C3A Cells Research Articles

Liver-lobule-mimicking patterning via dielectrophoresis and hydrogel photopolymerization

Patterning and cultivating heterogeneous cells on microfluidic chips is essential for engineering tissue regeneration. Current patterning technologies focus mainly on positioning cells at specific locations, but most do not mimic in-vivo cell patterns. Even few papers and reports care about maintaining cell patterns after the patterning process and several days of culture. Here, we report a liver-lobule-mimicking labchip consisting of three components: the concentration gradient generator, the dielectrophoretic cell-patterning, and four cell-culturing chambers providing culturing microenvironment. NIH/3T3 (fibroblast cell) and C3A cells (human hepatocyte) were patterned via dielectrophoresis to reconstruct the liver-lobule’s radial actinomorphic organization and pattern at each cell-culture chamber. Subsequently, GelMA at different concentrations was perfused to form GelMA culturing-microenvironment coverings with different concentrations of 5 %, 10 %, 15 % GelMA and 0 % GelMA for the control group. Cell viability was maintained 95 % at 5 % GelMA chamber after cultured for 72 h. For the 15 % GelMA chamber, cell viability was 78 % on 72-hrs culturing. Urea assays revealed that the co-cultured cells in 5 % GelMA maintained the high activity, which has the highest urea secretion, with the preserved biomimicking liver-lobule pattern compared with other culturing-microenvironments. By cultivating cells in our liver-lobule-mimicking labchip with cellular micropatterning and hydrogel covering, the liver-lobule-like cell pattern was not affected by the difference in cell growth rate and the streamline of the perfused culture medium. We also observed that the appropriate GelMA culturing-microenvironment not only preserves the biomimicking liver-lobule pattern but also enhances liver function related to urea production.

In vitro evaluation of the anti-diabetic potential of Helichrysum petiolare Hilliard & B.L. Burtt using HepG2 (C3A) and L6 cell lines.

Background:Helichrysum petiolare Hilliard & B.L. Burtt has been listed in a survey of plants used in traditional medicine for the treatment of type 2 diabetes in the Eastern Cape of South Africa. In this study, the antidiabetic potentials of ethanol, cold aqueous (CAQ) and boiled aqueous (BAQ) extracts of H. petiolare were investigated. Methods: The cytotoxic and glucose utilization effects of the extracts were evaluated using L6 myocytes and HepG2 (C3A) hepatocytes. α-amylase, α-glucosidase and lipase inhibition assays were also carried out. Results: The ethanol extract showed significant cytotoxic effects in the treated cells. Both BAQ and CAQ extracts significantly increased glucose uptake in L6 and C3A cell lines. The CAQ extract enhanced glucose uptake more in the L6 myocytes than in the C3A cell-lines hepatocytes. The BAQ extract showed higher levels of inhibition on α-amylase and α-glucosidase than CAQ. The activities were not significantly different from acarbose. However, BAQ showed lower lipase inhibition than acarbose (p<0.05). Conclusions: The BAQ and CAQ extracts of H. petiolare may, therefore, contain pharmacologically active and relatively non-toxic hypoglycaemic chemicals, which may be effective substitutes in the treatment of diabetes mellitus.

In vitro evaluation of the anti-diabetic potential of Helichrysum petiolare Hilliard & B.L. Burtt using HepG2 (C3A) and L6 cell lines

Background: Helichrysum petiolare Hilliard & B.L. Burtt has been listed in a survey of plants used in traditional medicine for the treatment of type 2 diabetes in the Eastern Cape of South Africa. In this study, the antidiabetic potentials of ethanol, cold aqueous (CAQ) and boiled aqueous (BAQ) extracts of H. petiolare were investigated. Methods: The cytotoxic and glucose utilization effects of the extracts were evaluated using L6 myocytes and HepG2 (C3A) hepatocytes. α-amylase, α-glucosidase and lipase inhibition assays were also carried out. Results: The ethanol extract showed significant cytotoxic effects in the treated cells. Both BAQ and CAQ extracts significantly increased glucose uptake in L6 and C3A cell lines. The CAQ extract enhanced glucose uptake more in the L6 myocytes than in the C3A cell-lines hepatocytes. The BAQ extract showed higher levels of inhibition on α–amylase and α-glucosidase than CAQ. The activities were not significantly different from acarbose. However, BAQ showed lower lipase inhibition than acarbose (p<0.05). Conclusions: The BAQ and CAQ extracts of H. petiolare may, therefore, contain pharmacologically active and relatively non-toxic hypoglycaemic chemicals, which may be effective substitutes in the treatment of diabetes mellitus.

Potent Clastogenicity of Bisphenol Compounds in Mammalian Cells-Human CYP1A1 Being a Major Activating Enzyme.

Bisphenols (BPs) are environmental pollutants with relevant DNA damage in human population; however, they are generally inactive in standard mutagenicity assays, possibly due to insufficient metabolic activation. In this study, induction of micronuclei and double-strand DNA breaks by BPA, BPF, and BPS in Chinese hamster V79-derived cell lines expressing various human CYP enzymes and a human hepatoma (C3A) (metabolism-proficient) cell line were investigated. Molecular docking of BPs to human CYPs indicated some substrate-enzyme potentials, including CYP1A1 for each compound, which did not induce micronuclei in V79-derived cell lines expressing human CYP1A2, 2E1, or 3A4 but became positive in human CYP1A1-expressing (V79-hCYP1A1) cells. In V79-hCYP1A1 and C3A cells, all compounds induced double-strand DNA breaks and micronuclei formation, which were blocked/significantly attenuated by 1-aminobenzotriazole (CYP inhibitor) or 7-hydroxyflavone (selective CYP1A1 inhibitor). Coexposure of C3A cells to pentachlorophenol (sulfotransferase 1 inhibitor) or ketoconazole (UDP-glucuronosyltransferase 1A inhibitor) potentiated micronuclei induction by each compound, with thresholds lowered from 2.5-5.0 to 0.6-1.2 μM. Immunofluorescence staining of centromere protein B with micronuclei formed in C3A cells by each compound indicated pure clastogenic effects. In conclusion, BPs are potently clastogenic in mammalian cells, which require activation primarily by human CYP1A1 and are negatively modulated by phase II metabolism.

Potent aneugenicity of 1-methylpyrene in human cells dependent on metabolic activation by endogenous enzymes.

1-Methylpyrene (1-MP) is a common environmental pollutant and animal carcinogen. After sequential activation by cytochromes P450 and sulfotransferases, it induced gene mutations and micronuclei in mammalian cells. The type of micronuclei formed, entire chromosomes or fragments, was not analysed. In this study, 1-MP and its primary metabolite, 1-hydroxymethylpyrene (1-HMP), were investigated for the induction of centromere-positive and -negative micronuclei in the human hepatoma cell line HepG2 and its derivative C3A, expressing relevant enzymes at higher levels. Under a short-exposure (9h)/long-recovery regime (2 cell cycles in total), 1-MP and 1-HMP provided negative test results in HepG2 cells. However, they induced micronuclei in C3A cells, the effect being blocked by 1-aminobenzotriazole (inhibitor of cytochromes P450s) and reduced by pentachlorophenol (inhibitor of sulfotransferases). Immunofluorescence staining of centromere protein B in the micronuclei revealed purely clastogenic effects under this regime. Unexpectedly, 1-MP and 1-HMP at concentrations 1/5-1/4 of that required for micronuclei formation led to mitotic arrest and spindle aberrations, as detected by immunofluorescence staining of β- and γ-tubulin. Following extended exposure (72h, 2 cell cycles, no recovery), damage to the spindle apparatus and centrosomes was detected at even lower concentrations, with concurrent formation of micronuclei. At low concentrations (1-8µM 1-MP, 0.25-0.5µM 1-HMP), the micronuclei induced were unexceptionally centromere-positive. Thus, the chromosome-damaging mechanism of 1-MP was regime and concentration dependent: potently aneugenic under persistent exposure, while clastogenic at higher concentrations following a short-exposure/long-recovery regime. This is a convincing evidence for the existence of metabolic activation-dependent aneugens.

In vitro evaluation of the anti-diabetic potential of Helichrysum petiolare Hilliard & B.L. Burtt using HepG2 (C3A) and L6 cell lines.

Background:Helichrysum petiolare Hilliard & B.L. Burtt has been listed in a survey of plants used in traditional medicine for the treatment of type 2 diabetes in the Eastern Cape of South Africa. In this study, the antidiabetic potentials of ethanol, cold aqueous (CAQ) and boiled aqueous (BAQ) extracts of H. petiolare were investigated. Methods: The cytotoxic and glucose utilization effects of the extracts were evaluated using L6 myocytes and HepG2 (C3A) hepatocytes. α-amylase, α-glucosidase and lipase inhibition assays were also carried out. Results: The ethanol extract showed significant cytotoxic effects in the treated cells. Both BAQ and CAQ extracts significantly increased glucose uptake in L6 and C3A cell lines. The CAQ extract enhanced glucose uptake more in the L6 myocytes than in the C3A cell-lines hepatocytes. The BAQ extract showed higher levels of inhibition on α-amylase and α-glucosidase than CAQ. The activities were not significantly different from acarbose. However, BAQ showed lower lipase inhibition than acarbose (p<0.05). Conclusions: The BAQ and CAQ extracts of H. petiolare may, therefore, contain pharmacologically active and relatively non-toxic hypoglycaemic chemicals, which may be effective substitutes in the treatment of diabetes mellitus.

Effects of genetically modified human skin fibroblasts, stably overexpressing hepatocyte growth factor, on hepatic functions of cocultured C3A cells.

Diseases leading to terminal hepatic failure are among the most common causes of death worldwide. Transplant of the whole organ is the only effective method to cure liver failure. Unfortunately, this treatment option is not available universally due to the serious shortage of donors. Thus, alternative methods have been developed that are aimed at prolonging the life of patients, including hepatic cells transplantation and bridging therapy based on hybrid bioartificial liver devices. Parenchymal liver cells are highly differentiated and perform many complex functions, such as detoxification and protein synthesis. Unfortunately, isolated hepatocytes display a rapid decline in viability and liver-specific functions. A number of methods have been developed to maintain hepatocytes in their highly differentiated state in vitro, amongst them the most promising being 3D growth scaffolds and decellularized tissues or coculture with other cell types required for the heterotypic cell-cell interactions. Here we present a novel approach to the hepatic cells culture based on the feeder layer cells genetically modified using lentiviral vector to stably produce additional amounts of hepatocyte growth factor and show the positive influence of these coculture conditions on the preservation of the hepatic functions of the liver parenchymal cells' model-C3A cells.

Enhanced cellular functions of hepatocytes in the hyaluronate-alginate-chitosan microcapsules.

The study aimed to develop a biocompatible microcapsule for hepatocytes and create a bio-mimic microenvironment for maintaining hepatic-specific functions of hepatocytes in vitro. The work is proposed for the bioartificial liver system in the treatment of liver failure. In this study, microcapsules were prepared with hyaluronate (HA)/sodium alginate (SA) as an inner core and an outer chitosan (CS) shell via one-step spraying method. C3A cells were encapsulated in microcapsules to examine the biocompatibility of HA-SA-CS microcapsules. MTT and fluorescence microscopy indicated that C3A cells had high viability in the HA-SA-CS microcapsules. The liver-specific functions, such as urea and albumin synthesis, and CYP1A2 and CYP3A4 activities from encapsulated cells were increased in the HA-SA-CS microcapsules compared to the SA-CS microcapsules. The gene expressions of CYP450 related genes were also increased by HA on day 3. The study suggests that HA-SA-CS microcapsules have good biocompatibility and can maintain a favorable environment for hepatocytes. This approach has improved the preservation of liver cells' metabolic functions and could be a candidate for the bioartificial liver system.

Sorafenib reduces steatosis-induced fibrogenesis in a human 3D co-culture model of non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) affects around 25% of the worldwide population. Non-alcoholic steatohepatitis (NASH), the more progressive variant of NAFLD, is characterized by steatosis, cellular ballooning, lobular inflammation, and may culminate on hepatic stellate cell (HSC) activation, thus increasing the risk for fibrosis, cirrhosis, and HCC development. Conversely, the antifibrotic effects of sorafenib, an FDA-approved drug for HCC treatment, have been demonstrated in 2D cell cultures and animal models, but its mechanisms in a NAFLD-related microenvironment in vitro requires further investigation. Thus, a human 3D co-culture model of fatty hepatocytes and HSC was established by culturing hepatoma C3A cells, pre-treated with 1.32 mM oleic acid, with HSC LX-2 cells. The fatty C3A/LX-2 spheroids showed morphological and molecular hallmarks of altered lipid metabolism and steatosis-induced fibrogenesis, similarly to the human disease. Sorafenib (15 μM) for 72 hours reduced fatty spheroid viability, and upregulated the expression of lipid oxidation- and hydrolysis-related genes, CPT1 and LIPC, respectively. Sorafenib also inhibited steatosis-induced fibrogenesis by downregulating COL1A1, TGFB1, PDGF, and TIMP1 and by decreasing protein levels of IL-6, TGF-β1, and TNF-α in fatty spheroids. The demonstration of the antifibrotic properties of sorafenib on steatosis-induced fibrogenesis in a 3D in vitro model of NAFLD supports its clinical use as a therapeutic agent for the treatment of NAFLD/NASH patients.

Discovery of Eicosapentaenoic Acid Esters of Hydroxy Fatty Acids as Potent Nrf2 Activators.

Branched fatty acid esters of hydroxy fatty acids (FAHFAs) are a recently discovered class of biologically active lipids with anti-inflammatory and anti-diabetic properties. Despite the possible link between endogenous FAHFA levels and nuclear factor erythroid 2-related factor 2 (Nrf2), their possible function as antioxidants and the mechanisms involved in this are unknown. Here, we investigate FAHFAs’ plausible antioxidant potential with reference to their effect on the Nrf2 levels, oxidative stress, and lipid droplet oxidation in human hepatocytes (C3A). Six authentic FAHFAs were chemically synthesized and performed activity-based screening by reporter gene assay. Among them, eicosapentaenoic acid (EPA) esterified 12-hydroxy stearic acid (12-HSA) and 12-hydroxy oleic acid (12-HOA) FAHFAs showed less cytotoxicity compared to their free fatty acids and potent activators of Nrf2. To define their mode of action, relative levels of nuclear Nrf2 were determined, which found a higher amount of Nrf2 in nucleus of cells treated with 12-EPAHSA compared to the control. Furthermore, 12-EPAHSA increased the expression of Nrf2-dependent antioxidant enzyme genes (NQO1, GCLM, GCLC, SOD-1, and HO-1). Fluorescence imaging analysis of linoleic-acid-induced lipid droplets (LDs) in C3A cells treated with 12-EPAHSA revealed the strong inhibition of small-size LD oxidation. These results suggest that EPA-derived FAHFAs as a new class of lipids with less cytotoxicity, and strong Nrf2 activators with plausible antioxidant effects via the induction of cytoprotective proteins against oxidative stress, induced cellular damage.

Human CYP2E1-activated mutagenicity of dioxin-like PCBs 105 and 118—Experimental data consistent with molecular docking results

Polychlorinated biphenyls (PCBs) are persistent organic pollutants with human carcinogenicity. Many lower chlorinated and non-dioxin-like PCBs have been observed to be mutagenic following activation by human CYP2E1, while activation of dioxin-like (DL-) PCBs by this enzyme has never been evidenced. In this study, each DL-PCB was analyzed by molecular docking to human CYP2E1 protein for predicting a substrate interaction. All compounds demonstrated high affinities with the active site of human CYP2E1, binding energy being −8.7 ∼ −9.7 kcal/mol. However, most compounds demonstrated ligand-heme distances as ≥ 6.8 Å, while the values for 2,3,3′,4,4′- (PCB 105) and 2,3′,4,4′,5-pentachlorobiphenyl (PCB 118) were 5.3 and 5.4 Å, respectively (valid for electron transfer). Experimentally, both PCB 105 and 118 induced micronuclei in a V79-derived cell line engineered for expression of human CYP2E1 at low micromolar concentrations, while inactive or weakly positive in V79-Mz control cells; these effects were blocked or reduced by 1-aminobenzotriazole, a suicide CYP inhibitor. However, DL-PCBs 77, 81 and 126 were all negative in both cell lines. In a human hepatoma (C3A) cell line, PCB 105 and 118 induced micronuclei marginally, while with ethanol pretreatment (to stabilize CYP2E1) both compounds induced micronuclei efficiently, and co-exposure to trans-1,2-dichloroethylene (a selective CYP2E1 inhibitor) led to clearly negative results with both compounds. Finally, both PCB 105 and 118 induced PIG-A gene mutations in C3A cells, which was blocked by trans-1,2-dichloroethylene. In summary, in silico and experimental results consistently suggest that DL- PCBs 105 and 118 may be activated by human CYP2E1 for mutagenic activities.

Phytochemical screening, antioxidant, anticoagulant and in vitro toxic and genotoxic properties of aerial parts extracts of Fumaria officinalis L. growing in Tunisia

Fumaria is an important plant species with many applications in herbal medicine. In this paper we report on the investigation of the phytochemical composition, antioxidant, anticoagulant and in vitro genotoxic properties of Fumaria officinalis L. which is amongst others widely used in Tunisia. Petroleum ether, ethyl acetate, chloroform and methanol extracts were made and investigated with respect to their total phenolics and flavonoids contents using standard methods. Antioxidant activity was evaluated by DPPH and ABTS methods. The anticoagulant activity was evaluated by prothrombin time (PT) and activated partial thromboplastin time (aPTT) tests. In vitro toxic and genotoxic properties with respectively the Neutral red uptake method and the bacterial Vitotox test and comet assay in human C3A cells. This study showed that methanolic extract had the highest total phenolic and flavonoids contents. The same extract showed surprising anticoagulant and antioxidant activities. The Vitotox test revealed absence of genotoxicity for all extracts in the bacterial Vitotox test. This was confirmed in the comet assay which was conducted on human C3A cells, except for the methanolic extract where DNA damage appeared to be significantly increased at nontoxic concentrations. Thus, showing the potential of the plant as a new source of bioactive molecules for therapeutic purposes, with particular emphasis on the treatment of cardiovascular diseases.

Genetically modified C3A cells with restored urea cycle for improved bioartificial liver

The bioartificial liver, a hybrid device aimed at improving the survival of patients with fulminant liver failure, requires a cell source to replicate human liver function. However, liver support systems that utilize porcine or human hepatoma-derived cells felt short of expectations in clinical trials. Here we present engineered C3A cells, with a restored function of the urea cycle, which can be used in an efficacious bioartificial liver. The genetic modification was performed using a lentiviral-mediated gene transfer which led to effective integration of the transgenes, coding for arginase I and ornithine transcarbamylase, into the target cell genomes. The engineered cells are more resistant to the oxidative/nitrosative stress induced by the presence of high concentrations of ammonia cations and produce more urea than their unmodified counterparts. Interestingly, the genetically modified cells secrete more albumin than control C3A cells and the synthesis of the protein is induced by increasing concentrations of ammonia. Although the physiological capabilities of the new cell line need to be further examined, at this stage of our study we may conclude that the genetically modified cells are able to convert ammonia to urea more effectively than regular C3A cells.

Identification of Novel Proteins Interacting with Proprotein Convertase Subtilisin/Kexin 9.

High levels of cholesterol, especially as low-density lipoprotein (LDL), are a well-known risk factor for atherosclerotic-related diseases. The key atherogenic property of LDL is its ability to form atherosclerotic plaque. Proprotein convertase subtilisin/kexin-9 (PCSK9) is an indirect regulator of plasma LDL levels by controlling the number of LDL receptor molecules expressed at the plasma membrane, especially in the liver. Herein, we performed a combination of affinity chromatography, mass spectrometry analysis and identification, and gene expression studies to identify proteins that interact with PCSK9. Through these studies, we identified three proteins, alpha-1-antitrypsin (A1AT), alpha-1-microglobulin/bikunin precursor (AMBP), and apolipoprotein H (APOH) expressed by C3A cells that interact with PCSK9. The expression levels of A1AT and APOH increased in cells treated with MITO+ medium, a condition previously shown to affect the function of PCSK9, as compared to treating with Regular (control) medium. However, AMBP expression did not change in response to the treatments. Additional studies are required to determine which of these proteins can modulate the expression/function of PCSK9. The identification of endogenous modulators of PCSK9’s function could lead to the development of novel diagnostic tests or treatment options for patients suffering hypercholesterolemia in combination with other chronic metabolic diseases.

Identification of a hormone response element that mediates suppression of APOF by LXR and PPARα agonists

Apolipoprotein F (ApoF) regulates cholesteryl ester transfer protein activity. We previously observed that hepatic APOF mRNA levels are decreased by high fat, cholesterol-enriched diets. Here we show in human liver C3A cells that APOF mRNA levels are reduced by agonists of LXR and PPARα nuclear receptors. This negative regulation requires co-incubation with the RXR agonist, retinoic acid. Bioinformatic analysis of the ~2 kb sequence upstream of the APOF promoter identified one potential LXR and 4 potential PPARα binding sites clustered between nucleotides −2007 and −1961. ChIP analysis confirmed agonist-dependent binding of LXRα, PPARα, and RXRα to this hormone response element complex (HREc). A luciferase reporter containing the 2 kb 5′ APOF sequence was negatively regulated by LXR and PPARα ligands as seen in cells. This regulation was maintained in constructs lacking the ~1700 nucleotides between the HREc and the APOF proximal promoter. Mutations of the HREc that disrupted LXRα and PPARα binding led to the loss of reporter construct inhibition by agonists of these nuclear receptors. siRNA knockdown studies showed that APOF gene regulation by LXRα or PPARα agonists did not require an interaction between these two nuclear receptors. Thus, APOF is subject to negative regulation by agonist-activated LXR or PPARα nuclear receptors binding to a regulatory element ~1900 bases 5′ to the APOF promoter. High fat, cholesterol-enriched diets likely reduce APOF gene expression via these receptors interacting at this regulatory site.

Human CYP1B1-dependent genotoxicity of dioxin-like polychlorinated biphenyls in mammalian cells

Polychlorinated biphenyls (PCBs) are persistent organic pollutants and human carcinogens. It was reported that rat CYP1A1 and catfish CYP1A can hydroxylate 3,3',4,4',5-pentachlorobiphenyl (PCB 126) and 3,3',4,4'-tetrachlorobiphenyl (PCB 77), while potential roles of other CYP1 enzymes in the metabolism of dioxin-like (DL) PCBs remain unconfirmed. In this study, three representative DL-PCBs, i.e., PCB 77, PCB 126, and 3,4,4',5-tetrachlorobiphenyl (PCB 81), were investigated on their genotoxicity in Chinese hamster V79-derived cell lines genetically engineered for the expression of human CYP1A1, 1A2 and 1B1, and in the human hepatoma C3A cell line, which endogenously expresses various CYPs. Under both 6 h/18 h and 18 h/6 h (exposure/recovery) regimes, PCB 77 and 81 induced micronuclei in V79-hCYP1B1 cells at micromolar levels, with slightly higher potency in the latter regime, while they were inactive in the parental V79-Mz cells and the V79-derived cell lines expressing human CYP1A1 and 1A2. However, PCB 126 was negative in each cell line. Likewise, PCB 77 and 81 induced micronuclei formation in C3A cells, which expressed CYP1B1. This effect was blocked by co-exposure to tetramethoxystilbene (30 nM), a selective CYP1B1 inhibitor. Immuno-fluorescent staining of centromere protein B in the micronuclei in PCB-treated cultures showed a predominance of centromere-negative micronuclei, which indicated a clastogenic effect. Moreover, all three PCBs elevated the level of γ-H2AX protein (indicating DNA double-strand breaks) in C3A cells, and these effects were blocked by tetramethoxystilbene (10 nM). This study demonstrates that some DL-PCBs are clastogenic in mammalian cells following metabolic activation by human CYP1B1.

Pharmacological evaluation of hydro-ethanol and hot water leaf extracts of Bauhinia galpinii (Fabaceae): A South African ethnomedicinal plant

Bauhinia galpinii is one of several plant species native to South Africa that has been reported to be used traditionally to manage ailments including gastrointestinal disorders, infectious diseases and inflammation. This study was designed to evaluate the in vitro antibacterial, antifungal, antioxidant, cytotoxicity and anti-inflammatory activities of the hydro-ethanol (70% ethanol) and hot water extracts of Bauhinia galpinii leaves. The antibacterial and antifungal activities of the extracts were determined using the serial microplate dilution technique. The minimum inhibitory concentrations (MIC) of seven bacterial strains (Staphylococcus aureus, Escherichia coli, Enterococcus faecalis, Salmonella enterica serovar Typhimurium (S. Typhimurium), Salmonella enterica Serotype Dublin (S. Dublin), Pseudomonas aeruginosa and Klebsiella pneumoniae) and three clinical fungal isolates (Aspergillus fumigatus, Candida albicans and Cryptococcus neoformans) were determined. The extraction yield and MIC values were used to determine the total activity (TA) of the extracts. Radical scavenging potential of the extracts was determined using the 2, 2-diphenyl-1-picrylhydrazyl (DPPH) assay while the lipoxygenase (LOX) enzyme inhibitory assay was used to ascertain the anti-inflammatory activity. The safety of the extracts was determined using the colorimetric mitochondrial viability assay against Vero, Caco-2 and C3A cells. UPLC-MS analysis was also conducted to detect and identify the compounds present in the plant. Results from the study showed that the extracts had inhibitory activities against the growth of all tested pathogens with MIC values ranging from 0.04 to 1.25 mg/ml. Both extracts were most active against S. Typhimurium with MIC = 0.16 mg/ml, while the hot water extract was very active at 48 h incubation against Candida albicans with MIC = 0.04 mg/ml. The hydro-ethanol and hot water extracts had high total antibacterial activity (TAA) of 1000 and 937 ml/g against Salmonella Typhimurium respectively. The highest total antifungal activity of 3750 ml/g was recorded against Candida albicans by the hot water extract. The extracts showed strong DPPH radical scavenging activity with the hydro-ethanol extract displaying higher scavenging activity (IC50 = 3.72 mg/ml) than the hot water extract (IC50 = 10.82 mg/ml). Both extracts had moderate lipoxygenase inhibitory activity. The extracts had relatively low cytotoxic to non-cytotoxic effects with LC50 values of 0.094 to ˃1.000 mg/ml and selectivity index values of 0.075 to 22.698. Three compounds with known biological activity (2′'-O-rhamnosylvitexin, myricetin 3-O-galactopyranoside and quercetin 3-O-galactopyranoside) were tentatively identified from UPLC-MS analysis. Therefore, this study supports the folkloric use of the plant in treating gastrointestinal disorders, inflammation and infectious diseases.

Assessment of biological functions for C3A cells interacting with adverse environments of liver failure plasma

BackgroundFor its better differentiated hepatocyte phenotype, C3A cell line has been utilized in bioartificial liver system. However, up to now, there are only a few of studies working at the metabolic alternations of C3A cells under the culture conditions with liver failure plasma, which mainly focus on carbohydrate metabolism, total protein synthesis and ureagenesis. In this study, we investigated the effects of acute liver failure plasma on the growth and biological functions of C3A cells, especially on CYP450 enzymes. MethodsC3A cells were treated with fresh DMEM medium containing 10% FBS, fresh DMEM medium containing 10% normal plasma and acute liver failure plasma, respectively. After incubation, the C3A cells were assessed for cell viabilities, lactate dehydrogenase leakage, gene transcription, protein levels, albumin secretion, ammonia metabolism and CYP450 enzyme activities. ResultsCell viabilities decreased 15%, and lactate dehydrogenase leakage had 1.3-fold elevation in acute liver failure plasma group. Gene transcription exhibited up-regulation, down-regulation or stability for different hepatic genes. In contrast, protein expression levels for several CYP450 enzymes kept constant, while the CYP450 enzyme activities decreased or remained stable. Albumin secretion reduced about 48%, and ammonia accumulation increased approximately 41%. ConclusionsC3A cells cultured with acute liver failure plasma showed mild inhibition of cell viabilities, reduction of albumin secretion, and increase of ammonia accumulation. Furthermore, CYP450 enzymes demonstrated various alterations on gene transcription, protein expression and enzyme activities.

HOTAIR regulates lipopolysaccharide-induced inflammatory response in hepatocytes.

It has been widely accepted that long-noncoding RNA (lncRNA) HOX transcript antisense intergenic RNA (HOTAIR) emerges as a crucial mediator in inflammation. Here, we first detected HOTAIR in lipopolysaccharide (LPS)-treated normal human liver cell line (L02) and hepatocellular carcinoma cell lines (C3A, HepG2, and SMMC-7721). Further, we explored the biological function of HOTAIR in LPS-induced hepatocytes (L02 and C3A) lesions and investigated the molecular mechanisms. Besides, we focused on inflammatory signaling crosstalk. The inflammatory insults were assayed by cell counting kit-8 (CCK-8), cell cycle and apoptosis analysis kit, and immunoblotting assay. HOTAIR level was examined by reverse-transcription polymerase chain reaction. To determine the effect of HOTAIR silence or overexpression in inflammation, we applied quantitative reverse-transcription polymerase chain reaction, immunoblotting assay, and enzyme-linked immuno sorbent assay. Regulator inhibitors of Janus kinase/signal transducer and activator of transcription (JAK2/STAT3; AG490) and nuclear factor κB (NF-κB; BAY-11-7082) were applied to treat cells. Our results suggested that LPS induced the overexpression of HOTAIR in L02, C3A, HepG2, and SMMC-7721 cells. LPS repressed viability, induced apoptosis, and facilitated the expression of interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α in L02 and C3A cells. IL-1β, IL-6, and TNF-α were upregulated by HOTAIR overexpression while downregulated by HOTAIR knockdown in LPS-treated cells. We further observed that HOTAIR overexpression accelerated LPS-induced phosphorylation whereas HOTAIR silence blocked this progress. Inhibition of JAK/STAT and NF-κB contributed to the suppression of cytokines which was evoked by LPS. Collectively, our findings indicated that HOTAIR exerted a crucial role in cytokines expression by activating JAK/STAT and NF-κB.

PES1 is regulated by CD44 in liver cancer stem cells via miR-105-5p.

Pescadillo (PES1) is a key molecule for ribosome formation in mammalian cells. In this study, human hepatoma C3A cells were reprogrammed by four transcription factors, Oct4, Sox2, Klf4 and c-Myc, into induced cancer stem cells, termed C3A-induced cancer stem cells (C3A-iCSCs). We found that PES1 was up-regulated in C3A-iCSCs and promoted cell proliferation. Moreover, the cancer stem cell marker CD44, which is located in the cytomembrane, translocated to the nucleus and was up-regulated in C3A-iCSCs. Our results suggest that CD44 has a negative effect on miR-105-5p. We found that PES1 is a direct target of, and was negatively regulated by, miR-105-5p. In summary, CD44 regulates PES1 in liver cancer stem cells via miR-105-5p to promote cell growth.