The impact and mechanism of TET3 overexpression on the progression of hepatic fibrosis.

Aims: To investigate whether TET3 regulates hepatic stellate cell apoptosis and understand the role of demethylation in hepatic fibrosis (HF). Methods: LX-2T cells were infected with TET3 lentivirus. After TET3 adenovirus infection, the degree of HF in each group was analyzed. Chromatin immunoprecipitation was used to verify the targeting relationship between TET3 and CBP, and finally the expression of various proteins was detected. Results: TET3 overexpression activated the CBP/FOXO1-BIM pathway, increased the expression of apoptotic proteins and accelerated the apoptosis of activated LX-2 cells. The degree of HF was improved in the TET3 upregulation group. Conclusion: TET3 can activate the CBP/FOXO1-BIM pathway to accelerate the apoptosis of activated hepatic stellate cells and ultimately alleviate HF.

TET3 as a non-invasive screening tool for the detection of fibrosis in patients with chronic liver disease.

Ten-eleven translocation protein 3 (TET3) is one of the key enzymes in DNA demethylation which can be expressed in liver tissues. However, the clinical value of TET3 for diagnosis and treatment of chronic liver disease have not been reported previously. We investigated the diagnostic accuracy of serum TET3 as a non-invasive screening tool for liver fibrosis. 212 patients with chronic liver disease from were enrolled in this study. Enzyme-linked immunosorbent assay was used to measure the serum levels of TET3. Receiver operating characteristics (ROC) were determined to examine the diagnostic accuracy of TET3 and combination model for diagnosis fibrosis. Serum TET3 level in fibrosis cases was significantly higher than that in non-fibrosis and controls, respectively. The areas under the ROC curve of the TET3 and fibrosis-4 index for liver fibrosis were 0.863 and 0.813, and 0.916 and 0.957 for liver cirrhosis. The combination of TET3 and fibrosis-4 index had a highly promising positive predictive value for detecting liver fibrosis and cirrhosis different stages of (93.5% and 100%) as compared with each diagnostic tool alone. TET3 is related to the development of liver fibrosis and cirrhosis. The TET3-fibrosis-4 model enhances discriminatory power and represents a promising non-invasive tool for the diagnosis and screening of liver fibrosis.

[Rat serum containing oxymatrine inhibits the proliferation of LX2 human hepatic stellate cells induced by sodium arsenite via blocking PI3K/AKT pathway].

Objective To investigate the effect of rat serum containing oxymatrine (OM) on the activation of LX2 human hepatic stellate cells induced by sodium arsenite and its mechanism. Methods SD rats were gavaged with 100 mg/kg OM or equal volume of normal saline to prepare OM-containing serum and blank serum. LO2 human embryonic liver cell line was treated with 100 µmol/L sodium arsenite for 24 hours, and then the supernatant was collected. LX2 cells were incubated with the mixture of the supernatant and normal medium at the ratio of 1:4 for 24 hours to establish the cell model of indirect arsenic exposure. Blank serum group (160 mL/L blank serum), indirect arsenic exposure group (160 mL/L blank serum with arsenic exposure), low-dose OM-containing serum group (80 mL/L blank serum and 80 mL/L OM-containing serum with arsenic exposure), high-dose OM-containing serum group (160 mL/L medicated serum with arsenic exposure) were set up. MTT assay and flow cytometry were used to detect cell proliferation and cell cycle, respectively. Western blot analysis was performed to detect the protein expressions of α-SMA, Bcl2, BAX, cyclin D1, PI3K, and phospho-AKT (p-AKT) in LX2 cells. Results After indirect arsenic treatment, the proliferation rate of LX2 cells increased, the proportion of G1 phase decreased, the proportion of apoptosis decreased, the expression of α-SMA, PI3K, p-AKT, cyclin D1, Bcl2 were significantly up-regulated, and the expression of BAX decreased. After OM-containing serum treatment, the proportion of cells in G1 phase increased, the proportion of apoptosis increased, the expression of BAX protein increased significantly, and the expression of other proteins were significantly down-regulated, especially in the high-dose group. Conclusion OM-containing serum can effectively inhibit the proliferation of LX2 hepatic stellate cells induced by arsenite and promote their apoptosis, which may be related to the blocking of PI3K/AKT signaling pathway.

A RAC-alpha serine/threonine-protein kinase (CgAKT1) involved in the synthesis of CgIFNLP in oyster Crassostrea gigas.

The RAC-alpha serine/threonine-protein kinase (AKT) is one of the most important protein kinases involved in many biological processes in eukaryotes. In the present study, a novel AKT homologue named CgAKT1 was identified from the Pacific oyster Crassostrea gigas. The open reading frame (ORF) of CgAKT1 cDNA was of 1482 bp encoding a peptide with 493 amino acid residues. There were classical domains in the predicted CgAKT1 protein, including an N-terminal pleckstrin homology domain, a central catalytic domain and a C-terminal hydrophobic domain. The mRNA transcripts of CgAKT1 were detected in all the examined tissues of C. gigas with higher level in gills (8.24-fold of that in mantle, p < 0.05) and haemocytes (3.62-fold of that in mantle, p < 0.05). After poly (I:C) stimulation, the mRNA expression of CgAKT1 decreased significantly in haemocytes from 3 h (0.44-fold of that in the control group, p < 0.001) to 24 h (0.20-fold of that in the control group, p < 0.001), and then increased significantly at 48 h (3.65-fold of that in the control group, p < 0.05). The expression level of CgAKT1 mRNA increased significantly at 6 h after rCgIFNLP stimulation, which was 3.60-fold of that in the control group (p < 0.001). The Alexa Fluor 488 positive signals of CgAKT1 protein were found to be distributed in the cytoplasm and cell membrane of haemocytes, while those in the cytoplasm became weaker after poly (I:C) stimulation. In CgAKT1-RNAi oysters, the mRNA expression of cyclic GMP-AMP synthase (CgcGAS) and TANK-binding kinase 1 (CgTBK1) did not change significantly, but the mRNA expression level of stimulator of interferon gene (CgSTING), interferon regulatory factor-1 (CgIRF-1), interferon regulatory factor-8 (CgIRF-8) and IFN-like protein (CgIFNLP) increased significantly, which was 1.40-fold, 1.53-fold, 1.72-fold and 1.99-fold of that in EGFP-RNAi oysters (p < 0.05), respectively. In CgIFNLP-RNAi oysters, the transcripts of CgAKT1 decreased significantly compared to those in EGFP-RNAi oysters (0.16-fold, p < 0.01). Moreover, the expression of p-CgTBK1, CgSTING and CgIFNLP at the protein level in the oysters treated with p-AKT1 activator (SC-79) was significantly suppressed after poly (I:C) stimulation. After the transfection of CgAKT1, the expression of p-cGAS protein in HEK293T cells increased significantly, while the cyclic GMP-AMP in the cells and the interferon (IFN-ß) in the cell culture fluid decreased significantly compared with that in the control group. These results indicated that CgAKT1 might play a negative role in antiviral immunity of oyster by regulating the synthesis of CgIFNLP.

Cortisol modulates glucose metabolism and oxidative response after acute high temperature stress in Pacific oyster Crassostrea gigas.

Cortisol is the main stress hormone that plays crucial roles in energy metabolism and immune response in vertebrates. In the present study, the homologues of 11ß-hydroxysteroid dehydrogenase type 1 (designated Cg11ß-HSD1) and 5α-reductase 1 (designated Cg5αR1), the key enzymes related to cortisol metabolism, were identified from Pacific oyster Crassostrea gigas. The Cg11ß-HSD1 harbored a conserved SDR domain, and Cg5αR1 contained a Steroid_dh domain and three transmembrane domains. The mRNA transcripts of Cg11ß-HSD1 and Cg5αR1 were constitutively expressed in all the examined tissues of oysters, with the highest expression level in haemocytes and labial palp, respectively. After acute high temperature stress (28 °C), the mRNA expression level of Cg11ß-HSD1 in hepatopancreas significantly up-regulated at 6 h and 12 h, and that of Cg5αR1 significantly up-regulated at 6 h, compared with the Blank group (11 °C). The concentration of cortisol and glucose, as well as the activities of superoxide dismutase (SOD) and catalase (CAT) in hepatopancreas all significantly up-regulated after acute high temperature stress, while the glycogen concentration in adductor muscle decreased significantly at 6 h and 12 h. After the blockage of Cg11ß-HSD1 with metyrapone, the cortisol concentration and the activities of SOD and CAT significantly decreased after acute high temperature stress, the glucose concentration in hepatopancreas significantly increased at 24 h, and the glycogen concentration in adductor muscle significantly increased at 6 h. These results collectively suggested that cortisol played a crucial role in regulating glucose metabolism and oxidative response in oysters upon acute high temperature stress.

[Endoplasmic reticulum stress in hepatic stellate cells induced by tunicamycin promotes apoptosis and cell cycle arrest].

Objective To investigate the effect of endoplasmic reticulum stress (ERS) induced by tunicamycin on proliferation, activation, and apoptosis of HSC-T6 rat hepatic stellate cells and its possible mechanism. Methods With the expression level of glucose regulated protein 78 (GRP78) as an indicator to explore the optimal concentration and time, a cell model of tunicamycin-induced ERS in HSC-T6 cells was established. HSC-T6 cells were randomized into control group, treatment group with 1 mL/L of dimethyl sulfoxide (DMSO), and treatment group with 1 µg/mL of tunicamycin, and the cells were treated for 12 h. MTT assay was used to detect cell proliferation, flow cytometry to detect apoptosis and cell cycle, and Western blot to detect the protein expressions of α-smooth muscle actin (α-SMA), C/EBP cAMP homologous protein (CHOP), caspase-12, and cyclin D1. Results The optimal dose of tunicamycin to induce ERS in HSC-T6 cells was 1 µg/mL and the optimal time was 12 hours. Compared with the control group and treatment group with DMSO, the treatment group with 1 µg/mL of tunicamycin had no significant change in cell proliferation, but the expression of α-SMA was up-regulated with the apoptosis increased, the proportion of G1 phase cells was significantly increased and that of S phase cells decreased, the ERS induced apoptosis related signal proteins CHOP and caspase-12 were significantly up-regulated, and the expression of cyclin D1 was significantly down-regulated. Conclusion Tunicamycin treatment of HSC-T6 cells for 12 hours induces significant ERS and activation of the cells. The insignificant change in the number of cells during the activation may be related to the increased apoptosis and the cell cycle arrest induced by the activation of the GRP78/CHOP/caspase-12 pathway.