Hepatocyte Membrane Research Articles

Quantitative contributions of hepatic and renal organic cation transporters to the clinical pharmacokinetic cimetidine-metformin interaction.

The widely prescribed oral anti-diabetic drug metformin is eliminated unchanged in the urine primarily through active tubular secretion. This process is mediated by organic cation transporter 2 (OCT2), an uptake transporter expressed on the basolateral membrane of renal proximal tubule cells. Metformin uptake into the liver, the site of action, is mediated by OCT1, which is expressed on the sinusoidal membrane of hepatocytes. Sixteen healthy adults participated in a clinical pharmacokinetic drug-drug interaction study in which they were orally administered metformin (50 mg) as a dual OCT1/2 substrate alone (baseline) and with cimetidine (400 mg) as an OCT inhibitor. Relative to baseline, metformin systemic plasma exposure increased by 24% ( p <0.05) in the presence of cimetidine, which was accompanied by a disproportional decrease (8%) in metformin renal clearance ( p =0.005). Genetic variants of OCT1 and OCT2 moderately impacted the significance and magnitude of the interaction. Collectively, we hypothesized that the cimetidine-metformin interaction involves inhibition of hepatic OCT1 as well as renal OCT2. We tested this hypothesis by developing a physiologically based pharmacokinetic (PBPK) model and assessing potential OCT biomarkers in plasma and urine to gain mechanistic insight into the transporters involved in this interaction. The PBPK model predicted that cimetidine primarily inhibits hepatic OCT1 and, to a lesser extent, renal OCT2. The unchanged renal clearance of potential OCT2 biomarkers following cimetidine exposure supports a minimal role for renal OCT2 in this interaction.

Enhanced delivery of CRISPR/Cas9 system based on biomimetic nanoparticles for hepatitis B virus therapy

The persistent presence of covalently closed circular DNA (cccDNA) in hepatocyte nuclei poses a significant obstacle to achieving a comprehensive cure for hepatitis B virus (HBV). Current applications of CRISPR/Cas9 for targeting and eliminating cccDNA have been confined to in vitro studies due to challenges in stable cccDNA expression in animal models and the limited non-immunogenicity of delivery systems. This study addresses these limitations by introducing a novel non-viral gene delivery system utilizing Gemini Surfactant (GS). The developed system creates stable and targeted CRISPR/Cas9 nanodrugs with a negatively charged surface through modification with red blood cell membranes (RBCM) or hepatocyte membranes (HCM), resulting in GS-pDNA@Cas9-CMs complexes. These GS-pDNA complexes demonstrated complete formation at a 4:1 w/w ratio. The in vitro transfection efficiency of GS-pDNA-HCM reached 54.61%, showing homotypic targeting and excellent safety. Additionally, the study identified the most effective single-guide RNA (sgRNA) from six sequences delivered by GS-pDNA@Cas9-HCM. Using GS-pDNA@Cas9-HCM, a significant reduction of 96.47% in in vitro HBV cccDNA and a 52.34% reduction in in vivo HBV cccDNA were observed, along with a notable decrease in other HBV-related markers. The investigation of GS complex uptake by AML-12 cells under varied time and temperature conditions revealed clathrin-mediated endocytosis (CME) for GS-pDNA and caveolin-mediated endocytosis (CVME) for GS-pDNA-HCM and GS-pDNA-RBCM. In summary, this research presents biomimetic gene-editing nanovectors based on GS (GS-pDNA@Cas9-CMs) and explores their precise and targeted clearance of cccDNA using CRISPR/Cas9, demonstrating good biocompatibility both in vitro and in vivo. This innovative approach provides a promising therapeutic strategy for advancing the cure of HBV.

Chronic aluminium chloride exposure induces redox imbalance, metabolic distress, DNA damage, and histopathologic alterations in Wistar rat liver.

Aluminium, a ubiquitous environmental toxicant, is distinguished for eliciting a broad range of physiological, biochemical, and behavioural alterations in laboratory animals and humans. The present work was conducted to study the functional and structural changes induced by aluminium in rat liver. Twenty five adult male Wistar rats (150-200 g) were randomly divided into five groups; control group and four Al-treated groups viz: Al 1 (25mg AlCl3/kg b.wt), Al 2 (35mg AlCl3/kg b.wt), Al 3 (45mg AlCl3/kg b.wt), and Al 4 (55mg AlCl3/kg b.wt). Rats in the aluminium-treated groups were administered AlCl3 for 30 days through oral gavage. Aluminium significantly increased the serum levels of liver function markers (ALT, AST, and ALP), phospholipids, and cholesterol. The activities of hepatocyte membrane (ALP, GGT, and LAP) and carbohydrate metabolic (G6P, F16BP, HK, LDH, MDH, ME, and G6PDH) enzymes were significantly altered by AlCl3 administration. Prolonged Al exposure induced oxidative stress in the liver, as evident by significant hepatocellular DNA damage, increased lipid peroxidation, and decreased non-enzymatic and enzymatic antioxidants. The toxic effects observed in this study were AlCl3 dose-dependent. Histopathological examination of liver sections revealed enlargement of sinusoidal spaces, derangement of the hepatic chord, loss of discrete hepatic cell boundaries, congestion of hepatic sinusoids, and degeneration of hepatocytes in Al-intoxicated rats. In conclusion, aluminium causes severe hepatotoxicity by inhibiting the hepatocyte membrane enzymes and disrupting the liver's energy metabolism and antioxidant defence.

Activation of pregnane X receptor protects against cholestatic liver injury by inhibiting hepatocyte pyroptosis.

Our previous study shows that activation of pregnane X receptor (PXR) exerts hepatoprotection against lithocholic acid (LCA)-induced cholestatic liver injury. In this study we investigated whether PXR activation could inhibit hepatocyte pyroptosis, as well as the underlying mechanisms. Male mice were treated with mouse PXR agonist pregnenolone 16α-carbonitrile (PCN, 50 mg·kg-1·d-1, i.p.) for 7 days, and received LCA (125 mg/kg, i.p., bid) from D4, then sacrificed 12 h after the last LCA injection. We showed that LCA injection resulted in severe cholestatic liver injury characterized by significant increases in gallbladder size, hepatocellular necrosis, and neutrophil infiltration with a mortality rate of 68%; PCN treatment significantly inhibited hepatocyte pyroptosis during LCA-induced cholestatic liver injury, as evidenced by reduced serum lactic dehydrogenase (LDH) levels, TUNEL-positive cells and hepatocyte membrane damage. Furthermore, PXR activation suppressed both the NOD-like receptor protein 3 (NLRP3) inflammasome-induced canonical pyroptosis and the apoptosis protease activating factor-1 (APAF-1) pyroptosome-induced non-canonical pyroptosis. Inhibition of the nuclear factor kappa B (NF-κB) and forkhead box O1 (FOXO1) signaling pathways was also observed following PXR activation. Notably, dual luciferase reporter assay showed that PXR activation inhibited the transcriptional effects of NF-κB on NLRP3, as well as FOXO1 on APAF-1. Our results demonstrate that PXR activation protects against cholestatic liver injury by inhibiting the canonical pyroptosis through the NF-κB-NLRP3 axis and the non-canonical pyroptosis through the FOXO1-APAF-1 axis, providing new evidence for PXR as a prospective anti-cholestatic target.

Abcb4-defect cholangitis mouse model with hydrophobic bile acid composition by in vivo liver-specific gene deletion

Progressive familial intrahepatic cholestasis (PFIC) is a liver disease that occurs during childhood and requires liver transplantation. ABCB4 is localized along the canalicular membranes of hepatocytes, transports phosphatidylcholine into bile, and its mutation causes PFIC3. Abcb4 gene-deficient mice established as animal models of PFIC3 exhibit cholestasis-induced liver injury. However, their phenotypes are often milder than those of human PFIC3, partly because of the existence of large amounts of less toxic hydrophilic bile acids synthesized by the rodent-specific enzymes Cyp2c70 and Cyp2a12. Mice with double deletions of Cyp2c70/Cyp2a12 (CYPDKO mice) have a human-like hydrophobic bile acid composition. PFIC-related gene mutations were induced in CYPDKO mice to determine whether these triple-gene-deficient mice are a better model for PFIC. To establish a PFIC3 mouse model using CYPDKO mice, we induced abcb4 gene deletion in vivo using adeno-associated viruses expressing SaCas9 under the control of a liver-specific promoter and abcb4-target gRNAs. Compared to Abcb4-deficient wild-type mice, Abcb4-deficient CYPDKO mice showed more pronounced liver injury along with an elevation of inflammatory and fibrotic markers. The proliferation of intrahepatic bile ductal cells and hematopoietic cell infiltration were also observed. CYPDKO/abcb4-deficient mice show a predominance of taurine-conjugated chenodeoxycholic acid and lithocholic acid in the liver. In addition, phospholipid levels in the gallbladder bile were barely detectable. Mice with both human-like bile acid composition and Abcb4-defect exhibit severe cholestatic liver injury and are useful for studying human cholestatic diseases and developing new treatments.

Identification of new correctors for traffic-defective ABCB4 variants by a high-content screening approach

ABCB4 is located at the canalicular membrane of hepatocytes and is responsible for the secretion of phosphatidylcholine into bile. Genetic variations of this transporter are correlated with rare cholestatic liver diseases, the most severe being progressive familial intrahepatic cholestasis type 3 (PFIC3). PFIC3 patients most often require liver transplantation. In this context of unmet medical need, we developed a high-content screening approach to identify small molecules able to correct ABCB4 molecular defects. Intracellularly-retained variants of ABCB4 were expressed in cell models and their maturation, cellular localization and function were analyzed after treatment with the molecules identified by high-content screening. In total, six hits were identified by high-content screening. Three of them were able to correct the maturation and canalicular localization of two distinct intracellularly-retained ABCB4 variants; one molecule was able to significantly restore the function of two ABCB4 variants. In addition, in silico molecular docking calculations suggest that the identified hits may interact with wild type ABCB4 residues involved in ATP binding/hydrolysis. Our results pave the way for their optimization in order to provide new drug candidates as potential alternative to liver transplantation for patients with severe forms of ABCB4-related diseases, including PFIC3.

Impact of OATP2B1 on Pharmacokinetics of Atorvastatin Investigated in rSlco2b1-Knockout and SLCO2B1-Knockin Rats.

The organic anion transporting polypeptide (OATP) 2B1 is considered an emerging drug transporter that is found expressed in pharmacokinetically relevant organs such as the liver, small intestine, and kidney. Despite its interaction with various substrate drugs, the understanding of its in vivo relevance is still limited. In this study, we first validated the interaction of atorvastatin with rat OATP2B1 using transiently transfected HeLa cells. Moreover, we characterized our rSlco2b1-knockout and SLCO2B1-knockin rats for mRNA, protein expression, and localization of OATP2B1 in the liver, small intestine, and kidney. The transporter showed the highest expression in the liver followed by the small intestine. In humanized rats, human OATP2B1 is localized on the sinusoidal membrane of hepatocytes. In enterocytes of wild-type and humanized rats, the transporter was detected in the luminal membrane with the vast majority being localized subapical. Subsequently, we assessed atorvastatin pharmacokinetics in male wild-type, rSlco2b1-knockout, and SLCO2B1-knockin rats after a single-dose administration (orally and intravenously). Investigating the contribution of rat OATP2B1 or human OATP2B1 to oral atorvastatin pharmacokinetics revealed no differences in concentration-time profiles or pharmacokinetic parameters. However, when comparing the pharmacokinetics of atorvastatin after intravenous administration in SLCO2B1-humanized rats and knockout animals, notable differences were observed. In particular, the systemic exposure (area under the curve) decreased by approximately 40% in humanized animals, whereas the clearance was 57% higher in animals expressing human OATP2B1. These findings indicate that human OATP2B1 influences pharmacokinetics of atorvastatin after intravenous administration, most likely by contributing to the hepatic uptake. SIGNIFICANCE STATEMENT: Wild-type, rSlco2b1-knockout, and SLCO2B1-humanized Wistar rats were characterized for the expression of rat and human SLCO2B1/OATP2B1. Pharmacokinetic studies of atorvastatin over 24 hours were conducted in male wild-type, rSlco2b1-knockout, and SLCO2B1-humanized rats. After a single-dose intravenous administration, a lower systemic exposure and an increase in clearance were observed in SLCO2B1-humanized rats compared with knockout animals indicating a contribution of OATP2B1 to the hepatic clearance.

Assessing the degree of hepatic ischemia-reperfusion injury using physiologically based pharmacokinetic modeling of sodium fluorescein disposition in ex vivo machine-perfused livers.

Ischemia-reperfusion injury (IRI) is an intrinsic risk associated with liver transplantation. Ex vivo hepatic machine perfusion (MP) is an emerging organ preservation technique that can mitigate IRI, especially in livers subjected to prolonged warm ischemia time (WIT). However, a method to quantify the biological response to WIT during MP has not been established. Previous studies used physiologically based pharmacokinetic (PBPK) modeling to demonstrate that a decrease in hepatic transport and biliary excretion of the tracer molecule sodium fluorescein (SF) could correlate with increasing WIT in situ. Furthermore, these studies proposed intracellular sequestration of the hepatocyte canalicular membrane transporter multidrug resistance-associated protein 2 (MRP2) leading to decreased MRP2 activity (maximal transport velocity; Vmax) as the potential mechanism for decreased biliary SF excretion. We adapted an extant PBPK model to account for ex vivo hepatic MP and fit a six-parameter version of this model to control time-course measurements of SF in MP perfusate and bile. We then identified parameters whose values were likely insensitive to changes in WIT and fixed them to generate a reduced model with only three unknown parameters. Finally, we fit the reduced model to each individual biological replicate SF time course with differing WIT, found the mean estimated value for each parameter, and compared them using a one-way ANOVA. We demonstrated that there was a significant decrease in the estimated value of Vmax for MRP2 at the 30-min WIT. These studies provide the foundation for future studies investigating real-time assessment of liver viability during ex vivo MP.NEW & NOTEWORTHY We developed a computational model of sodium fluorescein (SF) biliary excretion in ex vivo machine perfusion and used this model to assess changes in model parameters associated with the activity of MRP2, a hepatocyte membrane transporter, in response to increasing warm ischemia time. We found a significant decrease in the parameter value describing MRP2 activity, consistent with a role of decreased MRP2 function in ischemia-reperfusion injury leading to decreased secretion of SF into bile.

Coupling the role of lipids to the conformational dynamics of the ABC transporter P-glycoprotein

The ATP-binding cassette transporter P-glycoprotein (P-gp) is a multidrug efflux pump that is overexpressed in a variety of cancers and associated with the drug-resistance phenomenon. P-gp structures were previously determined in detergent and in nanodiscs, in which different transmembrane helix conformations were found, “straight” and “kinked,” respectively, indicating a possible role of the lipid environment on the P-gp structural ensemble. Here, we investigate the dynamic conformational ensembles and protein-lipid interactions of two human P-gp inward-open conformers, straight and kinked, employing all-atom molecular dynamics (MD) simulations in asymmetric multicomponent lipid bilayers that mimic the highly specialized hepatocyte membrane in which P-gp is expressed. The two conformers are found to differ in terms of the accessibility of the substrate cavity. The MD simulations show how cholesterol and different lipid species wedge, snorkel, and partially enter into the cavity of the straight P-gp conformer solved in detergent. However, access to the cavity of the kinked P-gp conformer solved in nanodiscs is restricted. Furthermore, the volume and dynamic fluctuations of the substrate cavity largely differ between the two P-gp conformers and are modulated by the presence (or absence) of cholesterol in the membrane and/or of ATP. From the mechanistic perspective, the findings indicate that the straight conformer likely precedes the kinked conformer in the functional working cycle of P-gp, with the latter conformation representing a post substrate-bound state. The inaccessibility of the main transmembrane cavity in the kinked conformer might be crucial in preventing substrate disengagement and transport withdrawal. Remarkably, in our unbiased MD simulations, one transmembrane helix (TM10) of the straight conformer underwent a spontaneous transition to a kinked conformation, underlining the relevance of both conformations in a native phospholipid environment and revealing structural descriptors defining the transition between the two P-gp conformers.

First-in-Human Predictions of Hepatic Clearance for Drugs With the Well-Stirred Model: Comparative Assessment Between Models of Fraction Unbound Based Either on the Free Drug Hypothesis, Albumin-Facilitated Hepatic Uptake or Dynamic Binding Kinetics

The well-stirred model (WSM) is commonly used to predict the hepatic clearance in vivo (CLH) of drugs. The necessary intrinsic clearance of the unbound drug (CLint-in vitro-unbound) is generated in the in vitro assays in the presence of microsomes or hepatocytes but in the absence of plasma proteins. The value of CLint-in vitro-unbound can be extrapolated with the fraction unbound determined in vitro in plasma (fup) only if the fraction unbound in vivo in liver is the same. However, this approach resulted to a systematic underprediction bias of CLH. With the goal of reducing this bias, two new models of fraction unbound were published in this journal. These models estimate the binding kinetics of the rates of association and dissociation of the drug-protein complex and propose that more dissociation in the liver compared to plasma will increase the fraction unbound available for the metabolism. Consequently, these two models generated higher values of fraction unbound, implying a lower underprediction bias of CLH with the WSM. The first model was developed by Poulin et al. and is referring to the value of fup that is adjusted (fu-adjusted) to quantify the effect of a full dissociation of the drug-protein complex at the hepatocyte membrane in accordance with the theory of the albumin-facilitated hepatic uptake. A second model was developed by Yan et al. who presented a dynamic fraction unbound (fu-dynamic) measuring the real dissociation kinetics of the drug-protein complex with a new in vitro assay in the presence and absence of a recombinant liver enzyme in plasma. Therefore, the objective of this study was to make the first comparative assessment between these two models. The results indicate that, in general, the WSM combined with the values of fu-adjusted was the most accurate approach for predicting CLH. The WSM combined with the values of fu-dynamic has underperformed particularly with the acidic and neutral drugs binding to the albumin and presenting a low metabolic turnover in vitro. Therefore, the new in vitro assay for fu-dynamic gave an underprediction bias of CLH for these drug properties. However, the values of fu-adjusted are significantly higher than those values of fu-dynamic, and, this resulted to no underprediction bias, which is reinforcing the theory of the ALB-facilitated hepatic uptake. For the other neutral and acidic drugs, the models of fu-dynamic and fu-adjusted are in closer agreement. Finally, for the basic drugs, the models of fu-adjusted and fu-dynamic as well as a third model only considering a pH gradient effect on fup are almost accurately equivalent.

Cholestasis in Chronic Liver Disease and the Role of Ademetionine in its Treatment (Literature Review and Expert Panel Resolution)

Aim: to analyse the principles of diagnosis and treatment of intrahepatic cholestasis in chronic liver diseases, to present data on the effectiveness of ademetionine in the treatment of chronic liver diseases with intrahepatic cholestasis and the materials of the Expert Meeting held in 2023. Key points. During the Expert Meeting, the problems of diagnostics and treatment of intrahepatic cholestasis in various chronic liver diseases were discussed, the effectiveness of ademetionine was clarified, and optimal regimens for its administration were determined. The relevance of the existing algorithm for diagnosing cholestasis in real clinical practice was assessed. The effectiveness of ademetionine in the treatment of various liver diseases occurring with intrahepatic cholestasis (cholestatic forms of drug-induced liver damage, alcoholic liver disease, non-alcoholic liver disease, primary biliary cholangitis) was demonstrated, manifested by a decrease in clinical and laboratory signs of cholestasis. The anticholestatic mechanisms of ademetionine action were clarified, which consist in normalizing the fluidity of hepatocyte membranes, regulating the activity of Nrf2, a key transcription factor, suppressing lipid peroxidation and the resulting damage to hepatocytes and cholangiocytes. Optimal regimens for prescribing ademetionine for various clinical situations were considered. Conclusions. Ademetionine is an effective drug that, due to its pleiotropic action and favourable safety profile, can be used in various chronic liver diseases accompanied by cholestasis, including as a part of the complex therapy.

Synthesis and biological evaluation of a novel 68Ga-labeled GalNAc-PET probe for asialoglycoprotein receptor imaging

PurposeThe asialoglycoprotein receptor (ASGPR), expressed exclusively on mammalian hepatocyte membranes, recognizes ligands terminated with β-D-galactose (Gal) and N-acetylglucosamine (GalNAc). This study aims to develop a 68Ga-labeled monovalent GalNAc derivative that particularly interacts with ASGPR. It also aims to assess the potential utility of this derivative in visualizing ASGPR-related liver dysfunction using positron emission tomography (PET)/ computed tomography (CT) imaging. MethodsThe PET imaging probe 68Ga-NOTA-GalNAc was developed and characterized with regard to radiochemical purity, biocompatibility, biodistribution patterns, and specific ASGPR-targeting ability. Groups of normal mice, mice with acute liver injury (ALI) induced by CCl4, mice with early-stage liver fibrosis induced by CCl4, mice with nonalcoholic fatty liver disease (NAFLD), and orthotopic HepG2 hepatoma-bearing mice were imaged with 68Ga-NOTA-GalNAc PET to evaluate the potential of this technique in monitoring ASGPR-related liver dysfunction. Results68Ga-NOTA-GalNAc, a hydrophilic compound with high radiochemical purity (>99 %) and good liver targeting ability, particularly binds ASGPR on the surface of hepatocytes with moderate affinity (KD = 6.82 μM). Compared with control, ASGPR-related liver dysfunction groups, even the group of mice with ALI (P <0.0001), revealed sensitive distinctions in the liver uptake value of 68Ga-NOTA-GalNAc. The 68Ga-NOTA-GalNAc absorbed in the livers of mice with early-stage liver fibrosis and NAFLD group is significantly less than that absorbed in the livers of mice in the normal group (P <0.0001). Hepatoma can be visualized in orthotopic HepG2 hepatoma-bearing mice because of different radioactivity accumulated in the nontumor region and lesion region, corresponding to the ASGPR expression confirmed with immunohistochemical staining. Conclusion68Ga-NOTA-GalNAc has the potential to be a specific, noninvasive, and sensitive PET imaging agent for ASGPR-related liver dysfunction.

Mitochondrial dysfunction of the inner membrane of hepatocytes in the development of glutamate-induced steatohepatosis and its correction

Elucidation of the mechanisms of the development of liver steatosis, which are at the heart of the pathogenesis of nonalcoholic fatty liver disease (NAFLD), will allow the introduction of new effective treatment methods into medical practice, as well as the development of new measures for the correction of this disease and accompanying pathologies. The purpose of the research is to establish the enzymatic activity of the complexes of the electron transport chain of the mitochondrial membrane of rat hepatocytes and to evaluate the corrective effect of the multiprobiotic “Symbiter acidophilic” concentrated or nanocrystalline cerium dioxide on the formation of steatohepatosis induced by neonatal sodium glutamate administration. The experiments were carried out on 50 white non-linear male rats; the direction included the study of the mechanisms of the development of steatohepatosis in 4-month-old rats, which were administered monosodium glutamate in the neonatal period, and the study of the functional state of the liver in rats after the neonatal administration of monosodium glutamate against the background of periodic administration of a multiprobiotic or nanocrystalline dioxide cerium. It was established that neonatal administration of monosodium glutamate causes metabolic changes in 4-month-old rats, manifested in the disproportionate accumulation of fat with the development of visceral obesity without hyperphagia, dyslipidemia, and steatohepatosis. In 4-month-old rats, after neonatal administration of sodium glutamate, the development of steatohepatosis was accompanied by mitochondrial dysfunction, which was manifested by changes in the lipid composition of the inner membrane of hepatocyte mitochondria with an increase in oxidized products and a change in the enzymatic activity of all complexes of the respiratory chain. In rats injected with monosodium glutamate in the neonatal period, periodic use of the multiprobiotic “Symbiter acidophilic” concentrated or nanocrystalline cerium dioxide significantly restored the functional state of the liver, reduced the manifestations of oxidative stress and prevented the development of steatohepatosis, which indicates the antioxidant effect of these drugs and the possibility of their use for prevention of steatohepatosis.

АНАЛИЗ ФАРМАЦЕВТИЧЕСКОГО РЫНКА ПРЕПАРАТОВ, СОДЕРЖАЩИХ ЭКСТРАКТИВНЫЕ БИОЛОГИЧЕСКИ АКТИВНЫЕ ВЕЩЕСТВА РАСТОРОПШИ ПЯТНИСТОЙ

Natural flavolignans of milk thistle are widely used in pharmaceutical practice in the form of herbal remedies for the treatment of various hepatopathologies. In medical practice, medications (medicines) and dietary supplements containing extractive substances of milk thistle fruits are used. The therapeutic effect of drugs containing extractive substances of milk thistle is due to the stabilizing effect of flavolignans on hepatocyte membranes and neutralization of free radicals in the liver. By having a stabilizing effect on cell membranes, the extractive components of milk thistle prevent the release of intracellular components (transaminases) and promote the regeneration of liver cells. The work carried out a marketing analysis of the pharmaceutical market of drugs in Volgograd containing extractive substances of milk thistle and an assessment of their therapeutic effect in medical practice. The conducted research showed the presence on the pharmaceutical market of Volgograd of a wide range of over-the-counter medications containing extract of milk thistle fruits, both domestic and foreign produced. It was revealed that prescription drugs are not available in pharmacy organizations in Volgograd. It is shown that the main segment contains preparations containing milk thistle extractives in the form of dietary supplements. The analyzed price segment of drugs and dietary supplements is defined as average.

Characterization and Applications of Permeabilized Hepatocytes in Drug Discovery.

Hepatocytes are one of the most physiologically relevant in vitro liver systems for human translation of clearance and drug-drug interactions (DDI). However, the cell membranes of hepatocytes can limit the entry of certain compounds into the cells for metabolism and DDI. Passive permeability through hepatocytes can be differentin vitro and in vivo, which complicates the human translation. Permeabilized hepatocytes offer a useful tool to probe mechanistic understanding of permeability-limited metabolism and DDI. Incubation with saponin of 0.01% at 0.5 million cells/mL and 0.05% at 5 million cells/mL for 5 min at 37°C completely permeabilized the plasma membrane of hepatocytes, while leaving the membranes of subcellular organelles intact. Permeabilized hepatocytes maintained similarenzymatic activity as intact unpermeabilized hepatocytes and can be stored at -80°C for at least 7 months. This approach reduces costs by preserving leftover hepatocytes. The relatively low levels of saponin in permeabilized hepatocytes had no significant impact on the enzymatic activity. As the cytosolic contents leak out from permeabilized hepatocytes, cofactors need to be added to enable metabolic reactions. Cytosolic enzymes will no longer bepresent if the media are removed after cells are permeabilized. Hence permeabilized hepatocytes with and without media removal may potentially enable reaction phenotyping of cytosolic enzymes. Although permeabilized hepatocytes work similarly as human liver microsomes and S9 fractions experimentally requiring addition of cofactors, they behave more like hepatocytes maintaining enzymatic activities for over 4 h. Permeabilized hepatocytes are a great addition to the drug metabolism toolbox to provide mechanistic insights.

Innovative prophylactic and therapeutic approaches in liver cirrhosis.

Liver cirrhosis is the consequence of chronicisation and of the evolution of untreated liver diseases. The complexity of the disease and the complications it can cause have been and are still intensively researched, aiming to discover new therapies or improve existing ones for the effective management of liver cirrhosis. Currently, the treatment used is directed against the cause that caused the disease, if it is known; in advanced cases, liver transplantation is the only valid therapeutic option. Hepatoprotectors that are currently on the market are numerous, having as common properties the antioxidant, anti-inflammatory, stabilizing properties of the hepatocytic membrane; A few examples: the ethanolic extract of Curcuma longa, the extract from the plant called Sophora flavescens, the extract of Glycyrrhiza glabra, silymarin (extracted from Sylibum marianum), the extract of Ganoderma lucidum, etc. Liver cirrhosis is accompanied by generalized hypovitaminosis, so supplementing the diet with hydro- and liposoluble vitamins is mandatory. Protein-caloric malnutrition can be prevented by a hyperprotein diet, especially beneficial being the supplementation with branched-chain amino acids, which are also applicable in the prophylaxis and treatment of hepatic encephalopathy. Nanoparticles are a state-of-the-art therapeutic option, proving increased bioavailability, for example polydopamine nanoparticles loaded with l-arginine have been tested as therapy in liver cirrhosis. Among the innovative treatment directions in liver cirrhosis are hybrid products (e.g. hybrid polymer nanoparticles loaded with caffeic acid), cell cultures and artificial or bioartificial liver support.

Evolutionary analysis of SLC10 family members and insights into function and expression regulation of lamprey NTCP.

The Na ( +)-taurocholate cotransporting polypeptide (NTCP) is a member of the solute carrier family 10 (SLC10), which consists of 7 members (SLC10a1-SLC10a7). NTCP is a transporter localized to the basolateral membrane of hepatocytes and is primarily responsible for the absorption of bile acids. Although mammalian NTCP has been extensively studied, little is known about the lamprey NTCP (L-NTCP). Here we show that L-NTCP follows the biological evolutionary history of vertebrates, with conserved domain, motif, and similar tertiary structure to higher vertebrates. L-NTCP is localized to the cell surface of lamprey primary hepatocytes by immunofluorescence analysis. HepG2 cells overexpressing L-NTCP also showed the distribution of L-NTCP on the cell surface. The expression profile of L-NTCP showed that the expression of NTCP is highest in lamprey liver tissue. L-NTCP also has the ability to transport bile acids, consistent with its higher vertebrate orthologs. Finally, using a farnesoid X receptor (FXR) antagonist, RT-qPCR and flow cytometry results showed that L-NTCP is negatively regulated by the nuclear receptor FXR. This study is important for understanding the adaptive mechanisms of bile acid metabolism after lamprey biliary atresia based on understanding the origin, evolution, expression profile, biological function, and expression regulation of L-NTCP.

Effect of Zinc Oxide Nanoparticles on Liver Functions in Albino Mice.

An alarming number of zinc oxide nanoparticles (ZnO-NPs) have leaked into the environment, endangering the tissues of many living creatures, due to the recent surge in their use in several items. Through intra-peritoneal injection, this research intends to examine the impact of ZnO-NPs on the hepatic and gastrointestinal structures of male albino mice. For seven and 14 days, animals were given 0.1 ml of 100 and 200 mg kg-1 of 50 nm-size ZnO-NPs, respectively. In contrast, those in the control group were given only water and food. The results demonstrated that the treated mice's livers underwent functional changes and histological damage. After seven and 14 days, there was a notable rise in the average levels of the glutamate-oxaloacetate transaminase and glutamate-pyruvate transaminase enzymes in comparison to the control group (p≤0.05). Concentration time determines the magnitude of this impact. When enzyme levels vary, it means the liver isn't working properly. Histological changes in the liver, such as necrosis, destruction of hepatocyte membranes, widening of sinusoidal spaces and vacuolation of their cytoplasm, vascular congestion, and an increased number of Kupffer cells, were induced in mice treated with ZnO-NPs at two studied concentrations (100 and 200 mg/kg) for seven and 14 days, respectively. These effects were time-dose-dependent, according to the results of hematoxylin-eosin staining of liver tissue images.

Chishao (Paeoniae Radix Rubra) alleviates intra-hepatic cholestasis by modulating NTCP in rats.

Background: Cholestasis is a common pathological manifestation dominated by accumulation of potentially toxic biliary compounds. Na+-taurocholate cotransporting polypeptide (NTCP) plays a critical role in protection from cholestasis and can be targeted therapeutically. Chishao (Paeoniae Radix Rubra) is a clinically efficacious agent for treating cholestasis, but the underlying mechanism has not been fully clarified. Objective: To evaluate the effects of Chishao on the expression of NTCP in rats with alpha-naphthylisothiocyanate (ANIT)-induced cholestasis. Methods: Chishao extracts were obtained by water decoction. Cholestasis model induced by ANIT in rats were established. Thirty rats were divided into five groups: control group (C), ANIT model group (M), 10g/kg Chishao group (LD), 20g/kg Chishao group (MD) and 40g/kg Chishao group (HD). The levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TB), direct bilirubin (DB), alkaline phosphatase (ALP) and total bile acid (TBA) were detected. The mRNA and protein expression of NTCP, multidrug resistance associated protein 2 (MRP2) and bile salt export pump (BSEP) were detected by reverse transcription qPCR and Western blotting respectively. To assess the effects of Chishao on NTCP, MRP2 and BSEP localized at the membrane of hepatocytes, an in vitro experiment involving primary hepatocytes was conducted via the utilization of laser scanning confocal microscopy. Results: The extracts of Chishao significantly improved serum ALT, AST, ALP, TB, DB and TBA (p < 0.05), especially ALP in the HD group (p < 0.01). The histological pathological findings were also reversed in LD, MD and HD groups. The mRNA level of MRP2 was significantly downregulated after treatment with ANIT, whereas it was reversed in MD and HD groups (p < 0.05). The mRNA expression of NTCP was significantly downregulated after ANIT treatment, but dramatically upregulated in the HD group. The expressions of BSEP and MRP2 were similar, but that of NTCP decreased after ANIT treatment, which was reversed significantly by Chishao extracts in a dose-dependent manner. The expression of NTCP in hepatocytes from rats increased dose-dependently after Chishao treatment in vitro. Conclusion: Chishao extracts can improve the serum and histological performances of intra-hepatic cholestasis caused by ANIT, probably by working on transport proteins in liver cell membranes.

Natural Product Baohuoside I Impairs the Stability and Membrane Location of MRP2 Reciprocally Regulated by SUMOylation and Ubiquitination in Hepatocytes.

Epimedii Folium (EF) is a botanical dietary supplement to benefit immunity. Baohuoside I (BI), a prenylated flavonoid derived from EF, has exhibited the cholestatic risk before. Here, the mechanism of BI on the stability and membrane localization of liver MRP2, a bile acid exporter in the canalicular membrane of hepatocytes, was investigated. The fluorescent substrate of MRP2, CMFDA was accumulated in sandwich-cultured primary mouse hepatocytes (SCH) under BI stimulation, followed by reduced membrane MRP2 expression. BI triggered MRP2 endocytosis associated with oxidative stress via inhibition of the NRF2 signaling pathway. Meanwhile, BI promoted the degradation of MRP2 by reducing its SUMOylation and enhancing its ubiquitination level. Co-IP and fluorescence colocalization experiments all proved that MRP2 was a substrate protein for SUMOylation for SUMO proteins. CHX assays showed that SUMO1 prolonged the half-life of MRP2 and further increased its membrane expression, which could be reversed by UBC9 knockdown. Correspondingly, MRP2 accumulated in the cytoplasm by GP78 knockdown or under MG132 treatment. Additionally, the SUMOylation sites of MRP2 were predicted by the algorithm, and a conversion of lysines to arginines at positions 940 and 953 of human MRP2 caused its decreased stability and membrane location. K940 was further identified as the essential ubiquitination site for MRP2 by an in vitro ubiquitination assay. Moreover, the decreased ubiquitination of MRP2 enhanced the SUMOylation MRP2 and vice versa, and the crosstalk of these two modifiers could be disrupted by BI. Collectively, our findings indicated the process of MRP2 turnover from the membrane to cytoplasm at the post-translational level and further elucidated the novel toxicological mechanism of BI.