-lowering medication in China. Nevertheless, there have been few papers that have explored the mechanism behind ECG's hypolipidemic characteristics from the perspective of treating nonalcoholic fatty liver disease (NAFLD). AIM OF STUDY: The purpose of our study was to confirm the therapeutic and preventative effects of ECG in NAFLD by regulating lipid accumulation and iron metabolism, and to explore the specific mechanism of ECG in enhancing hepatic iron transport and excretion capabilities. STUDY DESIGN: We constructed a NAFLD model by feeding male C57BL/6 J mice with a high-fat diet for 12 weeks. Mice were gavaged with ECG beginning in the seventh week of modeling, and three dosage gradients were established: low dose group (2.5 g/kg/d), medium dose group (5 g/kg/d) y, and high dose group (10 g/kg/d) until the end of model construction in week 12. MATERIALS AND METHODS: We used network pharmacology to analyze the relationship between ECG and NAFLD. In addition, we constructed a nonalcoholic fatty liver disease model by feeding male C57BL/6 J mice a high-fat diet for 12 weeks. Finally, lipid accumulation, iron accumulation, inflammation and oxidative stress were evaluated by serological index detection, histological detection, immunofluorescent and immunohistochemical staining, and western blotting. RESULTS: Network pharmacology confirmed the treatment effect of ECG in NAFLD. Three active components of ECG, including Naringenin, Naringin and Neohesperidin, were detected by UHPLC-HRMS analysis. The results of serum TC, TG, LDL concentration, HE staining, Oil red staining and Nile red staining demonstrated that ECG could improve lipid metabolism disorders. The results of serum iron concentration, liver tissue iron concentration, iron metabolism-related proteins Ferritin light chain, Ferroportin1, Transferrin receptor, and Transferrin demonstrated that ECG improved the iron transport and storage capacities of hepatic cells. CONCLUSIONS: Our results demonstrated that ECG relieved liver injury by inhibiting lipid accumulation and iron accumulation in NAFLD.

RAGE promotes dysregulation of iron and lipid metabolism in alcoholic liver disease.

Alcoholic liver disease (ALD) is associated with hepatic inflammatory activation and iron overload. The receptor for advanced glycation end products (RAGE) is an important metabolic mediator during the development of ALD. The aim of this study was to determine the effect of RAGE on iron homeostasis in ALD. We found increased circulating transferrin, hepcidin and ferritin in ALD patients and positively correlated with RAGE level. RAGE knockout (RAGE-/-) and wild-type mice were subjected to chronic alcoholic feeding for 6 weeks to induce ALD, and RAGE inhibitor, iron chelator or lipid peroxidation inhibitor were administered. We showed that chronic alcohol administration triggered hepatic steatosis, inflammation, and oxidative stress, which were eliminated by deficiency or inhibition of RAGE. Surprisingly, pathways of hepatic iron metabolism were significantly altered, including increased iron uptake (Tf/TfR) and storage (Ferritin), as well as decreased iron export (FPN1/Hepcidin). In vitro experiments confirmed that RAGE had different effects on the mechanism of iron metabolism of hepatocytes and macrophages respectively. In conclusion, our data revealed preclinical evidence for RAGE inhibition as an effective intervention for alleviating alcohol-induced liver injury.

Poria cocos polysaccharides rescue pyroptosis-driven gut vascular barrier disruption in order to alleviates non-alcoholic steatohepatitis.

ETHNOPHARMACOLOGICAL RELEVANCE: Poria cocos polysaccharides (PCP) are abundant in Poria cocos (Schw.) Wolf (Poria). This is a common traditional Chinese medicine used to treat gastrointestinal and liver diseases. Poria cocos dispel dampness and enhance gastrointestinal functions, strongly affecting the treatment of non-alcoholic fatty liver disease. Still, the mechanism is not yet clear. AIM OF THE STUDY: The latest research found that protecting the integrity of the intestinal barrier can slow down the progression of non-alcoholic fatty liver disease (NAFLD). Hence, our research ought to explore the protective mechanism of PCP on the intestinal barrier under a high-fat diet and to clarify the relationship between intestinal barrier damage and steatohepatitis. MATERIALS AND METHODS: H&E staining was done to evaluate pathological damage, whereas Nile red and oil red O staining was conducted to evaluate hepatic fat infiltration. Immunofluorescence staining and immunohistochemical staining were used to detect protein expression and locations. Bone marrow-derived macrophages were isolated for in vitro experiments. ONOO- and ROS fluorescent probes and MDA, SOD, and GSH kits assessed the levels of nitrogen and oxidative stress. LPS levels were detected with a Limulus Amebocyte Lysate assay. The Western blot analysis and reverse transcription-quantitative PCR detected the expression of related proteins and genes. The Elisa kit detected the level of the inflammatory factors in the cell supernatant. For the vivo NAFLD experiments, in briefly, mice were randomly chosen to receive either a High-fat diet or control diet for 12 weeks. Drug treatments started after 4 weeks of feeding. Zebrafish larvae were raised separately in fish water or 7 mM thioacetamide as the control or model group for approximately 72 h. In the therapy groups, different concentrations of PCP were added to the culture environment at the same time. RESULTS: In zebrafish, we determined the safe concentration of PCP and found that PCP could effectively reduce the pathological damage in the liver and intestines induced by the NAFLD model. In mice, PCP could slow down weight gain, hyperlipidemia, and liver steatosis caused by a high-fat diet. More importantly, PCP could reduce the destruction of the gut-vascular barrier and the translocation of endotoxins caused by a high-fat diet. Further, we found that PCP could inhibit intestinal pyroptosis by regulating PARP-1. Pyroptosis inhibitors, such as MCC950, could effectively protect the intestinal and liver damage induced by a high-fat diet. We also found that pyroptosis mainly occurred in intestinal macrophages. PCP could effectively improve the survival rate of bone marrow-derived macrophages in a high-fat environment and inhibit pyroptosis. CONCLUSIONS: These results indicated that PCP inhibited the pyroptosis of small intestinal macrophages to protect the intestinal barrier integrity under a high-fat diet. This resulted in decreased endotoxin translocation and progression of steatohepatitis.