Activation of PPAR-α attenuates myocardial ischemia/reperfusion injury by inhibiting ferroptosis and mitochondrial injury via upregulating 14-3-3η.

This study aimed to explore the effects of peroxisome proliferator-activated receptor α (PPAR-α), a known inhibitor of ferroptosis, in Myocardial ischemia/reperfusion injury (MIRI) and its related mechanisms. In vivo and in vitro MIRI models were established. Our results showed that activation of PPAR-α decreased the size of the myocardial infarct, maintained cardiac function, and decreased the serum contents of creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH), and Fe2+ in ischemia/reperfusion (I/R)-treated mice. Additionally, the results of H&E staining, DHE staining, TUNEL staining, and transmission electron microscopy demonstrated that activation of PPAR-α inhibited MIRI-induced heart tissue and mitochondrial damage. It was also found that activation of PPAR-α attenuated MIRI-induced ferroptosis as shown by a reduction in malondialdehyde, total iron, and reactive oxygen species (ROS). In vitro experiments showed that intracellular contents of malondialdehyde, total iron, LDH, reactive oxygen species (ROS), lipid ROS, oxidized glutathione disulphide (GSSG), and Fe2+ were reduced by the activation of PPAR-α in H9c2 cells treated with anoxia/reoxygenation (A/R), while the cell viability and GSH were increased after PPAR-α activation. Additionally, changes in protein levels of the ferroptosis marker further confirmed the beneficial effects of PPAR-α activation on MIRI-induced ferroptosis. Moreover, the results of immunofluorescence and dual-luciferase reporter assay revealed that PPAR-α achieved its activity via binding to the 14-3-3η promoter, promoting its expression level. Moreover, the cardioprotective effects of PPAR-α could be canceled by pAd/14-3-3η-shRNA or Compound C11 (14-3-3η inhibitor). In conclusion, our results indicated that ferroptosis plays a key role in aggravating MIRI, and PPAR-α/14-3-3η pathway-mediated ferroptosis and mitochondrial injury might be an effective therapeutic target against MIRI.

Intermittent hypoxia exacerbates metabolic dysfunction-associated fatty liver disease by aggravating hepatic copper deficiency-induced ferroptosis.

Intermittent hypoxia (IH) is an independent risk factor for metabolic dysfunction-associated fatty liver disease (MAFLD). Copper deficiency can disrupt redox homeostasis, iron, and lipid metabolism. Here, we investigated whether hepatic copper deficiency plays a role in IH-associated MAFLD and explored the underlying mechanism(s). Male C57BL/6 mice were fed a western-type diet with adequate copper (CuA) or marginally deficient copper (CuD) and were exposed separately to room air (RA) or IH. Hepatic histology, plasma biomarkers, copper-iron status, and oxidative stress were assessed. An in vitro HepG2 cell lipotoxicity model and proteomic analysis were used to elucidate the specific targets involved. We observed that there were no differences in hepatic phenotypes between CuA-fed and CuD-fed mice under RA. However, in IH exposure, CuD-fed mice showed more pronounced hepatic steatosis, liver injury, and oxidative stress than CuA-fed mice. IH induced copper accumulation in the brain and heart and exacerbated hepatic copper deficiency and secondary iron deposition. In vitro, CuD-treated cells with IH exposure showed elevated levels of lipid accumulation, oxidative stress, and ferroptosis susceptibility. Proteomic analysis identified 360 upregulated and 359 downregulated differentially expressed proteins between CuA and CuD groups under IH; these proteins were mainly enriched in citrate cycle, oxidative phosphorylation, fatty acid metabolism, the peroxisome proliferator-activated receptor (PPAR)α pathway, and ferroptosis. In IH exposure, CuD significantly upregulated the ferroptosis-promoting factor arachidonyl-CoA synthetase long chain family member (ACSL)4. ACSL4 knockdown markedly eliminated CuD-induced ferroptosis and lipid accumulation in IH exposure. In conculsion, IH can lead to reduced hepatic copper reserves and secondary iron deposition, thereby inducing ferroptosis and subsequent MAFLD progression. Insufficient dietary copper may worsen IH-associated MAFLD.

Current View on PPAR-α and Its Relation to Neurosteroids in Alzheimer's Disease and Other Neuropsychiatric Disorders: Promising Targets in a Therapeutic Strategy.

Peroxisome proliferator-activated receptors (PPARs) may play an important role in the pathomechanism/pathogenesis of Alzheimer's disease (AD) and several other neurological/neuropsychiatric disorders. AD leads to progressive alterations in the redox state, ion homeostasis, lipids, and protein metabolism. Significant alterations in molecular processes and the functioning of several signaling pathways result in the degeneration and death of synapses and neuronal cells, leading to the most severe dementia. Peroxisome proliferator-activated receptor alpha (PPAR-α) is among the processes affected by AD; it regulates the transcription of genes related to the metabolism of cholesterol, fatty acids, other lipids and neurotransmission, mitochondria biogenesis, and function. PPAR-α is involved in the cholesterol transport to mitochondria, the substrate for neurosteroid biosynthesis. PPAR-α-coding enzymes, such as sulfotransferases, which are responsible for neurosteroid sulfation. The relation between PPAR-α and cholesterol/neurosteroids may have a significant impact on the course and progression of neurodegeneration/neuroprotection processes. Unfortunately, despite many years of intensive studies, the pathogenesis of AD is unknown and therapy for AD and other neurodegenerative diseases is symptomatic, presenting a significant goal and challenge today. This review presents recent achievements in therapeutic approaches for AD, which are targeting PPAR-α and its relation to cholesterol and neurosteroids in AD and neuropsychiatric disorders.

Solanum nigrum L. berries extract ameliorated the alcoholic liver injury by regulating gut microbiota, lipid metabolism, inflammation, and oxidative stress.

Solanum nigrum L. (SN) berry is an edible berry containing abundant polyphenols and bioactive compounds, which possess antioxidant and antiinflammatory properties. However, the effects of SN on alcohol-induced biochemical changes in the enterohepatic axis remain unclear. In the current study, a chronic ethanol-fed mice ALD model was used to test the protective mechanisms of SN berries. Microbiota composition was determined via 16S rRNA sequencing, we found that SN berries extract (SNE) improved intestinal imbalance by reducing the Firmicutes to Bacteroides ratio, restoring the abundance of Akkermansia microbiota, and reducing the abundance of Allobaculum and Shigella. SNE restored the intestinal short-chain fatty acids content. In addition, liver transcriptome data analysis revealed that SNE primarily affected the genes involved in lipid metabolism and inflammatory responses. Furthermore, SNE ameliorated hepatic steatosis in alcohol-fed mice by activating AMP-activated protein kinase (AMPK), acetyl-CoA carboxylase (ACC), peroxisome proliferator-activated receptor α (PPAR-α). SNE reduced the expression of toll-like receptor 4 (TLR4), myeloid differentiation factor-88 (MyD88) nuclear factor kappa-B (NF-κB), which can indicate that SNE mainly adjusted LPS/TLR4/MyD88/NF-κB pathway to reduce liver inflammation. SNE enhanced hepatic antioxidant capacity by regulating NRF2-related protein expression. SNE alleviates alcoholic liver injury by regulating of gut microbiota, lipid metabolism, inflammation, and oxidative stress. This study may provide a reference for the development and utilization of SN resources.

A novel Alisma orientale extract alleviates non-alcoholic steatohepatitis in mice via modulation of PPARα signaling pathway.

Non-alcoholic fatty liver disease (NAFLD), particularly advanced non-alcoholic steatohepatitis (NASH), leads to irreversible liver damage. This study investigated the therapeutic effects and potential mechanism of a novel extract from traditional Chinese medicine Alisma orientale (Sam.) Juzep (AE) on free fatty acid (FFA)-induced HepG2 cell model and high-fat diet (HFD) + carbon tetrachloride (CCl4)-induced mouse model of NASH. C57BL/6 J mice were fed a HFD for 10 weeks. Subsequently, the mice were injected with CCl4 to induce NASH and simultaneously treated with AE at daily doses of 50, 100, and 200 mg/kg for 4 weeks. At the end of the treatment, animals were fasted for 12 h and then sacrificed. Blood samples and liver tissues were collected for analysis. Lipid profiles, oxidative stress, and histopathology were examined. Additionally, a polymerase chain reaction (PCR) array was used to predict the molecular targets and potential mechanisms involved, which were further validated in vivo and in vitro. The results demonstrated that AE reversed liver damage (plasma levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), hepatocyte ballooning, hepatic steatosis, and NAS score), the accumulation of hepatic lipids (TG and TC), and oxidative stress (MDA and GSH). PCR array analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that AE protects against NASH by regulating the adipocytokine signaling pathway and influencing nuclear receptors such as PPARα. Furthermore, AE increased the expression of peroxisome proliferator-activated receptor gamma coactivator-1α (PPARGC1α) and reversed the decreased expression of PPARα in NASH mice. Moreover, in HepG2 cells, AE reduced FFA-induced lipid accumulation and oxidative stress, which was dependent on PPARα up-regulation. Overall, our findings suggest that AE may serve as a potential therapeutic approach for NASH by inhibiting lipid accumulation and reducing oxidative stress specifically through the PPARα pathway.

Low GPR81 in ER+ breast cancer cells drives tamoxifen resistance through inducing PPARα-mediated fatty acid oxidation.

AIMS: The intricate molecular mechanisms underlying estrogen receptor-positive (ER+) breast carcinogenesis and resistance to endocrine therapy remain elusive. In this study, we elucidate the pivotal role of GPR81, a G protein-coupled receptor, in ER+ breast cancer (BC) by demonstrating low expression of GPR81 in tamoxifen (TAM)-resistant ER+ BC cell lines and tumor samples, along with the underlying molecular mechanisms. MAIN METHODS: Fatty acid oxidation (FAO) levels and lipid accumulation were explored using MDA and FAßO assay, BODIPY 493/503 staining, and Lipid TOX staining. Autophagy levels were assayed using CYTO-ID detection and Western blotting. The impact of GPR81 on TAM resistance in BC was investigated through CCK8 assay, colony formation assay and a xenograft mice model. RESULTS: Aberrantly low GPR81 expression in TAM-resistant BC cells disrupts the Rap1 pathway, leading to the upregulation of PPARα and CPT1. This elevation in PPARα/CPT1 enhances FAO, impedes lipid accumulation and lipid droplet (LD) formation, and subsequently inhibits cell autophagy, ultimately promoting TAM-resistant BC cell growth. Moreover, targeting GPR81 and FAO emerges as a promising therapeutic strategy, as the GPR81 agonist and the CPT1 inhibitor etomoxir effectively inhibit ER+ BC cell and tumor growth in vivo, re-sensitizing TAM-resistant ER+ cells to TAM treatment. CONCLUSION: Our data highlight the critical and functionally significant role of GPR81 in promoting ER+ breast tumorigenesis and resistance to endocrine therapy. GPR81 and FAO levels show potential as diagnostic biomarkers and therapeutic targets in clinical settings for TAM-resistant ER+ BC.

Effects of exercise training with intermittent hyperoxic intervention on endurance performance and muscle metabolic properties in male mice.

This study aimed to investigate how intermittent hyperoxic exposure (three cycles of 21% O2 [10 min] and 30% O2 [15 min]) affects exercise performance in mice. Three hours after the acute exposure, there was an observed increase in mRNA levels of phosphofructokinase (Bayes factor [BF] ≥ 10), mitochondrial transcription factor-A (BF ≥10), PPAR-α (BF ≥3), and PPAR-γ (BF ≥3) in the red gastrocnemius muscle (Gr). Four weeks of exercise training under intermittent (INT), but not continuous (HYP), hyperoxia significantly (BF ≥30) increased maximal exercise capacity compared to normoxic exercise-trained (ET) group. INT group exhibited significantly higher activity levels of 3-hydroxyacyl-CoA-dehydrogenase (HAD) in Gr (BF = 7.9) compared to ET group. Pyruvate dehydrogenase complex activity levels were significantly higher in INT group compared to ET group in white gastrocnemius, diaphragm, and left ventricle (BF ≥3). NT-PGC1α protein levels in Gr (BF = 7.7) and HAD activity levels in Gr (BF = 6.9) and soleus muscles (BF = 3.3) showed a significant positive correlation with maximal work values. These findings suggest that exercise training under intermittent hyperoxia is a beneficial strategy for enhancing endurance performance by improving fatty acid and pyruvic acid utilization.

Topical and oral peroxisome proliferator-activated receptor-α agonist ameliorates diabetic corneal neuropathy.

Diabetic corneal neuropathy (DCN) is a common diabetic ocular complication with limited treatment options. In this study, we investigated the effects of topical and oral fenofibrate, a peroxisome proliferator-activated receptor-α agonist, on the amelioration of DCN using diabetic mice (n = 120). Ocular surface assessments, corneal nerve and cell imaging analysis, tear proteomics and its associated biological pathways, immuno-histochemistry and western blot on PPARα expression, were studied before and 12 weeks after treatment. At 12 weeks, PPARα expression markedly restored after topical and oral fenofibrate. Topical fenofibrate significantly improved corneal nerve fibre density (CNFD) and tortuosity coefficient. Likewise, oral fenofibrate significantly improved CNFD. Both topical and oral forms significantly improved corneal sensitivity. Additionally, topical and oral fenofibrate significantly alleviated diabetic keratopathy, with fenofibrate eye drops demonstrating earlier therapeutic effects. Both topical and oral fenofibrate significantly increased corneal ß-III tubulin expression. Topical fenofibrate reduced neuroinflammation by significantly increasing the levels of nerve growth factor and substance P. It also significantly increased ß-III-tubulin and reduced CDC42 mRNA expression in trigeminal ganglions. Proteomic analysis showed that neurotrophin signalling and anti-inflammation reactions were significantly up-regulated after fenofibrate treatment, whether applied topically or orally. This study concluded that both topical and oral fenofibrate ameliorate DCN, while topical fenofibrate significantly reduces neuroinflammation.

A chemical modification of a peroxisome proliferator-activated receptor pan agonist produced a shift to a new dual alpha/gamma partial agonist endowed with mitochondrial pyruvate carrier inhibition and antidiabetic properties.

New analogs of the PPAR pan agonist AL29-26 encompassed ligand (S)-7 showing potent activation of PPARα and -γ subtypes as a partial agonist. In vitro experiments and docking studies in the presence of PPAR antagonists were performed to help interpretation of biological data and investigate the main interactions at the binding sites. Further in vitro experiments showed that (S)-7 induced anti-steatotic effects and enhancement of the glucose uptake. This latter effect could be partially ascribed to a significant inhibition of the mitochondrial pyruvate carrier demonstrating that (S)-7 also acted through insulin-independent mechanisms. In vivo experiments showed that this compound reduced blood glucose and lipid levels in a diabetic mice model displaying no toxicity on bone, kidney, and liver. To our knowledge, this is the first example of dual PPARα/γ partial agonist showing these combined effects representing, therefore, the potential lead of new drugs for treatment of dyslipidemic type 2 diabetes.

Efficacy and mechanism of action of Yanxiao Di'naer formula for non-alcoholic steatohepatitis treatment based on metabolomics and RNA sequencing.

ETHNOPHARMACOLOGICAL RELEVANCE: Nonalcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disease worldwide. Nonalcoholic steatohepatitis (NASH) is a crucial component of this disease spectrum. The Yanxiao Di'naer formula (YXDNE) is an Uyghur medical extract that has been used in folk medicine to treat hepatitis for a long time. However, the role and mechanism of action of YXDNE in NASH treatment remains unclear. OBJECTIVE: The objective of this study was to assess the effectiveness of YXDNE in treating NASH induced by injections of carbon tetrachloride combined with a high-fat high-cholesterol diet (HFHCD), and to clarify the underlying mechanisms. METHODS: The compounds in the YXDNE extract were analysed for classification and proportions using ultra-performance liquid chromatography-mass spectrometry. The efficacy of YXDNE in treating abnormal lipid metabolism was evaluated in L02 cells in vitro. In addition, a C57BL/6 mouse model of NASH was established to evaluate the therapeutic efficacy of YXDNE in vivo. Metabolomics and RNA sequencing were used to analyse the therapeutic effects of YXDNE on the liver. The corresponding signalling pathways were found to target AMPKα1, PPARα, and NF-κB. The efficacy of YXDNE was validated using inhibitors or silencing RNA (siRNA) against AMPKα1 and PPARα. RESULTS: This study confirmed that YXDNE treatment ameliorated NASH in a murine model of this disease. Metabolomics analysis suggested that YXDNE efficacy was associated with fatty acid catabolism and AMPK signalling pathways. RNA sequencing results showed that YXDNE efficacy in treating NASH was highly correlated with the AMPK, PPARα and NF-κB pathways. Both in vitro and in vivo experimental data demonstrated that YXDNE affected the expression of p-AMPKα1, PPARα, p-NF-κB, IκB, and p-IκB. The efficacy of YXDNE in treating NASH in vitro was cancelled when AMPK was inhibited with Compound C or PPARα was modulated via siRNA. CONCLUSIONS: YXDNE may have a therapeutic effect on abnormal lipid metabolism in L02 cells and in a murine model of NASH by affecting the AMPKα1/PPARα/NF-κB signalling pathway. Therefore, YXDNE has the potential for clinical application in the prevention and treatment of NASH.

Impact of Peroxisome Proliferator-Activated Receptor Agonists on Myosteatosis in the Context of Metabolic Dysfunction-Associated Steatotic Liver Disease.

BACKGROUND: Metabolic dysfunction-associated steatotic liver disease (MASLD), and more specifically steatohepatitis may be associated with fat infiltration of skeletal muscles which is known as myosteatosis. Pan-peroxisome proliferator-activated receptor (PPAR) agonists have been shown to promote metabolic dysfunction-associated steatohepatitis (MASH) remission. However, the effect of PPAR agonists on myosteatosis remains to be determined. The aim of this review is to evaluate the effect that PPAR agonists alone or in combination, have on myosteatosis in the context of MASLD. METHODS: Original research reports concerning the impact of PPAR agonists on muscle fat in MASLD were screened from PUBMED and EMBASE databases following the PRISMA methodology. RESULTS: Eleven original manuscripts were included in this review. Two preclinical studies assessed the impact of the PPARα agonist on fat content in the quadriceps muscle and the liver by extracting triglycerides in rats fed a high-fat diet and in insulin-resistant mice. Both models showed muscle and liver triglyceride content reduction using WY14643. Fenofibrate had no significant impact on soleus intramyocellular lipids or liver fat content in insulin-resistant subjects based on proton magnetic resonance spectroscopy. Treatment with PPARδ agonists increased the expression of genes involved in fatty acid oxidation in two studies on muscle cell culture. PPARγ agonists were investigated in two preclinical studies and one clinical study using spectroscopy and computed tomography respectively. In the first preclinical study in Zucker diabetic fatty rats, rosiglitazone reduced muscle lipids and hepatic steatosis. In a second preclinical study using the same animal model, pioglitazone reduced tibialis anterior intramyocellular lipids. In contrast, computed tomography analyses in patients with type 2 diabetes revealed a surface area increase of low-density muscles (suggesting an increase in muscle fat content) after a one-year treatment with rosiglitazone. Varying combinations of PPAR agonists (cevoglitazar, fenofibrate/pioglitazone and muraglitazar) were evaluated in two preclinical studies and one clinical study. In rats, these treatments showed variable results for muscle and liver depending on the combinations studied. In type 2 diabetic patients, treatment with muraglitazar (a PPARα/γ agonist) reduced the intramyocellular lipid content of tibialis anterior as well as liver fat content following spectroscopy assessment. CONCLUSION: The combination of different PPAR agonists could have a positive impact on reducing myosteatosis, in addition to their effect on the liver. Some discrepancies could be explained by the different techniques used to assess muscle lipid content, the muscles assessed and the possible adipogenic effect of PPARγ agonists. Further clinical research is needed to fully assess the efficacy of these treatments on both MASLD progression and associated myosteatosis.

(+)Alpha-Lipoic Acid Regulates Lipid Metabolism Gene Expression and Lipidic Profile in a Cellular Model of Fatty Acid Overload.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is a prevalent condition characterized by hepatic fat accumulation, often progressing to severe liver injury, for which approved treatments are currently lacking. This study explores the potential therapeutic impact of alpha-lipoic acid (ALA), a natural compound crucial in lipid metabolism, on NAFLD using an in vitro model. METHODS: HepG2 cells were treated with a palmitic acid:oleic acid (PA:OA) mixture, representing a cellular model of steatosis. Subsequent treatment with ALA at concentrations of 1 µM and 5 µM aimed to evaluate its effects on lipid content and metabolism. Real-time polymerase chain reaction (PCR), BODIPY staining, cytofluorimetric analysis, and lipidomics were used to assess gene expression, lipid droplet accumulation, and fatty acid profiles. RESULTS: Our results showed that ALA significantly reduced lipid droplets in PA:OA-treated HepG2 cells, with a concentration-dependent effect. Analysis of fatty acid profiles demonstrated a decrease in palmitic acid levels with ALA treatment, while oleic acid reduction was observed only at the higher concentration. Moreover, ALA modulated the expression of genes involved in cholesterol biosynthesis and low-density lipoprotein (LDL) metabolism, indicating a potential role in lipid homeostasis. Further insights into molecular mechanisms revealed that ALA modulated peroxisome proliferator activated receptors (PPARs), specifically PPAR-alpha and PPAR-gamma, involved in fatty acid metabolism and insulin sensitivity. Finally, ALA counteracted the overexpression of thermogenic genes induced by exogenous fatty acids, suggesting a regulatory role in energy dissipation pathways. CONCLUSION: In conclusion, this study highlights ALA as a therapeutic agent in mitigating lipid accumulation and dysregulation in NAFLD.

CBFA2T3 Is PPARA Sensitive and Attenuates Fasting-Induced Lipid Accumulation in Mouse Liver.

Peroxisome proliferator-activated receptor alpha (PPARA) is a ligand-activated transcription factor that is a key mediator of lipid metabolism and metabolic stress in the liver. Accumulating evidence shows that PPARA regulates the expression of various protein coding and non-coding genes that modulate metabolic stress in the liver. CBFA2/RUNX1 partner transcriptional co-repressor 3 (CBFA2T3) is a DNA-binding transcription factor that belongs to the myeloid translocation gene family. Many studies have shown that CBFA2T3 is associated with acute myeloid leukemia. Especially, CBFA2T3-GLIS2 fusion is a chimeric oncogene associated with a poor survival rate in pediatric acute megakaryocytic leukemia. A previous study identified that PPARA activation promoted Cbfa2t3 induction in liver and that Cbfa2t3 may have a modulatory role in metabolic stress. However, the effect of CBFA2T3 gene expression on metabolic stress is not understood. In this study, the PPARA ligand WY14643 activated Cbfa2t3 expression in mouse liver. Glucose tolerance test and insulin tolerance test data showed that insulin resistance is increased in Cbfa2t3-/- mice compared to Cbfa2t3+/+ mice. Hepatic CBFA2T3 modulates heat shock protein family A member 1b and carbonic anhydrase 5a expression. Histology analysis revealed lipid droplet and lipid accumulation in the liver of fasting Cbfa2t3-/- mice but not Cbfa2t3+/+ mice. The expression of lipid accumulation-related genes, such as Cd36, Cidea, and Fabp1, was increased in the liver of fasting Cbfa2t3-/- mice. Especially, basal expression levels of Cidea mRNA were elevated in the liver of Cbfa2t3-/- mice compared to Cbfa2t3+/+ mice. Much higher induction of Cidea mRNA was seen in the liver of Cbfa2t3-/- mice after WY14643 administration. These results indicate that hepatic CBFA2T3 is a PPARA-sensitive gene that may modulate metabolic stress in mouse liver.

Cannabidiol alleviates carbon tetrachloride-induced liver fibrosis in mice by regulating NF-κB and PPAR-α pathways.

Liver fibrosis has become a serious public health problem that can develop into liver cirrhosis and hepatocellular carcinoma and even lead to death. Cannabidiol (CBD), which is an abundant nonpsychoactive component in the cannabis plant, exerts cytoprotective effects in many diseases and under pathological conditions. In our previous studies, CBD significantly attenuated liver injury induced by chronic and binge alcohol in a mouse model and oxidative bursts in human neutrophils. However, the effects of CBD on liver fibrosis and the underlying mechanisms still need to be further explored. A mouse liver fibrosis model was induced by carbon tetrachloride (CCl4) for 10 weeks and used to explore the protective properties of CBD and related molecular mechanisms. After the injection protocol, serum samples and livers were used for molecular biology, biochemical and pathological analyses. The results showed that CBD could effectively improve liver function and reduce liver damage and liver fibrosis progression in mice; the expression levels of transaminase and fibrotic markers were reduced, and histopathological characteristics were improved. Moreover, CBD inhibited the levels of inflammatory cytokines and reduced the protein expression levels of p-NF-κB, NF-κB, p-IκBα, p-p38 MAPK, and COX-2 but increased the expression level of PPAR-α. We found that CBD-mediated protection involves inhibiting NF-κB and activating PPAR-α. In conclusion, these results suggest that the hepatoprotective effects of CBD may be due to suppressing the inflammatory response in CCl4-induced mice and that the NF-κB and PPAR-α signaling pathways might be involved in this process.

Development of an analytical platform for the affinity screening of natural extracts by SEC-MS towards PPARα and PPARγ receptors.

BACKGROUND: Peroxisome proliferator-activated receptors (PPARs) belong to the superfamily of nuclear receptors and represent the targets for the therapeutical treatment of type 2 diabetes, dyslipidemia and hyperglycemia associated with metabolic syndrome. Some medicinal plants have been traditionally used to treat this kind of metabolic diseases. Today only few drugs targeting PPARs have been approved and for this reason, the rapid identification of novel ligands and/or chemical scaffolds starting from natural extracts would benefit of a selective affinity ligand fishing assay. RESULTS: In this paper we describe the development of a new ligand fishing assay based on size exclusion chromatography (SEC) coupled to LC-MS for the analysis of complex samples such as botanical extracts. The known PPARα and PPARγ ligands, WY-14643 and rosiglitazone respectively, were used for system development and evaluation. The system has found application on an Allium lusitanicum methanolic extract, containing saponins, a class of chemical compounds which have attracted interest as PPARs ligands because of their hypolipidemic and insulin-like properties. SIGNIFICANCE: A new SEC-AS-MS method has been developed for the affinity screening of PPARα and PPARγ ligands. The system proved to be highly specific and will be used to improve the throughput for the identification of new selective metabolites from natural souces targeting PPARα and PPARγ.

Prenatal Choline Supplementation Improves Glucose Tolerance and Reduces Liver Fat Accumulation in Mouse Offspring Exposed to Ethanol during the Prenatal and Postnatal Periods.

Prenatal alcohol exposure (AE) affects cognitive development. However, it is unclear whether prenatal AE influences the metabolic health of offspring and whether postnatal AE exacerbates metabolic deterioration resulting from prenatal AE. Choline is a semi-essential nutrient that has been demonstrated to mitigate the cognitive impairment of prenatal AE. This study investigated how maternal choline supplementation (CS) may modify the metabolic health of offspring with prenatal and postnatal AE (AE/AE). C57BL/6J female mice were fed either a Lieber-DeCarli diet with 1.4% ethanol between embryonic day (E) 9.5 and E17.5 or a control diet. Choline was supplemented with 4 × concentrations versus the control throughout pregnancy. At postnatal week 7, offspring mice were exposed to 1.4% ethanol for females and 3.9% ethanol for males for 4 weeks. AE/AE increased hepatic triglyceride accumulation in male offspring only, which was normalized by prenatal CS. Prenatal CS also improved glucose tolerance compared to AE/AE animals. AE/AE suppressed hepatic gene expression of peroxisome proliferator activated receptor alpha (Ppara) and low-density lipoprotein receptor (Ldlr), which regulate fatty acid catabolism and cholesterol reuptake, respectively, in male offspring. However, these changes were not rectified by prenatal CS. In conclusion, AE/AE led to an increased risk of steatosis and was partially prevented by prenatal CS in male mice.

Protective effects and mechanism of Sangyu granule on acetaminophen-induced liver injury in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: The Sang Yu granule (SY), a traditional Chinese medicine prescription of Xijing Hospital, was developed based on the Guanyin powder in the classical prescription "Hong's Collection of Proven Prescriptions" and the new theory of modern Chinese medicine. It has been proved to have a certain therapeutic effect on drug-induced liver injury (DILI), but the specific mechanism of action is still unclear. AIM OF STUDY: Aim of the study was to explore the effect of SangYu granule on treating drug-induced liver injury induced by acetaminophen in mice. MATERIALS AND METHODS: The chemical composition of SY, serum, and liver tissue was analyzed using ultrahigh-performance liquid chromatography quadrupole time-of-flight mass spectrometry. To assess hepatic function, measurements were taken using kits for total bile acids, as well as serum AST, ALT, and ALP activity. Concentrations of IL-1ß and TNF-α in serum were quantified using ELISA kits. Transcriptome Sequencing Analysis and 2bRAD-M microbial diversity analysis were employed to evaluate gene expression variance in liver tissue and fecal microbiota diversity among different groups, respectively. Western blotting was performed to observe differences in the activation levels of FXR, SHP, CYP7A1 and PPARα in the liver, and the levels of FXR and FGF-15 genes and proteins in the ileum of mice. Additionally, fecal microbiota transplantation (FMT) experiments were conducted to investigate the potential therapeutic effect of administering the intestinal microbial suspension from mice treated with SY on drug-induced liver injury. RESULTS: SY treatment exhibited significant hepatoprotective effects in mice, effectively ameliorating drug-induced liver injury while concurrently restoring intestinal microbial dysbiosis. Furthermore, SY administration demonstrated a reduction in the concentration of total bile acids, the expression of FXR and SHP proteins in the liver was up-regulated, CYP7A1 protein was down-regulated, and the expressions of FXR and FGF-15 proteins in the ileum were up-regulated. However, no notable impact on PPARα was observed. Furthermore, results from FMT experiments indicated that the administration of fecal suspensions derived from mice treated with SY did not yield any therapeutic benefits in the context of drug-induced liver injury. CONCLUSION: The aforementioned findings strongly suggest that SY exerts a pronounced ameliorative effect on drug-induced liver injury through its ability to modulate the expression of key proteins involved in bile acid secretion, thereby preserving hepato-enteric circulation homeostasis.

A comparative study of the hypolipidemic effects and mechanisms of action of Laminaria japonica- and Ascophyllum nodosum-derived fucoidans in apolipoprotein E-deficient mice.

The structural characteristics of fucoidans exhibit species and regional diversity. Previous studies have demonstrated that Laminaria japonica- and Ascophyllum nodosum-derived fucoidans have type I and type II fucosyl chains, respectively. These chemical differences may contribute to distinct hypolipidemic effects and mechanisms of action. Chemical analysis demonstrated that the percentage contents of sulfate, glucuronic acid, and galactose were higher in L. japonica-derived fucoidans than those of A. nodosum-derived fucoidans. In hyperlipidemic apolipoprotein E-deficient mice, both A. nodosum- and L. japonica-derived fucoidans significantly decreased the plasma and hepatic levels of total cholesterol and triglyceride, leading to the reduction of atherosclerotic plaques. Western blotting experiments demonstrated that these fucoidans significantly enhanced the expression and levels of scavenger receptor B type 1, cholesterol 7 alpha-hydroxylase A1, and peroxisome proliferator-activated receptor (PPAR)-α, contributing to circulating lipoprotein clearance and fatty acid degradation, respectively. Differentially, L. japonica-derived fucoidan significantly increased the LXR/ATP-binding cassette G8 signaling pathway in the small intestine, as revealed by real-time quantitative PCR, which may lead to further cholesterol and other lipid excretion. Collectively, these data are useful for understanding the hypolipidemic mechanisms of action of seaweed-derived fucoidans, and their potential application for the prevention and/or treatment of atherosclerotic cardiovascular diseases.

Ketogenic diet time-dependently prevents NAFLD through upregulating the expression of antioxidant protein metallothionein-2.

BACKGROUND & AIMS: The past few decades have witnessed a rapid growth in the prevalence of nonalcoholic fatty liver disease (NAFLD). While the ketogenic diet (KD) is considered for managing NAFLD, the safety and efficacy of the KD on NAFLD has been a controversial topic. Here, we aimed to investigate the effect of KD of different durations on metabolic endpoints in mice with NAFLD and explore the underlying mechanisms. METHODS: NAFLD mice were fed with KD for 1, 2, 4 and 6 weeks, respectively. The blood biochemical indexes (blood lipids, AST, ALT and etc.) and liver fat were measured. The LC-MS/MS based proteomic analysis was performed on liver tissues. Metallothionein-2 (MT2) was knocked down with adeno-associated virus (AAV) or small interfering RNA (siRNA) in NAFLD mice and AML-12 cells, respectively. H&E, BODIPY and ROS staining were performed to examine lipid deposition and oxidative stress. Furthermore, MT2 protein levels, nucleus/cytoplasm distribution and DNA binding activity of peroxisome proliferators-activated receptors α (PPARα) were evaluated. RESULTS: KD feeding for 2 weeks showed the best improvement on NAFLD phenotype. Proteomic analysis revealed that MT2 was a key candidate for different metabolic endpoints of NAFLD affected by different durations of KD feeding. MT2 knockdown in NAFLD mice blocked the effects of 2 weeks of KD feeding on HFD-induced steatosis. In mouse primary hepatocytes and AML-12 cells, MT2 protein levels were induced by ß-hydroxybutyric acid (ß-OHB). MT2 Knockdown blunted the effects of ß-OHB on alleviating PA-induced lipid deposition. Mechanistically, 2 weeks of KD or ß-OHB treatment reduced oxidative stress and upregulated the protein levels of MT2 in nucleus, which subsequently increased its DNA binding activity and PPARα protein expression. CONCLUSIONS: Collectively, these findings indicated that KD feeding prevented NAFLD in a time dependent manner and MT2 is a potential target contributing to KD improvement on steatosis.

Constitutive Androstane Receptor and Peroxisome Proliferator-Activated Receptor α Do Not Perform Liquid-Liquid Phase Separation in Cells.

Constitutive androstane receptor (CAR) and peroxisome proliferator-activated receptor α (PPARα) are members of the nuclear receptor superfamily, which regulates various physiologic and pathologic processes. Phase separation is a dynamic biophysical process in which biomacromolecules form liquid-like condensates, which have been identified as contributors to many cellular functions, such as signal transduction and transcription regulation. However, the possibility of phase separation for CAR and PPARα remains unknown. This study explored the potential phase separation of CAR and PPARα The computational analysis utilizing algorithm tools examining the intrinsically disordered regions of CAR and PPARα suggested a limited likelihood of undergoing phase separation. Experimental assays under varying conditions of hyperosmotic stress and agonist treatments confirmed the absence of phase separation for these receptors. Additionally, the optoDroplets assay, which utilizes blue light stimulation to induce condensate formation, showed that there was no condensate formation of the fusion protein of Cry2 with CAR or PPARα Furthermore, phase separation of CAR or PPARα did not occur despite reduced target expression under hyperosmotic stress. In conclusion, these findings revealed that neither the activation of CAR and PPARα nor hyperosmotic stress induces phase separation of CAR and PPARα in cells. SIGNIFICANCE STATEMENT: Constitutive androstane receptor (CAR) and peroxisome proliferator-activated receptor α (PPARα) are key regulators of various functions in the body. This study showed that CAR and PPARα do not exhibit phase separation under hyperosmotic stress or after agonist-induced activation. These findings provide new insights into the CAR and PPARα biology and physiology.