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Abstract To assess the ameliorative effect of ursodeoxycholic acid (UDCA) on hydrogen peroxide (H2O2)-induced hepatocyte injury. In our in vivo experiments, we modelled hyperlipidemia in ApoE−/− mice subjected to a 3-month high-fat diet and found that HE staining of the liver showed severe liver injury and excessive H2O2 was detected in the serum. We modelled oxidative stress injury in L02 cells by H2O2 in vitro and analyzed the levels of reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD) and related genes. UDCA significantly improved the level of oxidative stress in H2O2-injured L02 cells (P

Objective: In order to better screen targeted drugs, a microfluidic chip that can culture both diseased and normal cells is designed. Methods: With Human hepatocellular carcinomas (HepG2) and Human normal liver cells (LO2) as the cell models, Polydimethylsiloxane-glass (PDMS-glass) chip as the carrier, and potential anticancer drug sanguinarine as the research object, two cells were co-cultured on the chip and the drug acted on both cells simultaneously in a diffusion manner. Results: In co-cultured cell chips, the apoptosis rate of HepG2 cells was significantly higher than that of LO2 cells under the action of sanguinarine. Conclusion: The chip diffusion perfusion form does not damage the cells, and can achieve cell staining and in situ observation more flexibly and conveniently, and more realistically reflects the selective effect of drugs on different cells, which has the advantages of simple operation and low cost.

Seven new withanolides (1−7) and four known ones (8−11) were isolated from the calyxes and fruits of Nicandra physaloides, an edible and medicinal plant. Their structures were identified by extensive spectroscopic analyses or comparison with literature data. The antioxidant effects and hepatoprotective mechanisms of compounds 1–11 on hydrogen peroxide (H2O2)-induced oxidative stress injury were investigated. Among them, compound 11 showed significant activity with EC50 value of 83.78 ± 0.30 μM. Further, 11 was found to markedly increase cellular survivals and activities of SOD, CAT and GSH, and to reduce the accumulation of reactive oxygen species (ROS) in H2O2-induced LO2 cells. In addition, compound 11 significantly activated Nrf2 nuclear translocation and enhanced the expression of target gene HO-1, suggesting its preliminary mechanism of protective effects. In summary, withanolide 11 might be used as a lead compound for developing a hepatopathy medicine owing to its antioxidative and hepatoprotective effects.

Background/Aims: Zinc finger protein 667 (ZNF667) is a member of C2H2 zinc finger protein family. For the first time, we aim to analyze the expression pattern of ZNF667 in hepatocellular carcinoma (HCC) tissues; to explore its role in HCC tumorigenesis. Methods: Immuno-histochemistry was carried out to characterize the ZNF667 expression in paraffin-embedded HCC samples. The relationship between ZNF667 expression and the clinical, pathological data of the patients were analyzed. Human normal hepatocyte cells LO2 over expressing ZNF667 (LO2-ZNF667 cells), ZNF667 depleted hepatocellular carcinoma HepG2 cells (HepG2-shZNF667 cells) were set up, their proliferation, migration and invasion abilities were analyzed. Xenograft nude mice were used to analyze the malignancy of HepG2-shZNF667 cells in vivo. Western blot was performed to analyze the expression of Bcl-2 and BAX in LO2-ZNF667 and HepG2-shZNF667 cells. Results: Increased ZNF667 was found via immuno-histochemistry in HCC. Enhanced ZNF667 expression was associated with tumor size, clinical stage and tumor differentiation. LO2-ZNF667 cells displayed increased and HepG2-shZNF667 cells decreased cell proliferation, migration and invasion. Xenograft experiments proved reduced malignancy of HepG2-shZNF667 cells in vivo. LO2-ZNF667 cells displayed increased Bcl-2 and decreased BAX protein expression. HepG2-shZNF667 cells displayed enhanced BAX and inhibited BCL-2 expression. Conclusions: ZNF667 is shown to be a new oncogene in HCC and it may serve as a new therapeutic target for HCC via enhancing BCL-2 and decreasing BAX expression.

Seven new withanolides ( 1 − 7 ) and four known ones ( 8 − 11 ) were isolated from the calyxes and fruits of Nicandra physaloides , an edible and medicinal plant. Their structures were identified by extensive spectroscopic analyses or comparison with literature data. The antioxidant effects and hepatoprotective mechanisms of compounds 1 – 11 on hydrogen peroxide (H 2 O 2 )-induced oxidative stress injury were investigated. Among them, compound 11 showed significant activity with EC 50 value of 83.78 ± 0.30 μM. Further, 11 was found to markedly increase cellular survivals and activities of SOD, CAT and GSH, and to reduce the accumulation of reactive oxygen species (ROS) in H 2 O 2 -induced LO2 cells. In addition, compound 11 significantly activated Nrf2 nuclear translocation and enhanced the expression of target gene HO-1, suggesting its preliminary mechanism of protective effects. In summary, withanolide 11 might be used as a lead compound for developing a hepatopathy medicine owing to its antioxidative and hepatoprotective effects. [ABSTRACT FROM AUTHOR]

Oxidative stress, as mediated by ROS (reactive oxygen species), is a significant factor in initiating the cells damaged by affecting cellular macromolecules and impairing their biological functions; SelX, a selenoprotein also known as MsrB1 belonging to the methionine sulfoxide reductase (Msr) family, is the redox repairing enzyme and involved in redox-related functions. In order to more precisely analyze the relationship between oxidative stress, cell oxidative damage, and SelX, we stably overexpressed porcine Selx full-length cDNA in human normal hepatocyte (LO2) cells. Cell viability, cell apoptosis rate, intracellular ROS, and the expression levels of mRNA or protein of apoptosis-related genes under HO-induced oxidative stress were detected. We found that overexpression of SelX can prevent the oxidative damage caused by HO and propose that the main mechanism underlying the protective effects of SelX is the inhibition of LO2 cell apoptosis. The results revealed that overexpressed SelX reduced the HO-induced intracellular ROS generation, inhibited the HO-induced upregulation of Bax and downregulation of Bcl-2, and increased the mRNA and protein ratio of Bcl-2/Bax. Furthermore, it inhibited HO-induced p38 MAPK phosphorylation. Taken together, our findings suggested that SelX played important roles in protecting LO2 cells against oxidative damage and that its protective effect is partly via the p38 pathway by acting as a ROS scavenger. [ABSTRACT FROM AUTHOR]

To determine the roles of selenoprotein X gene ( Selx) in protecting liver cells against oxidative damage, the influences of Selx knockdown on HO-induced apoptosis in human normal hepatocyte (LO2) cells were studied. pSilencer 3.1 was used to develop knockdown vector targeting the 3′-UTR of human Selx. The Selx knockdown and control cells were further exposed to HO, and cell viability, cell apoptosis rate, and the expression levels of mRNA and protein of apoptosis-related genes were detected. The results showed that vector targeting the 3′-UTR of Selx successfully silenced mRNA or protein expression of SelX in LO2 cells. Selx knockdown resulted in decreased cell viability, increased percentage of early apoptotic cells, decreased Bcl2A1 and Bcl-2 expression, and increased phosphorylation of P38 in LO2 cells. When Selx knockdown LO2 cells were exposed to HO, characteristics of HO-induced cell dysfunctions were further exacerbated. Taken together, our findings suggested that SelX played important roles in protecting LO2 cells against oxidative damage and reducing HO-induced apoptosis in liver cells. [ABSTRACT FROM AUTHOR]

Exosomes have been described as a messenger between cells' communication and contain various information (lipids, proteins, mRNAs, microRNAs, LncRNAs). It has been proved that Linc-ROR was enriched in exosomes released by HepG2 cells. Our aim was to investigate whether exosomes released by HepG2 cells could affect the biological behaviors of LO2 cells and whether Linc-ROR played an important role in this process. Exosomes-derived from HepG2 cells were isolated and characterized. Real-time PCR assessed expression level of Linc-ROR in specimens of cancerous tissues and carcinoma-adjacent tissues. The Linc-ROR expression level in HepG2, Huh7, SMMC-7721, Bel-7402, LO2 cells and exosomes was detected by real-time PCR. Knockdown the expression of Linc-ROR in HepG2 by using effective siRNA. Cell counting method was used to test LO2 cells proliferative activities. Flow cytometry was performed to quantify the apoptosis rates of LO2 cells cocultured with exosomes. The expression levels of OCT4, NANOG, SOX2, P53 and CD133 were assessed by western blot. Linc-ROR was enriched in exosomes released by HepG2 cells. Exosomes derived from HepG2 cells promoted the proliferation and suppressed the apoptosis of LO2 cells suffering nutrient deficiency. Knockdown the expression of Linc-ROR in HepG2 or LO2 cells could significantly impaired the exosomes' effects on LO2 cells. In addition, the long-term coculture with exosomes would obviously change the biological behaviors of LO2 cells. Our experiments indicated the HepG2 cells could transfer its Linc-ROR to the LO2 cells via exosomes, then influenced the recipient cells. • Linc-ROR was enriched in exosomes released by HepG2 cell. • Exosomes derived from HepG2 cells promoted LO2 cells proliferation. • Knockdown of the expression of Linc-ROR in HepG2 cells could significantly impair the exosomes' effects on LO2 cells. • Linc-ROR may affect the behavior of LO2 cells by affecting the proteins associated with tumor stem cells. [ABSTRACT FROM AUTHOR]

N-Glycosylation, being one of the most common and complex protein post-translational modifications (PTMs), is known to have microheterogeneity with the presence of different N-glycan structures at a single specific glycosite. These different structures may have exactly the same monosaccharide composition but totally different differential expressions and pathological relevance. Mass spectrometry-based N-glycoproteomics has so far been successful in large-scale characterization of these N-glycans at the composition level, and structure-level identification and quantitation is urgently needed. Here we report our development of the intact N-glycopeptide search engine GPSeeker and the GPSeeker-centered quantitative structural N-glycoproteomics pipeline. In benchmark characterization of differentially expressed N-glycosylation in hepatocellular carcinoma HepG2 cells relative to LO2 cells, 5 405 and 1 081 intact N-glycopeptides with putative linkage structures were identified and quantified with isotopic dimethyl labeling and 2D liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Among the 5 405 IDs, 837 were identified with no less than one structure diagnostic fragment ion for the N-glycan moieties. Besides double isomers of sialic acid linkages and fucose sequences, quadruple isomers from combination of two linages and two sequences were chromatographically separated and confidently identified; microheterogeneity with different differentially expressions were observed on 183 out of the 231 quantified N-glycosites. This GPSeeker-centered quantitative structural N-glycoproteomics pipeline can be widely applied to precise qualitative and quantitative characterization of N-glycosylation with physiological and pathological relevance.

Background/aims: Zinc finger protein 667 (ZNF667) is a member of C2H2 zinc finger protein family. For the first time, we aim to analyze the expression pattern of ZNF667 in hepatocellular carcinoma (HCC) tissues; to explore its role in HCC tumorigenesis., Methods: Immuno-histochemistry was carried out to characterize the ZNF667 expression in paraffin-embedded HCC samples. The relationship between ZNF667 expression and the clinical, pathological data of the patients were analyzed. Human normal hepatocyte cells LO2 over expressing ZNF667 (LO2-ZNF667 cells), ZNF667 depleted hepatocellular carcinoma HepG2 cells (HepG2-shZNF667 cells) were set up, their proliferation, migration and invasion abilities were analyzed. Xenograft nude mice were used to analyze the malignancy of HepG2-shZNF667 cells in vivo. Western blot was performed to analyze the expression of Bcl-2 and BAX in LO2-ZNF667 and HepG2-shZNF667 cells., Results: Increased ZNF667 was found via immuno-histochemistry in HCC. Enhanced ZNF667 expression was associated with tumor size, clinical stage and tumor differentiation. LO2-ZNF667 cells displayed increased and HepG2-shZNF667 cells decreased cell proliferation, migration and invasion. Xenograft experiments proved reduced malignancy of HepG2-shZNF667 cells in vivo. LO2-ZNF667 cells displayed increased Bcl-2 and decreased BAX protein expression. HepG2-shZNF667 cells displayed enhanced BAX and inhibited BCL-2 expression., Conclusions: ZNF667 is shown to be a new oncogene in HCC and it may serve as a new therapeutic target for HCC via enhancing BCL-2 and decreasing BAX expression., (© 2017 The Author(s). Published by S. Karger AG, Basel.)