Xanthohumol, a prenylated chalcone, regulates lipid metabolism by modulating the LXRα/RXR-ANGPTL3-LPL axis in hepatic cell lines and high-fat diet-fed zebrafish models.

Angiopoietin-like 3 (ANGPTL3) acts as an inhibitor of lipoprotein lipase (LPL), impeding the breakdown of triglyceride-rich lipoproteins (TGRLs) in circulation. Targeting ANGPTL3 is considered a novel strategy for improving dyslipidemia and atherosclerotic cardiovascular diseases (ASCVD). Hops (Humulus lupulus L.) contain several bioactive prenylflavonoids, including xanthohumol (Xan), isoxanthohumol (Isoxan), 6-prenylnaringenin (6-PN), and 8-prenylnaringenin (8-PN), with the potential to manage lipid metabolism. The aim of this study was to investigate the lipid-lowering effects of Xan, the effective prenylated chalcone in attenuating ANGPTL3 transcriptional activity, both in vitro using hepatic cells and in vivo using zebrafish models, along with exploring the underlying mechanisms. Xan (10 and 20 µM) significantly reduced ANGPTL3 mRNA and protein expression in HepG2 and Huh7 cells, leading to a marked decrease in secreted ANGPTL3 proteins via hepatic cells. In animal studies, orally administered Xan significantly alleviated plasma triglyceride (TG) and cholesterol levels in zebrafish fed a high-fat diet. Furthermore, it reduced hepatic ANGPTL3 protein levels and increased LPL activity in zebrafish models, indicating its potential to modulate lipid profiles in circulation. Furthermore, molecular docking results predicted that Xan exhibits a higher binding affinity to interact with liver X receptor α (LXRα) and retinoic acid X receptor (RXR) than their respective agonists, T0901317 and 9-Cis-retinoic acid (9-Cis-RA). We observed that Xan suppressed hepatic ANGPTL3 expression by antagonizing the LXRα/RXR-mediated transcription. These findings suggest that Xan ameliorates dyslipidemia by modulating the LXRα/RXR-ANGPTL3-LPL axis. Xan represents a novel potential inhibitor of ANGPTL3 for the prevention or treatment of ASCVD.

The Neuroprotective Effects of Flavonoid Fisetin against Corticosterone-Induced Cell Death through Modulation of ERK, p38, and PI3K/Akt/FOXO3a-Dependent Pathways in PC12 Cells.

The overactive hypothalamic-pituitary-adrenal (HPA) axis is believed to trigger the overproduction of corticosterone, leading to neurotoxicity in the brain. Fisetin is a flavonoid commonly found in fruits and vegetables. It has been suggested to possess various biological activities, including antioxidant, anti-inflammatory, and neuroprotective effects. This study aims to explore the potential neuroprotective properties of fisetin against corticosterone-induced cell death and its underlying molecular mechanism in PC12 cells. Our results indicate that fisetin, at concentrations ranging from 5 to 40 µM, significantly protected PC12 cells against corticosterone-induced cell death. Fisetin effectively reduced the corticosterone-mediated generation of reactive oxygen species (ROS) in PC12 cells. Fisetin treatments also showed potential in inhibiting the corticosterone-induced apoptosis of PC12 cells. Moreover, inhibitors targeting MAPK/ERK kinase 1/2 (MEK1/2), p38 MAPK, and phosphatidylinositol 3-kinase (PI3K) were found to significantly block the increase in cell viability induced by fisetin in corticosterone-treated cells. Consistently, fisetin enhanced the phosphorylation levels of ERK, p38, Akt, and c-AMP response element-binding protein (CREB) in PC12 cells. Additionally, it was found that the diminished levels of p-CREB and p-ERK by corticosterone can be restored by fisetin treatment. Furthermore, the investigation of crosstalk between ERK and CREB revealed that p-CREB activation by fisetin occurred through the ERK-independent pathway. Moreover, we demonstrated that fisetin effectively counteracted the corticosterone-induced nuclear accumulation of FOXO3a, an apoptosis-triggering transcription factor, and concurrently promoted FOXO3a phosphorylation and its subsequent cytoplasmic localization through the PI3K/Akt pathway. In conclusion, our findings indicate that fisetin exerts its neuroprotective effect against corticosterone-induced cell death by modulating ERK, p38, and the PI3K/Akt/FOXO3a-dependent pathways in PC12 cells. Fisetin emerges as a promising phytochemical for neuroprotection.

Pinostrobin modulates FOXO3 expression, nuclear localization, and exerts antileukemic effects in AML cells and zebrafish xenografts.

Acute myeloid leukemia (AML) is a disease characterized by abnormal cell proliferation in the bone marrow and is the most common quickly progressive leukemia in adults. Pinostrobin, a flavonoid phytochemical, has been reported to exhibit antioxidant, anti-inflammatory, and anticancer properties. In this study, we aimed to investigate the antileukemic effects of pinostrobin and its molecular mechanisms in human AML cells. Our study found that pinostrobin (0-80 µM) significantly reduced the viability of human AML cells, with the pronounced cytotoxic effects observed in MV4-11 > MOLM-13 > HL-60 > U-937 > THP-1 cells. Pinostrobin was found to suppress leukemia cell proliferation, modulate cell cycle progression, promote cell apoptosis, and induce monocytic differentiation in MV4-11 cells. In animal studies, pinostrobin significantly suppressed the growth of leukemia cells in a zebrafish xenograft model. Microarray-based transcriptome analysis showed that the differentially expressed genes (DEGs) in pinostrobin-treated cells were strongly associated with enriched Gene Ontology (GO) terms related to apoptotic process, cell death, cell differentiation, cell cycle progression, and cell division. Combining DisGeNET and STRING database analysis revealed that pinostrobin upregulates forkhead box 3 (FOXO3), a tumor suppressor in cancer development, and plays an essential role in controlling AML cell viability. Our study demonstrated that pinostrobin increases FOXO3 gene expression and promotes its nuclear translocation, leading to the inhibition of cell growth. Finally, the study found that pinostrobin, when combined with cytarabine, synergistically reduces the viability of AML cells. Our current findings shed light on pinostrobin's mechanisms in inhibiting leukemia cell growth, highlighting its potential as a chemotherapeutic agent or nutraceutical supplement for AML prevention or treatment.

The Citrus Flavonoid Nobiletin Downregulates Angiopoietin-like Protein 3 (ANGPTL3) Expression and Exhibits Lipid-Modulating Effects in Hepatic Cells and Adult Zebrafish Models.

Nobiletin, a dietary citrus flavonoid, exerts biological activities against hyperlipidemia, obesity, and atherosclerotic cardiovascular diseases (ASCVDs). The aim of this study was to explore the lipid-lowering effects of nobiletin and the underlying molecular mechanisms in vitro in hepatic cells and in vivo in zebrafish models. Transcriptome and gene ontology (GO) analyses of differentially expressed genes (DEGs) by gene set enrichment analysis (GSEA) showed that a set of twenty-eight core enrichment DEGs associated with "GO BP regulation of lipid metabolic process" (GO: 0019216) were significantly downregulated in nobiletin-treated cells. Among these genes, angiopoietin-like 3 (ANGPTL3), an inhibitor of lipoprotein lipase (LPL) activity that regulates TG-rich lipoprotein (TGRL) metabolism in circulation, was the protein most markedly downregulated by nobiletin. Nobiletin (20 and 40 µM) significantly reduced the levels of ANGPTL3 mRNA and intracellular and secreted ANGPTL3 proteins in hepatic cell lines. Furthermore, alleviation of secreted ANGPTL3 production by nobiletin was found to reinstate LPL catalytic activity. Nobiletin significantly inhibited ANGPTL3 promoter activity and attenuated the transcription factor liver X receptor-α (LXRα)-mediated ANGPTL3 transcription. Molecular docking analysis predicted that nobiletin could bind to the ligand-binding domain of LXRα, thereby counteracting LXRα activation. In animal studies, orally administered nobiletin significantly alleviated the levels of plasma triglycerides (TGs) and cholesterol in zebrafish fed a high-fat diet. Moreover, nobiletin significantly reduced the amounts of hepatic ANGPTL3 protein in zebrafish. Our findings suggest that nobiletin may regulate the LXRα-ANGPTL3-LPL axis and exhibit lipid-modulating effects in vitro and in vivo. Thus, nobiletin is a potential ANGPTL3 inhibitor for the regulation of lipid metabolism to ameliorate dyslipidemia and ASCVDs.

The Lipid-Modulating Effect of Tangeretin on the Inhibition of Angiopoietin-like 3 (ANGPTL3) Gene Expression through Regulation of LXRα Activation in Hepatic Cells.

The excessive accumulation of TG-rich lipoproteins (TGRLs) in plasma is associated with dyslipidemia and atherosclerotic cardiovascular diseases (ASCVDs). Tangeretin is a bioactive pentamethoxyflavone mainly found in citrus peels, and it has been reported to protect against hyperlipidemia, diabetes, and obesity. The aim of this study was to investigate the lipid-modulating effects and the underlying mechanisms of tangeretin action in hepatic cells. Transcriptome and bioinformatics analyses with the Gene Ontology (GO) database showed that tangeretin significantly regulated a set of 13 differentially expressed genes (DEGs) associated with the regulation of lipoprotein lipase (LPL) activity. Among these DEGs, angiopoietin-like 3 (ANGPTL3), an essential inhibitor of LPL catalytic activity that regulates TGRL metabolism in plasma, was markedly downregulated by tangeretin. We demonstrated that tangeretin significantly inhibited the mRNA expression of ANGPTL3 in HepG2 and Huh-7 cells. Tangeretin treatment of hepatic cells also reduced the levels of both intracellular and secreted ANGPTL3 proteins. Moreover, we found that inhibition of ANGPTL3 production by tangeretin augmented LPL activity. We further demonstrated that the transcriptional activity of the ANGPTL3 promoter was significantly attenuated by tangeretin, and we identified a DNA element located between the -250 and -121 positions that responded to tangeretin. Furthermore, we found that tangeretin did not alter the levels of the nuclear liver X receptor α (LXRα) protein, an essential transcription factor that binds to the tangeretin-responsive element, but it can counteract LXRα-mediated ANGPTL3 transcription. On the basis of molecular docking analysis, tangeretin was predicted to bind to the ligand-binding domain of LXRα, which would result in suppression of LXRα activation. Our findings support the hypothesis that tangeretin exerts a lipid-lowering effect by modulating the LXRα-ANGPTL3-LPL pathway, and thus, it can be used as a potential phytochemical for the prevention or treatment of dyslipidemia.

Angiopoietin-Like Protein 3 (ANGPTL3) Modulates Lipoprotein Metabolism and Dyslipidemia.

Dyslipidemia is characterized by increasing plasma levels of low-density lipoprotein-cholesterol (LDL-C), triglycerides (TGs) and TG-rich lipoproteins (TGRLs) and is a major risk factor for the development of atherosclerotic cardiovascular disorders (ASCVDs). It is important to understand the metabolic mechanisms underlying dyslipidemia to develop effective strategies against ASCVDs. Angiopoietin-like 3 (ANGPTL3), a member of the angiopoietin-like protein family exclusively synthesized in the liver, has been demonstrated to be a critical regulator of lipoprotein metabolism to inhibit lipoprotein lipase (LPL) activity. Genetic, biochemical, and clinical studies in animals and humans have shown that loss of function, inactivation, or downregulated expression of ANGPTL3 is associated with an obvious reduction in plasma levels of TGs, LDL-C, and high-density lipoprotein-cholesterol (HDL-C), atherosclerotic lesions, and the risk of cardiovascular events. Therefore, ANGPTL3 is considered an alternative target for lipid-lowering therapy. Emerging studies have focused on ANGPTL3 inhibition via antisense oligonucleotides (ASOs) and monoclonal antibody-based therapies, which have been carried out in mouse or monkey models and in human clinical studies for the management of dyslipidemia and ASCVDs. This review will summarize the current literature on the important role of ANGPTL3 in controlling lipoprotein metabolism and dyslipidemia, with an emphasis on anti-ANGPTL3 therapies as a potential strategy for the treatment of dyslipidemia and ASCVDs.

Tanshinone IIA Downregulates Lipogenic Gene Expression and Attenuates Lipid Accumulation through the Modulation of LXRα/SREBP1 Pathway in HepG2 Cells.

Abnormal and excessive accumulation of lipid droplets within hepatic cells is the main feature of steatosis and nonalcoholic fatty liver disease (NAFLD) or metabolic-associated fatty liver disease (MAFLD). Dysregulation of lipogenesis contributes to hepatic steatosis and plays an essential role in the pathological progress of MAFLD. Tanshinone IIA is a bioactive phytochemical isolated from Salvia miltiorrhiza Bunge and exhibits anti-inflammatory, antiatherosclerotic and antihyperlipidemic effects. In this study, we aimed to investigate the lipid-lowering effects of tanshinone IIA on the regulation of lipogenesis, lipid accumulation, and the underlying mechanisms in hepatic cells. We demonstrated that tanshinone IIA can significantly inhibit the gene expression involved in de novo lipogenesis including FASN, ACC1, and SCD1, in HepG2 and Huh 7 cells. Tanshinone IIA could increase phosphorylation of ACC1 protein in HepG2 cells. We further demonstrated that tanshinone IIA also could suppress the fatty-acid-induced lipogenesis and TG accumulation in HepG2 cells. Furthermore, tanshinone IIA markedly downregulated the mRNA and protein expression of SREBP1, an essential transcription factor regulating lipogenesis in hepatic cells. Moreover, we found that tanshinone IIA attenuated liver X receptor α (LXRα)-mediated lipogenic gene expression and lipid droplet accumulation, but did not change the levels of LXRα mRNA or protein in HepG2 cells. The molecular docking data predicted tanshinone IIA binding to the ligand-binding domain of LXRα, which may result in the attenuation of LXRα-induced transcriptional activation. Our findings support the supposition that tanshinone IIA possesses a lipid-modulating effect that suppresses lipogenesis and attenuates lipid accumulation by modulating the LXRα/SREBP1 pathway in hepatic cells. Tanshinone IIA can be potentially used as a supplement or drug for the prevention or treatment of MAFLD.

Xanthohumol Suppresses NPC1L1 Gene Expression through Downregulation of HNF-4α and Inhibits Cholesterol Uptake in Caco-2 Cells.

Xanthohumol (Xan) is a prenylated chalcone mainly found in hops; it has been demonstrated to function against hypercholesterolemia, hyperlipidemia, and atherosclerosis. In this study, we focused on the hypocholesterolemic effect of Xan on cholesterol uptake and the underlying molecular mechanisms of Xan in human intestinal Caco-2 cells. The microarray data showed that Niemann-Pick C1-like 1 (NPC1L1), an essential transporter for dietary cholesterol absorption, was significantly downregulated in Xan-treated Caco-2 cells. We demonstrated that Xan (10 and 20 µM) suppressed the mRNA and protein expression of NPC1L1 by 0.65 ± 0.12-fold and 0.54 ± 0.15-fold and 0.72 ± 0.04-fold and 0.44 ± 0.12-fold, respectively, compared to that of the vehicle-treated Caco-2 cells. Moreover, Xan (10 and 20 µM) significantly inhibited cholesterol uptake by approximately 12 and 32% in Caco-2 cells. NPC1L1 promoter activity was significantly suppressed by Xan, and a DNA element within the NPC1L1 promoter involved in Xan-mediated NPC1L1 reduction located between the -120 and -20 positions was identified. Moreover, Xan markedly decreased the mRNA and protein levels of hepatocyte nuclear factor 4α (HNF-4α), a critical activator of NPC1L1 transcription, and subsequently attenuated HNF-4α/NPC1L1 promoter complex formation, resulting in the suppression of NPC1L1 gene expression. Finally, we demonstrated that Xan markedly abolished lovastatin-induced NPC1L1 overexpression in Caco-2 cells. These findings reveal that Xan suppresses NPC1L1 expression via downregulation of HNF-4α and exerts inhibitory effects on cholesterol uptake in the intestinal Caco-2 cells. Our findings suggest Xan could serve as a potential cholesterol-lowering agent and supplement for statin therapy.

The Cholesterol-Modulating Effect of Methanol Extract of Pigeon Pea (Cajanus cajan (L.) Millsp.) Leaves on Regulating LDLR and PCSK9 Expression in HepG2 Cells.

Pigeon pea (Cajanus cajan (L.) Millsp.) is a legume crop consumed as an indigenous vegetable in the human diet and a traditional medicinal plant with therapeutic properties. The current study highlights the cholesterol-modulating effect and underlying mechanisms of the methanol extract of Cajanus cajan L. leaves (MECC) in HepG2 cells. We found that MECC increased the LDLR expression, the cell-surface LDLR levels and the LDL uptake activity in HepG2 cells. We further demonstrated that MECC suppressed the proprotein convertase subtilisin/kexin type 9 (PCSK9) mRNA and protein expression, but not affected the expression of other cholesterol or lipid metabolism-related genes including inducible degrader of LDLR (IDOL), HMG-CoA reductase (HMGCR), fatty acid synthase (FASN), acetyl-CoA carboxylase (ACC1), and liver X receptor-α (LXR-α) in HepG2 cells. Furthermore, we demonstrated that MECC down-regulated the PCSK9 gene expression through reducing the amount of nuclear hepatocyte nuclear factor-1α (HNF-1α), a major transcriptional regulator for activation of PCSK9 promoter, but not that of nuclear sterol-responsive element binding protein-2 (SREBP-2) in HepG2 cells. Finally, we identified the cajaninstilbene acid, a main bioactive stilbene component in MECC, which significantly modulated the LDLR and PCSK9 expression in HepG2 cells. Our current data suggest that the cajaninstilbene acid may contribute to the hypocholesterolemic activity of Cajanus cajan L. leaves. Our findings support that the extract of Cajanus cajan L. leaves may serve as a cholesterol-lowering agent.

Nobiletin Down-Regulates c-KIT Gene Expression and Exerts Antileukemic Effects on Human Acute Myeloid Leukemia Cells.

Nobiletin, a dietary citrus flavonoid, has been reported to possess several biological activities such as antioxidant, anti-inflammatory, and anticancer properties. The aim of this study was to investigate the antileukemic effects of nobiletin and its underlying mechanisms on human acute myeloid leukemia (AML) cells. We demonstrated that nobiletin (0-100 µM) significantly reduced cell viability from 100.0 ± 9.6% to 31.1 ± 2.8% in human AML THP-1 cell line. Nobiletin arrested cell cycle progression in G1 phase and induced myeloid cell differentiation in human AML cells. Microarray analysis showed that mRNA expression of the c- KIT gene, a critical proto-oncogene associated with leukemia progression, was dramatically reduced in nobiletin-treated AML cells. Furthermore, we verified that AML cells treated with nobiletin (40 and 80 µM) for 48 h markedly suppressed c-KIT mRNA expression (from 1.00 ± 0.07-fold to 0.62 ± 0.08- and 0.30 ± 0.05-fold) and reduced the level of c-KIT protein expression (from 1.00 ± 0.11-fold to 0.60 ± 0.15- and 0.34 ± 0.05-fold) by inhibition of KIT promoter activity. The knockdown of c-KIT expression by shRNA attenuated cancer cell growth and induced cell differentiation. Moreover, we found that the overexpression of c-KIT abolished nobiletin-mediated cell growth inhibition in leukemia cells. These results indicate that nobiletin exerts antileukemic effects through the down-regulation of c-KIT gene expression in AML cells. Finally, we demonstrated that the combination of a conventional AML chemotherapeutic agent, cytarabine, with nobiletin resulted in more reduction of cell viability in AML cells. Our current findings suggest that nobiletin is a novel c-KIT inhibitor and may serve as a chemo-preventive or -therapeutic agent against human AML.

Pinostrobin Inhibits Proprotein Convertase Subtilisin/Kexin-type 9 (PCSK9) Gene Expression through the Modulation of FoxO3a Protein in HepG2 Cells.

Pinostrobin, a flavonoid phytochemical found in variety of plants, has been demonstrated to possess numerous bioactivities such as antioxidant, anti-inflammatory, anticancer, and neuroprotective properties. The aim of this study was to investigate the hypocholesterolemic effect of pinostrobin on the regulation of the gene expression of PCSK9 and its underlying mechanisms in hepatic cells. We found that pinostrobin (20 and 40 µM) significantly inhibited the PCSK9 promoter activity from 1.00 ± 0.16 (fold) to 0.85 ± 0.06 and 0.54 ± 0.05, respectively, as well as the suppression of PCSK9 mRNA expression from 1.00 ± 0.11 (fold) to 0.81 ± 0.07 and 0.58 ± 0.07, respectively, in HepG2 cells. Pinostrobin significantly reduced the mature form of the PCSK9 protein, inhibited the catalytic activity of PCSK9, and increased the protein level of LDLR and the LDL uptake activity in HepG2 cells. We further demonstrated that pinostrobin markedly increased the level of nuclear forkhead box O3a (FoxO3a) protein, enhanced FoxO3a/PCSK9 promoter complexes formation, and attenuated the promoter binding capacity of nuclear HNF-1α. The knockdown of FoxO3a in HepG2 cells by small interference RNA (siRNA) abolished the pinostrobin-mediated PCSK9 reduction. Finally, we demonstrated that pinostrobin attenuated simvastatin-induced PCSK9 overexpression in HepG2 cells. Our current findings reveal that pinostrobin is a PCSK9 inhibitor and down-regulates the PCSK9 gene expression through the up-regulation of the FoxO3a level in hepatic cells. Pinostrobin with potential PCSK9 inhibitory activity may serve as a novel agent for cholesterol regulation and lipid management.