Rat Hepatocytes Research Articles

Mitochondria-Targeted Polyamidoamine Dendrimer-Curcumin Construct for Hepatocellular Cancer Treatment.

Mitochondrial malfunction plays a crucial role in cancer development and progression. Cancer cells show a substantially higher mitochondrial activity and greater mitochondrial transmembrane potential than normal cells. This concept can be exploited for targeting cytotoxic drugs to the mitochondria of cancer cells using mitochondrial-targeting compounds. In this study, a polyamidoamine dendrimer-based mitochondrial delivery system was prepared for curcumin using triphenylphosphonium ligands to improve the anticancer efficacy of the drug in vitro and in vivo. For the in vitro evaluations, various methods, such as viability assay, confocal microscopy, flow cytometry, reactive oxygen species (ROS), and real-time polymerase chain reaction analyses, were applied. Our findings showed that the targeted-dendrimeric curcumin (TDC) could successfully deliver and colocalize the drug to the mitochondria of the cancer cells, and selectively induce a potent apoptosis and cell cycle arrest at G2/M. Moreover, at a low curcumin dose of less than 25 μM, TDC significantly reduced adenosine triphosphate and glutathione, and increased the ROS level of the isolated rat hepatocyte mitochondria. The in vivo studies on the Hepa1-6 tumor-bearing mice also indicated a significant tumor suppression effect and the highest median survival days (Kaplan-Meier survival estimation and log-rank test) after treatment with the TDC construct compared to the free curcumin and untargeted construct. Besides its targeted nature and safety, the expected improved solubility and stability represent the prepared targeted-dendrimeric construct as an up-and-coming candidate for cancer treatment. The results of this study emphasize the promising route of mitochondrial targeting as a practical approach for cancer therapy, which can be achieved by optimizing the delivery method.

Metabolic profiling of ligustilide and identification of the metabolite in rat and human hepatocytes by liquid chromatography combined with high‐resolution mass spectrometry

Ligustilide is one of the most abundant bioactive ingredients in Rhizoma Chuanxiong that has been widely prescribed for medicinal purposes in China. To better understand the disposition and action of ligustilide, it is necessary to investigate the metabolic profiles. The in vitro metabolism was elucidated through incubating ligustilide with human and rat hepatocytes at 37°C. The incubation samples were collected at predefined time points to determine the metabolic stability. Upon metabolite identification and profiling, the incubation samples were analyzed by ultra-high-performance liquid chromatography combined with diode array detector and high-resolution mass spectrometry. The structures of the metabolites were characterized based on their mass spectrometry spectra, tandem mass spectrometry spectra, and fragmentation patterns. Ligustilide showed fast metabolism with high intrinsic clearance both in rat and human hepatocyte incubations. The half-lives of ligustilide in rat and human hepatocyte incubations were 8.0 and 15.0min, respectively. Most of the parent (>90%) was biotransformed into the metabolites. Among these metabolites, M1 (senkyunolide I) was the major metabolite both in rat and human hepatocytes with the percentage of 42 and 70%, respectively. The metabolic pathways of ligustilide included epoxidation, epoxide hydrolysis, aromatization, hydroxylation, and glutathionylation. This work provided an overview of the metabolic profiles of ligustilide, which would be helpful for us to understand the action of this compound.

Low-polar extract from seed of Cleistocalyx nervosum var. paniala modulates initiation and promotion stages of chemically-induced carcinogenesis in rats

BackgroundCleistocalyx nervosum var. paniala is a local fruit mainly cultivated in the north of Thailand. Our previous study has reported that the methanol extract of C. nervosum seed presented antimutagenicity in a Salmonella mutation assay. The present study focused on the effect of a low-polar extract of C. nervosum seed on the early stages of diethylnitrosamine (DEN)- and dimethylhydrazine (DMH)-induced carcinogenesis in rats. MethodsDried C. nervosum seed powder was extracted using dichloromethane. To study its effect on the initiation stage of carcinogenesis of rats, they were fed with various doses of C. nervosum seed extract (CSE) for 21 days. DEN injection was used to initiate hepatocarcinogenesis and partial hepatectomy was performed to amplify mutated hepatocytes resulting in micronucleated hepatocyte formation. To study the role of CSE on the promotion stage, rats were injected with DEN and DMH to induce preneoplastic lesions and the numbers of glutathione S-transferase placental form (GST-P) positive foci in the liver and aberrant crypt foci (ACF) in the colon were measured. This was followed by CSE administration for 10 weeks. The inhibitory mechanisms of CSE on initiation and promotion stages, including xenobiotic metabolism, cell proliferation and apoptosis, were investigated. ResultsThe total phenolic content in CSE was 80.34 ± 2.29 mg gallic acid equivalents (GAE) per g of extract and 2,4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone was found to be a major flavonoid. The main terpenoids in CSE were β-selinene, α-selinene, γ-selinene and o-cymene while 24(Z)-methyl-25-homocholesterol was a major phytosterol. CSE significantly decreased the number of micronucleated hepatocytes in DEN-initiated rats and enhanced the activities of hepatic glutathione S-transferase and UDP-glucuronyltransferase. Furthermore, the formation of preneoplastic lesions in the liver and colon was statistically reduced by CSE. CSE also diminished cell proliferation in the liver and colon indicated by the number of PCNA positive cells. However, CSE did not alter the numbers of apoptotic hepatocytes and colonocytes in DEN- and DMH-initiated rats. ConclusionsThe dichloromethane extract of C. nervosum seed demonstrated chemopreventive effects on chemically-induced carcinogenesis in both initiation and promotion stages in rats. The inhibitory mechanism might be involved in the modulation of hepatic detoxifying enzymes and suppression of hepatocyte and colonocyte proliferation.

Effects of a citrus fruit extract on nitric oxide production and inducible nitric oxide synthase expression In Interleukin-1β-treated rat hepatocytes

Rationale: Citrus fruits contain many nutrients and have been eaten for a health-promoting diet. The immature dried fruits of Citrus aurantium var. daidai, C. aurantium, and C. natsudaidai have also been used in several formulae of Japanese herbal (Kampo) medicines to treat liver diseases. Here we investigated the efficacy of crude fractions from a Citrus fruit extract by monitoring the production of nitric oxide (NO), an inflammatory mediator, in rat hepatocytes.

Heart and liver connexin expression related to the first stage of aging: A study on naturally aged animals

Connexins are membrane-spanning proteins that form membrane channels and hemichannels. They are involved in the cellular communication and in the maintenance of tissue homeostasis. Recent studies in humans and animals have demonstrated that the expression and distribution of Cx43, the most studied connexin, can change during aging. However, the research on the involvement of the other connexins in cardiac and hepatic aging is, at present, still very poor. Hence, the aim of this study is to evaluate the expression of Cx43 and Cx26 in the heart as well as Cx26 and Cx32 in the liver of a rat model that aged naturally, rather than prematurely because of genetic mutations or age-related diseases.The results obtained in the present study have demonstrated that these connexins decrease in rat cardiomyocytes and in rat hepatocytes as they age. This change was revealed only at protein level, as connexin-mRNAs remained unchanged during aging. Moreover, the aged rats showed an increase in body fat, whose subcutaneous layer tended to be higher.Finally, how these changes could represent signs of physiological adaptation in successful aging was discussed.

Novel testing strategy for prediction of rat biliary excretion of intravenously administered estradiol-17\u03b2 glucuronide

The aim of the present study was to develop a generic rat physiologically based kinetic (PBK) model that includes a novel testing strategy where active biliary excretion is incorporated using estradiol-17β glucuronide (E217βG) as the model substance. A major challenge was the definition of the scaling factor for the in vitro to in vivo conversion of the PBK-model parameter Vmax. In vitro values for the Vmax and Km for transport of E217βG were found in the literature in four different studies based on experiments with primary rat hepatocytes. The required scaling factor was defined based on fitting the PBK model-based predicted values to reported experimental data on E217βG blood levels and cumulative biliary E217βG excretion. This resulted in a scaling factor of 129 mg protein/g liver. With this scaling factor the PBK model predicted the in vivo data for blood and cumulative biliary E217βG levels with on average of less than 1.8-fold deviation. The study provides a proof of principle on how biliary excretion can be included in a generic PBK model using primary hepatocytes to define the kinetic parameters that describe the biliary excretion.

Ultra-high-performance liquid chromatography - high-resolution mass spectrometry profiling and hepatoprotective activity of purified saponin and flavonoid fractions from the aerial parts of wild spinach (Chenopodium bonus-henricus L.).

An ultra-high-performance liquid chromatography- high-resolution mass spectrometry profiling method was used for a comprehensive study of flavonoid and saponin-rich fractions from the aerial parts of wild spinach (Chenopodium bonus-henricus L.). Thirty-six compounds, respectively, 22 saponins of eight sapogenins (phytolaccagenin, bayogenin, medicagenic acid, 2β-hydroxygypsogenin, 2β-hydroxyoleanoic acid, 2-hydroxy-30-nor-gypsogenin, 2-hydroxyakebonic acid, and akebonic acid) together with 12flavonoid glycosides of 6-methoxykaempferol, isorhamnetin, patuletin, spinacetin as well as two ecdysteroids (20-hydroxyecdysone and polypodine B) were detected. The occurrence of sapogenins 2-hydroxy-30-nor-gypsogenin, 2-hydroxyakebonic acid, and akebonic acid in the Chenopodium genus is reported here for the first time. The flavonoid and saponin-rich fractions showed in vitro hepatoprotective and antioxidant activity comparable to those of flavonoid complex silymarin (60μg/mL) in a model of metabolic bioactivation, induced by CCl4. All tested fractions, compared to silymarin, significantly reduced the cellular damage caused by CCl4 in rat hepatocytes, preserved cell viability and GSH level, decreased LDH leakage, and reduced lipid damage. The results showed that saponin-rich fractions F3A and F3B possessed better hepatoprotective activity than flavonoid-rich fractions (F2A and F2B). The most active was fraction F3B and this is probably due to the synergism between the saponins and some acylated flavonol glycosides found there.

Cellular uptake and toxicity of gold nanoparticles on two distinct hepatic cell models

Gold nanoparticles (AuNPs) have huge potential for various biomedical applications, but their successful use depends on their uptake and possible toxicity in the liver, their main site for accumulation. Therefore, in this work we compared the cytotoxic effects induced by AuNPs with different size (~ 15 nm and 60 nm), shape (nanospheres and nanostars) and capping [citrate- or 11-mercaptoundecanoic acid (MUA)], in human HepaRG cells or primary rat hepatocytes (PRH) cultivated with serum-free or Foetal Bovine Serum (FBS)-supplemented media.The safety assessment of the AuNPs demonstrated that overall they present low toxicity towards hepatic cells. Among all the tested AuNPs, the smaller 15 nm spheres displayed the highest toxicity. The toxicological effect was capping, size and cell-type dependent with citrate-capping more toxic than MUA (PRH with FBS), the 15 nm AuNPs more toxic than 60 nm counterparts and PRH more sensitive, as compared to the HepaRG cells. The incubation with FBS-free media produced aggregation of AuNPs while its presence greatly influenced the toxicity outcomes. The cellular uptake of AuNPs was shape, size and capping dependent in PRH cultivated in FBS-supplemented media, and significantly different between the two types of cells with extensively higher internalization of AuNPs in PRH, as compared to the HepaRG cells.These data show that the physical-chemical properties of AuNPs, including size and shape, as well as the type of cellular model, greatly influence the interaction of the AuNPs with the biological environment and consequently, their toxicological effects.

Creating rat hepatocyte organoid as an in vitro model for drug testing.

BACKGROUNDLiver organoids have recently been applied as models for liver disease and drug screening, especially when combined with liver-on-a-chip technologies. Compared to hepatocyte-like cells, primary hepatocytes have high functionality but cannot maintain their function when cultured in vitro. Mesenchymal stem cells (MSCs) enhance hepatocyte function and maintain hepatocyte metabolism when co-cultured with hepatocytes. MSCs can help induced pluripotent stem cells to generate an organoid structure via the MSC-based traction force triggered by extracellular matrix (ECM) proteins. In this study, primary hepatocytes were co-cultured with MSCs on a liver-derived ECM to generate liver organoids within a short duration.AIMTo create hepatocyte organoids by co-culturing primary hepatocytes with MSCs on a porcine liver extracellular matrix (PLECM) gel. METHODSPerfusion and enzymatic hydrolysis were used to form the PLECM gel. Rat hepatocytes and human MSCs were mixed and plated on pre-solidified PLECM gel in a 48-well plate for 48 h to generate organoids. Generated organoids were evaluated through hematoxylin and eosin, periodic acid-Schiff, immuno-histological, and immunofluorescence staining, and quantitative PCR for alb, CYP450 gene markers, and urea cycle genes. Culture medium was collected to detect albumin (ALB) and urea production on days 2, 4, 6, 8, 14, and 20.RESULTSThe whole porcine liver was perfused and enzymatically hydrolyzed to form a PLECM gel. The structural components and basement membrane composition of the ECM, such as collagen type I, collagen type IV, fibronectin, and laminin, were demonstrated to be retained. Through interaction of human MSCs with the liver-derived ECM, primary hepatocytes and human MSCs assembled together into a 3D construction and generated primary hepatocyte organoids for 48 h. The mRNAs of the gene alb, the CYP450 gene markers cyp1a1, cyp1a2, and cyp3a2 as well as urea cycle genes arg-1, asl, ass-1, cps-1, nags were highly expressed in hepatocyte organoids. Long-term survival of the primary hepatocyte organoids, as well as stable functionality, was demonstrated via ALB and urea production in vitro.CONCLUSIONOur new method of creating primary hepatocyte organoids by co-culturing hepatocytes with MSCs on liver-derived ECM hydrogels could be used to develop models for liver disease and for drug screening.

Aspirin Eugenol Ester Attenuates Paraquat-Induced Hepatotoxicity by Inhibiting Oxidative Stress.

Aspirin eugenol ester (AEE) is a new potential drug with anti-inflammatory and antioxidant stress pharmacological activity. Paraquat (PQ) is an effective and commercially important herbicide that is widely used worldwide. However, paraquat is highly toxic and can cause various complications and acute organ damage, such as liver, kidney and lung damage. The purpose of this study was to investigate whether AEE has a protective effect on hepatotoxicity induced by PQ in vivo and in vitro. Cell viability, apoptosis rate, mitochondrial function and intracellular oxidative stress were detected to evaluate the protective effect of AEE on PQ-induced BRL-3A (normal rat hepatocytes) cytotoxicity in vitro. In vivo, AEE pretreatment could attenuate oxidative stress and histopathological changes in rat liver induced by PQ. The results showed that AEE could reduce the hepatotoxicity induced by PQ in vivo and in vitro. AEE reduced PQ-induced hepatotoxicity by inhibitingoxidative stress and maintaining mitochondrial function. This study proved that AEE is an effective antioxidant and can reduce the hepatotoxicity of PQ.

Emodin inhibited NADPH-quinone reductase competitively and induced cytotoxicity in rat primary hepatocytes

Consumption of Cassia occidentalis (CO) seeds, a ubiquitously distributed weed plant, is responsible for a pathological condition known as hepato-myo-encephalopathy (HME). The toxicity of CO seeds is largely attributed to the presence of anthraquinones (AQs). Here, we report that Emodin, a CO anthraquinone, inhibits the enzymatic activity of NADPH-Quinone reductase, which is an intracellular enzyme fundamentally involved in the detoxification of quinone containing compounds. Emodin binds to the active site of the enzyme and acts as a competitive inhibitor with respect to 2, 6-Dichlorophenolindophenol, a known substrate of NADPH-Quinone reductase. Moreover, our in-vitro study further revealed that Emodin was cytotoxic to primary rat hepatocytes.

Hepatoprotective potential of Indigofera tirunelvelica Sanjappa: in vitro and in vivo studies on CCl4 induced wistar albino rats

The hepatoprotective efficiency of Indigofera tirunelvelica Sanjappa whole plant against CCl4 induced hepatotoxicity was examined. Rat hepatocyte monolayer culture and wistar albino rats were exercised as in vitro and in vivo screening models of protective agent for liver. In in vitro analyses, the whole plant ethanolic extract of Indigofera tirunelvelica Sanjappa were inspected. Silymarin was chosen as a standard treatement drug. In vitro, free radical scavenging property was also evaluated. In animal studies, hepatotoxicity was produced in Wistar albino rats by dispensing CCl4. The degree of hepatotoxicity was examined by determining the ranges of serum enzyme. The antioxidant parameters such as superoxide dismutase, catalase, reduced glutathione, and malondialdehyde of the hepatocytes were also evaluated. In in vitro studies, ethanol extract of I. tirunelvelica whole plant was identified to be the most active than other assessed extracts. Besides, whole plant ethanol extract of I. tirunelvelica was noticed to be rich in phenolic and flavonoids. It exhibited expressive free radical scavenging property versus diphenylpicryl hydrazyl (DPPH) and superoxide ion radicals. In the animals studies, whole plant ethanolic extract of I. tirunelvelica at a ranges of doses (100, 200 and 400 mg/kg body weight) revealed considerable amount of protection against CCl4 induced hepatotoxicity as evident by the protection of CCl4 induced changes biochemical parameters. The results of the present study suggested that the significant hepatoprotective property of whole plant ethanol extract of I. tirunelvelica against CCl4 induced hepatotoxicity and intimates its use as a potential medicinal drug for liver diseases.

Identification of the metabolites of piperine via hepatocyte incubation and liquid chromatography combined with diode-array detection and high-resolution mass spectrometry.

Piperine, an alkaloid isolated from Piper nigrum L., has been demonstrated to have many pharmacological effects and several health benefits. The aim of this work was to study the metabolic profiles of piperine in mouse, rat, dog and human hepatocytes. The biotransformation was carried out by incubating piperine with hepatocytes at 37°C. After incubation for 2 h, the samples were pretreated and analyzed using liquid chromatography combined with diode-array detection and high-resolution mass spectrometry (LC/DAD-HRMS). The structures of the metabolites were assigned through a comparison of their accurate masses and product ions with those of the parent compound. A total of 20 metabolites were detected, and the structures were proposed. Piperine was metabolized through the following pathways: (a) oxidation to form a catechol derivative, which further underwent methylation, glucuronidation, glutathione (GSH) conjugation, and hydroxylation followed by opening of the piperidine ring; (b) hydroxylation to form a carbinolamine intermediate followed by opening of the piperidine ring and the formation of alcohol and acid derivatives; and (c) hydroxylation to form stable hydroxylated metabolites. In mouse, the formation of the catechol derivative (M12) and hydroxylation (M11) were the major metabolic pathways; in rat, the formation of the catechol derivative (M12) and glucuronidation (M9) were the main pathways; and in dog and human, the formation of the catechol derivative (M12) was the predominant pathway. No human-specific metabolite was observed. This study provided some new information on the metabolic profiles of piperine, which should be of great importance in the study of the pharmacology and toxicity of this compound.

Exosomes from Placenta-Derived Mesenchymal Stem Cells Are Involved in Liver Regeneration in Hepatic Failure Induced by Bile Duct Ligation.

Although the liver has a regenerative capacity, hepatic failure is a severe and irreversible chronic disease. Placenta-derived mesenchymal stem cells (PD-MSCs) have distinctive features, such as recycling of the placenta waste after birth, ease of accessibility, abundant cell numbers, and strong immunosuppressive properties. Previously, we reported that PD-MSCs can regenerate the liver in hepatic failure through antifibrotic and autophagic mechanisms. Many reports have investigated whether exosomes, which are formed by the budding of vesicular bodies and are emitted into the blood, from stem cells have therapeutic potential in various diseases. C-reactive protein (CRP) is produced in hepatocytes and secreted via vessels. Therefore, the objectives of this study were to compare the expression of CRP in exosomes of a hepatic failure rat model (bile duct ligation, BDL) and to evaluate the therapeutic effect by their correlation between CRP and angiogenesis depending on PD-MSC transplantation. The exosomes were analyzed in a BDL rat model with transplantation of PD-MSCs through LC-MS analysis and precipitation solution. The exosomes, CRP, and factors related to these molecules were evaluated and quantified in exosomes as well as investigated by real-time PCR, Western blot, and immunofluorescence (IF) in vivo and in vitro. CRP was present in exosomes from serum of a rat model and increased by PD-MSC transplantation. In the exosomes, CRP upregulated the factors related to the Wnt signaling pathway and angiogenesis in the BDL rat liver-transplanted PD-MSCs. Also, CRP regulated the Wnt pathway and vascularization in rat hepatocytes by interacting with endothelial cells. Therefore, our findings indicate that CRP in exosomes excreted by PD-MSCs functions in angiogenesis via the Wnt signaling pathway.

Involvement of HO-1 and Autophagy in the Protective Effect of Magnolol in Hepatic Steatosis-Induced NLRP3 Inflammasome Activation In Vivo and In Vitro.

Magnolol (MG) is the main active compound of Magnolia officinalis and exerts a wide range of biological activities. In this study, we investigated the effects of MG using tyloxapol (Tylo)-induced (200 mg/kg, i.p.) hyperlipidemia in rats and palmitic acid (PA)-stimulated (0.3 mM) HepG2 cells. Our results showed that Tylo injection significantly increased plasma levels of triglyceride and cholesterol as well as superoxide anion in the livers, whereas MG pretreatment reversed these changes. MG reduced hepatic lipogenesis by attenuating sterol regulatory element-binding protein-1c (SREBP-1c) and fatty acid synthase (FAS) proteins and Srebp-1, Fas, Acc, and Cd36 mRNA expression as well as upregulated the lipolysis-associated genes Hsl, Mgl, and Atgl. Furthermore, MG reduced plasma interleukin-1β (IL-1β) and protein expression of NLR family pyrin domain-containing 3 (NLRP3), apoptosis-associated speck-like protein (ASC), and caspase 1 as well as upregulated nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and induction of heme oxygenase-1 (HO-1) in hepatocytes of Tylo-treated rats. Enhanced autophagic flux by elevation of autophagy related protein 5-12 (ATG5-12), ATG7, Beclin1, and microtubule-associated protein light chain 3 B II (LC3BII)/LC3BI ratio, and reduction of sequestosome-1 (SQSTM1/p62) and phosphorylation of mTOR was observed by MG administration. However, autophagy inhibition with 3-methyladenine (3-MA) in HepG2 cells drastically abrogated the MG-mediated suppression of inflammation and lipid metabolism. In conclusion, MG inhibited hepatic steatosis-induced NLRP3 inflammasome activation through the restoration of autophagy to promote HO-1 signaling capable of ameliorating oxidative stress and inflammatory responses.

The Action of Cytomegalovirus (CMV) Promoter on Expression of Genetically Engineered Insulin in Rat Hepatocytes

Background: Insulin is a hormone produce in the pancreas that stimulates glucose uptake from blood to enter the body's cells, where it is converted into energy needed by muscles and tissues to function. The pancreatic cells are responsible for the secretion of insulin. In diabetes, the body cannot produce enough insulin or cannot use insulin effectively.
 Objectives: In this study, we attempted to activate the hepatic cells to secrete insulin instead of the pancreatic cells.
 Methodology: This was done by gene therapy; an excellent strategy to treat diabetes by supplying the correct wild type copy of a furin cleavable sites preproinsulin. The preproinsulin was extracted from the rat DNA, cloned and mutated to generate the two furin cleavable sites responsible for removal of C-peptide to form the two chains (A and B) for mature insulin production. This mutated insulin was derived by Cytomegalovirus (CMV) promoter to express insulin by its transfection inside the primary rat hepatocytes using a non-viral vehicle to keep the hepatic cells healthier against the transfection. In vitro, the rat hepatocytes could not divide well as in vivo, but with special hormones like insulin and dexamethasone lived longer and kept their function.
 Results: The CMV promoter is strong and lead to over expression of mature insulin inside rat hepatocytes leading to deterioration, toxicity of hepatocytes and finally cell death. Hepatocytes in vitro were more fragile and needed some modification to adapt with secretion of insulin.
 Conclusion: Glucokinase or glucose transporter promoters were much more perfect than CMV. They can activate the hepatocytes to modulate the glucose level and so limiting the amount of insulin secreted. These promoters are weaker than CMV but much more perfect for hepatocytes.

Effect of carnosine and α-lipoic acid on hepatocyte apoptosis and the cytokine profile in induced fatty liver disease in Wistar rats

Non-alcoholic fatty liver disease (NAFLD) is now a common liver disease affecting about a third of the world's population. In this regard, the issue of studying the pathogenetic factors of the development of this disease in order to select adequate drug therapy and biologically active substances with antioxidant properties regulating the balance of pro- and anti-inflammatory cytokines is of particular relevance. The aim of the study was to assess the effect of minor biologically active substances - carnosine and α-lipoic acid on hepatocyte apoptosis and the cytokine profile in the experimental model of the initial stage of NAFLD. Material and methods. The studies were performed on male Wistar rats with initial body weight of 150±10 g. Animals were divided into 5 groups of 8 rats each. Within 8 weeks, rats of the 1st group (control) received a complete modified diet AIN93M, in which soybean oil was replaced with sunflower oil and lard (1:1). Rats of the experimental groups consumed high-calorie choline-deficient diet (HCCDD), in which fat content was 45%, fructose content - 20% of the energy value of the diet. Rats of the 2nd group were fed HCCDD without any supplements, the 3rd group - with the addition of carnosine (75 mg/kg body weight), the 4th group - with the addition of α-lipoic acid (75 mg/kg body weight), the 5th group - with the addition of carnosine and α-lipoic acid in a total dose of 150 mg/kg body weight. The study of rat hepatocyte apoptosis was performed by flow cytometry. Hepatocytes were stained with annexin V and vital dye 7-aminoactinomycin, followed by detection on an flow cytometer. The content of cytokines and chemokines (IL-1α, IL-10, IL-17А, M-CSF, MIP-1α, MIP-3α, RANTES) in the cytoplasmic fraction of liver tissue was determined by multiplex immunoassay. Results and discussion. On the model of the initial stage of development of NAFLD in male Wistar rats the еnrichment of HCCDD with carnosine and α-lipoic acid had demonstrated a protective effect on hepatocytes with a decrease in apoptosis intensity to the level in control rats. Under the influence of HCCDD, an increase in the content of M-CSF and MIP-1α and a decrease in the levels of MIP-3α and RANTES, stimulating the migration and differentiation of various immunoregulatory populations to the parenchyma at an early stage of the formation of fatty hepatosis, in the cytoplasmic fraction of liver tissue were detected. Moreover, a decrease in the level of proinflammatory cytokines IL-17A and IL-1α and an increase in IL-10 produced mainly by Treg-populations indicate the absence of pronounced inflammatory changes in the liver of male Wistar rats at the initial stages of development of fatty dystrophy. Conclusion. Enrichment of HCCDD with both carnosine and α-lipoic acid in Wistar rats had a protective effect on hepatocytes with a decrease in apoptosis to a level in control rats. The increase in the IL-10/IL-17A ratio indicates the activation of anti-inflammatory mechanisms due to the functional predominance of Treg-cells over Th1/Th17 lymphocytes.

Vildagliptin, a DPP-4 inhibitor, attenuates carbon tetrachloride-induced liver fibrosis by targeting ERK1/2, p38α, and NF-κB signaling

Mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-ĸB signaling have been recognized for their causal connection with liver fibrosis. Hence, it is encouraging to discover drugs that can modify the interactions between these signaling cascades. It has been suggested that glucagon-like peptide-1 receptors (GLP-1Rs) might have a role in the observed hepatoprotection of dipeptidyl peptidase-4 inhibitors other than vildagliptin (VLD). Consequently, we aimed to elucidate the mechanisms underlying its potential antifibrotic activity in a CCl4-intoxicated mouse model. VLD increased the percentage of viable CCl4-intoxicated primary rat hepatocytes in vitro. It also attenuated hepatic fibrosis, improved liver function, and prolonged survival of CCl4-intoxicated mice in a dose-dependent manner. This hepatoprotection might be mediated mainly through interference with extracellular signal-regulated protein kinase 1/2 phosphorylation, the most downstream signal of the MAPK pathway. In addition, VLD hepatoprotective activity could be partially mediated through inhibition of p38α phosphorylation and phosphorylation-induced NF-ĸB activation. As a result, VLD downregulated profibrogenic mediators, such as tumor necrosis factor α, transforming growth factor β, tissue inhibitor of metalloproteinase 1 and platelet-derived growth factor BB. Consequently, decreased expression levels of fibrosis markers, such as hydroxyproline and α smooth muscle actin, were confirmed. VLD showed a strong trend toward increasing the antioxidant defense machinery of fibrotic tissue, and we confirmed that GLP-1Rs were not implicated in the observed hepatoprotection. Since VLD poses little risk of hypoglycemia and is a safe drug for patients with liver injury, it may be a hopeful candidate for adjuvant treatment of liver fibrosis in humans.

Differential Cytotoxicity of Rooibos and Green Tea Extracts against Primary Rat Hepatocytes and Human Liver and Colon Cancer Cells – Causal Role of Major Flavonoids

Differential anti-proliferative and pro-apoptotic effects of aqueous extracts of green rooibos (Rg; Aspalathus linearis) and green tea (GT; Camellia sinensis) and an aspalathin-enriched extract of green rooibos (GRE), were investigated in primary rat hepatocytes (PH) and human liver (HepG2) and colon (HT-29) cancer cells. Rooibos flavonoids, aspalathin and luteolin, and the green tea flavanol, epigallocatechin gallate (EGCG), were included to assess their contribution relative to their extract concentrations. GRE was the most effective in reducing cell growth parameters which was associated with a high total polyphenol content and high ferric reducing potential. Differential cell responses were noticed with HepG2 cells more sensitive than PH toward the induction of apoptosis by GRE. Luteolin induced apoptosis in PH and HepG2 cells while aspalathin lacked any effect. EGCG induced apoptosis in HepG2 cells while PH were resistant. HT-29 cells were resistant to apoptosis induction by the tea and pure flavonoids. Differences existed in the individual effects of the major rooibos and GT flavonoids against cell growth parameters compared to their equivalent concentrations in the extract mixtures. Diversity of the flavonoid constituents, physicochemical properties and cellular redox status governing cell survival are likely to explain the differential cell responses.

In Vitro Metabolism of DWP16001, a Novel Sodium-Glucose Cotransporter 2 Inhibitor, in Human and Animal Hepatocytes.

DWP16001 is currently in a phase 2 clinical trial as a novel anti-diabetes drug for the treatment of type 2 diabetes by selective inhibition of sodium-glucose cotransporter 2. This in vitro study was performed to compare the metabolism of DWP16001 in human, dog, monkey, mouse, and rat hepatocytes, and the drug-metabolizing enzymes responsible for the metabolism of DWP16001 were characterized using recombinant human cytochrome 450 (CYP) and UDP-glucuronosyltransferase (UGT) enzymes expressed from cDNAs. The hepatic extraction ratio of DWP16001 in five species ranged from 0.15 to 0.56, suggesting that DWP16001 may be subject to species-dependent and weak-to-moderate hepatic metabolism. Five phase I metabolites (M1–M5) produced by oxidation as well as three DWP16001 glucuronides (U1–U3) and two hydroxy-DWP16001 (M1) glucuronides (U4, U5), were identified from hepatocytes incubated with DWP16001 by liquid chromatography-high resolution mass spectrometry. In human hepatocytes, M1, M2, M3, U1, and U2 were identified. Formation of M1 and M2 from DWP16001 was catalyzed by CYP3A4 and CYP2C19. M3 was produced by hydroxylation of M1, while M4 was produced by hydroxylation of M2; both hydroxylation reactions were catalyzed by CYP3A4. The formation of U1 was catalyzed by UGT2B7, but UGT1A4, UGT1A9, and UGT2B7 contributed to the formation of U2. In conclusion, DWP16001 is a substrate for CYP3A4, CYP2C19, UGT1A4, UGT1A9, and UGT2B7 enzymes. Overall, DWP16001 is weakly metabolized in human hepatocytes, but there is a potential for the pharmacokinetic modulation and drug–drug interactions, involved in the responsible metabolizing enzymes of DWP16001 in humans.