Liver Cell Line BRL-3A Research Articles

Taurine Alleviates Cadmium-Induced Hepatotoxicity by Regulating Autophagy Flux.

Our previous studies have confirmed that cadmium (Cd) exposure causes hepatotoxicity; it also induces autophagy and blocks the autophagy flux. Therefore, we hypothesized that Cd hepatotoxicity could be alleviated through nutritional intervention. Taurine (Tau) has various biological functions such as acting as an antioxidant, acting as an anti-inflammatory, and stabilizing cell membranes. In order to explore the protective effect and internal mechanism of Tau on Cd-induced hepatotoxicity, normal rat liver cell line BRL3A cells were treated with Cd alone or in combination with Tau to detect cell injury and autophagy-related indexes in this study. We found that Tau can alleviate Cd-induced cell-proliferation decline and morphological changes in the cell. In addition, Tau activates autophagy and alleviates the blockage of Cd-induced autophagy flux. In this process, lysosome acidification and degradation were enhanced, and autophagosomes were further fused with lysosomes. Then, we found that Tau alleviated autophagic flux block by promoting the transfer of membrane fusion proteins STX17 and SNAP29 to autophagosomes and the translocation of VAMP8 to lysosomes, which in turn attenuated the hepatocyte injury induced by Cd exposure. This will further reveal the hepatotoxicity mechanism of Cd and provide the theoretical basis for the prevention and treatment of Cd poisoning.

Hydrogen sulfide alleviates ischemia induced liver injury by repressing the SPHK1/S1P pathway.

Ischemia/reperfusion (I/R) induced liver injury is a severe pathological process which frequently occurs during clinical hepatic operations. The current study investigated the protective function and underlying mechanisms of hydrogen sulfide (H2S) in I/R induced liver injury. The effects of H2S were examined using the fibroblast-like rat liver cell line BRL-3A (the name of normal hepatocytes in rats) cultured under hypoxic conditions and an I/R rat model. The viability of BRL-3A cells was assessed using the methylthiazolyldiphenyl-tetrazolium (MTT) assay and Hoechst analysis. The expression of C/EBP homologous protein (CHOP), sphingosine kinase 1 (SPHK1), and sphingosine 1-phosphate (S1P) were determined in hypoxic BRL-3A cells with or without H2S treatment. CHOP was overexpressed in hypoxic BRL-3A cells to further evaluate whether H2S protected the liver against I/R injury by decreasing endoplasmic reticulum (ER) stress. Finally, the inflammation levels in the serum and the histopathological changes of liver were examined in the I/R rat model to evaluate the therapeutic function of H2S on I/R induced liver injury in vivo. H2S alleviated hypoxic damage in BRL-3A cells. In addition, hypoxia increased the expression of CHOP, SPHK1, and S1P in BRL-3A cells, and this was abolished by H2S pretreatment. Notably, overexpression of CHOP significantly inhibited the effect of H2S on the viability of BRL-3A cells during hypoxia. Overall, H2S effectively protected against I/R induced liver injury, decreased the inflammatory responses, and attenuated apoptosis of hepatocyte via inhibiting the ER stress response. These findings demonstrated that pre-treatment of H2S protected against I/R induced liver injury by repressing the SPHK1/S1P pathway via inhibition of ER stress, suggesting an effective therapeutic method for the treatment of I/R induced liver injury.

ACC1 is overexpressed in liver cancers and contributes to the proliferation of human hepatoma Hep G2 cells and the rat liver cell line BRL 3A.

Acetyl-coenzyme A carboxylase 1 (ACC1) serves a major role in fatty acid synthesis. Previous reports have indicated that ACC1 is a promising drug target for treating human diseases, particularly cancers and metabolic diseases; however, the role of ACC1 in liver cancer and normal liver function remains unknown. In the present study, bioinformatics analysis indicated that ACC1 is overexpressed in liver cancer. Kaplan-Meier survival analysis revealed that the expression levels of ACC1 are highly associated with the prognosis of patients with liver cancer. To determine the role of ACC1 in cancer and normal liver cells, ACC1 expression was downregulated in human hepatoma Hep G2 cells and the rat liver cell line BRL 3A using RNA interference technology, which demonstrated that silencing of ACC1 significantly suppressed the cell viability in the two cell lines. Additionally, ACC1 knockdown decreased the mRNA and protein expression levels of the cell proliferation-associated genes MYCN, JUN, cyclin D1 (CCND1) and cyclin A2 (CCNA2) in BRL 3A. Furthermore, the number of cells in division phase (G2/M) was significantly reduced in the interference group, as detected by flow cytometry. Thus, ACC1 may bind and activate CCNA2, CCND1, MYCN and JUN to promote BRL 3A proliferation. In summary, the results of present study indicated that overexpression of ACC1 is significantly associated with the survival time of patients with liver cancer, and may provide insight into the association between ACC1 and cell proliferation in BRL 3A cells.

The novel protein C9orf116 promotes rat liver cell line BRL-3A proliferation.

Our previous study has proved that the chromosome 9 open reading frame 116 (C9orf116) (NM_001106564.1) was significantly up-regulated in the proliferation phase of liver regeneration. To study its possible physiological function, we analyzed the effect of C9orf116 on BRL-3A cells via over-expression and interference technique. MTT results showed that the cell viability of the interference group was significantly lower than the control group at 48h after transfection (P<0.05), whereas it was significantly higher in the over-expression group (P<0.05). The flow cytometry results showed that C9orf116 knockdown or over-expression had little effect on BRL-3A cell apoptosis. However, the number of cells in division phase (G2/M) was significantly reduced in the interference group (P<0.05), but significantly increased in the over-expression group (P<0.01). Furthermore, the expressions of cell proliferation-related genes CCNA2, CCND1 and MYC both at mRNA and protein levels were down-regulated in the interference group and up-regulated in the over-expression group. Therefore, we concluded that C9orf116 may promote cell proliferation by modulating cell cycle transition and the expression of key genes CCNA2, CCND1 and MYC in BRL-3A cells.

Amphipathic silica nanoparticles induce cytotoxicity through oxidative stress mediated and p53 dependent apoptosis pathway in human liver cell line HL-7702 and rat liver cell line BRL-3A

The aim of this study was to evaluate the potential cytotoxicity and the underlying mechanism of amphipathic silica nanoparticles (SiO2 NPs) exposure to human normal liver HL-7702 cells and rat normal liver BRL-3A cells. Prior to the cellular studies, transmission electron microscopy (TEM), dynamic light scattering (DLS), and X ray diffraction (XRD) were used to characterize SiO2 NPs, which proved the amorphous nature of SiO2 NPs with TEM diameter of 19.8±2.7nm. Further studies proved that exposure to SiO2 NPs dose-dependently induced cytotoxicity as revealed by cell counting kit (CCK-8) and lactate dehydrogenase (LDH) assays, with more severe cytotoxicity in HL-7702 cells than BRL-3A cells. Reactive oxygen species (ROS) and glutathione (GSH) assays showed elevated oxidative stress in both cells. Morphological studies by microscopic observation, Hochest 33258 and AO/EB staining indicated significant apoptotic changes after the cells being exposed to SiO2 NPs. Further studies by western blot indicated that SiO2 NPs exposure to both cells up-regulated p53, Bax and cleaved caspase-3 expression and down-regulated Bcl-2 and caspase-3 levels. Activated caspase-3 activity detected by colorimetric assay kit and caspase-3/7 activity detected by fluorescent real-time detection kit were significantly increased by SiO2 NPs exposure. In addition, antioxidant vitamin C significantly attenuated SiO2 NPs-induced caspase-3 activation, which indicated that SiO2 NPs-induced oxidative stress was involved in the process of HL-7702 and BRL-3A cell apoptosis. Taken together, these results suggested that SiO2 NPs-induced cytotoxicity in HL-7702 and BRL-3A cells was through oxidative stress mediated and p53, caspase-3 and Bax/Bcl-2 dependent pathway and HL-7702 cells were more sensitive to SiO2 NPs-induced cytotoxicity than BRL-3A cells.

SPINK3: A novel growth factor that promotes rat liver regeneration

Serine peptidase inhibitor, Kazal type 3 (SPINK3) is a trypsin inhibitor, and also a growth factor that has an identical structure to epidermal growth factor (EGF), which could combine with epidermal growth factor receptor (EGFR) to promote cell proliferation. To shed light on the role and regulation mechanism of SPINK3 in rat liver regeneration (LR), Rat Genome 230 2.0 assay was used to detect the expression profiles of LR genes after partial hepatectomy (PH). The results showed that Spink3 was significantly up-regulated at 2-24 h and 72-168 h after PH. In the present study, RT-PCR and immunoblotting were used to validate the assay results. Ingenuity Pathway Analysis 9.0 (IPA) software was used to build the SPINK3 signaling regulating LR and analyze the possible mechanism. And then the expression of cell proliferation-associated gene Ccna2 was examined by RT-PCR in normal rat liver cell line BRL-3A in which Spink3 was overexpressed. The results showed that Ccna2 was significantly up-regulated in BRL-3A in which Spink3 was over-expressed. SPINK3 combining with EGFR accelerated cell proliferation during rat liver regeneration via P38, PKC, JAK-STAT and AKT pathways. Thus, SPINK3 was likely to promote hepatocytes proliferation in LR through P38, PKC, JAK-STAT and AKT.

YImpact of exogenous TGF-β1 on cell cycle progression and apoptosis in rat liver cell line BRL-3A

YImpact of exogenous TGF-β1 on cell cycle progression and apoptosis in rat liver cell line BRL-3A

Intermedilysin plays a key role in the invasion of human cells by Streptococcus intermedius

Streptococcus intermedius, which causes endogenous infections leading to abscesses, produces intermedilysin (ILY), a human-specific cytolysin. Because of the significant correlation between higher ILY production levels by S. intermedius and deep-seated abscesses, we constructed an ily knockout mutant and two complementation strains, (i) with the wild-type ily gene and (ii) with an ILY SS-mutant gene (the latter was not cytolytic without reductant due to an intramolecular SS bridge between domains 2 and 3), from ILY high level expressing strain UNS38, in order to investigate the role of ILY in deep-seated infections. Infection with the wild strain and the complementation strain secreting cytolytic ILY reduced the viability of human liver cell line HepG2 at infection but not of rat liver cell line BRL3A, and increased intracellularly concomitantly with increasing HepG2 cell damage. Isogenic ily knockout mutant strain and the complementation strain secreting ILY SS-mutant were not cytotoxic and showed no proliferation in either cell line. Masking of cytotoxic strains with anti-ILY antibody drastically decreased adherence and invasion of HepG2. Moreover, coating ily knockout mutant strain with ILY partially restored adherence to HepG2 but without subsequent bacterial growth. These results indicate that: 1) surface-bound ILY on S. intermedius is an important factor for invasion of human cells by this bacterium; 2) secretion of ILY within host cells is essential for subsequent host cell death; and 3) cell membrane poration by ILY is required in infection steps. These strongly suggest that ILY is an important factor in the pathogenesis of abscesses in vivo by this streptococcus.

Intermedilysin Is Essential for the Invasion of Hepatoma HepG2 Cells by Streptococcus intermedius

Streptococcus intermedius causes endogenous infections leading to abscesses. This species produces intermedilysin (ILY), a human-specific cytolysin. Because of the significant correlation between higher ILY production levels by S. intermedius and deep-seated abscesses, we constructed ily knockout mutant UNS38 B3 and complementation strain UNS38 B3R1 in order to investigate the role of ILY in deep-seated infections. Strain UNS38 reduced the viability of human liver cell line HepG2 at infection but not of rat liver cell line BRL3A. Isogenic mutant strain UNS38 B3 was not cytotoxic in either cell line. Quantification of S. intermedius revealed that in infected HepG2 cells UNS38 but not UNS38 B3 increased intracellularly concomitantly with increasing cell damage. This difference between UNS38 and UNS38 B3 was not observed with UNS38 B3R1. Invasion and proliferation in BRL3A cells was not observed. Masking UNS38 or UNS38 B3R1 with ILY antibody drastically decreased adherence and invasion of HepG2. Moreover, coating strain UNS38 B3 with ILY partially restored adherence to HepG2 but without subsequent bacterial growth. At 1 day post-infection, many intact UNS38 were detected in the damaged phagosomes of HepG2 with bacterial proliferation observed in the cytoplasm of dead HepG2 after an additional 2 day incubation. These results indicate that surface-bound ILY on S. intermedius is an important factor for invasion of human cells by this bacterium and that secretion of ILY within host cells is essential for subsequent host cell death. These data strongly implicate ILY as an important factor in the pathogenesis of abscesses in vivo by this streptococcus.

C19 odd-chain polyunsaturated fatty acids (PUfas) are metabolized to C21-PUfas in a rat liver cell line, and curcumin, gallic acid, and their related compounds inhibit their desaturation.

It was demonstrated that the rat liver cell line BRL-3A converted exogenous C19 odd chain-polyunsaturated fatty acids (PUFAs) into the corresponding C21- and C23-PUFAs as follows: 21:3n-8, 21:4n-8, 23:3n-8, and 23:4n-8 (from 19:3n-8); 21:4n-5, 21:5n-5, 23:4n-5, and 23:5n-5 (from 19:4n-5); 21:5n-2, 21:6n-2, 23:5n-2, and 23:6n-2 (from 19:5n-2). It presumed that these C19 PUFAs were converted through the mimic route to docosahexaenoic acid (22:6n-3) from eicosapentaenoic acid (20:5n-3). In addition, the characterization of the change of fatty acid composition of cellular lipids in rat liver cells were examined, using 19:4n-5 and several fatty acid desaturation inhibitors. Curcumin related compounds, curcumin, capsaicin, isoeugenol, 4-(4-hydroxy-3-methoxyphenyl)-3-buten-2-one, and gallic acid esters with near five carbon numbered alcohol had great changes of fatty acid composition of cellular lipids based on inhibition of the A6 desaturation of C24-PUFAs in rat liver cells.

Use of reverse ligation-PCR to identify transcriptional start sites in GC-rich TATA-less genes: application to the rat IGFBP-2 gene.

TATA-less genes are often GC-rich in the region of transcriptional initiation and the corresponding mRNAs are prone to the formation of secondary structure. These properties have made it difficult to determine unambigously the start sites of transcription for some of these genes by conventional assays such as primer extension and nuclease protection. Using the TATA-less rat IGFBP-2 gene, we demonstrate that tobacco acid pyrophosphatase-reverse ligation polymerase chain reaction (TAP-RLPCR), a novel and sensitive assay, can be used to map the start sites of these genes. First, the validity of TAP-RLPCR was demonstrated by mapping the transcription start site of the rat insulin-like growth factor 1 (IGFBP-1) gene to the correct position (nucleotides -173 relative to ATG, +1). Using total RNA obtained from the rat liver cell line BRL-3A, the transcription start sites of the rat IGFBP-2 gene were mapped to a narrow cluster extending from nucleotides -86 to -90 (ATG, +1), 39 bp downstream of three adjacent GC boxes that are essential to the transcriptional activity of the gene. The assay was also used to map the start sites of a luciferase reporter gene driven by the fragment -1,295 to -32 of the rat IGFBP-2 promoter after transfection in the human embryonic kidney cell line 293. The hybrid gene utilized the same transcription start sites as the rat IGFBP-2 gene, indicating that the elements required for positioning of the transcription initiation complex are contained within the 3' end terminating at nucleotide -32.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and characterization of the receptor for insulin-like growth factor I.

The receptor for insulin-like growth factor I (IGF-I) was purified from the rat liver cell line BRL-3A by a combination monoclonal anti-receptor antibody column and a wheat germ agglutinin column. Analyses of these receptor preparations on reduced sodium dodecyl sulfate-polyacrylamide gels yielded protein bands of Mr 136K (alpha subunit) and Mr 85K and 94K (beta subunit). These receptor preparations bound 5 times more IGF-I than insulin, and the binding of both labeled ligands was more potently inhibited by unlabeled IGF-I than by insulin. These results indicate that these receptor preparations contained predominantly the IGF-I receptor. This highly purified receptor preparation was found to possess an intrinsic kinase activity; autophosphorylation of the receptor beta subunit was stimulated by low concentrations of IGF-I (half-maximal stimulation at 0.4 nM IGF-I). Twentyfold higher concentrations of insulin were required to give comparable levels of stimulation. A monoclonal antibody that inhibits the insulin receptor kinase was found to inhibit the IGF-I receptor kinase with the same potency with which it inhibits the insulin receptor. In contrast, monoclonal antibodies to other parts of the insulin receptor only poorly recognized the IGF-I receptor. A comparison of V8 protease digests of the insulin and IGF-I receptors again revealed some similarities and also some differences in the structures of these two receptors. Thus, the IGF-I receptor is structurally, antigenically, and functionally similar to but not identical with the insulin receptor.