Antioxidant, Anti-Inflammatory, and Antidiabetic Activities of Bioactive Compounds from the Fruits of Livistona chinensis Based on Network Pharmacology Prediction.

In this study, a chemical investigation on the fruits of Livistona chinensis (FLC) led to the isolation and identification of 45 polyphenols and 5 alkaloids, including two new compounds (Livischinol (1) and Livischinine A (46)), an undescribed compound (47) and 47 known compounds. FLC was predicted with novel potential antidiabetic function by collecting and analyzing the potential targets of the ingredients. Compound 32 exhibited significant α-glucosidase inhibitory activity (IC50 = 5.71 µM) and 1, 6, and 44 showed the PTP1B inhibitory activity with IC50 values of 9.41-22.19 µM, while that of oleanolic acid was 28.58 µM. The competitive inhibitors of PTP1B (compounds 1 and 44) formed strong binding affinity, with catalytic active sites, proved by kinetic analysis, fluorescence spectra measurements, and computational simulations, and stimulated glucose uptake in the insulin-resistant HepG2 cells at the dose of 50 µM. In addition, FLC was rich in antioxidant and anti-inflammatory bioactive compounds so that they could be developed as nutraceuticals against diabetes.

Evaluation of potential herb-drug interactions based on the effect of Suxiao Jiuxin Pill on CYP450 enzymes and transporters.

ETHNOPHARMACOLOGY RELEVANCE: Suxiao jiuxin pill (SJP) is a Chinese medical drug with anti-inflammatory, anti-apoptotic, and vasodilatory function. It is widely used in combination with other drugs for the treatment of coronary heart disease (CHD) and angina. Nevertheless, the effect of SJP on Cytochrome P450 (CYP450) enzymes and transporters' activity related to drug metabolism is rarely studied. OBJECTIVE: The aim of this study was to investigate the effect of SJP on the activity of drug-metabolizing enzyme CYP450 and transporters. MATERIALS AND METHODS: Human primary hepatocytes were used in present study. Probe substrates of CYP450 enzymes were incubated in human liver microsomes (HLMs) with and without SJP while IC50 values were calculated. The inhibitory effect of SJP on the activity of CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4 was evaluated. The inducing effect of SJP on the activity of CYP1A2, 2B6 and 3A4 was accessed. The inhibition of SJP on human OATP1B1 was investigated through cell-based assay. The inhibition of SJP on human MDR1 and BCRP was also estimated by means of the vesicles assay. RESULTS: The results showed that the SJP under the concentration of 1000 µg/mL could inhibit the activity of CYP1A2, 2B6, 2C19, and 3A4, with IC50 values of 189.7, 308.2, 331.2 and 805.7 µg/mL, respectively. There was no inhibitory effect found in the other 3 liver drug enzyme subtypes. In addition, SJP showed no induction effect on CYP1A2, 2B6 and 3A4, however it had a significant inhibitory effect on human-derived OATP1B1 at the concentration of 100 and 1000 µg/mL, with the IC50 value of 21.9 µg/mL. Simultaneously, the SJP inhibited BCRP at high concentration of 1000 µg/mL but did not affect human MDR1. CONCLUSIONS: Based on these research results above, it is suggested that the SJP can affect some of the CYP450 enzymes and transporters' activity. When used in combination with related conventional drugs, potential herb-drug interactions should be considered.

Comparison of gene expression and biotransformation activity of HepaRG cells under static and dynamic culture conditions.

Flow conditions have been shown to be important in improving longevity and functionality of primary hepatocytes, but the impact of flow on HepaRG cells is largely unknown. We studied the expression of genes encoding CYP enzymes and transporter proteins and CYP1 and CYP3A4 activity during 8 weeks of culture in HepaRG cells cultured under static conditions (conventional 24-/96-well plate culture with common bicarbonate/CO2 buffering) and under flow conditions in an organ-on-chip (OOC) device. Since the OOC-device is a closed system, bicarbonate/CO2 buffering was not possible, requiring application of another buffering agent, such as HEPES. In order to disentangle the effects of HEPES from the effects of flow, we also applied HEPES-supplemented medium in static cultures and studied gene expression and CYP activity. We found that cells cultured under flow conditions in the OOC-device, as well as cells cultured under static conditions with HEPES-supplemented medium, showed more stable gene expression levels. Furthermore, only cells cultured in the OOC-device showed relatively high baseline CYP1 activity, and their gene expression levels of selected CYPs and transporters were most similar to gene expression levels in human primary hepatocytes. However, there was a decrease in baseline CYP3A4 activity under flow conditions compared to HepaRG cells cultured under static conditions. Altogether, the present study shows that HepaRG cells cultured in the OOC-device were more stable than in static cultures, being a promising in vitro model to study hepatoxicity of chemicals upon chronic exposure.

Influence of Proteome Profiles and Intracellular Drug Exposure on Differences in CYP Activity in Donor-Matched Human Liver Microsomes and Hepatocytes.

Human liver microsomes (HLM) and human hepatocytes (HH) are important in vitro systems for studies of intrinsic drug clearance (CLint) in the liver. However, the CLint values are often in disagreement for these two systems. Here, we investigated these differences in a side-by-side comparison of drug metabolism in HLM and HH prepared from 15 matched donors. Protein expression and intracellular unbound drug concentration (Kpuu) effects on the CLint were investigated for five prototypical probe substrates (bupropion-CYP2B6, diclofenac-CYP2C9, omeprazole-CYP2C19, bufuralol-CYP2D6, and midazolam-CYP3A4). The samples were donor-matched to compensate for inter-individual variability but still showed systematic differences in CLint. Global proteomics analysis outlined differences in HLM from HH and homogenates of human liver (HL), indicating variable enrichment of ER-localized cytochrome P450 (CYP) enzymes in the HLM preparation. This suggests that the HLM may not equally and accurately capture metabolic capacity for all CYPs. Scaling CLint with CYP amounts and Kpuu could only partly explain the discordance in absolute values of CLint for the five substrates. Nevertheless, scaling with CYP amounts improved the agreement in rank order for the majority of the substrates. Other factors, such as contribution of additional enzymes and variability in the proportions of active and inactive CYP enzymes in HLM and HH, may have to be considered to avoid the use of empirical scaling factors for prediction of drug metabolism.

Hydrogen-rich water protects against liver injury in nonalcoholic steatohepatitis through HO-1 enhancement via IL-10 and Sirt 1 signaling.

Nonalcoholic steatohepatitis (NASH) could progress to hepatic fibrosis in the absence of effective control. The purpose of our experiment was to investigate the protective effect of drinking water with a high concentration of hydrogen, namely, hydrogen-rich water (HRW), on mice with nonalcoholic fatty liver disease to elucidate the mechanism underlying the therapeutic action of molecular hydrogen. The choline-supplemented, l-amino acid-defined (CSAA) or the choline-deficient, l-amino acid-defined (CDAA) diet for 20 wk was used to induce NASH and fibrosis in the mice model and simultaneously treated with the high-concentration 7-ppm HRW for different periods (4 wk, 8 wk, and 20 wk). Primary hepatocytes were stimulated by palmitate to mimic liver lipid metabolism during fatty liver formation. Primary hepatocytes were cultured in a closed vessel filled with 21% O2 + 5% CO2 + 3.8% H2 and N2 as the base gas to verify the response of primary hepatocytes in a high concentration of hydrogen gas in vitro. Mice in the CSAA + HRW group had lower serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and milder histological damage. The inflammatory cytokines were expressed at lower levels in the HRW group than in the CSAA group. Importantly, HRW reversed hepatocyte fatty acid oxidation and lipogenesis as well as hepatic inflammation and fibrosis in preexisting hepatic fibrosis specimens. Molecular hydrogen inhibits the lipopolysaccharide-induced production of inflammation cytokines through increasing heme oxygenase-1 (HO-1) expression. Furthermore, HRW improved hepatic steatosis in the CSAA + HRW group. Sirtuin 1 (Sirt1) induction by molecular hydrogen via the HO-1/adenosine monophosphate activated protein kinase (AMPK)/peroxisome proliferator-activated receptor α (PPARα)/peroxisome proliferator-activated receptor γ (PPAR-γ) pathway suppresses palmitate-mediated abnormal fat metabolism. Orally administered HRW suppressed steatosis induced by CSAA and attenuated fibrosis induced by CDAA, possibly by reducing oxidative stress and the inflammation response.NEW & NOTEWORTHY The mRNA expression of inflammatory cytokines in the HRW group was lower than in the CSAA group. HRW reversed hepatocyte apoptosis as well as hepatic inflammation and fibrosis in NASH specimens. Molecular hydrogen inhibits LPS-induced inflammation via an HO-1/interleukin 10 (IL-10)-independent pathway. HRW improved hepatic steatosis in the CSAA + HRW group. Sirt1 induction by molecular hydrogen via the HO-1/AMPK/PPARα/PPARγ pathway suppresses palmitate-mediated abnormal fat metabolism.

Exploration of components and mechanisms of Polygoni Multiflori Radix-induced hepatotoxicity using siRNA -mediated CYP3A4 or UGT1A1 knockdown liver cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Polygoni Multiflori Radix, the dried root of Polygonum multiflorum Thunb., and its processed products have been used as restoratives for centuries in China. However, the reports of Polygoni Multiflori Radix-induced liver injury (PMR-ILI) have received wide attention in recent years, and the components and mechanism of PMR-ILI are not completely clear yet. Our previous studies found that the PMR-ILI was related to the down-regulation of some drug metabolism enzymes (DME). AIM OF THE STUDY: To explore the effect of the inhibition of CYP3A4 or UGT1A1 on PMR-ILI, screen the relevant hepatotoxic components and unveil its mechanism. METHODS: RT-qPCR was used to detect the effects of water extract of Polygoni Multiflori Radix (PMR) and its main components on the mRNA expression of CYP3A4 and UGT1A1 in human hepatic parenchyma cell line L02. High-performance liquid chromatography (HPLC) was employed to detect the content of major components in the PMR. And then, the stable CYP3A4 or UGT1A1 knockdown cells were generated using short hairpin RNAs (shRNA) in L02 and HepaRG cells. Hepatotoxic components were identified by cell viability assay when PMR and its four representative components, 2,3,5,4'-tetrahydroxy stilbene glycoside (TSG), emodin (EM), emodin-8-O-ß-D-glucoside (EG), and gallic acid (GA), acted on CYP3A4 or UGT1A1 knockdown cell lines. The PMR-ILI mechanism of oxidative stress injury and apoptosis in L02 and HepaRG cells were detected by flow cytometry. Finally, the network toxicology prediction analysis was employed to excavate the targets of its possible toxic components and the influence on the metabolic pathway. RESULTS: PMR and EM significantly inhibited the mRNA expression of CYP3A4 and UGT1A1 in L02 cells, while TSG and GA activated the mRNA expression of CYP3A4 and UGT1A1, and EG activated CYP3A4 expression while inhibited UGT1A1 expression. The contents of TSG, EG, EM and GA were 34.93 mg/g, 1.39 mg/g, 0.43 mg/g and 0.44 mg/g, respectively. The CYP3A4 or UGT1A1 knockdown cells were successfully constructed in both L02 and HepaRG cells. Low expression of CYP3A4 or UGT1A1 increased PMR cytotoxicity remarkably. Same as PMR, the toxicity of EM and GA increased in shCYP3A4 and shUGT1A1 cells, which suggested EM and GA may be the main components of hepatotoxicity in PMR. Besides, EM not only inhibited the expression of metabolic enzymes but also reduced the cytotoxicity threshold. EM and GA affected the level of ROS, mitochondrial membrane potential, Ca2+ concentration, and dose-dependent induced hepatocyte apoptosis in L02 and HepaRG cells. The network toxicology analysis showed that PMR-ILI was related to drug metabolism-cytochrome P450, glutathione metabolism, and steroid hormone biosynthesis. CONCLUSION: The inhibition of mRNA expression of CYP3A4 or UGT1A1 enhanced hepatotoxicity of PMR. EM and GA, especially EM, may be the main hepatotoxic components in PMR. The mechanism of PMR, EM and GA induced hepatotoxicity was proved to be related to elevated levels of ROS, mitochondrial membrane potential, Ca2+ concentration, and induction of apoptosis in liver cells.

A functional long-term 2D serum-free human hepatic in vitro system for drug evaluation.

Human in vitro hepatic models generate faster drug toxicity data with higher human predictability compared to animal models. However, for long-term studies, current models require the use of serum and 3D architecture, limiting their utility. Maintaining a functional long-term human in vitro hepatic culture that avoids complex structures and serum would improve the value of such systems for preclinical studies. This would also enable a more straightforward integration with current multi-organ devices to study human systemic toxicity to generate an alternative model to chronic animal evaluations. A human primary hepatocyte culture system was characterized for 28 days in 2D and serum-free defined conditions. Under the studied conditions, human primary hepatocytes maintained their characteristic morphology, hepatic markers and functions for 28 days. The acute and chronic administration of known drugs validated the sensitivity of the system for drug testing. This human 2D model represents a realistic system to evaluate hepatic function for long-term drug studies, without the need of animal serum, confounding variable in most models, and with less complexity and resultant cost compared to most 3D models. The defined culture conditions can easily be integrated into complex multi-organ in vitro models for studying systemic effects driven by the liver function for long-term evaluations.

Garcinia linii extracts exert the mediation of anti-diabetic molecular targets on anti-hyperglycemia.

Different portions (stem GIS and leaf GIL) of Garcinia linii were extracted by ethanol/water and crude extracts were employed to investigate the contents of total phenol and flavonoids, antioxidation activities, and inhibitory activities of α-amylase and α-glucosidase via enzymatic assay and OGTT and OSTT for lowering glucose levels. The data revealed that GlS and GlL contained different levels of flavonoids and total phenol. Furthermore, the results showed the extracts exhibited remarkable antioxidation activities and inhibitory activities of α-amylase and α-glucosidase. In silico docking studies were done using Gold software and the probable molecules retrieved from PubChem were docked with several anti-diabetic relate targets, the results showed several components of G. linii could potentially inhibit diabetic molecules when compared with clinic drugs. The cell glucose uptake data also confirmed that GlL and GlS could retain the active component in the regulation of insulin, AMPK, PPARγ, and DPP4. In vivo, the evidence showed G. linii extracts including syringaldehyde suppressed effect of hyperglycemia on OSTT and OGTT assays. These results suggest that G. linii extract has a potential therapeutic value for the treatment of diabetes in humans.

Cell-to-Medium Concentration Ratio Overshoot in the Uptake of Statins by Human Hepatocytes in Suspension, but Not in Monolayer: Kinetic Analysis Suggesting a Partial Loss of Functional OATP1Bs.

Suspended human hepatocytes (SHH) have long been used in assessing hepatic drug uptake, while plated human hepatocytes in short-term monolayer culture (PHH) have gained use in recent years. This study aimed to cross-evaluate SHH and PHH in measuring the hepatic uptake mediated by organic anion transporting polypeptide 1Bs (OATP1Bs). We compared the time courses of cell-to-medium (C/M) concentration ratios and initial uptake clearance values of the OATP1B substrates (pitavastatin, rosuvastatin, cerivastatin, pravastatin, dehydropravastatin, and SC-62807) between SHH and PHH. For all compounds except cerivastatin, the C/M ratios in SHH displayed an apparent overshoot (an initial increase followed by a decrease) during the 180-min uptake experiment, but not in PHH. Based on the literature evidence suggesting the possible internalization of OATP1Bs in primary hepatocytes, separate experiments measured the drug uptake after varying lengths of pre-incubation in the drug-free medium. The initial uptake clearances of pitavastatin and rosuvastatin declined in SHH beyond an apparent threshold time of 20-min drug-free pre-incubation, but not in PHH. Kinetic modeling quantitatively captured the decline in the active uptake clearance in SHH, and more than half of the active uptake clearances of pitavastatin and rosuvastatin were prone to loss during the 180-min uptake experiment. These results suggested a partial, time-delayed loss of the functional OATP1Bs in SHH upon prolonged incubation. Our results indicate that PHH is more appropriate for experiments where a prolonged incubation is required, such as estimation of unbound hepatocyte-to-medium concentration ratio (Kp,uu) at the steady-state.

Evaluation of the Disconnect between Hepatocyte and Microsome Intrinsic Clearance and In Vitro In Vivo Extrapolation Performance.

The use of in vitro in vivo extrapolation (IVIVE) from human hepatocyte (HH) and human liver microsome (HLM) stability assays is a widely accepted predictive methodology for human metabolic clearance (CLmet). However, a systematic underprediction of CLmet from both matrices appears to be universally apparent, which can be corrected for via an empirical regression offset. After physiological scaling, intrinsic clearance (CLint) for compounds metabolized via the same enzymatic pathway should be equivalent for both matrices. Compounds demonstrating significantly higher HLM CLint relative to HH CLint have been encountered, raising questions regarding how to predict CLmet for such compounds. Here, we determined the HLM:HH CLint ratio for 140 marketed drugs/compounds, compared this ratio as a function of physiochemical properties and drug metabolism enzyme dependence, and examined methodologies to predict CLmet from both matrices. The majority (78%) of compounds displaying a high HLM:HH CLint ratio were CYP3A substrates. Using HH CLint for CYP3A substrates, the current IVIVE regression offset approach remains an appropriate strategy to predict CLmet (% compounds overpredicted/correctly predicted/underpredicted 27/62/11, respectively). However, using the same approach for HLM significantly overpredicts CLmet for CYP3A substrates (% compounds overpredicted/correctly predicted/underpredicted 56/33/11, respectively), highlighting that a different IVIVE offset is required for CYP3A substrates using HLM. This work furthers the understanding of compound properties associated with a disproportionately high HLM:HH CLint ratio and outlines a successful IVIVE approach for such compounds. SIGNIFICANCE STATEMENT: Oral drug discovery programs typically strive for low clearance compounds to ensure sufficient target engagement. Human liver microsomes and isolated human hepatocytes are used to optimize and predict human hepatic metabolic clearance. After physiological scaling, intrinsic clearance for compounds of the same metabolic pathway should be equivalent between matrices. However, a disconnect in intrinsic clearance is sometimes apparent. The work described attempts to further understand this phenomenon, and by achieving a mechanistic understanding, improvements in clearance predictions may be realized.

A Preliminary Study on the Effect and Mechanism of Breviscapine for Improving Insulin Resistance in HepG2 Cells.

Insulin resistance (IR) is known to be a critical factor, which can lead to the onset of type 2 diabetes. Traditional Chinese medicine (TCM) has special advantages in treating IR, but the active components and action mechanisms of most TCM remain unclear. Therefore, the elucidation of the potential mechanisms is a major challenge in TCM research. In the study, we tried to elucidate the potential pharmacological efficacy and mechanism of breviacapine for improving IR through network analysis and validate the possible biological target for its quality evaluation. We computationally recognized the active components, potential targets, and the targets closely related to IR by using integrative analysis based on network pharmacology approach. We also established the active components-targets network, protein interactions network and analyzing the biological functions and pathways of targets to evaluate the links between components and pharmacological actions to help explain the action mechanisms of breviscapine. Based on the network analysis, our experimental data preliminarily confirmed that breviscapine could improve IR in HepG2 cells, which may be associated with the dynamic regulation of the PTP1B. This study combined network pharmacology with partial experiment validation to clarify the underlying mechanism of breviscapine in improving IR and thus laid the experimental foundation for the depth exploration of its functional mechanism.

Clinically Relevant Cytochrome P450 3A4 Induction Mechanisms and Drug Screening in Three-Dimensional Spheroid Cultures of Primary Human Hepatocytes.

Cytochrome P450 (CYP) 3A4 induction is an important cause of drug-drug interactions, making early identification of drug candidates with CYP3A4 induction liability in drug development a prerequisite. Here, we present three-dimensional (3D) spheroid cultures of primary human hepatocytes (PHHs) as a novel CYP3A4 induction screening model. Screening of 25 drugs (12 known CYP3A4 inducers in vivo and 13 negative controls) at physiologically relevant concentrations revealed a 100% sensitivity and 100% specificity of the system. Three of the in vivo CYP3A4 inducers displayed much higher CYP3A4 induction capacity in 3D spheroid cultures as compared with in two-dimensional (2D) monolayer cultures. Among those, we identified AZD1208, a proviral integration site for Moloney murine leukemia virus (PIM) kinase inhibitor terminated in phase I of development due to unexpected CYP3A4 autoinduction, as a CYP3A4 inducer only active in 3D spheroids but not in 2D monolayer cultures. Gene knockdown experiments revealed that AZD1208 requires pregnane X receptor (PXR) to induce CYP3A4. Rifampicin requires solely PXR to induce CYP3A4 and CYP2B6, while phenobarbital-mediated induction of these CYPs did not show absolute dependency on either PXR or constitutive androstane receptor (CAR), suggesting its ability to switch nuclear receptor activation. Mechanistic studies into AZD1208 uncovered an involvement of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway in CYP3A4 induction that is sensitive to the culture format used, as revealed by its inhibition of ERK1/2 Tyrosine 204 phosphorylation and sensitivity to epidermal growth factor (EGF) pressure. In line, we also identified lapatinib, a dual epidermal growth factor receptor/human epidermal growth factor receptor 2 (EGFR/HER2) inhibitor, as another CYP3A4 inducer only active in 3D spheroid culture. Our findings offer insights into the pathways involved in CYP3A4 induction and suggest PHH spheroids for preclinical CYP3A4 induction screening.

Biotransformation of phytoestrogens from soy in enzymatically characterized liver microsomes and primary hepatocytes of Atlantic salmon.

Efficient aquaculture is depending on sustainable protein sources. The shortage in marine raw materials has initiated a shift to "green aquafeeds" based on staple ingredients such as soy and wheat. Plant-based diets entail new challenges regarding fish health, product quality and consumer risks due to the possible presence of chemical contaminants, natural toxins and bioactive compounds like phytoestrogens. Daidzein (DAI), genistein (GEN) and glycitein (GLY) are major soy isoflavones with considerable estrogenic activities, potentially interfering with the piscine endocrine system and affecting consumers after carry-over. In this context, information on isoflavone biotransformation in fish is crucial for risk evaluation. We have therefore isolated hepatic fractions of Atlantic salmon (Salmo salar), the most important species in Norwegian aquaculture, and used them to study isoflavone elimination and metabolite formation. The salmon liver microsomes and primary hepatocytes were characterized with respect to phase I cytochrome P450 (CYP) and phase II uridine-diphosphate-glucuronosyltransferase (UGT) enzyme activities using specific probe substrates, which allowed comparison to results in other species. DAI, GEN and GLY were effectively cleared by UGT. Based on the measurement of exact masses, fragmentation patterns, and retention times in liquid chromatography high-resolution mass spectrometry, we preliminarily identified the 7-O-glucuronides as the main metabolites in salmon, possibly produced by UGT1A1 and UGT1A9-like activities. In contrast, the production of oxidative metabolites by CYP was insignificant. Under optimized assay conditions, only small amounts of mono-hydroxylated DAI were detectable. These findings suggested that bioaccumulation of phytoestrogens in farmed salmon and consumer risks from soy-containing aquafeeds are unlikely.

Agriophyllum oligosaccharides ameliorate hepatic injury in type 2 diabetic db/db mice targeting INS-R/IRS-2/PI3K/AKT/PPAR-γ/Glut4 signal pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Agriophyllum squarrosum (L.) Moq. is a traditional Mongol medicine generally used to treat diabetes. OBJECTIVE: To investigate the protective effects and potential mechanisms of Agriophyllum oligosaccharides (AOS) on liver injury in type 2 diabetic db/db mice. MATERIALS AND METHODS: The db/db mice were divided into the model group (Model), metformin group (MET), high-dose AOS group (HAOS), and low-dose AOS group (LAOS). Nondiabetic littermate control db/m mice were used as the normal control group (Control). Mice in AOS groups were treated with AOS (380 or 750 mg/kg) daily, for 8 weeks. At 8 weeks, blood samples were collected to detect lipid and enzyme parameters concerning hepatic function, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein (TP), albumin (ALB), globulin (GLB), triglyceride (TG), total cholesterol (TC), and high-density lipoprotein cholesterol (HDL-C). Random blood glucose (RBG) test, oral glucose tolerance test (OGTT), and oral maltose tolerance test (OMTT) were also conducted. Microscopy was used to observe morphological changes in the liver of AOS-treated groups. Real-time PCR was used to detect the mRNA expression, including insulin receptor substrate 2 (IRS-2), phosphatidylinositol 3 kinase (PI3K), protein kinase B (AKT), peroxisome proliferator-activated receptor (PPAR)-γ, insulin receptor (INS-R), and Glut4. Furthermore, western blotting was performed to identify proteins, including phosphorylation of IRS-2 (p-IRS-2), PI3K, p-AKT, PPAR-γ, INS-R, and Glut4. Hepatic protein expression of p-IRS-2, PI3K, p-AKT, PPAR-γ, INS-R, and Glut4 was observed using immunohistochemical staining. RESULTS: AOS treatment significantly decreased RBG, OGTT, and OMTT in mice, as well as serum ALT and AST activities. AOS groups demonstrated significantly higher expressions of p-IRS-2, PI3K, PPAR-γ, p-AKT, INS-R, and Glut4 protein and IRS-2, PI3K, AKT, PPAR-γ, INS-R, and Glut4 mRNA in the liver tissue of db/db mice; the degeneration and necrosis of hepatocytes and formation of collagen fibres markedly reduced, improving the structural disorder in the liver. CONCLUSION: The results suggest that AOS could protect the liver in type 2 diabetes, in part by activating insulin in the INS-R/IRS2/PI3K/AKT/Glut4/PPAR-γ signal pathway, facilitating hepatocyte proliferation, and further reducing the blood glucose levels.

Hydrogen Sulfide Protects against Paraquat-Induced Acute Liver Injury in Rats by Regulating Oxidative Stress, Mitochondrial Function, and Inflammation.

In addition to the lung, the liver is considered another major target for paraquat (PQ) poisoning. Hydrogen sulfide (H2S) has been demonstrated to be effective in the inhibition of oxidative stress and inflammation. The aim of this study was to investigate the protective effect of exogenous H2S against PQ-induced acute liver injury. The acute liver injury model was established by a single intraperitoneal injection of PQ, evidenced by histological alteration and elevated serum aminotransferase levels. Different doses of NaHS were administered intraperitoneally one hour before exposure to PQ. Analysis of the data shows that exogenous H2S attenuated the PQ-induced liver injury and oxidative stress in a dose-dependent manner. H2S significantly suppressed reactive oxygen species (ROS) generation and the elevation of malondialdehyde content while it increased the ratio of GSH/GSSG and levels of antioxidant enzymes including SOD, GSH-Px, HO-1, and NQO-1. When hepatocytes were subjected to PQ-induced oxidative stress, H2S markedly enhanced nuclear translocation of Nrf2 via S-sulfhydration of Keap1 and resulted in the increase in IDH2 activity by regulating S-sulfhydration of SIRT3. In addition, H2S significantly suppressed NLRP3 inflammasome activation and subsequent IL-1ß excretion in PQ-induced acute liver injury. Moreover, H2S cannot reverse the decrease in SIRT3 and activation of the NLRP3 inflammasome caused by PQ in Nrf2-knockdown hepatocytes. In summary, H2S attenuated the PQ-induced acute liver injury by enhancing antioxidative capability, regulating mitochondrial function, and suppressing ROS-induced NLRP3 inflammasome activation. The antioxidative effect of H2S in PQ-induced liver injury can at least partly be attributed to the promotion of Nrf2-driven antioxidant enzymes via Keap1 S-sulfhydration and regulation of SIRT3/IDH2 signaling via Nrf2-dependent SIRT3 gene transcription as well as SIRT3 S-sulfhydration. Thus, H2S supplementation can form the basis for a promising novel therapeutic strategy for PQ-induced acute liver injury.

Differential effects of red yeast rice, Berberis aristata and Morus alba extracts on PCSK9 and LDL uptake.

BACKGROUND AND AIMS: The novel nutraceutical combination containing red yeast rice (monacolin K 3.3 mg), Berberis aristata cortex extract (Berberine 531.25 mg) and Morus alba leaves extract (1-deoxynojirimycin 4 mg) is effective in the management of elevated plasma low-density lipoprotein cholesterol (LDL-C) levels. The aim of the present study was to investigate the effects of the three components on proprotein convertase subtilisin/kexin type 9 (PCSK9), a key regulator of LDL receptor (LDLR) expression, in hepatocyte cell lines and to compare their effects on LDL cellular uptake. METHODS AND RESULTS: HepG2 and Huh7 cells were incubated with B. aristata cortex extract (BCE), red yeast rice (RYR) and M. alba leaves extract (MLE) alone or in combination for 24 h. RYR (50 µg/mL) increased PCSK9 protein expression (Western blot analysis and ELISA), PCSK9 mRNA (qPCR) and its promoter activity (luciferase reporter assay). BCE (40 µg/mL) reduced instead PCSK9 expression, mRNA levels and promoter activity. MLE determined a concentration-dependent reduction of PCSK9 at the mRNA and protein levels, with a maximal reduction at 1 mg/mL, without significant changes of PCSK9 promoter activity. MLE also downregulated the expression of 3-hydroxy-3-methyl-3-glutaryl coenzyme A reductase and fatty acid synthase mRNA levels. The combination of RYR, BCE and MLE reduced the PCSK9 mRNA and protein levels, as well as the promoter activity. Finally, the single components and their combination induced LDL receptor and LDL uptake by the hepatocytes. CONCLUSION: The positive effect of MLE on PCSK9 supports the rationale of using the nutraceutical combination of RYR, BCE and MLE to control hyperlipidemic conditions.

Hdac1 Regulates Differentiation of Bipotent Liver Progenitor Cells During Regeneration via Sox9b and Cdk8.

BACKGROUND & AIMS: Upon liver injury in which hepatocyte proliferation is compromised, liver progenitor cells (LPCs), derived from biliary epithelial cells (BECs), differentiate into hepatocytes. Little is known about the mechanisms of LPC differentiation. We used zebrafish and mouse models of liver injury to study the mechanisms. METHODS: We used transgenic zebrafish, Tg(fabp10a:CFP-NTR), to study the effects of compounds that alter epigenetic factors on BEC-mediated liver regeneration. We analyzed zebrafish with disruptions of the histone deacetylase 1 gene (hdac1) or exposed to MS-275 (an inhibitor of Hdac1, Hdac2, and Hdac3). We also analyzed zebrafish with mutations in sox9b, fbxw7, kdm1a, and notch3. Zebrafish larvae were collected and analyzed by whole-mount immunostaining and in situ hybridization; their liver tissues were collected for quantitative reverse transcription polymerase chain reaction. We studied mice in which hepatocyte-specific deletion of ß-catenin (Ctnnb1flox/flox mice injected with Adeno-associated virus serotype 8 [AAV8]-TBG-Cre) induces differentiation of LPCs into hepatocytes after a choline-deficient, ethionine-supplemented (CDE) diet. Liver tissues were collected and analyzed by immunohistochemistry and immunoblots. We performed immunohistochemical analyses of liver tissues from patients with compensated or decompensated cirrhosis or acute on chronic liver failure (n = 15). RESULTS: Loss of Hdac1 activity in zebrafish blocked differentiation of LPCs into hepatocytes by increasing levels of sox9b mRNA and reduced differentiation of LPCs into BECs by increasing levels of cdk8 mRNA, which encodes a negative regulator gene of Notch signaling. We identified Notch3 as the receptor that regulates differentiation of LPCs into BECs. Loss of activity of Kdm1a, a lysine demethylase that forms repressive complexes with Hdac1, produced the same defects in differentiation of LPCs into hepatocytes and BECs as observed in zebrafish with loss of Hdac1 activity. Administration of MS-275 to mice with hepatocyte-specific loss of ß-catenin impaired differentiation of LPCs into hepatocytes after the CDE diet. HDAC1 was expressed in reactive ducts and hepatocyte buds of liver tissues from patients with cirrhosis. CONCLUSIONS: Hdac1 regulates differentiation of LPCs into hepatocytes via Sox9b and differentiation of LPCs into BECs via Cdk8, Fbxw7, and Notch3 in zebrafish with severe hepatocyte loss. HDAC1 activity was also required for differentiation of LPCs into hepatocytes in mice with liver injury after the CDE diet. These pathways might be manipulated to induce LPC differentiation for treatment of patients with advanced liver diseases.

Apoptosis in HepaRG and HL-7702 cells inducted by polyphyllin II through caspases activation and cell-cycle arrest.

Rhizoma Paridis, a traditional Chinese medicine, has shown promise in cancer prevention and therapy. Polyphyllin II is one of the most significant saponins in Rhizoma Paridis and it has toxic effects on kinds of cancer cells. However, our results in this study proved that the polyphyllin II has hepatotoxicity in vitro through caspases activation and cell-cycle arrest. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide results indicated polyphyllin II inhibited proliferation, induced apoptosis in HepaRG cells and HL-7702 cells and showed a concentration and time-dependent. Then, we selected the innovative cell model-HepaRG cells to explore the mechanism of hepatotoxicity. Our data showed the reactive oxygen species (ROS) increased and the mitochondrial membrane potential decreased in HepaRG cells after administration of polyphyllin II. Besides, with the increase of concentration, the release of lactate dehydrogenase increased and the S phase of the cell cycle was arrested. Nevertheless, when pretreatment with antioxidant N-acetylcysteine, apoptotic cells decreased significantly, inhibited the production of ROS and improved the decrease of membrane potential in HepaRG cells. Moreover, polyphyllin II treatment increased levels of Fas, Bax, cytochrome c, activated caspase-3, -8, -9, cleaved poly(ADP-ribose) polymerase and decreased Bcl-2 expression levels. Finally, we identified two signal pathways of apoptosis induced by polyphyllin II including the death receptor pathway and the mitochondria pathway. This study confirmed the hepatotoxicity of the polyphyllin II in vitro, which has never been discovered and gave a wake-up call for the clinical application of Rhizoma Paridis.

The metabolism and hepatotoxicity of ginkgolic acid (17 : 1) in vitro.

Pharmacological activities and adverse side effects of ginkgolic acids (GAs), major components in extracts from the leaves and seed coats of Ginkgo biloba L, have been intensively studied. However, there are few reports on their hepatotoxicity. In the present study, the metabolism and hepatotoxicity of GA (17 : 1), one of the most abundant components of GAs, were investigated. Kinetic analysis indicated that human and rat liver microsomes shared similar metabolic characteristics of GA (17 : 1) in phase I and II metabolisms. The drug-metabolizing enzymes involved in GA (17 : 1) metabolism were human CYP1A2, CYP3A4, UGT1A6, UGT1A9, and UGT2B15, which were confirmed with an inhibition study of human liver microsomes and recombinant enzymes. The MTT assays indicated that the cytotoxicity of GA (17 : 1) in HepG2 cells occurred in a time- and dose-dependent manner. Further investigation showed that GA (17 : 1) had less cytotoxicity in primary rat hepatocytes than in HepG2 cells and that the toxicity was enhanced through CYP1A- and CYP3A-mediated metabolism.