Simulated Microgravity Inhibits the Proliferation of Chang Liver Cells by Attenuation of the Major Cell Cycle Regulators and Cytoskeletal Proteins.

Simulated microgravity (SMG) induced the changes in cell proliferation and cytoskeleton organization, which plays an important factor in various cellular processes. The inhibition in cell cycle progression has been considered to be one of the main causes of proliferation inhibition in cells under SMG, but their mechanisms are still not fully understood. This study aimed to evaluate the effects of SMG on the proliferative ability and cytoskeleton changes of Chang Liver Cells (CCL-13). CCL-13 cells were induced SMG by 3D clinostat for 72 h, while the control group were treated in normal gravity at the same time. The results showed that SMG reduced CCL-13 cell proliferation by an increase in the number of CCL-13 cells in G0/G1 phase. This cell cycle phase arrest of CCL-13 cells was due to a downregulation of cell cycle-related proteins, such as cyclin A1 and A2, cyclin D1, and cyclin-dependent kinase 6 (Cdk6). SMG-exposed CCL-13 cells also exhibited a downregulation of α-tubulin 3 and ß-actin which induced the cytoskeleton reorganization. These results suggested that the inhibited proliferation of SMG-exposed CCL-13 cells could be associate with the attenuation of major cell cycle regulators and main cytoskeletal proteins.

Protective effect of ulinastatin on hepatic ischemia reperfusion injury through autophagy activation in Chang liver cells.

This study aimed to investigate the protective effect of ulinastatin in hepatic ischemia-reperfusion progress, involving its association with the role of autophagy during hypoxia-induced hypoxia-reoxygenation injury in vitro. The model of hepatic hypoxia/reoxygenation (H/R) injury in Chang liver cells was established. After treatment with ulinastatin at the doses of 10, 100, and 1000 U/mL in H/R liver cells, the cell proliferation was significantly increased, morphological damage was reduced, and the cell apoptosis rate was decreased. The protein levels of antiapoptotic myeloid cell leukemia-1 (Mcl-1) and caspase-3 were upregulated, and C-PARP protein was downregulated. Meanwhile, ulinastatin led to an increase in the messenger RNA and protein levels of autophagy maker Unc-like kinase 1 (ULK1), Beclin-1, and microtubule-associated protein 1 light chain 3 (LC-3) and a decrease in p62. Then, 3-methyladenine (3-MA), an inhibitor of autophagy, made morphological damage and cell apoptosis worsen in ulinastatin-treated H/R liver cells. And the expression levels of caspase-3, C-PARP, p62, Beclin-1, and LC-3, proteins were also reversed by 3-MA. Taken together, our results demonstrate that ulinastatin inhibited the hepatic H/R injury in Chang liver cells, which was, to some extent, related to the autophagy activation.

Different administration patterns of docosahexaenoic acid in combating cytotoxic manifestations due to arsenic trioxide (acute promyelocytic leukemia drug) induced redox imbalance in hepatocytes.

Docosahexaenoic acid (DHA) obtained from fish and plant sources is an essential dietary fatty acid and an important cell membrane structural component. The acute promyelocytic leukemia (APL) drug arsenic trioxide (As2O3), causes hepatotoxicity. We evaluated the protective potential of DHA as pre/combination/post-administration patterns against As2O3 induced toxicity. The therapeutic concentration of As2O3 (10 µM) resulted in cytotoxicity with a significant (p < 0.05) variation from the control group. Reduced cell viability, morphological alterations, enhanced LDH release and apoptosis were observed. The oxidative stress markers (lipid peroxidation, nitric oxide, and ROS) and hepatic enzymes (AST and ALT) and intracellular calcium levels were found to be elevated by the As2O3 administration. Reduction in levels of mitochondrial membrane potential, cellular free radical scavenging potential, intracellular proteins, ATPases and major antioxidants (catalase, SOD, GSH, and GPx) were also observed. Administration of DHA along with As2O3 as pre/combination administration patterns offered protection against As2O3 induced cytotoxicity at significant levels (p < 0.05) from As2O3 alone treated group. The cell viability and morphology were protected with reduced LDH release and apoptosis. The hepatic enzymes and oxidative stress markers were reduced with replenishment of mitochondrial membrane potential, free radical scavenging potential, intracellular proteins, ATPases and antioxidant levels. DHA pre/combination administration patterns showed protective potential against As2O3 with pre-treatment being the best and the post-treatment method failed to produce any protective effect.

Identification and characterisation of the anti-oxidative stress properties of the lamprey prohibitin 2 gene.

The highly conserved protein prohibitin 2 (PHB2) has been implicated as a cell-surface receptor in the regulation of proliferation, apoptosis, transcription, and mitochondrial protein folding. In the present study, we identified a Lampetra morii homologue of PHB2, Lm-PHB2, showing greater than 61.8% sequence identity with its homologues. Phylogenetic analysis indicated that the position of Lm-PHB2 is consistent with lamprey phylogeny. Expression of the Lm-PHB2 protein was nearly equivalent in the heart, liver, kidneys, intestines, and muscles of normal lampreys. However, the Lm-PHB2 protein was down-regulated in the myocardia of lampreys challenged for 5 days with adriamycin (Adr), followed by a significant up-regulation 10 days after treatment. In vitro, recombinant Lm-PHB2 (rLm-PHB2) protein could significantly enhance the H2O2-induced oxidative stress tolerance in Chang liver (CHL) cells. Further mechanism studies indicated that the nucleus-to-mitochondria translocation of Lm-PHB2 was closely involved in the oxidative stress protection. Our results suggests that the strategies to modulate Lm-PHB2 levels may constitute a novel therapeutic approach for myocardial injury and liver inflammatory diseases, conditions in which oxidative stress plays a critical role in tissue injury and inflammation.

Apoptosis induced by para-phenylenediamine involves formation of ROS and activation of p38 and JNK in chang liver cells.

para-Phenylenediamine (p-PD) is a suspected carcinogen, but it has been widely used as a component in permanent hair dyes. In this study, the mechanism of p-PD-induced cell death in normal Chang liver cells was investigated. The results demonstrated that p-PD decreased cell viability in a dose-dependent manner. Cell death via apoptosis was confirmed by enhanced DNA damage and increased cell number in the sub-G1 phase of the cell cycle, using Hoechst 33258 dye staining and flow cytometry analysis. Apoptosis via reactive oxygen species generation was detected by the dichlorofluorescin diacetate staining method. Mitogen-activated protein kinase (MAPK) activation was assessed by western blot analysis and revealed that p-PD activated not only stress-activated protein kinase (SAPK)/c-Jun N-terminal kinases (JNK) and p38 MAPK but also extracellular signal-regulated kinase (ERK). Cytotoxicity and apoptosis induced by p-PD were markedly enhanced by ERK activation and selectively inhibited by ERK inhibitor PD98059, thus indicating a negative role of ERK. In contrast, inhibition of p38 MAPK activity with the p38-specific inhibitor SB203580 moderately inhibited cytotoxicity and apoptosis induction by p-PD. Similarly, SP600125, an inhibitor of SAPK/JNK, moderately inhibited cytotoxicity and apoptosis induced by p-PD, thus implying that p38 MAPK and SAPK/JNK had a partial role in p-PD-induced apoptosis. Western blot analysis revealed that p-PD significantly increased phosphorylation of p38 and SAPK/JNK and decreased phosphorylation of ERK. In conclusion, the results demonstrated that SAPK/JNK and p38 cooperatively participate in apoptosis induced by p-PD and that a decreased ERK signal contributes to growth inhibition or apoptosis.

In vitro inhibition of histamine release behavior of cetirizine intercalated into Zn/Al- and Mg/Al-layered double hydroxides.

The intercalation of cetirizine into two types of layered double hydroxides, Zn/Al and Mg/Al, has been investigated by the ion exchange method to form CTZAN and CTMAN nanocomposites, respectively. The basal spacing of the nanocomposites were expanded to 31.9 Å for CTZAN and 31.2 Å for CTMAN, suggesting that cetirizine anion was intercalated into Layered double hydroxides (LDHs) and arranged in a tilted bilayer fashion. A Fourier transform infrared spectroscopy (FTIR) study supported the formation of both the nanocomposites, and the intercalated cetirizine is thermally more stable than its counterpart in free state. The loading of cetirizine in the nanocomposite was estimated to be about 57.2% for CTZAN and 60.7% CTMAN. The cetirizine release from the nanocomposites show sustained release manner and the release rate of cetirizine from CTZAN and CTMAN nanocomposites at pH 7.4 is remarkably lower than that at pH 4.8, presumably due to the different release mechanism. The inhibition of histamine release from RBL2H3 cells by the free cetirizine is higher than the intercalated cetirizine both in CTZAN and CTMAN nanocomposites. The viability in human Chang liver cells at 1000 µg/mL for CTZAN and CTMAN nanocomposites are 74.5 and 91.9%, respectively.

Increased glutathione synthesis following Nrf2 activation by vanadyl sulfate in human chang liver cells.

Jeju ground water, containing vanadium compounds, was shown to increase glutathione (GSH) levels as determined by a colorimetric assay and confocal microscopy. To investigate whether the effects of Jeju ground water on GSH were specifically mediated by vanadium compounds, human Chang liver cells were incubated for 10 passages in media containing deionized distilled water (DDW), Jeju ground water (S1 and S3), and vanadyl sulfate (VOSO(4)). Vanadyl sulfate scavenged superoxide anion, hydroxyl radical and intracellular reactive oxygen species. Vanadyl sulfate effectively increased cellular GSH level and up-regulated mRNA and protein expression of a catalytic subunit of glutamate cysteine ligase (GCLC), which is involved in GSH synthesis. The induction of GCLC expression by vanadyl sulfate was found to be mediated by transcription factor erythroid transcription factor NF-E2 (Nrf2), which critically regulates GCLC by binding to the antioxidant response elements (AREs). Vanadyl sulfate treatment increased the nuclear translocation of Nrf2 and the accumulation of phosphorylated Nrf2. Extracellular regulated kinase (ERK) contributed to ARE-driven GCLC expression via Nrf2 activation. Vanadyl sulfate induced the expression of the active phospho form of ERK. Taken together, these results suggest that the increase in GSH level by Jeju ground water is, at least in part, due to the effects of vanadyl sulfate via the Nrf2-mediated induction of GCLC.

HPLC profiling of phenolics and flavonoids of Adonidia merrillii fruits and their antioxidant and cytotoxic properties.

In this study the antioxidant and cytotoxicity activity of the Adonidia merrillii fruits were investigated using different solvent polarities (methanol, ethyl acetate and water). The results showed that the total phenolic and flavonoid contents of the methanolic extract was higher compare with other extract with respective values of 17.80 ± 0.45 mg gallic acid equivalents/g dry weight (DW) and 5.43 ± 0.33 mg rutin equivalents/g DW. Beside that The RP-HPLC analyses indicated the presence of gallic acid, pyrogallol, caffeic acid, vanillic acid, syringic acid, naringin and rutin. In the DPPH, NO2 and ABTS scavenging assays, the methanolic extract exhibited higher antioxidant activity as compared to the ethyl acetate and water extracts. The extracts exhibited moderate to weak cytotoxic activity in the assays using human hepatocytes (Chang liver cells) and NIH/3T3 (fibroblasts cell) cell lines. The findings showed the Adonidia merrillii fruit extracts to possess considerable antioxidant and cytotoxicity properties. The fruit, therefore, is a potential candidate for further work to discover antioxidant and cytotoxic drugs from natural sources.

cDNA Microarray Analysis and Influx Transporter OATP1B1 in Liver Cells After Exposure to Gadoxetate Disodium, a Gadolinium-based Contrast Agent in MRI Liver Imaging.

BACKGROUND/AIM: Gadoxetate disodium (Primovist or Eovist) is extensively used as a hepatospecific contrast agent during magnetic resonance imaging (MRI) examinations. However, there is no information determining whether gadoxetate disodium has a cytotoxic impact and/or affects relative gene expression on liver cells. In the current study, we investigated the effects of gadoxetate disodium on cytotoxicity and the levels of gene expression in human normal Chang Liver cells. MATERIALS AND METHODS: The cytotoxic effect was detected via methyl thiazolyl tetrazolium (MTT) assay and 4',6-diamidino-2-phenylindole (DAPI) staining. mRNA expression was monitored by cDNA microarray and quantitative PCR (qPCR) analysis. The protein levels were determined by western blotting. RESULTS: Gadoxetate disodium at 5 and 10 mM failed to induce any cell cytotoxicity and morphological changes in Chang Liver cells. Our data demonstrated that gadoxetate disodium significantly enhanced the expression of 29 genes and suppressed that of 27. The SLCO1C1 (solute carrier organic anion transporter family member 1C1) mRNA expression was also increased by 2.62-fold (p-value=0.0006) in gadoxetate disodium-treated cells. Furthermore, we also checked and found that gadoxetate disodium up-regulated organic anion transporter polypeptide 1B1 (OATP1B1) protein level and increased OATP uptake transporter gene SLCO1C1 mRNA expression. CONCLUSION: Our results provide evidence regarding that gadoxetate disodium might be no cytotoxic effects on liver cells.

Protection of 4-hydroxybenzyl-chitooligomers against inflammatory responses in Chang liver cells.

The aim of this study was to investigate anti-inflammatory activity of 4-hydroxybenzyl-chitooligomers (HB-COS) in Chang liver cells stimulated by a cytokine mixture. It was revealed that HB-COS decreased the level of nitric oxide and prostaglandin E2 (PGE2) production by diminishing the expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) without significant cytotoxicity. Moreover, HB-COS exerted inhibitory effects on the production of pro-inflammatory mediator (interleukin-6) in Chang liver cells. Notably, HB-COS exhibited anti-inflammatory activities via blocking degradation of inhibitory kappa B alpha (IκB-α), translocation of nuclear factor kappa B (NF-κB), and phosphorylation of mitogen-activated protein kinases (MAPKs) in a dose-dependent manner. Collectively, these findings indicated that HB-COS possessed potential anti-inflammatory effects in Chang liver cells, and could be a useful therapeutic agent for the treatment of hepatic inflammatory diseases.

Prevention of H2O2-induced oxidative stress in Chang liver cells by 4-hydroxybenzyl-chitooligomers.

In this study, a bioactive derivative of chitooligomers (1.0-3.0 kDa), 4-hydroxybenzyl-COS (HB-COS), was synthesized to enhance antioxidant activity. Hence, HB-COS was evaluated for its capabilities against H2O2-induced oxidative stress in human Chang liver cells. It was found that HB-COS possessed the free radical scavenging activity via decreasing the intracellular reactive oxygen species production. Furthermore, HB-COS significantly reduced the oxidation of DNA in a dose-dependent manner. Notably, HB-COS treatment upregulated the gene and protein expressions of antioxidative enzymes and thereby enhancing the intracellular antioxidant mechanisms. In addition, HB-COS treatment caused a remarkable blockade on degradation of inhibitory kappa B alpha (IκB-α) protein and translocation of nuclear factor kappa B (NF-κB). The current study demonstrated that HB-COS effectively attenuated hydrogen peroxide-induced oxidative stress in Chang liver cells by increasing levels of antioxidant enzymes and inhibiting reactive oxygen species generation, DNA oxidation and the NF-κB signaling pathway.

Prevention of oxidative stress in Chang liver cells by gallic acid-grafted-chitosans.

Gallic acid-grafted-chitosans (GA-g-chitosans) were prepared according to our previous method, and the in vitro protective effect of the various GA-g-chitosans on tert-butyl hydroperoxide (t-BHP)-induced oxidative stress was investigated in Chang liver cells. Pretreatment of the GA-g-chitosans decreased cell damage induced by t-BHP in a dose-dependent manner. In addition, t-BHP-induced reactive oxygen species (ROS) generation and lipid peroxidation were inhibited by the GA-g-chitosans. Also, intracellular glutathione (GSH) was increased in the presence of the GA-g-chitosans. And GA-g-chitosan (I), which had the highest GA content, showed the highest activity among the tested compounds. Moreover, antioxidant enzyme activities such as catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) were decreased by t-BHP-induction., while GA-g-chitosan (I) pretreatment increased levels of CAT, SOD, and GPx. Overall, we demonstrated that GA-g-chitosans effectively attenuated the oxidative stress induced by t-BHP in Chang liver cells by inhibiting ROS generation, lipid peroxidation, and increasing levels of antioxidant enzymes.

Antidiabetic and cytotoxicity screening of five medicinal plants used by traditional African health practitioners in the Nelson Mandela Metropole, South Africa.

Diabetes mellitus is a growing problem in South Africa and of concern to traditional African health practitioners in the Nelson Mandela Metropole, because they experience a high incidence of diabetic cases in their practices. A collaborative research project with these practitioners focused on the screening of Bulbine frutescens, Ornithogalum longibracteatum, Ruta graveolens, Tarchonanthus camphoratus and Tulbaghia violacea for antidiabetic and cytotoxic potential. In vitro glucose utilisation assays with Chang liver cells and C2C12 muscle cells, and growth inhibition assays with Chang liver cells were conducted. The aqueous extracts of Bulbine frutescens (143.5%), Ornithogalum longibracteatum (131.9%) and Tarchonanthus camphoratus (131.5%) showed significant increased glucose utilisation activity in Chang liver cells. The ethanol extracts of Ruta graveolens (136.9%) and Tulbaghia violacea (140.5%) produced the highest increase in glucose utilisation in C2C12 muscle cells. The ethanol extract of Bulbine frutescens produced the most pronounced growth inhibition (33.3%) on Chang liver cells. These findings highlight the potential for the use of traditional remedies in the future for the management of diabetes and it is recommended that combinations of these plants be tested in future.