AML12 Cells Research Articles

O-GlcNAcylation regulates long-chain fatty acid metabolism by inhibiting ACOX1 ubiquitination-dependent degradation

BackgroundCold as a common environmental stress, causes increased heat production, accelerated metabolism and even affects its production performance. How to improve the adaptability of the animal organism to cold has been an urgent problem. As a key hub of lipid metabolism, the liver can regulate lipid metabolism to maintain energy balance, and O-GlcNAcylation is a kind of important PTMs, which participates in a variety of signaling and mechanism regulation, and at the same time, is very sensitive to changes in stress and nutritional levels, and is the body's “stress receptors” and “nutrient receptors”. Therefore, the aim of this experiment was to investigate the effect of cold-induced O-GlcNAcylation on hepatic lipid metabolism, and to explore the potential connection between O-GlcNAcylation and hepatic lipid metabolism. MethodsTo investigate the loss of O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) and the precise impacts of additional cold-induced circumstances on liver mass, shape, and metabolic profile, C57 mice were used as an animal model. Using the protein interactions approach, the mechanism of O-GlcNAcylation, as well as the degradation pathway of acyl-Coenzyme A oxidase 1 (ACOX1), were clarified. Additional in vitro analyses of oleic acid (OA) and OGT inhibitor tetraoxan (Alloxan) (Sigma, 2244-11-3) on lipid breakdown in AML-12 cells. ResultsIn C57BL/6 mice, deletion of O-GlcNAcylation disrupted lipid metabolism, caused hepatic edema and fibrosis, and altered mitochondrial apoptosis. This group of modifications was made worse by cold induction. The accumulation of medium- and long-chain fatty acids is a hallmark of lipolysis, which is accelerated by the deletion of O-GlcNAcylation, whereas lipid synthesis is slowed down. The association between ACOX1 and OGT at the K48 gene precludes ubiquitinated degradation.

Astragaloside IV ameliorates cisplatin-induced liver injury by modulating ferroptosis-dependent pathways

Ethnopharmacological relevanceThe use of antineoplastic drugs, such as cisplatin, in clinical practice can cause adverse effects in patients, such as liver injury, which limits their long-term use. Therefore, there is an urgent need to develop alternative therapeutic strategies or drugs to minimize cisplatin-induced liver injury. Huangqi, the root of Astragalus membranaceus, is extensively used in traditional Chinese medicine (TCM) and has been employed in treating diverse liver injuries. Astragalus membranaceus contains several bioactive constituents, including triterpenoid saponins, one of which, astragaloside IV (ASIV), has been reported to have anti-inflammatory and antioxidant stress properties. However, its potential in ameliorating cisplatin-induced liver injury has not been explored. Aim of the studyThe objective of this study was to examine the mechanism by which ASIV protects against cisplatin-induced liver injury. Materials and methodsThis study established a model of cisplatin-induced liver injury in mice, followed by treatment with various doses of astragaloside IV (40 mg/kg, 80 mg/kg). In addition, a model of hepatocyte ferroptosis in AML-12 cells was established using RSL3. The mechanism of action of astragaloside IV was investigated using a range of methods, including Western blot assay, qPCR, immunofluorescence, histochemistry, molecular docking, and high-content imaging system. ResultsThe findings suggested a significant improvement in hepatic injury, inflammation and oxidative stress phenotypes with the administration of ASIV. Furthermore, network pharmacological analyses provided evidence that a major pathway for ASIV to attenuate cisplatin-induced hepatic injury entailed the cell death cascade pathway. It was observed that ASIV effectively inhibited ferroptosis both in vivo and in vitro. Subsequent experimental outcomes provided further validation of ASIV's ability to hinder ferroptosis through the inhibition of PPARα/FSP1 signaling pathway. The current findings suggest that ASIV could function as a promising phytotherapy composition to alleviate cisplatin-induced liver injury. ConclusionsThe current findings suggest that astragaloside IV could function as a promising phytotherapy composition to alleviate cisplatin-induced liver injury.

Abstract 7443: Investigating the impact of ferroptosis in a high-fat and high-oxidative stress hepatic steatosis environment

Abstract An iron-dependent mechanism of cell death, ferroptosis, occurs with intracellular iron accumulation and is associated with lipid peroxidation. Previous studies have shown that lipid peroxidation-induced ROS leads to DNA damage and promotes the progression of steatosis to advanced HCC. Therefore, we aim to study ferroptosis in a cell model with hepatocytes co-cultured with ACM (adipocyte culture medium) and low dose H2O2 for a high-fat/high ROS environment in vitro; an animal model with high-fat content and accompanying low dose carbon tetrachloride in vivo to observe the possibility of hepatocyte steatosis due to ferroptosis. Animal model: Mice were fed a Western diet and provided with high fructose/glucose-containing sugar drinking water (WFSD) for 7 months while receiving a monthly extra-low dose of CCl4. Tissue staining: Liver sections were performed to observe histological abnormalities in each group. Cell model: Pre-adipocytes, 3T3L1 were differentiated by adding IBMX, Dexamethasone, and Insulin (10 μg/ml). After differentiation, ACM was collected. The XTT assay was used to examine the relationship between cell death and ferroptosis by a ferroptosis inducer (Erastin) in high-fat and high-ROS environments.qPCR: To detect the expression of iron transport-related genes.Western Blot: To ascertain protein levels associated with ferroptosis. Mitochondrial assay: Mitochondrial membrane potential (MMP), mitochondrial dynamics-related proteins, and fluorescence mitochondrial tracking and staining. Ferritin, GPX4, IRP2, and ferroportin were upregulated when ACM co-culture with the hepatocytic AML12 cell line, and it also increased the sensitivity of ROS-related cell cytotoxicity, which was related to ferroptosis by the inducer, Erastin, in the high-fat/high-ROS cell environment. However, these results can be suppressed by UAMC3203, a ferroptosis inhibitor. As for the animal model with high-fat and carbon tetrachloride accompanied experiments, the mice had higher weight, Alanine Aminotransferase (ALT) levels, and developed tumors. Their liver sections showed lipid accumulation, histological fibrosis, and iron accumulation in both the WD and WD+CCl4 groups, which corresponds to the IRP1 and IRP2 upregulated in both groups by qPCR result. Our research findings demonstrate that ferroptosis correlates with high-fat and high-ROS-induced hepatocellular damage. The related mitochondria dynamics changes in liver tissue and high-fat and/or high ROS provide evidence for ferroptosis-related hepatic steatosis. We have established the effect of ferroptosis on hepatic steatosis in vivo and in vitro in nonalcoholic steatosis hepatitis (NASH). These results may enable the development of precise health prevention strategies by inhibiting ferroptosis to prevent the occurrence of liver diseases including hepatic steatosis and HCC. Citation Format: Chi-Sheng (Wayne) Chen, Shu-Chi Wang. Investigating the impact of ferroptosis in a high-fat and high-oxidative stress hepatic steatosis environment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7443.

Identification and validation of cuproptosis-related genes for diagnosis and therapy in nonalcoholic fatty liver disease.

In recent years, nonalcoholic fatty liver disease (NAFLD) has become a more serious public health issue worldwide. This study strived to investigate the molecular mechanism of pathogenesis of NAFLD and explore promising diagnostic and therapeutic targets for NAFLD. Raw data from GSE130970 were downloaded from the Gene Expression Omnibus database. We used the dataset to analyze the expression levels of cuproptosis-related genes in NAFLD patients and healthy controls to identify the differentially expressed cuproptosis-related genes (DECRGs). The relationship and potential mechanism between DECRGs and clinicopathological factors were examined by enrichment analysis and two consensus clustering methods. We screened key DECRGs based on Random Forest (RF), and then verified the key DECRGs in NAFLD patients, high-fat diet (HFD)-fed mice, and palmitic acid-induced AML12 cells. ROC analysis showed good diagnostic function of DECRGs in normal and NAFLD liver tissue. Two consensus clusters indicated the important role of cuproptosis in the development of NAFLD. We screened for key DECRGs (DLD, DLAT) based on RF and found a close relationship between the DECRGs and clinicopathological factors. We collected clinical blood samples to verify the differences in gene expression levels by qPCR. In addition, we further verified the expression levels of DLD and DLAT in HFD mice and AML12 cells, which showed the same results. This study provides a novel perspective on the pathogenesis of NAFLD. We identified two cuproptosis-related genes that are closely related to NAFLD. These genes may play a significant role in the molecular pathogenesis of NAFLD, which may be useful to make progress in the diagnosis and treatment of NAFLD.

Oleanolic acid promotes liver regeneration after partial hepatectomy via regulating pregnane X receptor signaling pathway in mice

Liver regeneration after liver tumor resection or liver transplantation is crucial, the remaining liver frequently fails to regenerate in some patients. Oleanolic acid (OA), a pentacyclic triterpenoid compound which has been shown to protect against various liver diseases. However, the effect of OA on liver regeneration after partial hepatectomy (PHx) is still unclear. In this study, the results showed that OA (50 mg/kg, twice daily) treatment induced liver mass restoration and increased the liver-to-body weight ratio of mice following PHx. Meanwhile, OA promoted hepatocyte proliferation and increased the number of BrdU-, Ki67-and PCNA-positive cells. Furthermore, OA increased the nuclear accumulation of PXR and induced the expression of PXR downstream proteins such as CYP3A11, UGT1A1 and GSTM2 in mice, as well as in AML12 and HepRG cells. Luciferase reporter assay and nuclear localization of PXR further demonstrated the effect of OA on PXR activation in vitro. Molecular docking simulation showed that OA could interact with the PXR active sites. Moreover, OA inhibited the expression of FOXO1, RBL2 and CDKN1B, and increased the expression of PCNA, CCND1 and CCNE1 in vivo and in vitro. Silencing of Pxr further confirmed that OA-mediated upregulation of proliferation-related proteins depended on PXR. The current study illustrated that OA exhibited a significant promoting effect on liver regeneration following PHx, potentially through regulation of the PXR signaling pathway to accelerate liver recovery.

Ethanol-Induced Hepatic Ferroptosis Is Mediated by PERK-Dependent MAMs Formation: Preventive Role of Quercetin.

Iron deposition is frequently observed in alcoholic liver disease (ALD), which indicates a potential role of ferroptosis in its development. This study aims to explore the effects of quercetin on ferroptosis in ALD and elucidates the underlying mechanism involving the formation of mitochondria-associated endoplasmic reticulum membranes (MAMs) mediated by protein kinase RNA-like endoplasmic reticulum kinase (PERK). C57BL/6J mice are fed either a regular or an ethanol-containing liquid diet (with 28% energy form ethanol) with or without quercetin supplementation (100mgkg-1 BW) for 12 weeks. Ethanol feeding or treatment induced ferroptosis in mice and AML12 cells, which is associated with increased MAMs formation and PERK expression within MAMs. Quercetin attenuates these changes and protects against ethanol-induced liver injury. The antiferroptotic effect of quercetin is abolished by ferroptosis inducers, but mimicked by ferroptosis inhibitors and PERK knockdown. The study demonstrates that PERK structure, rather than its kinase activity (transfected with the K618A site mutation that inhibits kinase activity-ΔK plasmid or protein C terminal knockout-ΔC plasmid of PERK), mediates the enhanced MAMs formation and ferroptosis during the ethanol exposure. Quercetin ameliorates ethanol-induced liver injury by inhibiting ferroptosis via modulating PERK-dependent MAMs formation.

Ferritinophagy Is Critical for Deoxynivalenol-Induced Liver Injury in Mice.

Background: Deoxynivalenol (DON) contamination, pervasive throughout all stages of food production and processing, presents a significant threat to human health. The degradation of ferritin mediated by nuclear receptor coactivator 4 (NCOA4), termed ferritinophagy, plays a crucial role in maintaining iron homeostasis and regulating ferroptosis. Aim: This study aims to elucidate the role of ferritinophagy and ferroptosis in DON-induced liver injury. Methods: Male mice and AML12 cells were subjected to varying doses of DON, serving as in vivo and in vitro models, respectively. Protein expression was assessed by using immunofluorescence and western blot techniques. Co-immunoprecipitation was employed to investigate the protein-protein interactions. Results: Our findings demonstrate that DON triggers hepatocyte ferroptosis in a ferritinophagy-dependent manner. Specifically, DON impedes the activation of the mammalian target of rapamycin complex 1 (mTORC1) by inhibiting RAC1's binding to mTOR, thereby ultimately inducing autophagy. Concurrently, DON amplifies NCOA4's affinity for ferritin by facilitating NCOA4 phosphorylation through the ataxia-telangiectasia mutated kinase (ATM), thus promoting the autophagy-dependent degradation of ferritin. Both autophagy inhibition and NCOA4 expression suppression ameliorate DON-induced ferroptosis. Conclusion: Our study concludes that DON facilitates NCOA4-mediated ferritinophagy via the ATM-NCOA4 pathway, subsequently inducing ferroptosis in the liver.

A novel HDAC6 inhibitor attenuate APAP-induced liver injury by regulating MDH1-mediated oxidative stress

Glutathione (GSH) depletion, mitochondrial damage, and oxidative stress have been implicated in the pathogenesis of acetaminophen (APAP) hepatotoxicity. Here, we demonstrated that the expression of histone deacetylase 6 (HDAC6) is highly elevated, whereas malate dehydrogenase 1 (MDH1) is downregulated in liver tissues and AML-12 cells induced by APAP. The therapeutic benefits of LT-630, a novel HDAC6 inhibitor on APAP-induced liver injury, were also substantiated. On this basis, we demonstrated that LT-630 improved the protein expression and acetylation level of MDH1. Furthermore, after overexpression of MDH1, an upregulated NADPH/NADP+ ratio and GSH level and decreased cell apoptosis were observed in APAP-stimulated AML-12 cells. Importantly, MDH1 siRNA clearly reversed the protection of LT-630 on APAP-stimulated AML-12 cells. In conclusion, LT-630 could ameliorate liver injury by modulating MDH1-mediated oxidative stress induced by APAP.

Dendrobium officinale regulate lipid metabolism in diabetic mouse liver via PPAR-RXR signaling pathway: Evidence from an integrated multi-omics analysis

Dendrobium officinale (DEN) is recognized as a kind of functional food that can effectively ameliorate endocrine and metabolic disruptions. This study delved into the pharmacological mechanism of DEN on hepatic lipotoxicity associated with Type II diabetes mellitus (T2DM). In vivo study experiments on db/db mice indicated that DEN treatment notably enhanced liver function, decreased blood lipid levels, and improved insulin sensitivity. Non-targeted metabolomics analysis revealed that DEN significantly ameliorated metabolism pathways, including lipoic acid, linoleic acid, bile secretion, and the alanine/aspartate/glutamate metabolism, as well as taurine and hypotaurine metabolism. Transcriptomics analysis demonstrated DEN treatment could modulate the expression of genes such as Cpt1b, Scd1, G6pc2, Fos, Adrb2, Atp2a1, Ppp1r1b, and Cyp7a1. Furthermore, Proteomics analysis indicated that the beneficial effect of DEN on lipid metabolism was linked to pathways like AMPK and PPAR signaling. The integrative analysis of multi-omics revealed that the PPAR-RXR signaling was critical to the therapeutic effect of DEN on T2DM-induced fatty liver. Additionally, in vitro study on AML-12 cells confirmed that DEN counteract PA-induced lipid accumulation by activating the PPAR-RXR pathway. Overall, these findings suggested that DEN exhibited the potential to mitigate T2DM-induced hepatic lipo-toxicity and manage lipid imbalances in T2DM.

Regeneration of Non-Alcoholic Fatty Liver Cells Using Chimeric FGF21/HGFR: A Novel Therapeutic Approach.

Non-alcoholic fatty liver disease (NAFLD) has emerged as a significant liver ailment attributed to factors like obesity and diabetes. While ongoing research explores treatments for NAFLD, further investigation is imperative to address this escalating health concern. NAFLD manifests as hepatic steatosis, precipitating insulin resistance and metabolic syndrome. This study aims to validate the regenerative potential of chimeric fibroblast growth factor 21 (FGF21) and Hepatocyte Growth Factor Receptor (HGFR) in NAFLD-afflicted liver cells. AML12, a murine hepatocyte cell line, was utilized to gauge the regenerative effects of chimeric FGF21/HGFR expression. Polysaccharide accumulation was affirmed through Periodic acid-Schiff (PAS) staining, while LDL uptake was microscopically observed with labeled LDL. The expression of FGF21/HGFR and NAFLD markers was analyzed by mRNA analysis with RT-PCR, which showed a decreased expression in acetyl-CoA carboxylase 1 (ACC1) and sterol regulatory element binding protein (SREBP) cleavage-activating protein (SCAP) with increased expression of hepatocellular growth factor (HGF), hepatocellular nuclear factor 4 alpha (HNF4A), and albumin (ALB). These findings affirm the hepato-regenerative properties of chimeric FGF21/HGFR within AML12 cells, opening novel avenues for therapeutic exploration in NAFLD.

Baicalin and N-acetylcysteine regulate choline metabolism via TFAM to attenuate cadmium-induced liver fibrosis

(Background): Cadmium is an environmental pollutant associated with several liver diseases. Baicalin and N-Acetylcysteine have antioxidant and hepatoprotective effects. (Purpose): However, it is unclear whether baicalin and N-Acetylcysteine can alleviate Cadmium -induced liver fibrosis by regulating metabolism, or whether they exert a synergistic effect. (Study design): We treated Cadmium-poisoned mice with baicalin, N-Acetylcysteine, or baicalin+ N-Acetylcysteine. We studied the effects of baicalin and N-Acetylcysteine on Cadmium-induced liver fibers and their specific mechanisms. (Methods): We used C57BL/6J mice, and AML12, and HSC-6T cells to establish in vitro assays and in vivo models. (Results): Metabolomics was used to detect the effect of baicalin and N-Acetylcysteine on liver metabolism, which showed that compared with the control group, the Cadmium group had increased fatty acid and amino acid levels, with significantly reduced choline and acetylcholine contents. Baicalin and N-Acetylcysteine alleviated these Cadmium-induced metabolic changes. We further showed that choline alleviated Cadmium -induced liver inflammation and fibrosis. In addition, cadmium significantly promoted extracellular leakage of lactic acid, while choline alleviated the cadmium -induced destruction of the cell membrane structure and lactic acid leakage. Western blotting showed that cadmium significantly reduced mitochondrial transcription factor A (TFAM) and Choline Kinase α(CHKα2) levels, and baicalin and N-Acetylcysteine reversed this effect. Overexpression of Tfam in mouse liver and AML12 cells increased the expression of CHKα2 and the choline content, alleviating and cadmium-induced lactic acid leakage, liver inflammation, and fibrosis. (Conclusion): Overall, baicalin and N-Acetylcysteine alleviated cadmium-induced liver damage, inflammation, and fibrosis to a greater extent than either drug alone. TFAM represents a target for baicalin and N-Acetylcysteine, and alleviated cadmium-induced liver inflammation and fibrosis by regulating hepatic choline metabolism.

Isolation of Calenduloside E from Achyranthes bidentata Blume and its effects on LPS/D-GalN-induced acute liver injury in mice by regulating the AMPK-SIRT3 signaling pathway

BackgroundAcute liver injury (ALI) is a frequent fatal liver disease with a high mortality. Calenduloside E (CE) is a pentacyclic triterpenoid derived from Achyranthes bidentata Blume. It has been found that liver injury is associated with mitochondrial dysfunction, and activation of the AMPK-SIRT3 signaling pathway protects the mitochondrial function to play a role in resistance to the disease. However, whether CE is protective against ALI through the AMPK-SIRT3 signaling pathway is unclear. PurposeTo clarify the influences of Calenduloside E (CE) isolated from Achyranthes bidentata Blume on LPS/D-GalN-induced Acute liver injury (ALI). MethodsA mouse model of ALI was developed, intraperitoneal injection of 10 μg/kg LPS and 700 mg/kg D-GalN, histopathological, oxidative stress, and immune inflammation of the mice were monitored. The mechanism of CE influencing liver injury was investigated by examining the gut microbiota, mitochondrial dysfunction, and the AMPK-SIRT3 signaling pathway. The antagonistic effects of specific AMPK and SIRT3 blocker, as well as AMPKα1, AMPKα2, SIRT3 transfection-mediated silencing were investigated to confirm the role of the AMPK-SIRT3 signaling pathway in this process. ResultsCE relieved liver pathological damage of mice and led to reduced oxidative stress and immune inflammation in mice, affected the balance of gut microbiota in mice with liver injury, as well as energy metabolism, and regulated mRNA and protein expressions of AMPK-SIRT3 signaling pathway. In addition, in vitro studies showed that CE relieved mitochondrial respiratory and protein expressions of AMPK-SIRT3 signaling pathway in LPS/D-GalN-induced AML12 and LX2 cells, and such effect was blocked by AMPK and SIRT3 inhibitors. Furthermore, silencing of AMPKα1, AMPKα2, and SIRT3 blocked the effects of CE. Overall, the influences of CE on mice with liver injury is tuned by the AMPK-SIRT3 signaling pathway. ConclusionCE mediates mitochondrial function and eventually regulate energy metabolism by regulating the AMPK-SIRT3 signaling pathway. The results of this study provide molecular evidences for application of CE in treatment of ALI and provide references to the drug development for ALI.

The effect of STAT1, miR-99b, and MAP2K1 in alcoholic liver disease (ALD) mouse model and hepatocyte.

Alcoholic liver disease (ALD) serves as the leading cause of chronic liver diseases-related morbidity and mortality, which threatens the life of millions of patients in the world. However, the molecular mechanisms underlying ALD progression remain unclear. Here, we applied microarray analysis and experimental approaches to identify miRNAs and related regulatory signaling that associated with ALD. Microarray analysis identified that the expression of miR-99b was elevated in the ALD mouse model. The AML-12 cells were treated with EtOH and the expression of miR-99b was enhanced in the cells. The expression of miR-99b was positively correlated with ALT levels in the ALD mice. The microarray analysis identified the abnormally expressed mRNAs in ALD mice and the overlap analysis was performed with based on the differently expressed mRNAs and the transcriptional factors of miR-99b, in which STAT1 was identified. The elevated expression of STAT1 was validated in ALD mice. Meanwhile, the treatment of EtOH induced the expression of STAT1 in the AML-12 cells. The expression of STAT1 was positively correlated with ALT levels in the ALD mice. The positive correlation of STAT1 and miR-99b expression was identified in bioinformatics analysis and ALD mice. The expression of miR-99b and pri-miR-99b was promoted by the overexpression of STAT1 in AML-12 cells. ChIP analysis confirmed the enrichment of STAT1 on miR-99b promoter in AML-12 cells. Next, we found that the expression of mitogen-activated protein kinase kinase 1 (MAP2K1) was negatively associated with miR-99b. The expression of MAP2K1 was downregulated in ALD mice. Consistently, the expression of MAP2K1 was reduced by the treatment of EtOH in AML-12 cells. The expression of MAP2K1 was negative correlated with ALT levels in the ALD mice. We identified the binding site of MAP2K1 and miR-99b. Meanwhile, the treatment of miR-99b mimic repressed the luciferase activity of MAP2K1 in AML-12 cells. The expression of MAP2K1 was suppressed by miR-99b in the cells. We observed that the expression of MAP2K1 was inhibited by the overexpression of STAT1 in AML-12 cells. Meanwhile, the apoptosis of AML-12 cells was induced by the treatment of EtOH, while miR-99b mimic promoted but the overexpression of MAP2K1 attenuated the effect of EtOH in the cells. In conclusion, we identified the correlation and effect of STAT1, miR-99b, and MAP2K1 in ALD mouse model and hepatocyte. STAT1, miR-99b, and MAP2K1 may serve as potential therapeutic target of ALD.

Regulation of the Nur77-P2X7r Signaling Pathway by Nodakenin: A Potential Protective Function against Alcoholic Liver Disease.

Alcoholic liver disease (ALD) is the main factor that induces liver-related death worldwide and represents a common chronic hepatopathy resulting from binge or chronic alcohol consumption. This work focused on revealing the role and molecular mechanism of nodakenin (NK) in ALD associated with hepatic inflammation and lipid metabolism through the regulation of Nur77-P2X7r signaling. In this study, an ALD model was constructed through chronic feeding of Lieber-DeCarli control solution with or without NK treatment. Ethanol (EtOH) or NK was administered to AML-12 cells, after which Nur77 was silenced. HepG2 cells were exposed to ethanol (EtOH) and subsequently treated with recombinant Nur77 (rNur77). Mouse peritoneal macrophages (MPMs) were treated with lipopolysaccharide/adenosine triphosphate (LPS/ATP) and NK, resulting in the generation of conditioned media. In vivo, histopathological alterations were markedly alleviated by NK, accompanied by reductions in serum triglyceride (TG), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) levels and the modulation of Lipin-1, SREBP1, and Nur77 levels in comparison to the EtOH-exposed group (p < 0.001). Additionally, NK reduced the production of P2X7r and NLRP3. NK markedly upregulated Nur77, inhibited P2X7r and Lipin-1, and promoted the function of Cytosporone B, a Nur77 agonist (p < 0.001). Moreover, Nur77 deficiency weakened the regulatory effect of NK on P2X7r and Lipin-1 inhibition (p < 0.001). In NK-exposed MPMs, cleaved caspase-1 and mature IL-1β expression decreased following LPS/ATP treatment (p < 0.001). NK also decreased inflammatory-factor production in primary hepatocytes stimulated with MPM supernatant. NK ameliorated ETOH-induced ALD through a reduction in inflammation and lipogenesis factors, which was likely related to Nur77 activation. Hence, NK is a potential therapeutic approach to ALD.

Effect of high NEFA concentration on lipid metabolism disorders in hepatocytes based on lipidomics.

Introduction: High concentrations of nonesterified fatty acids (NEFA) is the key of characteristic of fatty liver in dairy cows. Therefore, the aim of this study was to investigate the effect of high concentration of NEFA on lipid metabolism in hepatocytes through the lipidomic approach and molecular biology techniques. Methods: Stimulate AML-12 cells with different concentrations of NEFA, observe the cellular lipid accumulation, and select 0.6mM NEFA stimulation concentration for subsequent experiments. Collect cells for lipidomics analysis. Results: High concentration of NEFA (0.6-2.4mM) significantly reduced the cell viability in a concentration-dependent manner, indicating that high concentrations of NEFA have lipotoxicity on hepatocytes. In addition, NEFA promoted triglycerides (TAG) accumulation, increased the mRNA expression of the lipogenic molecules SREBP1c and FASN, and decreased the mRNA expression of lipolytic molecules CPT1A and HSL in hepatocytes. Mechanistically, high concentration of NEFA induced lipid metabolism disorders in hepatocytes by regulating metabolic pathways such as glycerol phospholipid metabolism, glycosyl phosphatidylinositol anchored biosynthesis, triglyceride metabolism, sphingolipid metabolism, and inositol phosphate metabolism. Discussion: High concentration of NEFA is lipotoxic to cells, promoting lipid accumulation. LPE (18:2), LPE (18:3), LPE (18:1) via glycerophospholipid metabolism, glycosylphosphatidylinositol (GPI)-anchor biosynthesis, glycerolipid metabolism, sphingolipid metabolism, and inositol phosphate metabolism, indicating their potential regulation role in the pathogenesis of fatty liver.

PCSK9 dysregulates cholesterol homeostasis and triglyceride metabolism in olanzapine-induced hepatic steatosis via both receptor-dependent and receptor-independent pathways.

Schizophrenia, affecting approximately 1% of the global population, is often treated with olanzapine. Despite its efficacy, olanzapine's prolonged use has been associated with an increased risk of cardiovascular diseases and nonalcoholic fatty liver disease (NAFLD); however, the underlying mechanism remains unclear. Proprotein convertase subtilisin kexin type 9 (PCSK9) plays a crucial role in lipid metabolism and is involved in NAFLD pathogenesis via an unknown mechanism. This study aims to investigate the role of PCSK9 in olanzapine-induced NAFLD. C57BL/6J mice and HepG2 and AML12 cell lines were treated with varying concentrations of olanzapine to examine the effects of olanzapine on PCSK9 and lipid metabolism. PCSK9 levels were manipulated using recombinant proteins, plasmids, and small interfering RNAs invitro, and the effects on hepatic lipid accumulation and gene expression related to lipid metabolism were assessed. Olanzapine treatment significantly increased PCSK9 levels in both animal and cell line models, correlating with elevated lipid accumulation. PCSK9 manipulation demonstrated its central role in mediating hepatic steatosis through both receptor-dependent pathways (impacting NPC1L1) and receptor-independent pathways (affecting lipid synthesis, uptake, and cholesterol biosynthesis). Interestingly, upregulation of SREBP-1c, rather than SREBP-2, was identified as a key driver of PCSK9 increase in olanzapine-induced NAFLD. Our findings establish PCSK9 as a pivotal factor in olanzapine-induced NAFLD, influencing both receptor-related and metabolic pathways. This highlights PCSK9 inhibitors as potential therapeutic agents for managing NAFLD in schizophrenia patients treated with olanzapine.

Dimethyl fumarate restores Ca2+ dyshomeostasis through activation of the SIRT1 signal to treat nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) is characterized by an excessive lipid accumulation in the liver, with a global prevalence of approximately 25 %. While early-stage steatosis is reversible and can be intervened upon, it has the potential to progress to some serious complications, including cirrhosis and even liver cancer. Dimethyl fumarate (DMF), a derivative of fumaric acid shows promise in intervening in certain diseases. However, the precise effect and underlying mechanism of DMF on hepatic steatosis remain unclear. In this study, we demonstrated that DMF mitigates hepatic steatosis in mice subjected to high-fat/high-cholesterol (HFHC) diets. Meanwhile, our in vivo and in vitro results showed that DMF relieves lipid accumulation, oxidative stress, and endoplasmic reticulum (ER) stress. Mechanically, our findings revealed that the effect of DMF on reducing lipid accumulation is linked to the restoration of Ca2+ homeostasis. Furthermore, we found that activation of the SIRT1 signal by DMF plays an important role in correcting the mishandling of the Ca2+ signal, and knockdown of SIRT1 expression reverses the beneficial role of DMF PA-incubated AML12 cells. In conclusion, our results suggested DMF's amelioration of hepatic steatosis is related to the activation of SIRT1-mediated Ca2+ signaling.

Sodium sulfite triggered hepatic apoptosis, necroptosis, and pyroptosis by inducing mitochondrial damage in mice and AML-12 cells

Sodium sulfite (SS) is a biological derivative of the air pollutant sulfur dioxide, and is often used as a food and pharmaceutical additive. Improper or excessive SS exposure in liver cell death. The phenomenon of simultaneous regulation of apoptosis, necroptosis, and pyroptosis is defined as PANoptosis. However, the specific types of programmed cell death (PCD) caused by SS and their interconnections remain unclear. In the present study, C57BL/6 mice were orally administered SS for 30 d, consecutively, to establish an in vivo mouse exposure model. AML-12 cells were treated with SS for 24 h to establish an in vitro exposure model. The results showed that SS-induced mitochondrial reactive oxygen species (mtROS) accumulation activated the BAX/Bcl-2/caspase 3 pathway to trigger apoptosis and RIPK1/RIPK3/p-MLKL to trigger necroptosis. Interestingly, ROS-activated p-MLKL perforated not the cell membrane as well as the lysosomal membrane. We determined that p-MLKL mediates lysosomal membrane permeabilization (LMP), resulting in cathepsin B (CTSB) release. Furthermore, knockdown of MLKL, a CTSB inhibitor (CA074-ME) and an NLRP3 inhibitor (MCC950) alleviated SS-induced pyroptosis. In summary, our study showed that SS induced apoptosis and necroptosis though mtROS accumulation, whereas the activation of p-MLKL mediated NLRP3-dependent pyroptosis by causing CTSB leakage through LMP. This study comprehensively explored the mechanism unerlying SS-induced PCD and provided an experimental basis for p-MLKL as a potential regulatory protein in PANoptosis.

Robinetin Alleviates Metabolic Failure in Liver through Suppression of p300-CD38 Axis.

Metabolic abnormalities in the liver are closely associated with diverse metabolic diseases such as non-alcoholic fatty liver disease, type 2 diabetes, and obesity. The aim of this study was to evaluate the ameliorating effect of robinetin (RBN) on the significant pathogenic features of metabolic failure in the liver and to identify the underlying molecular mechanism. RBN significantly decreased triglyceride (TG) accumulation by downregulating lipogenesis-related transcription factors in AML-12 murine hepatocyte cell line. In addition, mice fed with Western diet (WD) containing 0.025% or 0.05% RBN showed reduced liver mass and lipid droplet size, as well as improved plasma insulin levels and homeostatic model assessment of insulin resistance (HOMA-IR) values. CD38 was identified as a target of RBN using the BioAssay database, and its expression was increased in OPA-treated AML-12 cells and liver tissues of WD-fed mice. Furthermore, RBN elicited these effects through its anti-histone acetyltransferase (HAT) activity. Computational simulation revealed that RBN can dock into the HAT domain pocket of p300, a histone acetyltransferase, which leads to the abrogation of its catalytic activity. Additionally, knock-down of p300 using siRNA reduced CD38 expression. The chromatin immunoprecipitation (ChIP) assay showed that p300 occupancy on the promoter region of CD38 was significantly decreased, and H3K9 acetylation levels were diminished in lipid-accumulated AML-12 cells treated with RBN. RBN improves the pathogenic features of metabolic failure by suppressing the p300-CD38 axis through its anti-HAT activity, which suggests that RBN can be used as a new phytoceutical candidate for preventing or improving this condition.

Design, synthesis, and activity evaluation of novel multitargeted l-tryptophan derivatives with powerful antioxidant activity against Alzheimer's disease.

Alzheimer's disease (AD) is a multifactorial neurological disease, and the multitarget directed ligand (MTDL) strategy may be an effective approach to delay its progression. Based on this strategy, 27 derivatives of l-tryptophan, 3a-1-3d-1, were designed, synthesized, and evaluated for their biological activity. Among them, IC50 (inhibitor concentration resulting in 50% inhibitory activity) values of compounds 3a-18 and 3b-1 were 0.58 and 0.44 μM for human serum butyrylcholinesterase (hBuChE), respectively, and both of them exhibited more than 30-fold selectivity for human serum acetylcholinesterase. Enzyme kinetics studies showed that these two compounds were mixed inhibitors of hBuChE. In addition, these two derivatives possessed extraordinary antioxidant activity in OH radical scavenging and oxygen radical absorption capacity fluorescein assays. Meanwhile, these compounds could also prevent β-amyloid (Aβ) self-aggregation and possessed low toxicity on PC12 and AML12 cells. Molecular modeling studies revealed that these two compounds could interact with the choline binding site, acetyl binding site, and peripheral anionic site to exert submicromolar BuChE inhibitory activity. In the vitro blood-brain barrier permeation assay, compounds 3a-18 and 3b-1 showed enough blood-brain barrier permeability. In drug-likeness prediction, compounds 3a-18 and 3b-1 showed good gastrointestinal absorption and a low risk of human ether-a-go-go-related gene toxicity. Therefore, compounds 3a-18 and 3b-1 are potential multitarget anti-AD lead compounds, which could work as powerful antioxidants with submicromolar selective inhibitory activity for hBuChE as well as prevent Aβ self-aggregation.