Rat Hepatocytes Research Articles

In Silico and Chromatographic Methods for Analysis of Biotransformation of Prospective Neuroprotective Pyrrole-Based Hydrazone in Isolated Rat Hepatocytes.

In the current study, chromatographic and in silico techniques were applied to investigate the biotransformation of ethyl 5-(4-bromophenyl)-1-(2-(2-(2-hydroxybenzylidene) hydrazinyl)-2-oxoethyl)-2-methyl-1H-pyrrole-3-carboxylate (11b) in hepatocytic media. The initial chromatographic procedure was based on the employment of the conventional octadecyl stationary phase method for estimation of the chemical stability. Subsequently, a novel and rapid chromatographic approach based on a phenyl-hexyl column was developed, aiming to separate the possible metabolites. Both methods were performed on a Dionex 3000 ThermoScientific (ACM 2, Sofia, Bulgaria) device equipped with a diode array detector set up at 272 and 279 nm for analytes detection. An acetonitrile: phosphate buffer of pH 3.5: methanol (17:30:53 v/v/v) was eluted isocratically as a mobile phase with a 1 mL/min flow rate. A preliminary purification from the biological media was achieved by protein precipitation with methanol. A validation procedure was carried out, where the method was found to correspond to all ICH (Q2) and M10 set criteria. Additionally, an in silico-based approach with the online server BioTransformer 3.0 was applied in an attempt to predict the possible metabolites of the title compound 11b. It was hypothesized that four CYP450 isoforms (1A2, 2C9, 3A4, and 2C8) were involved in the phase I metabolism, resulting in the formation of 12 metabolites. Moreover, docking studies were conducted to evaluate the formation of stable complexes between 11b and the aforementioned isoforms. The obtained data indicated three metabolites as the most probable products, two of which (M9_11b and M10_11b) were synthesized by a classical approach for verification. Finally, liquid chromatography with a mass detector was implemented for comprehensive and summarized analysis, and the obtained results revealed that the metabolism of the 11b proceeds possibly with the formation of glucuronide and glycine conjugate of M11_11b.

Extra-corporeal non-liver transplant therapies for acute liver failure: Focus on plasma exchange and continuous renal replacement therapy.

The acute inflammatory milieu in patients with acute liver failure (ALF) results in 'toxic' blood in these patients. In vitro experiments have shown that the plasma obtained from ALF patients is toxic to rabbit hepatocytes and inhibits regeneration of rat hepatocytes. Treatments such as plasma exchange and continuous renal replacement therapy to cleanse the blood have improved survival in ALF patients. In the liver microcirculation, the exchange of fluid across fenestrae in liver sinusoidal endothelial cells (LSECs) is vital for proper functioning of hepatocytes. Clogging of the liver filter bed by inflammatory debris and cells ('traffic jam hypothesis') impeding blood flow in sinusoids may in turn reduce the exchange of fluid across LSEC fenestrae and cause dysfunction and necrosis of hepatocytes in ALF patients. In mouse model of paracetamol overdose, disturbances in microcirculation in the liver preceded the development of injury and necrosis of hepatocytes. This may represent a reversible pathophysiological mechanism in ALF which may be improved by the anti-inflammatory effect of plasma exchange. Wider access to urgent plasma exchange is a major advantage compared to urgent liver transplantation to treat ALF patients worldwide, especially so in resource constrained settings. Continuous hemo-filtration or dialysis is used toreduce ammonia levels and treat cerebral edema in ALF patients. In this review, we discuss the different modalities to cleanse the blood in ALF patients, with an emphasis on plasma exchange, from a hepatology perspective.

Activity refinement of aryl amino acetamides that target the P. falciparum STAR-related lipid transfer 1 protein

Malaria is a devastating disease that causes significant morbidity worldwide. The development of new antimalarial chemotypes is urgently needed because of the emergence of resistance to frontline therapies. Independent phenotypic screening campaigns against the Plasmodium asexual parasite, including our own, identified the aryl amino acetamide hit scaffold. In a prior study, we identified the STAR-related lipid transfer protein (PfSTART1) as the molecular target of this antimalarial chemotype. In this study, we combined structural elements from the different aryl acetamide hit subtypes and explored the structure-activity relationship. It was shown that the inclusion of an endocyclic nitrogen, to generate the tool compound WJM-715, improved aqueous solubility and modestly improved metabolic stability in rat hepatocytes. Metabolic stability in human liver microsomes remains a challenge for future development of the aryl acetamide class, which was underscored by modest systemic exposure and a short half-life in mice. The optimized aryl acetamide analogs were cross resistant to parasites with mutations in PfSTART1, but not to other drug-resistant mutations, and showed potent binding to recombinant PfSTART1 by biophysical analysis, further supporting PfSTART1 as the likely molecular target. The optimized aryl acetamide analogue, WJM-715 will be a useful tool for further investigating the druggability of PfSTART1 across the lifecycle of the malaria parasite.

Alleviation of taurine on liver injury of type 2 diabetic rats by improving antioxidant and anti-inflammatory capacity

Type 2 diabetes mellitus (T2DM) is a serious metabolic disease characterized by insulin resistance and reduced insulin production, which causes abnormally elevated blood glucose. It has been reported that T2DM can enhance oxidative stress and inflammatory responses, and stimulate a variety of complications including liver injury. Studies have shown that taurine has antioxidant and anti-inflammatory effects that can not only ameliorate diabetes but also alleviate liver injury caused by various diseases. However, its effect on liver injury in T2DM is not clear. In our study, a high-fat diet and intraperitoneal injection of streptozotocin (STZ) was used to induce liver injury in T2DM rats, and taurine was given as a treatment. Through the use of HE staining on paraffin sections, ELISA, and qRT-PCR, the effects of taurine on liver pathological alterations, antioxidant capacity, and inflammatory response were investigated. We found that: hepatic transaminase levels of rats were reduced significantly following taurine administration; histopathological observations revealed that the morphology of rat hepatocytes was close to normal, and the number of inflammatory cells around liver vessels was significantly reduced; antioxidant-related indicators were significantly increased, including SOD, CAT, GSH-Px and T-AOC, while related factors of the Nrf2 signalling pathway and its downstream HO-1, NQO1 and γ-GCS were significantly increased; the expression of the JAK2-STAT1 signalling pathway, TLR4/NF-κB signalling pathway and NLRP3 inflammatory vesicle-related factors were significantly reduced. Our results suggest taurine can alleviate T2DM-induced liver injury by improving the antioxidant capacity of the liver and inhibiting macrophage M1-type polarization and the inflammatory response mediated thereby.

Thermoresponsive block-copolymer brush-modified interfaces for effective fabrication of hepatocyte sheets

Hepatic tissue engineering has been investigated for the treatment of liver disease. One promising tissue is the hepatocyte sheet, cultured in temperature-responsive cell culture dishes. However, the preparation of hepatocyte sheets requires precoating of the dish with fetal bovine serum (FBS), which is not desirable for clinical applications. To overcome this issue, we developed a thermoresponsive polymer-modified glass with an affinity for hepatocytes. Poly(N-p-vinylbenzyl-O-β-d-galactopyranosyl-(1→4)-d-guliconamide)-b-poly(N-isopropylacrylamide) (PVLA-b-PNIPAAm) was prepared through two steps of atom transfer radical polymerization (ATRP). The prepared copolymer brushes were characterized using X-ray photoelectron spectroscopy, atomic force microscopy, gel permeation chromatography, contact angle measurements, and fluorescence labeled-fibronectin and -lectin adsorption assays. These characterizations showed that the smooth block copolymer layer was successfully modified by ATRP. The feasibility of using the prepared polymer brush-grafted glass as a hepatocyte sheet fabrication dish was investigated using primary rat hepatocytes. The PVLA-b-PNIPAAm brushes exhibited hepatocyte adhesion and proliferation without FBS precoating. By reducing the temperature from 37 °C to 20 °C, the hepatocyte sheet was recovered from the copolymer brush. These results indicated that the prepared PVLA-b-PNIPAAm brushes can be utilized for effective hepatocyte sheet fabrication without the need for FBS precoating.

Biochemical Evaluation of Adansonia Digitata Methanolic Leaf and Stem Bark Extract on Streptozotocin Induced Diabetic Rats

Several herbal preparations are used to treat diabetes; however, their overall biochemical effects other than hypoglycemic effects are scanty. This study therefore was designed to evaluate the effect of methanolic extract of leaves and stem-bark of Adansonia digitata on biochemical indices of streptozotocin-induced diabetic rats. Sixty-three wistar rats were distributed into 9 groups of 7 each. The test animals were intraperitoneally administered with single dose of 50 mg/kg streptozotocin and monitored for 72 hours for development of hyperglycemia. Diabetic rats were treated in 12 h cycles for three weeks with 100 mg/kg, 200 mg/kg and 300 mg/kg of both leaves and stem-bark of Adansonia digitata methanolic extract and metformin (50 mg/kg). Non-diabetic control rats received a lacebo of distilled water. Group 1 served as normal control, group 2 served as diabetic control, while groups 4-9 were diabetic rats treated orally with methanolic leave and stem-bark extracts of Adansonia digitata (100 mg/kg, 200 mg/kg and 300 mg/kg) for 21days. Group 3 animals were diabetic rats treated with anti-diabetic drug (metformin 50 mg/kg). The levels of fasting blood sugar (FBS), electrolytes (Na+, K+, Cl-), Urea and creatinine Triglyceride (TG), Cholesterol (CHOL), thiobarbituric acid reactive substance (TBARS), White Blood Cell (WBC) count, Red Blood Cell (RBC) count, Haemoglobin (HGB), Hematocrit (HCT), Platelet (PLT), Mean Cell Volume (MCV), Mean Cell Haemoglobin (MCH), Mean Cell Haemoglobin Concentration (MCHC), Lymphocyte (LYM), aspartatate aminotransfrease (AST), alanine aminotransferase (ALT), total protein (TP), albumin (ALB), catalase (CAT) and superoxide dismutase (SOD) activities total, total bilirubin (TB), direct bilirubin(DB) and indirect bilirubin (IB) concentrations were assayed. The results indicated that the concentrations of Na+, K+, Cl-, creatinine, urea TBARS, ALT, AST, ALB, CHOL and IB bilirubin were significantly (p<0.05) increased, while the levels of WBC, RBC, HGB, HCT, PLT, LYM, MCV SOD and CAT were reduced in the diabetic control (p<0.05). The Adansonia digitata methanolic leaves and stem-bark extract significantly increased WBC, RBC, HGB, HCT, PLT, MCV, LYM, TP, CAT and SOD activity and reduced the FBS, Na+, K+, Cl-, TBARS, ALT, AST, creatinine, urea and bilirubin concentrations significantly (p<0.05) compared to normal control. However, treatment with metformin showed slight modification in the changes observed compared to Adansonia digitata methanolic leaves and stem-bark extract. MCV and MCHC reduced non-significantly (p>0.05) in the diabetic animals as compared to the normal control and the extract-treated rats, while they increased non- significantly (p>0.05) in the test groups when compared to the diabetic control. Levels of TB, DB, and TG showed non-significant (p<0.05) increase in diabetic control, but treatment with extracts and metformin caused non-significant decrease (p<0.05). Diabetic control exhibited significantly (p<0.05) decreased ALB levels and non-significant TP decrease compared to normal control, while Adansonia digitata extracts and metformin significantly increased ALB and non-significantly increased TP levels compared to diabetic control. The study concluded that Adansonia digitata extracts reversed diabetes-induced oxidative stress in rat hepatocytes, potentially through beta cell regeneration or insulin release stimulation, suggesting their potential for managing diabetic complications.

Rat Pharmacokinetics and In Vitro Metabolite Identification of KM-819, a Parkinson's Disease Candidate, Using LC-MS/MS and LC-HRMS.

FAF1 (FAS-associated factor 1) is involved in the activation of Fas cell surface death receptors and plays a role in apoptosis and necrosis. In patients with Parkinson's disease, FAF1 is overexpressed in dopaminergic neurons in the substantia nigra. KM-819, an FAF1 inhibitor, has shown potential for preventing dopaminergic neuronal cell death, promoting the degradation of α-synuclein and preventing its accumulation. This study aimed to develop and validate a quantitative analytical method for determining KM-819 levels in rat plasma using liquid chromatography-tandem mass spectrometry. This method was then applied to pharmacokinetic (PK) studies in rats. The metabolic stability of KM-819 was assessed in rat, dog, and human hepatocytes. In vitro metabolite identification and metabolic pathways were investigated in rat, dog, and human hepatocytes. The structural analog of KM-819, namely N-[1-(4-bromobenzyl)-3,5-dimethyl-1H-pyrazol-4-yl]-2-(phenylsulfanyl) acetamide, served as the internal standard (IS). Proteins were precipitated from plasma samples using acetonitrile. Analysis was carried out using a reverse-phase C18 column with a mobile phase consisting of 0.1% formic acid in distilled water and 0.1% formic acid in acetonitrile. The analytical method developed for KM-819 exhibited linearity within the concentration range of 0.002-10 μg/mL in rat plasma. The precision and accuracy of the intra- and inter-day measurements were <15% for the lower limit of quantification (LLOQ) and all quality control samples. KM-819 demonstrated stability under various sample storage conditions (6 h at room temperature (25 °C), four weeks at -20 °C, three freeze-thaw cycles, and pretreated samples in the autosampler). The matrix effect and dilution integrity met the criteria set by the Food and Drug Administration and the European Medicines Agency. This sensitive, rapid, and reliable analytical method was successfully applied in pharmacokinetic studies in rats. Pharmacokinetic analysis revealed the dose-independent kinetics of KM-819 at 0.5-5 mg/kg, with a moderate oral bioavailability of ~20% in rats. The metabolic stability of KM-819 was also found to be moderate in rat, dog, and human hepatocytes. Metabolite identification in rat, dog, and human hepatocytes resulted in the discovery of six, six, and eight metabolites, respectively. Glucuronidation and mono-oxidation have been proposed as the major metabolic pathways. Overall, these findings contribute to a better understanding of the pharmacokinetic characteristics of KM-819, thereby aiding future clinical studies.

Structural Changes in Rat Hepatocyte Nucleolus under Nucleolar Stress Caused by Hypothermia.

Structural changes in rat hepatocyte nucleoli were studied during deep hypothermia simulated by immersion in water at 5°C for 40 min (ambient air temperature 7°C). In comparison with the control, phenomena of nucleolar stress occurred in rats during hypothermia: the number of fibrillar centers (FC) per nucleus (by 1.7 times) and per nucleolus (by 1.6 times), nucleolonemal nucleoli per nucleus (by 2.8 times), and the relative content of nucleolonemal nucleoli per nucleus (by 2.6 times) significantly decreased (p=0.0000001); the number of FC per nucleolonemal nucleolus also decreased by 1.4 times (p=0.01). In the hepatocyte nuclei, we observed an increase in the relative content of transitional type nucleoli per nucleus (by 1.3 times; p=0.01), the number of FC per transitional type nucleolus (by 1.4 times; p=0.003), the content of free FC per nucleus (by 3 times; p=0.00004), and the percentage of free FC per nucleus (by 3.5 times; p=0.00004). These changes can be considered as compensatory and adaptive reactions, and transitional type nucleoli can be attributed to the "reserve" nucleolar pool.

A computational model of endogenous hydrogen peroxide metabolism in hepatocytes, featuring a critical role for GSH

This paper presents an ordinary differential equation (ODE) model of endogenous H2O2metabolism in hepatocytes that is unique, at the time of writing, in its ability to accurately compute intracellular H2O2concentration during incidents of oxidative stress and in its usefulness for constructing PBPK/PD models for ROS-generating xenobiotics. Versions of the model are presented for rat hepatocytes in vitro and mouse liver in vivo. A generic method is given for using the model to create PBPK/PD models which predict intracellular H2O2concentration and oxidative-stress-induced hepatocyte death; these are identifiable from in vitro data sets reporting cell mortality following xenobiotic exposure at various levels. The procedure is demonstrated for the trivalent arsenical dimethylarsinous acid (DMAIII), which is produced in liver as part of the arsenic elimination pathway. This is the first model of H2O2metabolism in hepatocytes to feature values for the endogenous rates of H2O2production by mitochondria and other organelles which are inferred from the physiology literature, and to feature a detailed, realistic treatment of GSH metabolism; the latter is achieved by incorporating a minimal version of Reed and coworkers’ pioneering model of GSH metabolism in liver. Model simulations indicate that critical GSH depletion is the immediate trigger for intracellular H2O2rising to concentrations associated with apoptosis (>1μM), that this may only occur hours after the xenobiotic concentration peaks (“delay effect”), that when critical GSH depletion does occur, H2O2concentration rises rapidly in a sequence of two boundary layers, characterized by the kinetics of glutathione peroxidase (first boundary layer) and catalase (second boundary layer), and that intracellular H2O2concentration >1μM implies critical GSH depletion. There has been speculation that ROS levels in the range associated with apoptosis simply indicate, rather than cause, an apoptotic milieu. Model simulations are consistent with this view. In a result of interest to the wider physiology community, the delay effect is shown to provide a GSH-based mechanism by which cells can distinguish transient elevations in H2O2concentration, of use in intracellular signaling, from persistent ones indicative of either pathology or the presence of toxins, the second state of affairs eventually triggering apoptosis.

Epac2 activation mediates glucagon-induced glucogenesis in primary rat hepatocytes.

Glucagon plays an essential role in hepatic glucogenesis by enhancing glycogen breakdown, inducing gluconeogenesis, and suppressing glycogenesis. Moreover, glucagon increases cyclic adenosine monophosphate (cAMP) levels, thereby activating protein kinase A (PKA) and cAMP guanine nucleotide exchange factor (also known as Epac). Although the function of PKA in the liver has been studied extensively, the function of hepatic Epac is poorly understood. The aim of this study was to elucidate the role of Epac in mediating the action of glucagon on the hepatocytes. Epac mRNA and protein expression, localization, and activity in the hepatocytes were analyzed by reverse transcription polymerase chain reaction, western blotting, immunofluorescence staining, and Rap1 activity assay, respectively. Additionally, we investigated the effects of an Epac-specific activator, 8-CPT, and an Epac-specific inhibitor, ESI-05, on glycogen metabolism in isolated rat hepatocytes. Further mechanisms of glycogen metabolism were evaluated by examining glucokinase (GK) translocation and mRNA expression of gluconeogenic enzymes. Epac2, but not Epac1, was predominantly expressed in the liver. Moreover, 8-CPT inhibited glycogen accumulation and GK translocation and enhanced the mRNA expression of gluconeogenic enzymes. ESI-05 failed to reverse glucagon-induced suppression of glycogen storage and partially inhibited glucagon-induced GK translocation and the mRNA expression of gluconeogenic enzymes. Epac signaling plays a role in mediating the glucogenic action of glucagon in the hepatocytes.

Comparative metabolism of aschantin in human and animal hepatocytes.

Aschantin, a tetrahydrofurofuran lignan with a 1,3-benzodioxole group derived from Flos Magnoliae, exhibits antioxidant, anti-inflammatory, cytotoxic, and antimicrobial activities. This study compared the metabolic profiles of aschantin in human, dog, mouse, and rat hepatocytes using liquid chromatography-high-resolution mass spectrometry. The hepatic extraction ratio of aschantin among the four species was 0.46-0.77, suggesting that it undergoes a moderate-to-extensive degree of hepatic metabolism. Hepatocyte incubation of aschantin produced 4 phase 1 metabolites, including aschantin catechol (M1), O-desmethylaschantin (M2 and M3), and hydroxyaschantin (M4), and 14 phase 2 metabolites, including O-methyl-M1 (M5 and M6) via catechol O-methyltransferase (COMT), six glucuronides of M1, M2, M3, M5, and M6, and six sulfates of M1, M2, M3, M5, and M6. Enzyme kinetic studies using aschantin revealed that the production of M1, a major metabolite, via O-demethylenation is catalyzed by cytochrome 2C8 (CYP2C8), CYP2C9, CYP2C19, CYP3A4, and CYP3A5 enzymes; the formation of M2 (O-desmethylaschantin) is catalyzed by CYP2C9 and CYP2C19; and the formation of M4 is catalyzed by CYP3A4 enzyme. Two glutathione (GSH) conjugates of M1 were identified after incubation of aschantin with human and animal liver microsomes in the presence of nicotinamide adenine dinucleotide phosphate and GSH, but they were not detected in the hepatocytes of all species. In conclusion, aschantin is extensively metabolized, producing 18 metabolites in human and animal hepatocytes catalyzed by CYP, COMT, UDP-glucuronosyltransferase, and sulfotransferase. These results can help in clarifying the involvement of metabolizing enzymes in the pharmacokinetics and drug interactions of aschantin and in elucidating GSH conjugation associated with the reactive intermediate formed from M1 (aschantin catechol).

Citrus limon (L.) Osbeck Fruit Peel Extract Attenuates Carbon Tetrachloride-Induced Hepatocarcinogenesis in Sprague-Dawley Rats.

Traditional herbal medicine practitioners in the Ashanti region of Ghana use the fruit peels of Citrus limon (L.) Osbeck (C. limon) in preventive and curative treatment of many cancers including liver cancer. This ethnobotanical claim remains to be verified scientifically. Aim of the Study. This study investigated prophylactic hepatoprotective and anti-HCC effects of C. limon peel extract (LPE) in CCl4/olive oil-induced HCC-like rats. After preparation of LPE, it was subjected to phytochemical screening using standard phytochemical methods. A total of 30 healthy adult male Sprague-Dawley rats (weighing 150-200 g) were randomly assigned into six groups of 5 rats each. Rats in the control group received olive oil (5 mL/kg ip) twice weekly for 16 weeks. Rats in the model group received CCl4/olive oil (2 mL/kg, ip) twice weekly for 16 weeks. Rats in capecitabine (10 mg/kg po) and LPE (50, 100, and 200 mg/kg po) groups received CCl4/olive oil (2 mL/kg, i.p) in the morning and their respective treatments in the afternoon twice a week for 16 weeks. Rats in all groups had free access to food and water ad libitum. Body weight and survival rates were monitored. Rats were sacrificed under deep anesthesia, blood was collected, and liver and other organs were isolated. Aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), gamma-glutamyltransferase (GGT), prothrombin time, bilirubin, C-reactive protein (CRP), alpha- (α-) fetoprotein (AFP), and liver histology were assessed. Alkaloids, tannins, flavonoids, terpenoids, and saponins were detected in LPE. Model rats demonstrated increased serum levels of AFP, CRP, ALP, GGT, ALT, and AST, prothrombin time, total bilirubin, direct bilirubin, blood lymphocyte, and monocyte counts, but decreased serum albumin and total protein compared to control rats. Unlike the control, model rats demonstrated fat accumulation in periportal and centrilobular hepatocytes and neoplastic transformation. Semiquantitation of periodic acid Schiff- (PAS-) stained liver sections showed decreased glycogen storage in hepatocytes of model rats compared to control rats. Compared to the model, LPE treatment protected against CCl4-induced hepatocarcinogenesis, which was evidenced by decreased AFP, CRP, liver enzymes, total and direct bilirubin, prothrombin time, and blood lymphocyte and monocyte counts; attenuation of fat accumulation; and increased glycogen storage, albumin, and total protein. LPE abates CCl4-induced hepatocarcinogenesis by attenuating liver inflammation and improving metabolic, biosynthetic, and detoxification functions of the liver. The prophylactic hepatoprotective and anti-hepatocarcinogenic effects of LPE are attributable to its phytochemical composition raising hopes of finding potential anticancer bioactive compounds from C. limon fruit peels.

Effect of aqueous-alcoholic extract of Ducrosia Anethifolia Boiss on the fetal liver of diabetic rats

Objective: Moshgak (Ducrosia anethifolia) is a wild plant with medicinal value. The present study aimed to evaluate the effect of Moshgak on the liver tissue of the diabetic rat fetus. Materials and Methods: In this animal study, the aqueous-alcoholic extract of Moshgak was prepared in the standard method. Forty rats were divided randomly into five groups, including control, sham, and three diabetic groups. The rats were diabetic with intraperitoneal injection of a single dose of streptozotocin (80 mg) and 2 diabetic groups were treated with Moshgak extract (280 and 560 mg/kg/bw) for 19 days. The rats were anesthetized and their blood was taken to measure the blood glucose, insulin, and malondialdehyde. Then, their fetuses were removed. The fetal liver sections were obtained by using the stereological methods. The micrometry of the liver tissue was performed and data were analyzed. Results: The finding showed a statistically significant increase (P &lt; 0.01) in the total volume of liver, connective tissue, sinusoid, and hepatocytes in diabetic rats compared to control rats, while these parameters decreased significantly in treated groups with Moshgak. Hepatic cell count hepatic decreased in the treated groups. Furthermore, the changes in blood glucose, malondialdehyde, and insulin in diabetic rats were improved significantly by Moshgak treatment. The dilation of sinusoids, hepatocyte vacuolation, and mild lymphocytosis was observed in all diabetic groups except the treatment group with Moshgak 560 mg/kg/bw. Conclusions: According to obtained results, Moshgak extract was able to compensate partially the changes induced by diabetes in the fetal liver tissue. Therefore, due to the side effects of diabetes during pregnancy, further research on anti-diabetic properties of Moshgak is suggested.

Influence of drop on the ultrastructure lipid composition of liver hepatocytes of pregnant rats

The influence of a toxic agent on the structure of the liver is inevitable. Of interest are the effects of pesticides on intrauterine formation and their teratogenic and embryotoxic properties. The influence of teratogens may manifest itself at different levels of organization of a living organism, and some aspects of cell metabolism change. Using advanced electron microscopy techniques combined with modern knowledge of membranology and toxicology, we conducted a detailed study of morphological changes in the subcellular architecture of the liver after repeated dropplet exposure. Ultrastructural studies were carried out on pregnant rats and their embryos on the 3rd, 13th, 19th days of pregnancy and embryonic development. Poisoning of animals with the pesticide dropp causes clear degenerative processes in the cellular and subcellular structures of the liver.

A decellularized matrix enriched collagen microscaffold for a 3D in vitro liver model.

The development of liver scaffolds retaining their three-dimensional (3D) structure and extra-cellular matrix (ECM) composition is essential for the advancement of liver tissue engineering. We report the design and validation of an alginate-based platform using a combination of decellularized matrices and collagen to preserve the functionality of liver cells. The scaffolds were characterized using SEM and fluorescence microscopy techniques. The proliferation and functional behaviours of hepatocellular carcinoma HuH7 cells were observed. It was found that the decellularized skin scaffold with collagen was better for maintaining the growth of cells in comparison to other decellularized matrices. In addition, we observed a significant increase in the functional profile once exogenous collagen was added to the liver matrix. Our study also suggests that a cirrhotic liver model should have a different matrix composition as compared to a healthy liver model. When primary rat hepatocytes were used for developing a healthy liver model, the proliferation studies with hepatocytes showed a decellularized skin matrix as the better option, but the functionality was only maintained in a decellularized liver matrix with addition of exogenous collagen. We further checked if these platforms can be used for studying drug induced toxicity observed in the liver by studying the activation of cytochrome P450 upon drug exposure of the cells growing in our model. We observed a significant induction of the CYP1A1 gene on administering the drugs for 6 days. Thus, this platform could be used for drug-toxicity screening studies using primary hepatocytes in a short span of time. Being a microscaffold based system, this platform offers some advantages, such as smaller volumes of samples, analysing multiple samples simultaneously and a minimal amount of decellularized matrix in the matrix composition, making it an economical option compared to a completely dECM based platform.

Sulforaphane Is Protective against Warm Ischemia/Reperfusion Injury and Partial Hepatectomy in Rats.

Sulforaphane (SFN) has various beneficial effects on organ metabolism. However, whether SFN affects inflammatory mediators induced by warm hepatic ischemia/reperfusion injury (HIRI) is unclear. To investigate the hepatoprotective effects of SFN using an in vivo model of HIRI and partial hepatectomy (HIRI + PH), rats were subjected to 15 min of hepatic ischemia with blood inflow occlusion, followed by 70% hepatectomy and release of the inflow occlusion. SFN (5 mg/kg) or saline was randomly injected intraperitoneally 1 and 24 h before ischemia. Alternatively, ischemia was prolonged for 30 min to evaluate the effect on mortality. The influence of SFN on the associated signaling pathways was analyzed using the interleukin 1β (IL-1β)-treated primary cultured rat hepatocytes. In the HIRI + PH-treated rats, SFN reduced serum liver enzyme activities and the frequency of pathological liver injury, such as apoptosis and neutrophil infiltration. SFN suppressed tumor necrosis factor-alpha (TNF-α) mRNA expression and inhibited nuclear factor-kappa B (NF-κB) activation by HIRI + PH. Mortality was significantly reduced by SFN. In IL-1β-treated hepatocytes, SFN suppressed the expression of inflammatory cytokines and NF-κB activation. Taken together, SFN may have hepatoprotective effects in HIRI + PH in part by inhibiting the induction of inflammatory mediators, such as TNF-α, via the suppression of NF-κB in hepatocytes.

Empagliflozin: Potential Protective Effects on Hepatocytes and Liver Outcomes in Streptozotocin -Diabetic Rats.

The current study investigated the potential positive impact of EMPA, an antidiabetic medication, on hepatocytes and liver outcomes in STZ-induced diabetic rats. Male Wistar rats were randomly assigned into four groups: control, DM (received 40mg/kg streptozotocin IP injection), DM+EMPA (received 40mg/kg streptozotocin and 10 mg/kg EMPA), and EMPA (received 10 mg/kg EMPA). Here, liver functional tests were assessed spectrophotometrically, while histological analysis of liver tissues was evaluated using light microscopy. Treated diabetic rats significantly reduced AST levels compared to treated control rats (p &lt; 0.05). DM rats, with or without EMPA treatment, showed significantly elevated ALT levels compared to control rats (p &lt; 0.005). Also, LDH levels were found to be lower in both treated and untreated diabetic rats compared to control rats (p &lt; 0.0001; p &lt; 0.05, respectively), while ALP levels were higher in both groups of diabetic rats relative to control rats (p &lt; 0.0001; p &lt; 0.005). Interestingly, the data showed clear trends indicating that empagliflozin-treated diabetic rats had improved liver parameters compared to untreated diabetic rats, although statistically significant differences were not observed. Remarkably, histological examination showed significant sinusoidal dilation and infiltration of inflammatory cells in hepatocytes in diabetic rats, whereas treated diabetic rats exhibited a normal hepatocyte arrangement with minor sinusoidal dilation. Altogether, the observed results suggest that EMPA may possess a protective effect on hepatocytes, thereby highlighting its potential as a therapeutic intervention for diabetes-related liver complications.

Synergistic Suppression of NF1 Malignant Peripheral Nerve Sheath Tumor Cell Growth in Culture and Orthotopic Xenografts by Combinational Treatment with Statin and Prodrug Farnesyltransferase Inhibitor PAMAM G4 Dendrimers.

Neurofibromatosis type 1 (NF1) is a disorder in which RAS is constitutively activated due to the loss of the Ras-GTPase-activating activity of neurofibromin. RAS must be prenylated (i.e., farnesylated or geranylgeranylated) to traffic and function properly. Previous studies showed that the anti-growth properties of farnesyl monophosphate prodrug farnesyltransferase inhibitors (FTIs) on human NF1 malignant peripheral nerve sheath tumor (MPNST) cells are potentiated by co-treatment with lovastatin. Unfortunately, such prodrug FTIs have poor aqueous solubility. In this study, we synthesized a series of prodrug FTI polyamidoamine generation 4 (PAMAM G4) dendrimers that compete with farnesyl pyrophosphate for farnesyltransferase (Ftase) and assessed their effects on human NF1 MPNST S462TY cells. The prodrug 3-tert-butylfarnesyl monophosphate FTI-dendrimer (i.e., IG 2) exhibited improved aqueous solubility. Concentrations of IG 2 and lovastatin (as low as 0.1 μM) having little to no effect when used singularly synergistically suppressed cell proliferation, colony formation, and induced N-RAS, RAP1A, and RAB5A deprenylation when used in combination. Combinational treatment had no additive or synergistic effects on the proliferation/viability of immortalized normal rat Schwann cells, primary rat hepatocytes, or normal human mammary epithelial MCF10A cells. Combinational, but not singular, in vivo treatment markedly suppressed the growth of S462TY xenografts established in the sciatic nerves of immune-deficient mice. Hence, prodrug farnesyl monophosphate FTIs can be rendered water-soluble by conjugation to PAMAM G4 dendrimers and exhibit potent anti-tumor activity when combined with clinically achievable statin concentrations.

Comparison of in vitro thyroxine (T4) metabolism between Wistar rat and human hepatocyte cultures

In vitro assays remain relatively new in exploring human relevance of liver, in particular nuclear receptor-mediated perturbations of the hypothalamus-pituitary-thyroid axis seen in rodents, mainly in the rat. Consistent with in vivo data, we confirm that thyroid hormone thyroxine metabolism was 9 times higher in primary rat hepatocytes (PRH) than in primary human hepatocytes (PHH) cultured in a 2D sandwich (2Dsw) configuration. In addition, thyroxine glucuronide (T4-G) was by far the major metabolite formed in both species (99.1% in PRH and 69.7% in PHH) followed by thyroxine sulfate (T4-S, 0.7% in PRH and 18.1% in PHH) and triiodothyronine/reverse triiodothyronine (T3/rT3, 0.2% in PRH and 12.2% in PHH). After a 7-day daily exposure to orphan receptor-mediated liver inducers, T4 metabolism was strongly increased in PRH, almost exclusively through increased T4-G formation. These results were consistent with the inductions of glucuronosyltransferase Ugt2b1 and canalicular transporter Mrp2. PHH also responded to activation of the three nuclear receptors, with mainly induction of glucuronosyltransferase UGT1A1 and canalicular transporter MRP2. Despite this, T4 disappearance rate and secreted T4 metabolites were only slightly increased in PHH. Overall, our data highlight that cryopreserved hepatocytes in 2Dsw culture allowing long-term exposure and species comparison are of major interest in improving liver-mediated human safety assessment.

High throughput production and characterization of primary rat hepatocyte spheroids

Developing in vitro models to explore cell biology and physiology holds significant importance in biotechnology, cancer research, drug discovery, toxicity testing, and the emerging fields of tissue engineering and regenerative medicine. The conventional two-dimensional (2D) approaches of mammalian cell culture (2D) have limitations in replicating all of the mechanical and biochemical signals in vivo. Culturing cells as spheroids provides a three-dimensional environment that more accurately mimics physiological conditions compared to 2D culture. In this study, we established a method for high-throughput spheroid formation using primary rat hepatocytes (PRHs). Using a high throughput platform, we investigated the effect of varying concentrations of cell culture media supplements on spheroid formation. Additionally, we assessed different cell seeding densities and characterized their properties for 7 days. The average diameter and circularity of PRH spheroids remained stable on days 2, 4, and 7, regardless of the initial seeding density. Moreover, PRH spheroids demonstrated high viability (&gt; 90 %) for up to 7 days across all seeding cell densities. Our results demonstrated that this technique enables straightforward, large scale, consistent, and repeatable spheroid manufacturing and presents an alternative approach for future applications.