Search Results (358)

Background and Rationale: Tinospora crispa (L.) Hook.f. & Thomson (T. crispa) is a climbing medicinal plant with long-standing ethnopharmacological use, particularly in inflammatory and hepatic disorders and cancer-related conditions. There is a knowledge gap regarding how wild versus cultivated ecotypes differ in chemotype, bioactivity, and safety, and how this might support or refine traditional use. Study Objectives: This study aimed to compare wild and cultivated ecotypes of T. crispa from the Nile Delta (Egypt) in terms of quantitative and qualitative phytochemical profiles; selected in vitro biological activities (especially antioxidant and cytotoxic actions); genetic markers potentially associated with metabolic variation; and short-term oral safety in an animal model. Core Methodology: Standardized extraction of plant material from wild and cultivated ecotypes. Determination of total phenolics, total flavonoids, and major phytochemical classes (alkaloids, tannins, terpenoids). Metabolomic characterization using UHPLC-ESI-QTOF-MS, supported by NMR, to confirm key compounds such as berberine, palmatine, chlorogenic acid, rutin, and borapetoside C. In vitro bioassays including: Antioxidant activity (e.g., radical-scavenging assay with EC₅₀ determination). Cytotoxicity against human cancer cell lines, with emphasis on HepG2 hepatoma cells and calculation of IC₅₀ values. Targeted genetic analysis to detect single-nucleotide polymorphisms (SNPs) in the gen1 locus that differentiate ecotypes. A 14-day oral toxicity study in rats, assessing liver and kidney function markers and performing histopathology of liver and kidney tissues. Principal Results: The wild ecotype showed a 43–65% increase in total flavonoid and polyphenol content compared with the cultivated ecotype, as well as substantially higher levels of key alkaloids, particularly berberine (around 12.5 ± 0.8 mg/g), along with elevated chlorogenic acid and borapetoside C. UHPLC-MS and NMR analyses confirmed the identity of the main bioactive constituents and defined a distinct chemical fingerprint for the wild chemotype. Bioassays demonstrated stronger antioxidant activity of the wild extract than the cultivated one and selective cytotoxicity of the wild extract against HepG2 cells (IC₅₀ ≈ 85 µg/mL), being clearly more potent than extracts from cultivated plants. Genetic profiling detected a C → T SNP within the gen1 region that differentiates the wild ecotype and may be linked to altered biosynthetic regulation. The 14-day oral toxicity study (up to 600 mg/kg) revealed no evidence of hepatic or renal toxicity, with biochemical markers remaining within physiological limits and normal liver and kidney histology. Conclusions and Future Perspectives: The wild Nile-Delta ecotype of T. crispa appears to be a stress-adapted chemotype characterized by enriched levels of multiple bioactive metabolites, superior in vitro bioactivity, and an encouraging preliminary safety margin. These findings support further evaluation of wild T. crispa as a candidate source for standardized botanical preparations targeting oxidative stress-related and hepatic pathologies, while emphasizing the need for: More comprehensive in vivo efficacy studies. Cultivation strategies that deliberately maintain or mimic beneficial stress conditions to preserve phytochemical richness. Broader geographical and genetic sampling to assess how generalizable the present chemotypic and bioactivity patterns are across the species. Full article

Branched-chain amino acids (BCAAs) are essential amino acids in humans, with reported anti-proliferative effects on HepG2 hepatoma cells and the potential to reduce hepatocellular carcinoma (HCC) development in cirrhotic patients. PDCD4, a tumor suppressor that is downregulated in many cancers, is also suppressed by serum, EGF, or TPA treatment. This study examined the effect BCAA has on PDCD4 expression and related cellular pathways in Huh7 hepatoma cells. Cells were treated with different concentrations of BCAA, and analyzed by Western blotting, qRT-PCR, and immunofluorescence staining. Treatment with BCAA upregulated the protein levels of PDCD4, while downregulating its mRNA levels. BCAA enhanced the phosphorylation of mTORC1 substrate 4E-BP1, p70S6K1, and p70S6K1 substrate S6 ribosomal protein. BCAA also elevated the protein levels of autophagy factors p62 and ATG5 while reducing LC3-II particle formation, thus indicating impaired autophagy. ULK1 knockdown also upregulated the protein levels of PDCD4 and p62. Additionally, BCAA upregulated the phosphorylation of ULK1 at serine 757, which was inhibited by rapamycin. These findings suggest that BCAA inhibits autophagy through the mTORC1-mediated phosphorylation of ULK1 at serine 757, thereby impairing autophagosome formation and upregulating the PDCD4 protein levels by inhibiting its degradation via autophagy. Furthermore, FACS analysis showed that BCAA inhibited the proliferation of Huh7 cells. BCAA may have a preventive effect against tumor development through the modulation of autophagy and the tumor suppressor pathways. Full article

Lipid peroxide (LPO) accumulation and a depletion of intracellular antioxidants are hallmarks of ferroptosis, a controlled iron-dependent form of cell death. Iron chelators and radical scavengers can stop it, while erastin or iron overload can cause it. The main catechin in green tea extract (GTE), epigallocatechin-3-gallate (EGCG), has iron-chelating and antioxidant activities. Herein, we investigated the effects of EGCG-rich GTE on ferroptosis in iron-loaded hepatocytes. The contents of EGCG, total phenolics (TPC), and flavonoids (TFC), as well as ABTS^•+-scavenging activity and cytotoxicity, were determined. Human hepatoma (Huh7) cells were treated with ferric ammonium citrate (FAC) to induce ferroptosis and were co-treated with various concentrations of GTE. Labile iron pool (LIP), reactive oxygen species (ROS), LPO, glutathione (GSH), and glutathione peroxidase 4 (GPX-4) activity were then measured in the cells. One gram of GTE contained 26 mg of EGCG, with a TPC of 172.2 mg gallic acid equivalents and a TFC of 32.9 mg quercetin equivalents. GTE displayed concentration-dependent ABTS^•+-scavenging activity (IC₅₀ = 1.03 mg) that was equivalent to 0.29 mg of Trolox, reporting a Trolox-equivalent antioxidant capacity (TEAC) value of 0.29 mg. High-dose GTE (>100 µM EGCG equivalent) reduced cell viability below 80% (p < 0.05). Intracellular LIP, ROS, and LPO levels were markedly elevated, whereas GSH and GPX-4 activity levels were decreased (p < 0.05) in iron-loaded Huh7 cells. GTE treatment mitigated these alterations in a dose-dependent manner (p < 0.05). These cell-based in vitro findings indicate that EGCG-rich GTE can attenuate ferroptosis-associated oxidative stress in hepatocytes under iron-loading conditions. GTE may serve as a potential dietary antioxidant candidate; further mechanistic studies and in vivo experiments are required to determine its physiological relevance and translational applicability. Full article

Objectives: Aqueous mulberry leaf extract (MLE) contains 1-deoxynojirimycin (DNJ) and L-leucine (LL). This study investigated the effects of MLE, DNJ, and LL on lipid accumulation caused by palmitic acid (PA) in human hepatoma HepG2 cells, pro-inflammatory cytokine levels, and regulation of lipogenesis. Methods: PA was applied to HepG2 cells to generate a fatty liver in vitro model. Then, the cells were treated with MLE, DNJ, or LL for 24 h. Western blot analysis was performed to determine the protein expression levels of peroxisome proliferator-activated receptor gamma (PPAR-γ) and fatty acid synthase (FAS) in HepG2 cells. Results: Staining with Oil Red O (ORO) indicated that MLE, DNJ, and LL significantly decreased excessive lipid accumulation in HepG2 cells. Cytokine ELISA assay indicated that MLE, DNJ, and LL significantly decreased excessive pro-inflammatory cytokine levels in HepG2 cells. In addition, MLE, DNJ, and LL decreased the protein expression levels of PPAR-γ and FAS, suggesting a potential suppression of lipogenesis. Conclusions: Our results suggest that MLE, DNJ, and LL reduce lipid accumulation, pro-inflammatory cytokine levels, and the protein expressions of FAS and PPAR-γ in PA-induced fatty liver cells. Full article

Age-related macular degeneration (AMD) is driven in part by the accumulation of reactive metabolites like glyoxal (GO), which induces retinal pigment epithelium (RPE) degeneration. Here, we demonstrate that GO triggers ferroptosis in human ARPE-19 cells, as characterized by iron-dependent lipid peroxidation, glutathione depletion, and reactive oxygen species (ROS) accumulation. This ferroptotic cell death is coupled with profound mitochondrial dysfunction, featuring network fragmentation and the downregulation of the key regulators MFN2, PGC-1α, and SIRT1. We identify hepatoma-derived growth factor (HDGF) as a potent protector against GO-induced damage. HDGF operates through a dual mechanism: it activates the p38 MAPK/AKT and SIRT1/PGC-1α axes to restore mitochondrial biogenesis and homeostasis, while concurrently enhancing the glutathione/GPX4 antioxidant system to suppress ferroptosis. This cytoprotective action is mediated via the PGC-1α/Nrf2 pathway, which integrates the enhancement of antioxidant defenses with the preservation of mitochondrial integrity. Our findings establish HDGF as a novel therapeutic agent for AMD, uniquely capable of concurrently targeting the interconnected pathways of ferroptosis and mitochondrial dysfunction, thereby addressing a critical unmet need in retinal disease treatment. Full article

The efficacy of X-ray-induced photodynamic therapy (X-PDT) for deep tumors is often hindered by conventional scintillators, typically rare-earth nanoparticles plagued by long-term toxicity and suboptimal scintillation yields. Here, we introduce a copper iodide (Cu-I) cluster, Cu₂I₂(PPh₃)₂(pz), composed of earth-abundant elements, as an efficient and biocompatible energy transducer for X-PDT. A theranostic nanoplatform, CuI@PcNP, was engineered by co-encapsulating the Cu-I cluster and a phthalocyanine photosensitizer (Pc4OH) within a 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol-2000 (DSPE-PEG2K) matrix, which confers excellent physiological stability. This nano-architecture ensures nanoscale proximity between the cluster (donor) and photosensitizer (acceptor), facilitating efficient (58%) Förster resonance energy transfer (FRET) while overcoming aggregation-induced quenching. Upon X-ray irradiation, the platform effectively converted X-rays to visible light, activating Pc4OH to generate potent reactive oxygen species (ROS) and inducing significant dose-dependent cytotoxicity in human hepatocellular carcinoma (HepG2) cells. In a murine hepatoma model, enabling image-guided X-PDT that resulted in a 77.4% tumor inhibition rate with negligible systemic toxicity. Collectively, this work pioneers the integration of phthalocyanine with Cu-I clusters, providing a stable and versatile nanoplatform for image-guided X-PDT. Full article

Respiratory viral infections are a major cause of morbidity and mortality worldwide. The COVID-19 pandemic has evidenced the need for broad-spectrum antivirals and improved preclinical models that more accurately recapitulate human respiratory disease. These new strategies should also involve the search for drug targets in the infected cell that hamper the development of resistance and of potential efficacy against diverse viruses. Since many viruses reprogram cellular metabolism to support viral replication, we performed a comparative analysis of inhibitors targeting the PI3K/AKT/mTOR pathway, central to virus-induced metabolic adaptations, using MRC5 lung fibroblasts and Huh7 hepatoma cells. HCoV-229E infection in MRC5 cells caused the expected shift in the energy metabolism but the inhibitors had markedly different effects on the metabolic profile and antiviral activity in these two cell lines. Dichloroacetate (DCA), a clinically approved inhibitor of aerobic glycolysis, showed antiviral activity against HCoV-229E in MRC5 cells, but not in Huh7 cells, underscoring that the screening model is more critical than previously assumed. We further tested DCA in polarized human small airway epithelial cells cultured in air–liquid interface, a 3D model that mimics the human respiratory tract. DCA reduced the viral progeny of HCoV-229E, SARS-CoV-2, and respiratory syncytial virus by 2–3 orders of magnitude, even when administered after infection was established. Our work reinforces the need for advanced human preclinical screening models to identify antivirals that target host metabolic pathways frequently hijacked by respiratory viruses, and establishes DCA as a proof-of-concept candidate. Full article

Increased hepatic triacylglycerol (TG) storage in lipid droplets (LDs) is a hallmark of metabolic dysfunction-associated steatotic liver disease (MASLD) and metabolic dysfunction-associated steatohepatitis (MASH). Human carboxylesterase 1 (CES1) regulates TG storage and secretion in hepatocytes, but the mechanism remains to be elucidated. We performed studies in rat hepatoma McArdle RH7777 cells stably transfected with CES1 cDNA and in Ces1d-deficient mice using a variety of biochemical, pharmacological and cell biology approaches including the assessment of gene expression, confocal immunofluorescence microscopy, lipid synthesis measurements and quantitative mass spectrometry. CES1-expressing cells accrued more TG compared to cells lacking CES1 when incubated with oleic acid. CES1 increased the expression of Srebf1c, Nr1h3 and Nr1h2 encoding transcription factors (SREBP1c and LXRα and LXRβ, respectively) that regulate the expression of lipogenic genes. Additionally, CES1 increased the expression of Acsl1 encoding an enzyme catalyzing fatty acid activation and the expression of Dgat1 and Dgat2 encoding enzymes catalyzing TG synthesis. Treatment of CES1-expressing cells with PPARγ antagonist (GW9662), LXR antagonist (GSK2033) or CYP27A1 inhibitor Felodipine prevented CES1-mediated fatty acid esterification into TG. Ces1d-deficient mice fed high-fat diet (HFD) presented with decreased expression of Nr1h3, Nr1h2, Srebf1c and reduced hepatic TG content. Felodipine and GSK2033 treatment eliminated the differential effects on TG concentration between wild-type and Ces1d-deficient hepatocytes. The results suggest that CES1/Ces1d activates PPARγ, LXR and SREBP1c pathways, thereby increasing TG synthesis and LD storage by augmenting fatty acid esterification. Full article

Oral Candida infections result from the overgrowth of this opportunistic fungus in the oral mucosa. Risk factors include immunosuppression, antibiotic or corticosteroid use, xerostomia, and conditions such as diabetes mellitus. Fungal resistance in Candida spp. has become a significant challenge, especially due to the excessive use of conventional antifungals such as azoles, echinocandins, and polyenes. Therefore, this study aims to determine the spectrum of antifungal activity of Juglans regia and assess its cytotoxicity on hepatocytes. Thus, a broth microdilution test was conducted according to the CLSI (M27-A3) guidelines. After initial screening, biofilm tests were conducted using the crystal violet (CV) and metabolic activity assays (MTT). Cytotoxicity was evaluated on human hepatocytes (HEPG2). The J. regia extract showed dose-dependent antifungal activity. At a concentration of 200 mg/mL, inhibition was greater according to the CV test in Candida albicans (31%) and Candida tropicalis (30.4%), while the MTT assay indicated a greater reduction in viability in C. albicans (61%) and C. glabrata (53.5%). At 100 mg/mL, C. albicans remained sensitive (37.7% CV; 71.6% MTT), while C. krusei and C. dubliniensis showed low viability by MTT (18.4% and 11.8%, respectively). At 50 mg/mL, C. albicans remained affected (74.3% MTT), but C. krusei, C. dubliniensis, and C. guilliermondii showed the lowest viability values (≤19.4% MTT), suggesting greater sensitivity to lower concentrations. These results indicate variation in susceptibility between species, with C. albicans being consistently inhibited, while C. krusei and C. dubliniensis responded better to lower doses. The extract showed cytocompatibility when applied to human hepatoma cells (HEPG2) and therefore holds significant potential for developing a new therapeutic approach. Full article

Micromeria graeca (L.) Benth. ex Rchb. (Lamiaceae) is a promising medicinal plant valued for its antioxidant, anti-hyperglycemic, anti-hypertensive, antimicrobial, and anti-aflatoxigenic properties. It is rich in phenolic and flavonoid compounds, supporting its traditional use for digestive, respiratory, cardiovascular, and dermatological conditions. Plant tissue culture facilitates controlled in vitro propagation to study plant growth and bioactive properties. The effects of activated charcoal and varying subculture intervals on multiplication and biomass production in M. graeca shoot cultures were investigated. The phenolic composition of methanolic extracts from in vitro-grown plants was characterized using high-performance liquid chromatography (HPLC), identifying rosmarinic, caffeic, and syringic acids as the primary phenolic compounds. Antimicrobial activity against selected microbial strains was evaluated using a micro-well dilution assay. Anticancer activity of selected extracts was assessed in human hepatocellular carcinoma cell line HepG2, with flow cytometry (Annexin-V/PI staining) used to analyze cell death mechanisms, and compared to pure rosmarinic acid (RA). Activated charcoal showed no beneficial effects on multiplication or biomass production, but significantly increased phenolic acid content (up to 4-fold). RA dominated the phenolic profiles, with other phenolic acids present in lower amounts. Methanolic extracts exhibited negligible antimicrobial activity compared to reference antibiotics and fungicide. Extracts from 4-week-old shoot cultures displayed modest anti-hepatoma activity (IC50 values of CV assay ranging from 193 to 274 µg mL⁻¹), inducing HepG2 cell apoptosis via oxidative stress, independent of RA. Our results suggest that the metabolic output of M. graeca shoot cultures and consequently their biological activity can be modulated by varying in vitro culture conditions. These findings underscore the potential of their methanolic extracts for biotechnological production and therapeutic applications. Full article

Metabolic rewiring is a hallmark of both hepatic regeneration and malignant transformation, complicating the identification of cancer-specific traits. This study aimed to distinguish the metabolic profiles of proliferating hepatocytes and hepatocellular carcinoma (HCC) cells through integrated analyses of mRNA and protein expression, along with functional characterization. We compared non-malignant Upcyte^® hepatocytes (HepaFH3) cultured under proliferative and confluent conditions with primary human hepatocytes, primary human hepatoma cells, and hepatoma cell lines. Proliferating HepaFH3 cells exhibited features of metabolic reprogramming, including elevated glycolysis, increased HIF1A expression, and ketone body accumulation, while maintaining low c-MYC expression and reduced BDH1 levels, distinguishing them from malignant models. In contrast, HCC cells showed upregulation of HK2, c-MYC, and BDH1, reflecting a shift toward aggressive glycolytic and ketolytic metabolism. Functional assays supported the transcript and protein expression data, demonstrating increased glucose uptake, elevated lactate secretion, and reduced glycogen storage in both proliferating and malignant cells. These findings reveal that cancer-like metabolic changes also occur during hepatic regeneration, limiting the diagnostic utility of individual metabolic markers. HepaFH3 cells thus provide a physiologically relevant in vitro model to study regeneration-associated metabolic adaptation and may offer insights that contribute to distinguishing regenerative from malignant processes. Our findings highlight the potential of integrated metabolic profiling in differentiating proliferation from tumorigenesis. Full article

A dietary supplement, trans-resveratrol and hesperetin (tRES+HESP)—also known as GlucoRegulate—induces increased expression of glyoxalase 1 (Glo1) by activation of transcription factor Nrf2, countering accumulation of the reactive dicarbonyl glycating agent, methylglyoxal. tRES+HESP corrected insulin resistance and decreased fasting and postprandial plasma glucose and low-grade inflammation in overweight and obese subjects in a clinical trial. The aim of this study was to explore, for the first time, health-beneficial gene expression other than Glo1 induced by tRES+HESP in human endothelial cells and fibroblasts in primary culture and HepG2 hepatoma cell line and activity of cis-resveratrol (cRES) as a Glo1 inducer. We measured antioxidant response element-linked gene expression in these cells in response to 5 µM tRES+HESP by the NanoString method. tRES+HESP increases gene expression linked to the prevention of dicarbonyl stress, lipid peroxidation, oxidative stress, proteotoxicity and hyperglycemia-linked glycolytic overload. Downstream benefits were improved regulation of glucose and lipid metabolism and decreased inflammation, extracellular matrix remodeling and senescence markers. The median effective concentration of tRES was ninefold lower than cRES in the Glo1 inducer luciferase reporter assay. The GlucoRegulate supplement provides a new treatment option for the prevention of type 2 diabetes and metabolic dysfunction–associated steatotic liver disease and supports healthy aging. Full article

Objectives: The aim of this work was to determine the phenolic composition of sweet wormwood (Artemisia annua L., Asteraceae) from controlled cultivation in Serbia and to assess the potential antioxidant effects and cytotoxicity. Methods: High-performance liquid chromatography was used to determine the phenolic composition of Artemisia annua ethanolic extract. The antioxidant activity was studied using in vitro tests of inhibition of the neutralization of 2,2-diphenyl-1-picrylhydrazyl (DPPH), hydroxyl (OH), and nitroso (NO) radicals, as well as the process of inhibiting lipid peroxidation and the ferric reducing antioxidant power (FRAP). The cytotoxicity was evaluated by the effect on three cell lines (the rat pancreatic insulinoma cell line (Rin-5F), the rat hepatoma cell line (H4IIE), and human hepatocellular carcinoma (Hep G2)) using the MTT test of viability. Results: Ethanol extract showed the highest potency in inhibiting the DPPH radical, and the half maximal inhibitory concentration (IC₅₀) was 5.17 μg/mL. Chlorogenic acid was the dominant phenolic compound with an amount of 651 μg/g of dry extract. The results of the MTT viability test showed that the extract has the potential to inhibit the growth of the Rin-5F and Hep G2 cell lines, while no growth inhibition was observed on the H4IIE cell line. Conclusions: Undoubtedly, Artemisia annua is a powerful plant and a rich source of phenolic compounds. Inhibitory activity on causes of oxidative stress shows that the plant has a good antioxidant effect. Also, the anticancer activity shown through the inhibition of cell growth is not negligible. Full article

Changes in hepatic lipoprotein metabolism are responsible for the majority of metabolic dysfunction-associated disorders, including familial hypercholesterolemia (FH), metabolic syndrome (MetS), metabolic dysfunction-associated fatty liver disease (MAFLD), and age-related diseases such as atherosclerosis, a major health burden in modern society. This review aims to advance the understanding of state-of-the-art mechanistic concepts in lipoprotein metabolism, with a particular focus on lipoprotein uptake and secretion and their dysregulation in disease, and to provide a comprehensive overview of experimental models used to study these processes. Human lipoprotein research faces several challenges. First, significant differences in lipoprotein metabolism between humans and other species hinder the reliability of non-human model systems. Additionally, ethical constraints often limit studies on human lipoprotein metabolism using tracers. Lastly, while 2D hepatocyte cell culture systems are widely used, they are commonly of cancerous origins, limiting their physiological relevance and necessitating the use of more physiologically representative models. In this review, we will elaborate on key findings in lipoprotein metabolism, as well as limitations and challenges of currently available study tools, highlighting mechanistic insights throughout discussion of these models. These include human tracer studies, animal studies, 2D tissue culture-based systems derived from cancerous tissue as well as from induced pluripotent stem cells (iPSCs)/embryonic stem cells (ESCs). Finally, we will discuss precision-cut liver slices, liver-on-a-chip models, and, particularly, improved 3D models: (i) spheroids generated from either hepatoma cancer cell lines or primary human hepatocytes and (ii) organoids generated from liver tissues or iPSCs/ESCs. In the last section, we will explore future perspectives on liver-in-a-dish models in studying mechanisms of liver diseases, treatment options, and their applicability in precision medicine approaches. By comparing traditional and advanced models, this review will highlight the future directions of lipoprotein metabolism research, with a focus on the growing potential of 3D liver organoid models. Full article

Foodborne trematode infections are recognized as a significant risk factor for cholangiocarcinoma (CCA) in endemic regions. Infection with the liver fluke Opisthorchis felineus induces precursor lesions of CCA, including the biliary intraepithelial neoplasia. The mechanisms underlying liver-fluke-associated neoplasia remain poorly understood. This study aims to identify the role of EGFR and Toll-like receptor 4-associated signaling pathways in bile duct epithelial neoplasia linked to liver fluke infection in patients, animal models, and cell models. Elevated levels of EGFR and phosphorylated EGFR were observed in the bile duct epithelium of patients with cholangiocarcinoma, as well as in the bile duct epithelium of laboratory hamsters. The EGFR content correlated with the degree of bile duct epithelial neoplasia. Additionally, a significant increase in the cell proliferation and migration rates of human H69 cholangiocytes was found, whereas those of HepG2 hepatoma cells remained unaffected following the helminth excretory–secretory product (ESP) treatment. An EGFR inhibitor eliminated the enhanced cell proliferation (p = 0.005) and migration (p = 0.001) rates. Similar outcomes were achieved using Marimastat, an inhibitor of TLR-4-associated metalloproteinases. Thus, our study unveils novel avenues for exploring the mechanisms of helminth-associated carcinogenesis and for identifying key components of ESPs that mediate their mitogenic effects. Full article