Induced HepG2 Cells Research Articles

Thiazolidinedione-Conjugated Lupeol Derivatives as Potent Anticancer Agents Through a Mitochondria-Mediated Apoptotic Pathway.

To improve the potential of lupeol against cancer cells, a privileged structure, thiazolidinedione, was introduced into its C-3 hydroxy group with ester, piperazine-carbamate, or ethylenediamine as a linker, and three series of thiazolidinedione-conjugated compounds (6a-i, 9a-i, and 12a-i) were prepared. The target compounds were evaluated for their cytotoxic activities against human lung cancer A549, human breast cancer MCF-7, human hepatocarcinoma HepG2, and human hepatic LO2 cell lines, and the results revealed that most of the compounds displayed improved potency over lupeol. Compound 12i exhibited significant activity against the HepG2 cell line, with an IC50 value of 4.40 μM, which is 9.9-fold more potent than lupeol (IC50 = 43.62 μM). Mechanistic studies suggested that 12i could induce HepG2 cell apoptosis, as evidenced by AO/EB staining and annexin V-FITC/propidium iodide dual staining assays. Western blot analysis suggested that compound 12i can upregulate Bax expression, downregulate Bcl-2 expression, and activate the mitochondria-mediated apoptotic pathway. Collectively, compound 12i is worthy of further investigation to support the discovery of effective agents against cancer.

Fucoidan from Sargassum hemiphyllum (Turner) C. Agardh inhibits human hepatoma cell (HepG2) through cell cycle arrest and apoptotic induction

Cancer has become one of the most serious diseases threatening human health, while traditional chemotherapeutic drugs have serious side effects. Drugs of marine origin have attracted many scholars due to their remarkable effectiveness, low drug resistance and low adverse reactions. In this study, the fucoidan DF1 and DF2 extracted from Sargassum hemiphyllum (Turner) C. Agardh were used to explore their effects on induced HepG2 hepatoma cell apoptosis and their relevant molecular mechanisms of apoptosis in HepG2 cells were analyzed by flow cytometry and western blotting assay. DF1 and DF2 could induce excess intracellular reactive oxygen species (ROS) production in HepG2 cells and lead to stimulation of the mitochondrial apoptotic pathway, up-regulate the protein expressions of Bax, Cytochrome C and Cleaved-Caspase-3/8, and down-regulate the protein expressions of Bcl-2 and Caspase-3/8/9. Meanwhile, DF1 and DF2 could induce HepG2 cell cycle arrest in the S-phase and up-regulate the protein expression of p21, and down-regulate the expression of Cyclin A and CDK2. This study provides a scientific basis for the development of VC/H2O2-treated fucoidans as anticancer drugs or adjuvant drugs.

Mechanism investigation of anti-NAFLD of Shugan Yipi Granule based on network pharmacology analysis and experimental verification

As a classical traditional Chinese patent medicine, Shugan Yipi Granule is widely used in China to treat non-alcoholic fatty liver disease (NAFLD) recently. Our previous study confirmed that Shugan Yipi Granule are effective in NAFLD. However, its underlying mechanism is still unknown. This study aims to investigate the mechanism of Shugan Yipi Granule on NAFLD based on network pharmacology prediction, liquid chromatography-mass spectrometry (LC-MS) analysis and in vitro verification. We obtained the active ingredients and targets of Shugan Yipi Granule and NAFLD from 6 traditional Chinese medicine databases, and the crucial components and targets screened by protein-protein interaction (PPI) network were used for molecular docking. Plasma metabolomics of NAFLD patients treated with Shugan Yipi Granule for one month was analyzed using LC-MS methods and MetaboAnalyst 4.0 to obtain significant differential metabolites and pathways. Finally, free fatty acid (FFA) induced HepG2 cells were treated with different concentrations of quercetin and kaempferol, then oil red o (ORO) and triglyceride (TG) level were tested to verify the lipid deposition of the cell. Network pharmacology analysis showed that the main active ingredients of Shugan Yipi Granule include quercetin, kaempferol and other 58 ones, as well as 188 potential targets. PI3K/Akt signaling pathway was found to be the most relevant pathway for the treatment of NAFLD. Non-targeted metabolomics showed that quercetin and kaempferol were significantly up-regulated differential metabolites and were involved in metabolic pathways such as thyroid hormone signaling. In vitro results showed that quercetin, kaempferol were effective in reducing lipid deposition and TG content by inhibiting cellular fatty acid uptake. Ultimately, with the network pharmacology and serum metabolomics analysis, quercetin and kaempferol were found to be the important active ingredients and significantly up-regulated differential metabolites of Shugan Yipi Granule against NAFLD, which we inferred that they may regulate NAFLD through PI3K/Akt signaling pathway and thyroid hormone metabolism pathway. The in vitro experiment verification results showed that quercetin and kaempferol attenuated the lipid accumulation and TG content by inhibiting the fatty acid uptake in the FFA-induced HepG2 cell. Current study provides the necessary experimental basis for subsequent in-depth mechanism research.

Cardiac glycosides with cytotoxic activity from the seeds of Thevetia peruviana

Phytochemical investigation on the extract of the seeds of Thevetia peruviana resulted in the isolation of six new cardiac glycosides, namely theveperosides A-F (1–6), including a rare 19-nor-cardenolide (1), together with seven known analogues (7–13). The chemical structures of these compounds were determined based on detailed spectroscopic analysis. The cytotoxic activities of 1–13 were evaluated against MCF-7, HCT-116, HeLa, and HepG2 cancer cell lines, and their structure-activity relationships (SARs) were investigated. Compound 3 exhibited the significant cytotoxic effects with IC50 values ranging from 0.032 to 0.055 μΜ, which could induce HepG2 cells apoptosis in a dose-dependent manner.

Lappanolides A−N, fourteen undescribed sesquiterpenoids from Saussurea costus (Syn. Saussurea lappa) and their anti HBV activity

Lappanolides A−N (1–14), 14 undescribed sesquiterpenoids, along with 23 known ones (15–37), were isolated from the roots of Saussurea costus, which were primarily categorized into eudesmane, guaiane, and germacrane types. Lappanolide A (1) possessed an unprecedented pseudo-disesquiterpenoids. Their structures and absolute configurations were established using physical data analyses (HRESIMS, IR, 1D and 2D NMR) and ECD calculations. All isolated compounds were tested for anti-hepatitis B virus (anti-HBV) activity. Ten compounds (1, 9, 11, 12, 19, 22, 28, 29, 31, and 36) exhibited activities against HBsAg secretions as determined by ELISA assay, with IC50 values ranging from 5.2 to 45.7 μM. In particular, compounds 28 and 29 showed inhibition of HBsAg secretion with IC50 values of 5.28 and 5.30 μM, and CC50 values of 9.85 and 6.37 μM, respectively, though they all exhibited low selectivity. Several compounds displayed cytotoxicity in the MTT assay. Among them, compound 28 was the most notable and was chosen for further study using flow cytometry. The result showed that it significantly induced HepG2 cell arrest in the S phase and induced apoptosis.

Design and Synthesis of Gefitinib Derivatives as Potential Drugs fo r Cancer Treatment: Antiproliferative Activity, Molecular Docking, and ADMET Prediction

Background: Non-small cell lung cancer is one of the most common cancers worldwide, and targeted chemotherapy has become a kind of the main treatment. Gefitinib, the most widely studied targeted agent in non-small cell lung cancer, is an orally active tyrosine kinase inhibitor. However, gefitinib inevitably generates acquired drug resistance, leading to treatment failure. Objective: A new class of compounds containing 4-anilinoquinazoline lead structure was designed and synthesized by modifying the structure of gefitinib. These compounds are expected to exert better anticancer activity and better binding to the EGFR-TK domain, enrich the structure of 4-anilinoquinazoline derivatives and inspire further structural modifications. Methods: The antiproliferative activity of nine derivatives was determined in three cancer cell lines (A549, PC9, and HepG2) using the MTT method. The ADMET profile of all compounds was predicted, and the binding affinity of the compounds (5 and 6) to EGFR was predicted by Schrödinger. In addition, the effect of these compounds (3-6) in inducing apoptosis in HepG2 cells was also studied. Results: Four (3, 5, 6 and 9) of the newly synthesized derivatives exhibited superior antiproliferative activity against A549 to gefitinib (IC50 = 12.64 ± 3.59 μM), with compound 5 having the best activity (IC50 = 7.39 ± 1.24 μM). Moreover, the ability of compounds (3-6) to induce HepG2 cell apoptosis was significantly better than that of gefitinib. Conclusion: Nine structures (compounds 2-10) were synthesized and characterized, and compound 5 had the best antiproliferative activity. Compound 3 possessed the best ability to induce HepG2 apoptosis. Also, ADMET calculations were performed in silico, and the results revealed that compound 3 has more suitable characteristics as a potential drug candidate.

Construction of an in vitro simulated one compartment extravascular administration model and its comparison with classic in vitro administration model in copper chloride induced HepG2 cell death

In this study, we designed an in vitro administration device based on compartment model theory and utilized it to construct an in vitro simulated one compartment extravascular administration model of copper chloride. Within the Cmax range of 3.91–1000.00 μM, the measured concentration-time curves of the simulated one compartment extravascular administration model almost coincide with the corresponding theoretical curves. The measured values of toxicokinetic parameters, including ke, T1/2, ka, T1/2a, Tmax, Cmax, CL, and AUC0-∞ are close to the corresponding theoretical values. The fitting coefficients are >0.9990. In simulated one compartment extravascular administration and classic in vitro administration, copper chloride dose-dependently induced HepG2 cell death. When Cmax/administration concentration is equal, classic in vitro administration induces a higher cell death rate than simulated one compartment extravascular administration. However, there is no significant difference in inducing cell death between the two administration models when area under the curve is equal. In conclusion, the device designed in this study can be used to in vitro simulate one compartment extravascular administration, making in vitro toxicity testing more similar to in vivo scenarios. There are differences in copper chloride induced HepG2 cell death between simulated one compartment extravascular administration and classic in vitro administration.

Chlorogenic Acid and its Isomers Attenuate NAFLD by Mitigating Lipid Accumulation in Oleic Acid-Induced HepG2 Cells and High-Fat Diet- Fed Zebrafish.

Chlorogenic acid (Chl), isochlorogenic acid A (Isochl A), and isochlorogenic acid B (Isochl B) are naturally occurring phenolic compounds, which have been shown to exert a regulatory effect on lipid metabolism. However, the mechanism underlying this effect remains unclear. Herein, we investigated the inhibitory effects and underlying mechanisms of these three phenolic compounds on oleic acid (OA)-induced HepG2 cells and high-fat diet (HFD)-fed zebrafish. Lipid accumulation and triacylglycerol levels increased in OA-induced cells, which was attenuated by Chl, Isochl A, and Isochl B. Moreover, the levels of malondialdehyde (MDA) and reactive oxygen species (ROS) decreased, while superoxide dismutase (SOD) levels increased by Chl, Isochl A and Isochl B treatment. Western blot analysis demonstrated that Chl, Isochl A and Isochl B reduced the expression of lipogenesis-related protein, including fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC) and peroxisome proliferator-activated receptor gamma (PPARγ). Moreover, peroxisome proliferator-activated receptor alpha gamma (PPARα) was increased by Chl, Isochl A, and Isochl B treatment. In addition, our results indicated that Chl, Isochl A and Isochl B decreased lipid profiles and lipid accumulation in HFD-fed zebrafish. Thus, these findings highlight the potential of Chl, Isochl A, and Isochl B as effective agents for treating or/and ameliorating non-alcoholic fatty liver disease (NAFLD).

Design, synthesis, and biological evaluation of (E)-3-(3-methoxy-4-substituted phenyl)-acrylic acid derivatives: Role of compound S17 in regulating lipid accumulation via AMPK activation

Non-alcoholic fatty liver disease (NAFLD) is increasingly recognized as a global public health concern characterized by excessive lipid accumulation in the liver. Ferulic acid (FA), a common foodborne phenolic acid, has been shown to reduce lipid accumulation by activating adenosine monophosphate-activated protein kinase (AMPK) and suppressing the expression of sterol regulatory element-binding protein-1 (SREBP-1). In this study, we synthesized a new class of (E)-3-(3-methoxy-4-substituted phenyl)-acrylic acid derivatives as AMPK activators. Among these derivatives, compound S17 demonstrated a more potent inhibitory effect on lipid accumulation compared to its counterparts. Specifically, S17 notably diminished intracellular triglyceride (TG) levels and the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), which are indicators of liver function. In addition, S17 curtailed intracellular lipid accumulation by activating the AMPK signaling pathway and down-regulating the protein expression of SREBP-1 in free fatty acid (FFA)-induced HepG2 cells. Therefore, findings from this study strongly suggest that compound S17 may serve as a promising therapeutic candidate for the treatment of NAFLD.

Gentiopicroside ameliorates glucose and lipid metabolism in T2DM via targeting FGFR1

BackgroundThe suppression of the fibroblast growth factor 21/fibroblast growth factor receptor 1 (FGF21/FGFR1) signaling pathway is considered as a vital factor in the type 2 diabetes mellitus (T2DM) progression. Our previous study showed that gentiopicroside (GPS), the main active compound present in Gentiana macrophylla Pall., has the capacity to control disorders related to glucose and lipid metabolism in individuals with T2DM. Nevertheless, the specific mechanism remains unclear. PurposeIn light of the fact that the PharmMapper database suggests FGFR1 as the target of GPS, our investigation aims to determine if GPS can enhance glucose and lipid metabolism issues in T2DM by modulating the FGF21/FGFR1 signaling pathway. MethodsIn this study, we used palmitic acid (PA)-induced HepG2 cells and db/db mice to investigate the function and mechanism of GPS in the FGF21/FGFR1 signaling pathway. To examine the interaction between GPS and FGFR1, researchers performed Cellular Thermal Shift Assay (CETSA) and Surface Plasmon Resonance (SPR) analysis. ResultsThe results suggest that GPS activates the traditional metabolic pathways, including PI3K/AKT and AMPK, which are the subsequent stages of the FGF21/FGFR1 pathway. This activation leads to the enhancement of glucose and lipid metabolism issues in PA-treated HepG2 cells and db/db mice. Furthermore, the depletion of FGFR1 has been noticed to oppose the stimulation of PI3K/AKT and AMPK pathways by GPS in HepG2 cells subjected to PA. Notability, our research affirms that GPS binds directly to FGFR1, hindering the ubiquitinated degradation of FGFR1 by neural precursor cells expressing developmentally decreased protein 4 (NEDD4) and ultimately promoting FGF21 signal transduction. ConclusionThis study demonstrates that GPS targeting FGFR1 activates the PI3K/AKT and AMPK pathways, which is an important mechanism for its treatment of T2DM.

Molecular engineering to elevate reactive oxygen species generation for synergetic damage on lipid droplets and mitochondria

Photodynamic therapy (PDT), characterized by high treatment efficiency, absence of drug resistance, minimal trauma, and few side effects, has gradually emerged as a novel and alternative clinical approach compared to traditional surgical resection, chemotherapy and radiation. Whereas, considering the limited diffusion distance and short lifespan of reactive oxygen species (ROS), as well as the hypoxic tumor microenvironment, it is crucial to design photosensitizers (PSs) with suborganelle specific targeting ability and low-oxygen dependence for accurate and highly efficient photodynamic therapy. In this study, we have meticulously designed three PSs, namely CIH, CIBr, and CIPh, based on molecular engineering. Theoretical calculation demonstrate that the three compounds possess good molecular planarity with calculated S1-T1 energy gaps (ΔES1-T1) of 1.04 eV for CIH, 0.92 eV for CIBr, and 0.84 eV for CIPh respectively. Notably, CIPh showcases remarkable dual subcellular targeting capability towards lipid droplets (LDs) and mitochondria owing to the synergistic effect of lipophilicity derived from coumarin's inherent properties combined with electropositivity conferred by indole salt cations. Furthermore, CIPh demonstrates exclusive release of singlet oxygen (1O2)and highly efficient superoxide anion free radicals(O2⦁−) upon light irradiation supported by its smallest S1-T1 energy gap (ΔES1-T1 = 0.84 eV). This leads to compromised integrity of LDs along with mitochondrial membrane potential, resulting in profound apoptosis induction in HepG2 cells. This successful example of molecular engineering guided by density functional theory (DFT) provides valuable experience for the development of more effective PSs with superior dual targeting specificity. It also provides a new idea for the development of advanced PSs with efficient and accurate ROS generation ability towards fluorescence imaging-guided hypoxic tumor therapy.

Discovery of novel octahydroquinazoline scaffolds endowed with dual inhibition of tubulin polymerization/Eg5 against HCC: Apoptotic and radio-chemotherapeutic studies

Mitotic kinesin Eg5 isozyme as a motor protein plays a critical role in cell division of tumor cells. Kinesin Eg5 selective inhibitors and Colchicine binding site suppressors are essential targets for many anticancer drugs and radio chemotherapies. On this work, a new series of octahydroquinazoline as anti-mitotic candidates 2–13 has been synthesized with dual inhibition of tubulin polymerization/Eg5 against HCC cell line. All octahydroquinazolines have been in vitro assayed against HepG-2 cytotoxicity, Eg5 inhibitory and anti-tubulin polymerization activities. The most active analogues 7, 8, 9, 10, and 12 against HepG-2 were further subjected to in vitro cytotoxic assay against HCT-116 and MCF-7 cell lines. Chalcones 9, 10, and 12 displayed the most cytotoxic potency and anti-tubulin aggregation in comparable with reference standard colchicine and potential anti-mitotic Eg5 inhibitory activity in comparison with Monastrol as well. Besides, they exhibited cell cycle arrest at the G2/M phase. Moreover, good convinced apoptotic activities have been concluded as overexpression of caspase-3 levels and tumor suppressive gene p53 in parallel with higher induction of Bax and inhibition of Bcl-2 biomarkers. Octahydroquinazoline 10 displayed an increase in caspase-3 by 1.12 folds compared to standard colchicine and induce apoptosis and demonstrated cell cycle arrest in G2/M phase arrest by targeting p53 pathway. Analogue 10 has considerably promoted cytotoxic radiation activity and boosted apoptotic induction in HepG-2 cells by 1.5 fold higher than standard colchicine.

Protopanaxadiol Targeting Membrane Induces HepG2 Cell Apoptosis Via Raft-like Formation and Tubulation Disruption.

Protopanaxadiol (PPD), which has a molecular structure similar to cholesterol, is a potent anticancer agent that has been proposed to target the lipid membrane for the pharmacological effects. However, the underlying mechanism by which PPD modulates the cell membrane leading to cancer cell death is not be fully understood. In this work, we used single cell infrared spectroscopy, scanning electron microscopy and confocal microscopy to investigate the effects of PPD on human hepatocellular carcinoma (HepG2) cells, focusing on the change in membrane structure. We found that PPD significantly reduced the number of membrane tubules over the course of treatment. Interestingly, the addition of PPD could promote the formation of lipid raft-like domains (PPD rafts) and even restore the domain disruption caused by methyl-beta-cyclodextrin depletion of membrane cholesterol. In addition, PPD pre-treatment may increase the induction effect of FasL, which impairs cell viability, although it does not appear to be beneficial for Fas clustering in the PPD rafts. Collectively, these results highlight a non-classical mechanism by which PPD induces HepG2 apoptosis by directly affecting the physical properties of the cell membrane, providing a novel insight into understanding membrane-targeted therapy.

Hymoins A–C, three unusual polycyclic polyprenylated acylphloroglucinols with lipid-lowering activity from Hypericum monogynum

Hymoins A–C (1–3), three unusual polycyclic polyprenylated acylphloroglucinols (PPAPs) were isolated from the flowers of Hypericum monogynum. Hymoin A features the first intriguing 6/5/5/5/7 pentacyclic caged PPAP. Hymoin B is characterized by an unprecedented rearranged 5/6/8 tricyclic ring system, while hymoin C represents the first rearranged PPAP with a fantastic spirocyclic 5/6/7 ring system. Their structures were established by extensive spectroscopic analysis, X-ray crystallography, and computational methods. The plausible biosynthetic routes for the compounds were also proposed. In oleic acid (OA)-induced HepG2 cells, all compounds exhibited significant lipid-lowering activity at the concentrations of 2–8 μmol/L. Further mechanistic study implied that compound 1 exhibited excellent lipid-lowering activity in OA-induced HepG2 cells through inhibiting the proteins of free fatty acids synthesis and improving lipidolysis.

UPLC-Q-TOF-MS Analysis of Chemical Constituents of the Kadsura coccinea Extract with Effect of Attenuating Lipid Accumulation in Vitro

Objective/Background: This study aims to explore the Kadsura coccinea extract (KCE)'s effect on lipid accumulation in vitro and its chemical components characterizations, aiming at developing a new alternative plant medicinal resource to fight against non-alcoholic fatty liver disease (NAFLD). Methods: After toxicological evaluation of KCE on HepG2 cells, Oil red O staining model and intracellular TGs quantification kit were used to examine the effects of KCE on lipid accumulation in vitro. The chemical components characterizations and potential active chemical constituents of the bioactive KCE were analyzed using ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) technology. Results: Among the chosen non-toxic concentrations of KCE (5, 10, 20 μg/mL), KCE can reduce the number and volume of lipid droplets in the oleic acid induced HepG2 cells in a dose-dependent manner, and the results of TGs quantification were almost consistent with the results of the oil red O staining experiment. These data indicate the KCE has ameliorative effect on lipid accumulation in vitro. In addition, a total of 26 compounds from the KCE were tentatively identified, dibenzocyclooctadiene lignans (DCLs) including Kadsulignan L and Gomisin J could be the main supposed components. Conclusion: These findings support further investigation into Kadsura coccinea containing DCLs as a new alternative medicinal arsenal to battle against NAFLD.

Effects of dietary arsenic exposure on liver metabolism in mice

Arsenic, a ubiquitous environmental toxicant with various forms and complex food matrix interactions, can reportedly exert differential effects on the liver compared to drinking water exposure. To examine its specific liver-related harms, we targeted the liver in C57BL/6 J mice (n=48, 8-week-old) fed with arsenic-contaminated food (30 mg/kg) for 60 days, mimicking the rice arsenic composition observed in real-world scenarios (iAsV: 7.3%, iAsIII: 72.7%, MMA: 1.0%, DMA: 19.0%). We then comprehensively evaluated liver histopathology, metabolic changes, and the potential role of the gut-liver axis using human hepatocellular carcinoma cells (HepG2) and microbiota/metabolite analyses. Rice arsenic exposure significantly altered hepatic lipid (fatty acids, glycerol lipids, phospholipids, sphingolipids) and metabolite (glutathione, thioneine, spermidine, inosine, indole-derivatives, etc.) profiles, disrupting 33 metabolic pathways (bile secretion, unsaturated fatty acid biosynthesis, glutathione metabolism, ferroptosis, etc.). Pathological examination revealed liver cell necrosis/apoptosis, further confirmed by ferroptosis induction in HepG2 cells. Gut microbiome analysis showed enrichment of pathogenic bacteria linked to liver diseases and depletion of beneficial strains. Fecal primary and secondary bile acids, short-chain fatty acids, and branched-chain amino acids were also elevated. Importantly, mediation analysis revealed significant correlations between gut microbiota, fecal metabolites, and liver metabolic alterations, suggesting fecal metabolites may mediate the impact of gut microbiota and liver metabolic disorders. Gut microbiota and its metabolites may play significant roles in arsenic-induced gut-liver injuries. Overall, our findings demonstrate that rice arsenic exposure triggers oxidative stress, disrupts liver metabolism, and induces ferroptosis.

Non-oxidized and oxidized flaxseed orbitides differently induce HepG2 cell apoptosis: involvement of cellular uptake and membrane death receptor DR4.

Flaxseed orbitides have health-promoting properties, particularly potent anti-cancer activity. However, flaxseed orbitides containing a methionine structure, such as [1-9-NαC]-linusorb B2 (CLB), are easily oxidized to sulfoxide ([1-9-NαC],[1-Rs,Ss-MetO]-linusorb-B2 (CLC)) and sulfone ([1-9-NαC], [1-MetO]-linusorb B2 (CLK)), with CLC having less anti-cancer ability than CLB. It is unclear why oxidized flaxseed orbitides are less effective against cancer than non-oxidized flaxseed orbitide. Non-oxidized ([1-9-NαC]-linusorb-B3 (CLA) and CLB) and oxidized (CLC and CLK) flaxseed orbitides were found to significantly upregulate the levels of pro-apoptotic proteins, including Bax/Bcl-2, CytoC, caspase-3, and caspase-8, in a dose-dependent manner, with non-oxidized flaxseed orbitides being more effective than oxidized flaxseed orbitides. Mechanically, the cellular absorption of non-oxidized flaxseed orbitides was higher than that of oxidized flaxseed orbitides. Moreover, the significant fluorescence quenching of DR4 protein by flaxseed orbitides (especially non-oxidized orbitides) indicated the formation of a DR4-orbitide complex. Molecular docking demonstrated that non-oxidized orbitides could easily dock into the active cavity of DR4 protein. Further blocking DR4 significantly reduced the ability of non-oxidized flaxseed orbitides to stimulate caspase-3 expression, whereas oxidized flaxseed orbitides retained this ability. Non-oxidized flaxseed orbitides are more effective against cancer than oxidized flaxseed orbitides due to higher cellular uptake and activation of the DR4-mediated death receptor signaling pathway. © 2024 Society of Chemical Industry.

Enhanced Anti-Inflammatory and Non-Alcoholic Fatty Liver Disease (NAFLD) Improvement Effects of Bacillus subtilis-Fermented Fagopyrum tataricum Gaertner

In this study, we investigated the enhanced anti-inflammatory activity and the effects on non-alcoholic fatty liver disease (NAFLD) of fermented Fagopyrum tataricum (F. tataricum) Gaertner extract (FFT) through in vitro analysis. We utilized high-performance liquid chromatography (HPLC) to analyze the non-fermented F. tataricum Gaertner extract (NFT) and the marker components, rutin and quercetin in FFT, to confirm changes in composition due to fermentation. The anti-inflammatory activity of NFT and FFT was evaluated using a lipopolysaccharide (LPS)-induced RAW 264.7 cell inflammation model. Simultaneously, the NAFLD improvement effects were measured by evaluating lipid accumulation and the expression of lipid synthesis regulators in free fatty acid (FFA)-induced HepG2 cells. HPLC analysis confirmed an increase in rutin content after the fermentation of F. tataricum Gaertner. Upon treatment with NFT and FFT at a concentration of 400 μg/mL, LPS-induced nitric oxide (NO) production values in RAW 264.7 cells were reduced to 16.12 μM and 2.09 μM, respectively, indicating enhanced significant inhibition (p < 0.05) of NO production through fermentation. FFT demonstrated the significant inhibition (p < 0.05) of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) protein, and inflammatory cytokine mRNA expression through the nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways in LPS-induced RAW 264.7 cells. In FFA-induced HepG2 cells, FFT significant suppressed (p < 0.05) lipid accumulation and the expression of sterol regulatory element binding protein (SREBP)-1c, CCAAT/enhancer binding protein (C/EBP)α proteins, and acetyl-CoA carboxylase (ACC) mRNA. The results of this study suggest the potential utilization of FFT as a material for improving NAFLD.

Intervention effect of Cigu Xiaozhi prescription on ceramide lipoapoptosis in non-alcoholic fatty liver disease.

To explore the mechanism of the Chinese medicine Cigu Xiaozhi prescription (, CGXZ) in the treatment of the non-alcoholic fatty liver disease (NAFLD) by detoxification and phlegm-reducing, the effect of CGXZ prescription on ceramide-mediated lipid apoptosis in Hep G2 cells with NAFLD. The experiment was randomly divided into 6 groups: normal control group, model group, CGXZ prescription medicated serum high, medium, and low dose groups, and pioglitazone positive control group. Using 500 μmol/L free fatty acid (FFA) mixture to induce Hep G2 cells to establish NAFLD cell model, respectively, with 2%, 4%, and 6% concentration of CGXZ prescription medicated serum intervention for 24 h. The changes in organelles and lipid droplet accumulation were observed under the electron microscope. Furthermore, TdT-mediated dUTP Nick-End Labeling method was used to assay hepatocyte apoptosis; Biochemical determination of glutamic-pyruvic transaminase, glutamic oxalacetic transaminase, triglycerides, and FFA levels in Hep G2 cells; the content of ceramide was determined by high-performance thin-layer chromatography. Finally, Western Blot and quantitative real-time polymerase chain reaction (qRT-PCR) were used to determine the protein and gene expression levels, such as inducible nitric oxide synthase (iNOS), nuclear factor κB (NF-κB), B cell lymphoma 2 (Bcl-2) and Bcl-2-associated X (Bax). Under the electron microscope, the cells in the model group showed moderate-to-severe steatosis, and apoptotic bodies could be seen. The model group had greater improvements in the apoptosis rate (P < 0.01), and the levels of ceramide C2 and FFA in the cytoplasm (P < 0.01) than the normal control group. The protein expressions of NF-κB, iNOS, and Bax were significantly up-regulated (P < 0.05), while the Bcl-2 had no significant change (P > 0.05). Compared with the model group, the levels of ceramide C2 and FFA (P < 0.01), the protein expressions of NF-κB, iNOS, and Bax (P < 0.05) in the CGXZ prescription treatment group and pioglitazone positive control group were significantly decreased; Only the Bcl-2 protein was significantly up-regulated in the high-dose Chinese medicine group (P < 0.05). The down-regulation of Bax mRNA expression in each Chinese medicine treatment group was significantly better than in the pioglitazone positive control group (P < 0.01). The CGXZ prescription, formulated with the method of detoxification and phlegm, can inhibit lipoapoptosis in the NAFLD cell model by down-regulating the levels of ceramide C2 and FFA, which may be achieved by regulating ceramide/iNOS/NF-κB signaling pathway.

Ginsenoside Rg5 inhibits lipid accumulation and hepatocyte apoptosis via the Notch1 signaling pathway in NASH mice

BackgroundNon-alcoholic steatohepatitis (NASH) is a prevalent chronic liver disease that lacks an FDA-approved treatment medicine. Despite the known antitumor and hypoglycemic properties of Ginsenoside Rg5, its effects and underlying mechanisms in the context of NASH remain largely unexplored. PurposeThis study aims to investigate the effect of Rg5 on NASH mice induced by a high-fat diet and CCl4. Study designIn vivo experiments, a mouse NASH model was established by a HFHC diet plus intraperitoneal injection of low-dose CCl4. In vitro experiments, a cellular steatosis model was established using free fatty acids (FFA) induced HepG2 cells. In addition, a fibrogenesis model was established using HSC-LX2 cells. MethodsThe effects of Ginsenoside Rg5 on lipid accumulation and oxidative damage were analyzed by ELISA kit, H&E staining, Oil Red O staining, flow cytometry and Western blot. The effects of Ginsenoside Rg5 on liver fibrosis were analyzed by Masson staining, Sirus Red staining, immunohistochemistry and Western blot. The effect of Ginsenoside Rg5 on Notch1 signaling pathway in liver was studied by protein Oil Red staining, protein immunoblotting and immunofluorescence. ResultsIn terms of lipid accumulation, Rg5 has the ability to regulate key proteins related to lipogenesis, thereby inhibiting hepatic lipid accumulation and oxidative stress. Additionally, Rg5 can reduce the occurrence of hepatocyte apoptosis by regulating the p53 protein. Moreover, after Rg5 intervention, the presence of fibrotic proteins (α-SMA, Collagen 1, TGF-β) in the liver is significantly suppressed, thus inhibiting liver fibrosis. Lastly, Rg5 leads to a decrease in the expression levels of Notch1 and its ligand Jagged-1 in the liver. ConclusionIn summary, the regulatory effects of Rg5 on the Notch1 signaling pathway play a crucial role in modulating hepatic lipid metabolism and preventing hepatocyte apoptosis, thereby impeding the progression of NASH. These findings highlight the potential of Rg5 as a promising natural product for interventions targeting NASH.