Rat Hepatocytes Protect against Lead-Cadmium-Triggered Apoptosis Based on Autophagy Activation.

Lead and cadmium are foodborne contaminants that threaten human and animal health. It is well known that lead and cadmium produce hepatotoxicity; however, defense mechanisms against the co-toxic effects of lead and cadmium remain unknown. We investigated the mechanism of autophagy (defense mechanism) against the co-induced toxicity of lead and cadmium in rat hepatocytes (BRL-3A cells). Cultured rat liver BRL-3A cell lines were co-cultured with 10, 20, 40 µM lead and 2.5, 5, 10 µM cadmium alone and in co-culture for 12 h and exposed to 5 mM 3-Methyladenine (3-MA), 10 µM rapamycin (Rapa), and 50 nM Beclin1 siRNA to induce cellular autophagy. Our results show that treatment of BRL-3A cells with lead and cadmium significantly decreased the cell viability, increased intracellular reactive oxygen species levels, decreased mitochondrial membrane potential levels, and induced apoptosis, which are factors leading to liver injury, and cell damage was exacerbated by co-exposure to lead-cadmium. In addition, the results showed that lead and cadmium co-treatment induced autophagy. We further observed that the suppression of autophagy with 3-MA or Beclin1 siRNA promoted lead-cadmium-induced apoptosis, whereas enhancement of autophagy with Rapa suppressed lead-cadmium-induced apoptosis. These results demonstrated that co-treatment with lead and cadmium induces apoptosis in BRL-3A cells. Interestingly, the activation of autophagy provides cells with a self-protective mechanism against induced apoptosis. This study provides insights into the role of autophagy in lead-cadmium-induced apoptosis, which may be beneficial for the treatment of lead-cadmium-induced liver injury.

Lactoferrin Alleviates Ethanol-Induced Injury via Promoting Nrf2 Nuclear Translocation in BRL-3A Rat Liver Cells.

Our previous animal studies found that the preventive effects of lactoferrin (Lf) on alcoholic liver injury (ALI) are associated with nuclear factor E2-related factor 2 (Nrf2). To further explore the causality, experiments were performed using rat normal liver BRL-3A cells. Lf treatment reduced ethanol-induced death and apoptosis; meanwhile, Lf treatment alleviated excessive LDH release. These findings confirmed the protection of Lf against ethanol-induced injury in BRL-3A cells. Mechanistically, Lf treatment reversed the reduction in nuclear Nrf2 induced by ethanol without affecting the cytoplasmic Nrf2 level, which led to antioxidant enzyme activity restoration. However, the blocking of Nrf2 nuclear translocation by ML385 eliminated the protective effects of Lf. In a conclusion, Lf protects BRL-3A cells from ethanol-induced injury via promoting Nrf2 nuclear translocation.

Quercetin alleviates cadmium-induced BRL-3A cell apoptosis by inhibiting oxidative stress and the PERK/IRE1α/ATF6 signaling pathway.

Cadmium (Cd) is a highly toxic environmental pollutant. The liver is an important metabolic organ in the body and is susceptible to Cd toxicity attacks. Quercetin (Que) is a flavonoid compound with pharmacological activities of scavenging free radicals and antioxidant activity. Previous studies have shown that Que can alleviate Cd caused hepatocyte apoptosis in rats, but the specific mechanism remains unclear. To explore the specific mechanism, we established a model of Cd toxicity and Que rescue in BRL-3A cells and used 4-phenylbutyrate (4-PBA), an endoplasmic reticulum stress (ERS) inhibitor, as positive control. Set up a control group, Cd treatment group, Cd and Que co treatment group, Que treatment group, Cd and 4-PBA co treatment group, and 4-PBA treatment group. Cell Counting Kit-8 (CCK-8) method was employed to measure cell viability. Fluorescence staining was applied to observe cell apoptosis. Flow cytometry was performed to detect reactive oxygen species levels. Real-time quantitative polymerase chain reaction (qRT-PCR) and Western blot method was adopted to detect the mRNA and protein expression levels of ERS and apoptosis-related genes. The results showed that compared with the control group, the Cd treated group showed a significant decrease in cell viability (P < 0.01), an increase in intracellular ROS levels, and apoptosis. The mRNA and protein expression levels of ERS and apoptosis related factors such as GRP78, IRE1α, XBP1, ATF6, Caspase-12, Caspase-3 and Bax in the cells were significantly increased (P < 0.01), while the mRNA and protein expression levels of Bcl-2 were significantly reduced (P < 0.01). Compared with the Cd treatment group, the Cd and Que co treatment group and the Cd and 4-PBA co treatment group showed a significant increase in cell viability (P < 0.01), a decrease in intracellular ROS levels, a decrease in cell apoptosis, and a significant decrease in the expression levels of ERS and apoptosis related factors mRNA and protein (P < 0.01), as well as a significant increase in Bcl-2 mRNA and protein expression (P < 0.01). We confirmed that Que could alleviate the apoptosis caused by Cd in BRL-3A cells, and the effects of Que were similar to those of ERS inhibitor.

Alleviating effect of quercetin on cadmium-induced oxidative damage and apoptosis by activating the Nrf2-keap1 pathway in BRL-3A cells.

Cadmium (Cd) is a toxic heavy metal extensively used in industrial and agricultural production. Among the main mechanisms of Cd-induced liver damage is oxidative stress. Quercetin (QE) is a natural antioxidant. Herein, the protective effect of QE on Cd-induced hepatocyte injury was investigated. BRL-3A cells were treated with 12.5 µmol/L CdCl2 and/or 5 µmol/L QE for 24 h. The cells and medium supernatant were collected, and the ALT, AST, and LDH contents of the medium supernatant were detected. The activities or contents of SOD, CAT, GSH, and MDA in cells were determined. Intracellular ROS levels were examined by flow cytometry. Apoptosis rate and mitochondrial-membrane potential (ΔΨm) were detected by Hoechst 33,258 and JC-1 methods, respectively. The mRNA and protein expression levels of Nrf2, NQO1, Keap1, CytC, caspase-9, caspase-3, Bax, and Bcl-2 were determined by real-time PCR (RT-PCR) and Western blot methods. Results showed that Cd exposure injured BRL-3A cells, the activity of antioxidant enzymes decreased and the cell ROS level increased, whereas the ΔΨm decreased, and the expression of apoptotic genes increased. Cd inhibited the Nrf2-Keap1 pathway, decreased Nrf2 and NQO1, or increased Keap1 mRNA and protein expression. Through the combined action of Cd and QE, QE activated the Nrf2-Keap1 pathway. Consequently, antioxidant-enzyme activity decreased, cellular ROS level decreased, ΔΨm increased, Cd-induced BRL-3A cell damage was alleviated, and cell apoptosis was inhibited. After the combined action of QE and Cd, Nrf2 and NQO1 mRNA and protein expression increased, Keap1 mRNA and protein expression decreased. Therefore, QE exerted an antioxidant effect by activating the Nrf2-Keap1 pathway in BRL-3A cells.

Characterization of xanthine oxidase inhibitory activities of phenols from pickled radish with molecular simulation.

Pickled radish is a general source of natural bioactive compounds that include phenols. Here, we used molecular docking, fluorescence quenching, circular dichroism spectroscopy and molecular dynamics simulations to identify potential inhibitors against xanthine oxidase from a library of pickled radish compounds. The most effective compounds were selected for validation through in vitro experiments including enzyme activity inhibition tests, and cell-based assays. Molecular docking results revealed that 2,6-Dihydroxyacetophenone, 4-Hydroxyphenethyl alcohol, and 4-Hydroxybenzaldehyde exhibited significant effects on xanthine oxidase inhibition. Three phenols have varying degrees of inhibition on xanthine oxidase, which is driven by hydrophobic interactions and hydrogen bonds and affects the secondary structure and hydrophobic homeostasis of xanthine oxidase. The stability of xanthine oxidase inhibition by three phenols was analyzed by molecular dynamics simulation. Finally, cellular experiments confirmed that three phenols reduced uric acid levels by inhibiting the xanthine oxidase enzyme activity of BRL 3A cells.

[Effect of perfluorooctanoic acid on lipid accumulation in rat liver cells BRL-3A and its possible mechanism].

OBJECTIVE: To investigate the effect of perfluorooctanoic acid on rat hepatocytes BRL-3 A cell viability and the expression of transcription factor nuclear factor erythroid 2-related factor 2(Nrf2) and arginosuccinate synthase(Ass1). METHODS: Rat hepatocytes BRL-3 A were cultured and divided into control group(0 µmol/L PFOA), low-dose group(6.25 µmol/L PFOA), and medium-dose group(25 µmol/L PFOA), high-dose group(100 µmol/L PFOA). After 48 hours, cell viability was detected by MTT, ROS content was detected by free radical indicator H_2DCFDA, enzyme activity related to oxidative stress was detected by the kit, Nrf2 and Ass1 protein expression level was detected by Western blot and immunocytochemistry(ICC). RESULTS: Compared with the control group, with the increase of the PFOA concentration, the cell viability of the middle and high dose groups had a downward trend, but there was no statistical significance(P>0.05). The intracellular ROS content increased, among which in the middle and high dose groups significantly increased(P<0.05), and the average fluorescence intensity was(5417.66±161.09) and(5725.50±166.83), respectively. Compared with the control group, the content of intracellular TG, TC and MDA in the low and medium dose groups did not change significantly, and the content of TG, TC and MDA in the high dose group was significantly increased(P<0.05), which was(0.21±0.05) mmol/L, (14.5±6.07) mmol/L and(1.23±0.33) nmol/mL, respectively. According to the ICC and Western blot result, the expression level of Nrf2 protein increased significantly in the high-dose group(P<0.05), and the expression level of Ass1 protein increased significantly in the low-dose group(P<0.05). CONCLUSION: Exposure to a certain dose of PFOA can lead to the accumulation of ROS in BRL-3 A cells. Nrf2 and Ass1 can play a certain role in eliminating ROS and ammonia detoxification by increasing their expression under the oxidative damage of rat liver cells caused by PFOA.

Microbe-derived antioxidants attenuate cobalt chloride-induced mitochondrial function, autophagy and BNIP3-dependent mitophagy pathways in BRL3A cells.

Environmental excessive cobalt (Co) exposure increases risks of public health. This study aimed to evaluate the potential mechanism of microbe-derived antioxidants (MA) blend fermented by probiotics in attenuating cobalt chloride (CoCl2)-induced toxicology in buffalo rat liver (BRL3A) cells. Herein, results showed that some phenolic acids increased in MA compared with the samples before fermentation through UHPLC-QTOF-MS analysis. Also, the contents of essential and non-essential amino acids, their derivatives and minerals were rich in MA. The DPPH, O2-, OH- and ABTS+ scavenging ability of MA is comparable to those of vitamin C and better than mitoquinone mesylate (mitoQ). In vitro cell experiments showed that CoCl2 treatment increased the percentage of apoptosis cells, lactate dehydrogenase and genes involved in glycolysis, increased ATP production and decreased mitochondrial membrane potential, increased genes involved in canonical autophagy process (including initiation, autophagosomes maturation and fusion with lysosome) and BNIP3-dependent mitophagy pathways in BRL3A cells, while MA attenuated CoCl2-induced reactive oxygen species (ROS) production, apoptosis, mitochondrial protein expression and dysfunction, and BNIP3-dependent mitophagy. Collectively, these results provide insights into the role of MA in reversing CoCl2-induced toxicology in BRL3A cells, providing the promising constituents for decreasing Co-induced toxicology in functional foods.

Effects of bisphenols on proliferation and oxidative stress of BRL 3A rat liver cells and their mutagenicities / 预防医学

Objective@#To examine the effects of bisphenol A (BPA), bisphenol S ( BPS ), bisphenol F ( BPF ) and bisphenol AF ( BPAF ) on the proliferation and oxidative stress of BRL 3A rat liver cells, and to preliminarily evaluate their mutagenicities.@*Methods@#In vitro cultured BRL 3A rat liver cells were treated with BPA, BPS, BPF and BPAF at concentrations of 0, 5, 10, 25, 50, 100, 150 and 200 μmol/L for 48 h, respectively. Then, the cell viability was determined using the CCK-8 assay, and the half maximal inhibitory concentration ( IC50 ) was calculated. The minimum inhibitory concentration for BRL 3A cell proliferation was screened, and the intracellular reactive oxygen species ( ROS ) was measured in BRL 3A cells using the 2',7'-dichlorodihydrofluorescein diacetate ( DCFH-DA ) assay. In addition, the effects of BPA, BPS, BPF and BPAF at concentrations of 1 000, 200, 40, 8 and 1.6 μg/plate on the mutant colonies of histidine-deficient Salmonella typhimurium ( TA1535, TA97a, TA98, TA100 and TA102 ) were tested using the Ames test.@*Results@#Treatment with BPA and BPF at concentrations of 100 to 200 μmol/L and with BPAF at concentrations of 25 to 200 μmol/L inhibited BRL 3A cell survival at a concentration-dependent manner, while exposure to BPS at concentrations of 5 to 200 μmol/L resulted in no changes in BRL 3A cell survival. The IC50 values of BPA, BPS, BPF and BPAF were 131.7, >200, 187.5 and 21.6 μmol/L against BRL 3A cells, respectively. Treatment with BPS at 100 μmol/L or BPAF at 25 μmol/L caused no significant changes in the ROS level; however, exposure to BPA at 100 μmol/L and BPF at 100 μmol/L significantly increased the ROS level. Ames test showed that BPA, BPS, BPF and BPAF did not induce mutagenicity in TA1535, TA97a, TA98, TA100 or TA102 strains.@*Conclusions@#BPAF shows the highest cytotoxicity to BRL 3A cells, and low-concentration exposure to BPS has few effects on BRL 3A cells. The cytotoxicity of bisphenols against BRL 3A cells may be associated with the induction of oxidative stress. None of the four bisphenols show mutagenic effects under the present experimental conditions.

Toxic Effects of Docosahexaenoic Acid Treatment in the Rat Liver BRL-3A Cell.

The cytotoxicity of docosahexaenoic acid (DHA) on normal cells is still unclear. This study investigated the effects of DHA on the cytotoxicity and possible mechanism in the BRL-3A cell. The cultured rat liver BRL-3A cell line was treated with 50, 100 and 200 µM DHA for 24 h. The cell viability was increased in the 50 and 100 µM DHA treatments, but decreased in the 200 µM DHA treatment. The 50, 100 and 200 µM DHA treatments increased the proportion of the apoptotic cells, the levels of lactate dehydrogenase (LDH), alkaline phosphatase (AKP) and IL-6 in the supernatant, and the ratio of the phosphonated p38MAPK to the p38MAPK (p-p38/p38) protein in the cells. The expression of TGF beta-activated kinase 1 (TAK1), nuclear transcription factor-κB p65 (NF-κB p65) and the inhibitor of NF-κB alpha (IκBα) mRNA, and the ratio of the phosphonated IκBα (p-IκBα) to IκBα protein were increased in the 200 µM DHA treatment, while the ratio of phosphonated extracellular regulated protein kinases (p-ERK) to ERK protein was decreased in the 200 µM DHA treatment. These results indicate that DHA-treated (50, 100 and 200 µM) BRL-3A cells for 24 h promotes cell apoptosis and inflammatory response, and the p38 MAPK, ERK and NF-κB signal pathways were involved in mediating the apoptosis and inflammatory response.

Rosmarinic acid attenuates acrylamide induced apoptosis of BRL-3A cells by inhibiting oxidative stress and endoplasmic reticulum stress.

Acrylamide (AA) is a common endogenous contaminant in food, with a complex toxicity mechanism. The study on liver damage to experimental animals caused by AA has aroused a great attention. Rosmarinic acid (RosA) as a natural antioxidant shows excellent protective effects against AA-induced hepatotoxicity, but the potential mechanism is still unclear. In the current study, the protective effect of RosA on BRL-3A cell damage induced by AA was explored. RosA increased the activity of SOD and GSH, reduced the content of ROS and MDA, and significantly reduced the oxidative stress (OS) damage of BRL-3A cells induced by AA. RosA pretreatment inhibited the MAPK signaling pathway activated by AA, and down-regulated the phosphorylation of JNK, ERK and p38. RosA pretreatment also reduced the production of calcium ions caused by AA. In addition, the key proteins p-IRE1α, XBP-1s, TRAF2 of the IRE1 pathway, and the expression of endoplasmic reticulum stress (ERS) characteristic proteins GRP78, p-ASK1, Caspase-12 and CHOP were also down-regulated by RosA. NAC blocked the activation of the MAPK signaling pathway and inhibited the ERS pathway. RosA reduced the rate of apoptosis and down-regulated the expression of Bax/Bcl-2 and Caspase-3, thereby inhibiting AA-induced apoptosis. In conclusion, RosA reduced the OS and ERS induced by AA in BRL-3A cells, thereby inhibiting cell apoptosis, and it could be used as a potential protective agent against AA toxicity.

Development and evaluation of puerarin-loaded controlled release nanostructured lipid carries by central composite design.

The present work was aimed at developing optimized puerarin-loaded nanostructured lipid carrier (PA-NLC) on base of phospholipid complex. The puerarin phospholipid complex (PA-PC) was prepared by a solvent evaporation method and the formulation was confirmed according to the encapsulation efficiency (EE%). The hepatoprotective effect of PA-NLC on BRL 3A cell stimulated by ethanol was carried out using MTT assay, and cell imaging was done using an inverted phase contrast tissue culture microscope. The NLCs were produced by nanoemulsion method using glyceryl monostearate (GMS), olive oil, and Poloxamer 188 as the solid, liquid lipids, and surfactant. A single factor analysis determined the optimal ratio of solid lipid to liquid lipid. A three-factor, five-level central composite design (CCD) was used to predict response variables and construct 3D-response contour plots. The independent variables, which were the concentrations of PA-PC, total lipid, and surfactant affected particle size, surface charge of the nanoparticles, and the EE. An optimized NLC composition consisted of 31.25% PA-PC, 46.87% GMS, 9.38% olive oil, and 18.75% Poloxamer 188. The NLC had an average particle size of 159 ± 1.1 nm, zeta potential of -28.3 mV, EE% of 92.16%, and drug loading (DL%) of 5.75%. Differential scanning calorimetry (DSC), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR) studies showed that the formation of NLC was accompanied by changes in crystallinity and intermolecular interaction. The PA-NLC system showed an enhanced therapeutic effect on alcohol-induced cell injury of BRL-3A.

Antimalarial agent artesunate induces G0/G1 cell cycle arrest and apoptosis via increasing intracellular ROS levels in normal liver cells.

Artesunate (ARS) has been shown to be highly effective against chloroquine-resistant malaria. In vitro studies reported that ARS has anticancer effects; however, its detrimental action on cancer cells may also play a role in its toxicity toward normal cells and its potential toxicity has not been sufficiently researched. In this study, we investigated the possible cytotoxic effects using normal BRL-3A and AML12 liver cells. The results showed that ARS dose-dependently inhibited cell proliferation and arrested the G0/G1 phase cell cycle in both BRL-3A and AML12 liver cells. Western blotting demonstrated that ARS induced a significant downregulation of cyclin-dependent kinase-2 (CDK2), CDK4, cyclin D1, and cyclin E1 in various levels and then caused apoptosis when the Bcl-2/Bax ratio decreased. Conversely, the levels of intracellular reactive oxygen species (ROS) were increased. The ROS scavenger N-acetylcysteine can significantly inhibit cell cycle arrest and apoptosis induced by ARS. Thus, the data confirmed that ARS exposure impairs normal liver cell proliferation by inducing G0/G1 cell cycle arrest and apoptosis, and this detrimental action may be associated with intracellular ROS accumulation. Collectively, the possible side effects of ARS on healthy normal cells cannot be neglected when developing therapies.

Inhibition of Frizzled-2 by small interfering RNA protects rat hepatic BRL-3A cells against cytotoxicity and apoptosis induced by Hypoxia/Reoxygenation.

Frizzled-2 plays an important role in maintaining normal hepatic cell functionality. This study aimed to investigate the role of inhibition of Frizzled-2 in protecting rat liver BRL-3A cells from Hypoxia/Reoxygenation (H/R). In vitro H/R hepatic cell model was established by culturing BRL-3A cells under H/R condition. Frizzled-2 siRNA was transfected into BRL-3A cells to inhibit Frizzled-2 signaling. Wnt5a and Frizzled-2 were significantly increased in BRL-3A cells upon H/R treatment. H/R treatment induced cell cytotoxicity, the early apoptosis rate and the intracellular Ca2+ level in BRL-3A cells while silencing frizzled-2 gene decreased the H/R induced cell cytotoxicity, apoptosis and intracellular Ca2+ level. In vivo mice study further showed the up-regulation of Frizzled-2/Wnt 5 pathway and cleaved Caspase-3 expression in liver tissues under ischemia and reperfusion injury (IRI). In summary, inhibition of Frizzled-2 by its siRNA may protects BRL-3A cells by attenuating the H/R induced cell cytotoxicity and apoptosis.

circRNA-14723 promotes hepatocytes proliferation in rat liver regeneration by sponging rno-miR-16-5p.

Circular RNA (circRNA) is a subclass of noncoding RNA (ncRNA) detected within mammalian tissues and cells. However, its regulatory role during the proliferation phase of rat liver regeneration (LR) remains unreported. This study was designed to explore their regulatory mechanisms in cell proliferation of LR. The circRNA expression profile was detected by high-throughput sequencing. It was indicated that 260 circRNAs were differentially expressed during the proliferation phase of rat LR. Among them, circ-14723 displayed a significantly differential expression. We further explored its regulatory mechanism in rat hepatocytes (BRL-3A cells). First, EdU, flow cytometry and western blot (WB) indicated that knocking down circ-14723 inhibited BRL-3A cells proliferation. Second, RNA-Pulldown and dual-luciferase report assay showed that circ-14723 could sponge rno-miR-16-5p. At last, WB showed that the reported target genes of rno-miR-16-5p, CCND1, and CCNE1 were downregulated after knocking down circ-14723. In conclusion, we found that circ-14723 exerted a critical role in G1/S arrest to promote cell proliferation via rno-miR-16-5p/CCND1 and CCNE1 axis in rat LR. This finding further revealed the regulatory mechanisms of circRNA on cell proliferation of LR, and might provide a potential target for clinical problems.

Serine peptidase inhibitor Kazal type III (SPINK3) promotes BRL-3A cell proliferation by targeting the PI3K-AKT signaling pathway.

The serine protease inhibitor, Kazal type III (SPINK3), is a trypsin inhibitor associated with liver disease, which highly overexpresses in a variety of cancers. In one of our previous studies of our laboratory, Spink3 was observed to be significantly upregulated in rat liver regeneration (LR) via a gene expression profile. For the current study, rat hepatocyte BRL-3A cells were treated by gene addition/interference, and the addition of the exogenous rat recombinant protein SPINK3. It was revealed that both the overexpression of endogenous Spink3 and addition of exogenous rat recombinant SPINK3 (rrSPINK3) significantly promoted the cell proliferation of BRL-3A cells, whereas cell proliferation was inhibited when Spink3 was interfered. Furthermore, quantitative reverse transcription polymerase chain reaction and western blot results revealed that three signaling pathways, including extracellular-signal-regulated kinase 1/2 (ERK1/2), Janus kinase (JAK)-signal transducer and activator of transcription (STAT), and phosphatidylinositol-3-kinase (PI3K)-protein kinase B (AKT), as well as their related genes, were altered following endogenous Spink3 addition/interference. Also, the PI3K-AKT and SRC-p38 pathways and their related genes were modified following exogenous SPINK3 treatment. Among them, the common signaling pathway was PI3K-AKT pathway. We concluded that SPINK3 could activate the PI3K-AKT pathway by enhancing the expression of AKT1 to regulate the proliferation of BRL-3A cells. This study may contribute to shedding light on the potential mechanisms of SPINK3 that regulate the proliferation of BRL-3A cells.

In silico, in vitro: antioxidant and antihepatotoxic activity of gnetol from Gnetum ula Brongn.

Introduction: Gnetum ula is a notable medicinal plant used to cure various ailments. The stem part of the plant is used traditionally to treat jaundice and other disorders. The present work is to investigate the in vitro hepatoprotective and antioxidant activity of ethanol extract of stem of G. ula (GUE) and its isolated compound gnetol. Methods: Column chromatography was carried out for GUE and various column fractions were obtained. DPPH and reducing power assays were performed for GUE and column fractions. The potent fraction was characterized, interpreted and tested for in vitro hepatoprotective activity on the BRL3A cell line. In silico docking studies of gnetol compound on the protein TGF-ß (transforming growth factor - ß) and Peroxisome proliferator-activated receptor α (PPARα) was carried out. Results: DPPH scavenging and reducing power assay showed that the fourth column fraction has antioxidant potential than other fractions. The fourth column fraction was characterized to obtain gnetol compound. BRL3A cell line was used for the toxicity study of GUE and gnetol. Both, the extract and the isolated compound were found to be nontoxic with CTC50 value more than 1000 µg/mL. At the concentration of 200 µg/mL, GUE and gnetol offered cell protection of 50.2% and 54.3%, however, silymarin showed 77.15% protection at 200 µg/mL concentration against CCl4 treated BRL3A cell line. The docking results of the ligand molecule TGF-ß showed that gnetol has the binding affinity of -7.0 and standard silymarin being -6.8. TGF-ß showed good hydrophobic interactions and formed two hydrogen bonds with the amino acids. For PPARα protein, gnetol showed the binding affinity of -8.4 and silymarin with -6.5. Hydrogen bonding and good hydrophobic interactions against the amino acid molecules in relation to the PPARα protein are shown. Conclusion: Gnetum ula stem extract and its isolated compound are safe and offered significant hepatoprotection against CCl4 induced toxicity. Isolated compound gnetol exhibited a potent antioxidant activity offering protection to liver damage. However, in vivo studies need to be carried out to validate the traditional use of G. ula .

Screening of the Hepatotoxic Components in Fructus Gardeniae and Their Effects on Rat Liver BRL-3A Cells.

Fructus Gardeniae (FG) is a common Chinese medicine and food. However, the toxicity of FG has drawn increasing concern, especially its hepatotoxicity. The purpose of this study was to screen the hepatotoxic components of FG and evaluate their effects on rat liver BRL-3A cells. The chemical composition of FG was determined by HPLC-ESI-MS. CCK-8 assay was used to evaluate the cytotoxicity of ten chemical components from FG, and then the toxic components with significant inhibitory activity were selected for further study. The results showed that geniposide, genipin, genipin-1-gentiobioside, gardenoside, and shanzhiside all suppress cells viability. Apoptosis assays further indicated that geniposide and its metabolite genipin are the main hepatotoxic components of FG. Pretreatment of cells with geniposide or genipin increased the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP). The activities of superoxide dismutase (SOD) and glutathione (GSH) were decreased, while the malondialdehyde (MDA) level was increased. The cell contents of tumor necrosis factor (TNF-α), interleukin-6 (IL-6), and nitric oxide (NO) were also increased. Molecular docking simulations were used to investigate the mechanism of FG-induced hepatotoxicity, revealing that geniposide and genipin bind strongly to the pro-inflammatory factor TNFR1 receptor of the NF-κB and MAPK signaling pathways. The obtained results strongly indicate that the hepatotoxicity of FG is caused by iridoids compounds. Genipin had the most significant hepatotoxic effect. These toxic substances destroy the cell antioxidant defense system, increasing inflammatory injury to the liver cells and leading to apoptosis and even necrosis. Thus, this study lays a foundation for toxicology research into FG and its rational application.

PLCG2 promotes hepatocyte proliferation in vitro via NF-κB and ERK pathway by targeting bcl2, myc and ccnd1.

Phospholipase Cγ2 (PLCG2) has been implicated in the regulation of cell proliferation, transformation, and tumor growth. In this study, we investigate the mechanism of PLCG2 action using a short interference RNA (siRNA) method. The effects of PLCG2 on rat liver BRL-3A cells treated siRNA were studied by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT assay), bromodeoxyuridine (BrdU) labelling assay, flow cytometry method (FCM), quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. The results showed when PLCG2 was reduced, cell vitality and proliferation rate were significantly decreased (p < .05 vs. control). FCM analysis showed that the number of cell division phase (G2 + M) was declined (p < .05 vs. control). RT-PCR and western blot revealed that the expression of signalling related genes NF-κB, FOS, JUN and ELK, target genes BCL2, CCNB1 and CCND1 were remarkably down-regulated in cells treated with PLCG2 siRNAs. Based on these results, we conclude PLCG2 plays an important role in rat liver cell proliferation via ERK and NF-κB pathway by regulating the expression of BCl2, MYC and CCND1.

Osteopontin promotes rat hepatocyte proliferation both in vitro and in vivo.

Aim: This study aimed to examine the effects of osteopontin (OPN) on hepatocyte growth and liver regeneration (LR). Methods: A recombinant lentivirus expressing OPN and OPN-siRNAs were used to treat BRL-3A cells, while the adenovirus expressing OPN or OPN-targeted shRNA were applied for rat primary hepatocytes. Moreover, rrOPN and OPN-Ab were added to treat BRL-3A. Next, rrOPN was administrated into rat regenerating livers. Then in vitro and in vivo assays were performed to evaluate the biological function of OPN in hepatocyte growth and LR. Results: OPN overexpression facilitated proliferation and viability of BRL-3A cells and primary hepatocytes, while OPN silencing reversed these effects. Similarly, rrOPN stimulated cell cycle progression and viability, but OPN-Ab led to cell cycle arrest and decreased viability. OPN overexpression induced the expression of p-STAT3, p-AKT and CCND1, and OPN siRNA led to reduction of p-AKT and CCND1. Furthermore, rrOPN promoted the expression of p-STAT3 and p-AKT, while OPN-Ab and PI3K/Akt inhibitor LY294002 both inhibited the expressions of p-AKT and Bcl2. Moreover, LR rate, serum IL-6 and TNF-α, Ki-67+ proportion and the phosphorylation of STAT3, AKT and p65 were augmented by rrOPN treatment. Conclusion: OPN promotes hepatocyte proliferation both in vitro and in vivo through STAT3 and AKT signaling pathways.

Mono-2-ethylhexyl phthalate (MEHP) promoted lipid accumulation via JAK2/STAT5 and aggravated oxidative stress in BRL-3A cells.

Mono-2-ethylhexyl phthalate (MEHP), as the major metabolite of Di-(2-ethylhexyl) phthalate (DEHP), can induce lipid accumulation in hepatocytes and further leads to non-alcoholic fatty liver disease (NAFLD), while the underlying mechanism is unclear. We aim to clarify the effects of JAK2/STAT5 pathway on lipid accumulation induced by MEHP and the role of oxidation stress in NAFLD. BRL-3A hepatocytes were exposed to MEHP (0, 10, 50, 100 and 200 µM) for 24 h and 48 h. Then the lipid droplets in cells were observed by Oil-Red-O staining and quantified by isopropyl alcohol. The levels of TG, SOD, TBARS, AST and ALT were all detected by commercial kits. RT-PCR was used to detect mRNA expression, and western blotting was used to detect the expression of proteins encoded by JAK2/STAT5 pathway genes and lipid metabolism-related genes. As a result, MEHP promoted the lipid synthesis and accumulation in BRL-3A cells. MEHP down-regulated the expression and inhibited the activation of JAK2/STAT5. Moreover, the lipid metabolism-related kinases levels were elevated after MEHP exposure. In addition, the SOD levels were gradually decreased and the TBARS levels were increased in MEHP-treated groups. The lipid metabolism-related proteins levels were correlated with the oxidation stress levels. Furthermore, the ALT and AST levels were elevated after MEHP exposure. Therefore, we concluded that MEHP led to lipid accumulation through inhibiting JAK2/STAT5 pathway, resulted in damaging liver parenchyma and NAFLD by aggravating oxidation stress.