Rat Liver Cell Line Research Articles

MiR-148b Caused Liver Injury in Rats with Traumatic Hemorrhagic Shock by Inhibiting SIRT6 Expression.

The purpose of this study was to investigate the role of miR- 148b in liver injury in rats with traumatic hemorrhagic shock (THS) and to elucidate its potential mechanism. The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum of rats were detected by enzyme-linked immune sorbent assay (ELISA), and the injury of rat liver was analyzed by hematoxylin-eosin (H&E) staining. Apoptosis of rat hepatocytes and normal rat liver cell line (BRL3A) was identified by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay and flow cytometry, respectively. MiR-148b and sirtuin 6 (SIRT6) expression was measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot. Lactate dehydrogenase (LDH) content and cell viability were measured by commercial kits and cell counting kit-8 (CCK-8) assay, respectively. The binding sites of miR-148b and SIRT6 were predicted by the Starbase database and verified by dual luciferase reporter assay. MiR-148b expression in THS rats or ischemia-reperfusion (I/R)-treated cells was higher than in the control group. Overexpression of miR-148b further promoted the effects of I/R, which enhanced the levels of ALT, AST and LDH, cell apoptosis of liver tissue or BRL3A cells and decreased the expression of SITR6. Besides, miR-148b negatively correlated with SIRT6, and upregulated the expression of SIRT6 could partly reverse the effect of miR-148b. Hepatocyte injury induced by I/R was achieved by regulating miR-148b /SIRT6 axis.

Galactose-1-phosphate inhibits cytochrome c oxidase and causes mitochondrial dysfunction in classic galactosemia

Classic galactosemia is an inborn error of metabolism caused by mutations in the GALT gene resulting in the diminished activity of the galactose-1-phosphate uridyltransferase enzyme. This reduced GALT activity leads to the buildup of the toxic intermediate galactose-1-phosphate and a decrease in ATP levels upon exposure to galactose. In this work, we focused our attention on mitochondrial oxidative phosphorylation in the context of this metabolic disorder. We observed that galactose-1-phosphate accumulation reduced respiratory rates in vivo and changed mitochondrial function and morphology in yeast models of galactosemia. These alterations are harmful to yeast cells since the mitochondrial retrograde response is activated as part of the cellular adaptation to galactose toxicity. In addition, we found that galactose-1-phosphate directly impairs cytochrome c oxidase activity of mitochondrial preparations derived from yeast, rat liver, and human cell lines. These results highlight the evolutionary conservation of this biochemical effect. Finally, we discovered that two compounds - oleic acid and dihydrolipoic acid – that can improve the growth of cell models of mitochondrial diseases, were also able to improve galactose tolerance in this model of galactosemia. These results reveal a new molecular mechanism relevant to the pathophysiology of classic galactosemia - galactose-1-phosphate-dependent mitochondrial dysfunction - and suggest that therapies designed to treat mitochondrial diseases may be repurposed to treat galactosemia.

TFE3-mediated impairment of lysosomal biogenesis and defective autophagy contribute to fluoride-induced hepatotoxicity

Excessive fluoride exposure can cause liver injury, but the specific mechanisms need further investigation. We aimed to explore the role of impaired lysosomal biogenesis and defective autophagy in fluoride-induced hepatotoxicity and its potential mechanisms, focusing on the role of transcription factor E3 (TFE3) in regulating hepatocyte lysosomal biogenesis. To this end, we established a Sprague-Dawley (SD) rat model exposed to sodium fluoride (NaF) and a rat liver cell line (BRL3A) model exposed to NaF. The results showed that NaF exposure diminished liver function and led to apoptosis as well as autophagosome accumulation and impaired autophagic degradation. In addition, NaF exposure caused compromised lysosome biogenesis and decreased lysosomal degradation, and inhibited TFE3 nuclear translocation. Notably, the mTOR inhibitors rapamycin (RAPA) and Ad-TFE3 promoted lysosomal biogenesis and enhanced lysosomal degradation function. Furthermore, RAPA and Ad-TFE3 reduced NaF-induced apoptosis by alleviating impaired autophagic degradation. In conclusion, NaF impairs lysosomal biogenesis by inhibiting TFE3 nuclear translocation, decreasing lysosomal degradation function, resulting in impaired autophagic degradation, and ultimately inducing apoptosis. Therefore, TFE3 may be a promising therapeutic target for fluoride-induced hepatotoxicity.

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.

The role of ZNF143 overexpression in rat liver cell proliferation

BackgroundZinc finger protein 143(ZNF143), a member of the Krüppel C2H2-type zinc finger protein family, is strongly associated with cell cycle regulation and cancer development. A recent study suggested that ZNF143 plays as a transcriptional activator that promotes hepatocellular cancer (HCC) cell proliferation and cell cycle transition. However, the exact biological role of ZNF143 in liver regeneration and normal liver cell proliferation has not yet been investigated.MethodsIn our study, we constructed a stable rat liver cell line (BRL-3A) overexpressing ZNF143 and then integrated RNA-seq and Cleavage Under Targets and Tagmentation (CUT&Tag) data to identify the mechanism underlying differential gene expression.ResultsOur results show that ZNF143 expression is upregulated during the proliferation phase of liver regeneration after 2/3 partial hepatectomy (PH). The cell counting kit-8 (CCK-8) assay, EdU staining and RNA-seq data analyses revealed that ZNF143 overexpression (OE) significantly inhibited BRL-3A cell proliferation and cell cycle progression. We then performed CUT&Tag assays and found that approximately 10% of ZNF143-binding sites (BSs) were significantly changed genome-wide by ZNF143 OE. However, CCCTC-binding factor (CTCF) binding to chromatin was not affected. Interestingly, the integration analysis of RNA-seq and CUT&Tag data showed that some of genes affected by ZNF143 differential BSs are in the center of each gene regulation module. Gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses indicated that these genes are critical in the maintenance of cell identity.ConclusionThese results indicated that the expression level of ZNF143 in the liver is important for the maintenance of cell identity. ZNF143 plays different roles in HCC and normal liver cells and may be considered as a potential therapeutic target in liver disease.

DNA methylation mediated downregulation of histone H3 variant H3.3 affects cell proliferation contributing to the development of HCC

Chromatin alterations brought by histone variants and modifications potentially regulate gene transcription from tumor initiation to progression. Histone H3.3 variant is one such epigenetic player important for disease progression and development. Though many studies have implicated H3.3 role in cancer progression and metastasis, its regulation, importance of specific modifications and chaperones have been not understood yet. We report DNA methylation mediated downregulation of histone H3 variant H3.3 in HCC and a concomitant increase in the level of the H3.2 variant. The loss of H3.3 in cancer tissues correlates with a decrease in the histone modifications associated with active transcription like H3K9/K14/K27Ac and H3K4Me3. The ectopic overexpression of H3.3 and H3.2 did not affect global PTMs and cell physiology, probably owing to the deregulation of specific histone chaperones CAF-1 (for H3.2) and HIRA (for H3.3) as observed in HCC tissues. Notably, knockdown of P150, a subunit of CAF-1 leads to a cell cycle arrest in S-phase in a neoplastic rat liver cell line, possibly due to the decrease in the histone levels necessary for DNA packaging. Remarkably, modulation of H3.3 in pre-neoplastic rat liver cells lead to an increase in cell proliferation and a decreased transcription of tumor suppressor genes, recapitulating the tumor cell phenotype. Our data suggests, inhibition of DNA methylation and histone deacetylation leads to the restoration of histone H3 variant expression in tumor cells.

Diazinon toxicity in hepatic and spleen mononuclear cells is associated to early induction of oxidative stress

ABSTRACT Diazinon is an organophosphorus pesticide, which may have potential toxic effects on the liver and immune system; however, the underlying mechanisms remain mostly unidentified. This work is aimed at evaluating the oxidative stress and cell cycle alterations elicited by low-dose diazinon in a rat liver cell line (BRL-3A) and spleen mononuclear cells (SMC) from Wistar rats. Diazinon (10–50 μM) caused early reactive oxygen species (ROS) generation (from 4 h) as well as increased O2 •- level (from 0.5 h), which led to subsequent lipid peroxidation at 24 h, in BRL-3A cells. In SMC, diazinon (20 μM) produced similar increases in ROS levels, at 4 and 24 h, with the highest O2 •- level being found at 4 h. Low-dose diazinon induced G1-phase arrest and cell death in hepatic cells and SMC. Therefore, diazinon could affect the liver and the immunological system through the premature oxidative stress induction. Abbreviations: O2 •-: superoxide anion radical; ROS: reactive oxygen species; SMC: spleen mononuclear cells; TBARS: thiobarbituric acid reactive substances

MiR-27a-5p regulates acrylamide-induced mitochondrial dysfunction and intrinsic apoptosis via targeting Btf3 in rats

Acrylamide (AA), a potential carcinogen, is commonly formed in foods rich in carbohydrates at high heat. It is known that AA-induced mitochondrial dysfunction is responsible for its toxicity. Previously we found AA exposure increased miR-27a-5p expression in livers of SD rats. Here, the regulation mechanism of miR-27a-5p in mitochondrial dysfunction was investigated in rat liver cell lines (IAR20) and SD rats. The results showed that the overexpressed miR-27a-5p contributes to modulating mitochondrial dysfunction and Btf3 is identified as its target gene. The knockdown of Btf3 increases the cleaved PARP1 level and the phosphorylation of ATM and p53, which results in mitochondria-dependent apoptosis. Therefore, the miR-27a-5p-Btf3-ATM-p53 axis might play a vital role in the promotion of AA-induced cell apoptosis through disrupting mitochondrial structure and function. This would provide a potential target for the assessment and intervention of AA toxicity.

Acute acrylonitrile exposure inhibits endogenous H2S biosynthesis in rat brain and liver: The role of CBS/3-MPST-H2S pathway in its astrocytic toxicity

Hydrogen sulfide (H2S) as the third gasotransmitter molecule serves various biological regulatory roles in health and disease. Acrylonitrile (AN) is a common occupational toxicant and environmental pollutant, causing brain and liver damage in mammals. The biotransformation of AN is dependent-upon reduced glutathione (GSH), cysteine and other sulfur-containing compounds. However, the effects of AN on the endogenous H2S biosynthesis pathway have yet to be determined. Herein, we demonstrated that a single exposure to AN (at 25, 50, or 75 mg/kg for 1, 6 or 24 h) decreased the endogenous H2S content and H2S-producing capacity in a dose-dependent manner, both in the cerebral cortex and liver of rats in vivo. In addition, the inhibitory effects of AN (1, 2.5, 5, 10 mM for 12 h) on the H2S content and/or the expression of H2S-producing enzymes were also found both in primary rat astrocytes and rat liver cell line (BRL cells). Impairment in the H2S biosynthesis pathway was also assessed in primary rat astrocytes treated with AN. It was found that inhibition of the cystathionine-β-synthase (CBS)/3-mercaptopyruvate sulfurtransferase (3-MPST)-H2S pathway with the CBS inhibitor or 3-MPST-targeted siRNA significantly increased the AN-induced (5 mM for 12 h) cytotoxicity in astrocytes. In turn, CBS activation or 3-MPST overexpression as well as exogenous NaHS supplementation significantly attenuated AN-induced cytotoxicity. Taken together, endogenous H2S biosynthesis pathway was disrupted in rats acutely exposed to AN, which contributes to acute AN neurotoxicity in primary rat astrocytes.

Abstract 15555: Potential Cure for Hypertension? The Effect of Crispr Genome Editing

Hypertension is recognized as one of the largest contributors to the global burden of disease. Unfortunately, for the majority of patients, hypertension is poorly controlled due, in part, to poor medical compliance resulting from the need to take one or more drugs, often several times daily. An ideal therapy would be administered a single time but yield long-term blood pressure control. Under the appropriate condition, we hypothesize that this modality could potentially lead to a cure for hypertension. In this study, we investigated whether that sustained, and possibly life-long, reductions in blood pressure could be achieved via Crispr-Cas9 mediated disruption of a key gene in the RAAS, Angiotensinogen (AGT). In vitro, we demonstrated that expression of Crispr-Cas9 system in the BRL 3A rat liver cell line led to a significant reduction in AGT protein levels (75% reduction, N=3, P<0.01). Delivery of the Crispr-Cas9 system into the liver via systemic delivery of the hepatocyte-targeting AAV8 reduced both AGT mRNA (70% reduction, N=5, P<0.01) and circulating AGT protein (50% reduction, N=5, P<0.01) and Ang II levels (33% reduction, n=5, P<0.01). In the SHR model of hypertension, Crispr-Cas9 mediated loss of AGT expression reduced systolic blood pressure in adult animals with established hypertension (30-35mmHg decrease in both females and males, N=12, P<0.001). Importantly, AGT gene disruption also prevented the spontaneous development of hypertension in young pre-hypertensive SHR (SHR control: 55-60mmHg increase in systolic blood pressure both female and male SHR; SHR plus Crispr-Cas9: 30-33 mmHg increase. N=12. P<0.001). Importantly, in all the models studied, reductions in blood pressure were sustained for at least 4 months. Importantly, the partial disruption of the hepatic AGT gene was sufficient to control hypertension but did not block the renin-angiotensin response to cardiovascular stress such as sodium depletion. In summary, we have demonstrated that targeting the Crispr-Cas9 system to RAAS reduces blood pressure and may be a potential therapy to achieve safe and sustained, possibly life-long, control of hypertension and this approach maybe a cure for hypertension.

Prooxidation and Cytotoxicity of Selenium Nanoparticles at Nonlethal Level in Sprague-Dawley Rats and Buffalo Rat Liver Cells.

The effects of selenium nanoparticles (SeNPs) on the antioxidant capacity in Sprague-Dawley (SD) rats were investigated. The rats were given intragastric administration of an SeNP suspension at doses of 0, 2, 4, and 8 mg Se/kg BW for two weeks. The antioxidant capacity in serum and organic tissues (liver, heart, and kidney) and the gene expression levels of glutathione peroxidase 1 (GPX1) and glutathione peroxidase 4 (GPX4) in the liver were measured. Buffalo rat liver (BRL) cell lines were further constructed to explore the cytotoxicity mechanism induced by SeNPs through the determination of antioxidant capacity; cell activity; apoptosis; and Caspase-3, Caspase-8, and Caspase-9 family activities. The results showed that SeNP administration over 4.0 mg Se/kg BW decreased the antioxidant capacities in the serum, liver, and heart and downregulated mRNA expression of GPX1 and GPX4 in the liver. The BRL cell line experiments showed that treatment with over 24 μM SeNPs decreased the viability of the cells and damaged the antioxidant capacity. Flow cytometry analysis showed that decreased cell viability induced by SeNPs is mainly due to apoptosis, rather than cell necrosis. Caspase-3 and Caspase-8 activities were also increased when BRL cells were treated with 24 μM and 48 μM SeNPs. Taken together, a nonlethal level of SeNPs could impair the antioxidant capacity in serum and organic tissues of rats, and the liver is the most sensitive to the toxicity of SeNPs. A pharmacological dose of SeNPs could lead to cytotoxicity and induce cell death through apoptosis and extrinsic pathways contributing to SeNP-induced apoptosis in BRL cells.

Evaluation of carotenoid extract from Dunaliella salina against cadmium-induced cytotoxicity and transforming growth factor β1 induced expression of smooth muscle α-actin with rat liver cell lines

The carotenoid extract from Dunaliella salina was identified and quantitated. The effects against cadmium (Cd)-induced hepatotoxicity and transforming growth factor β1 (TGF-β1)-induced activation of hepatic stellate cells (HSCs) were evaluated. All-trans-β-carotene (474.82 mg/g extract) and 9- or 9′-cis-β-carotene (425.64 mg/g extract) were major carotenoids in the extract, accounting for 90.30% of total carotenoids (997.17 mg/g extract). Other carotenoids were all-trans-lutein (22.77 mg/g extract), all-trans-zeaxan-thin (39.26 mg/g extract), 13- or 13′-cis-β-carotene (17.18 mg/g extract), all-trans-α-carotene (9.26 mg/g extract) and 9- or 9′-cis-α-carotene (8.24 mg/g extract). The algal extract had a higher effect (p < 0.05) than all-trans-β-carotene in the reduction of cytotoxicity for CdCl2-treated Clone 9 cells and expression of smooth muscle α-actin (αSMA) for TGF-β1-treated HSC-T6 cells. These effects were shown to be concentration-dependent.

Genomic Study, Phytochemical Characterization,and Antiproliferative Activity of Two Different Genotypes of Jatropha curcas L. Obtained by a Breeding Program

Jatropha curcas (Euphorbiaceaefamily) is a multipurpose plant with considerable potential in biodiesel production, and in cosmetic and medicinal uses. The part of J. curcas usually used is the oil obtained from its seeds, whereas the leaves generally represent the waste material. The aim of this study was to characterize the composition and to preliminary investigate the biological activity ofJ. curcas leaves obtained from plants by a breeding program. To describe genomic structure, molecular markers were applied. For biological study, two genotypes (JA and JB) that are genetically divergent were selected by cluster analysis. A phytochemical analysis was carried out to characterize the chemical composition of the extracts, which resulted rich in biologically active compounds, whereas toxic phorbol esters were absent. Biological assays showed an antiproliferative effect on the Buffalo normal rat liver cell line (BRL-3A), with genotype B being more potent than that of the counterpart (JA).The purified compounds isolated did not show antiproliferative activity, suggesting that the effect observed was due to the phytocomplex and should involve several secondary metabolites. This study highlights that a plant of the same genus and same species that has been cultivated in the same soil and climatic conditions can be characterized by a high variability. This is what makes research in pharmacognosya complex process.

DNA methylation and RASSF4 expression are involved in T-2 toxin-induced hepatotoxicity

T-2 toxin is a secondary metabolite produced by Fusarium species and commonly contaminates food and animal feed. T-2 toxin can induce hepatotoxicity through apoptosis and oxidative stress; however, the underlying mechanism is not clear. Recent studies indicated that RASSF4, a member of the RASSF family, participates in cell apoptosis and some cancers due to its inactivation via DNA hypermethylation. However, its role in T-2 toxin-induced liver toxicity is poorly understood. Therefore, in this study, female Wistar rats were given a single dose of T-2 toxin at 2 mg/kg b.w. and were sacrificed at 1, 3 and 7 days post-exposure. A normal rat liver cell line (BRL) was exposed to different concentrations of T-2 toxin (10, 20, 40 nM) for 4, 8, 12 h, respectively. Histopathological analysis revealed with apoptosis in some liver cells and clear proliferation under T-2 toxin exposure. Expression analysis by immunohistochemical assays, quantitative real-time PCR (qPCR) and western blot demonstrated that T-2 toxin activated PI3K-Akt/Caspase/NF-κB signaling pathways. Additionally, DNA methylation assays revealed that the expression of RASSF4 was silenced by promoter hypermethylation after exposure to T-2 toxin for 1 and 3 days as compared to the control group. Moreover, joint treatment of 5-Aza-2′-deoxycytidine (DAC) (5 μM) and T-2 toxin (40 nM) increased expression of RASSF4 and PI3K-Akt/caspase/NF-κB signaling pathways-related genes, inducing cell apoptosis. These findings for the first time demonstrated that DNA methylation regulated the RASSF4 expression under T-2 toxin, along with the activation of its downstream pathways, resulting in apoptosis.

DNA methylation is involved in pro-inflammatory cytokines expression in T-2 toxin-induced liver injury

Currently, T-2 toxin has been reported to cause liver toxicity with the effects of oxidative stress and inflammation; however, the underlying mechanism of T-2 toxin-induced liver injury is not fully understood. Increasing lines of evidence show that DNA methylation affects the expression of inflammatory cytokine, and plays a crucial role in autoimmune diseases. Nevertheless, the potential role of DNA methylation in the hepatotoxicity of T-2 toxin has not been explored. In this study, female Wistar rats were given a single dose of T-2 toxin at 2 mg/kg b.w. and were sacrificed at 1, 3 and 7 days post-exposure. In vitro, a normal rat liver cell line (BRL) was exposed to different concentrations of T-2 toxin. Histopathological analysis was used to investigate damage to the liver, which was detected at the molecular level by RT-PCR, Western blot and immunohistochemical assays, methylation-specific PCR (MSP), bisulfite sequencing (BSP), and flow cytometry. The results showed that T-2 toxin significantly increased the levels of DNA methyltransferases (DNMT1, DNMT3A), which were mainly concentrated at the site of liver injury. The 5-methylcytosine (5-mC) level of genomic DNA was also raised in T-2 toxin-treated rat livers. The expression of inflammatory cytokines (IL-6, IL-1β, IL-11, IL-1α, and TNF-α) increased both in vivo and in vitro under T-2 toxin treatment. Notably, DNA demethylation directly increased the expression of cytokines IL-11, IL-6, IL-α, and TNF-α under T-2 toxin exposure. DNA methylation inhibitors combined with T-2 toxin directly or indirectly induced the production of inflammatory cytokines and aggravate cell apoptosis. Our study uncovered for the first time that DNA methylation is related to the expression of inflammatory cytokines in T-2 toxin-induced liver injury. These findings suggested that DNA methylation is a potential mechanism of T-2 toxin-induced hepatotoxicity.

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.

Involvement of gap junctions in propylthiouracil-induced cytotoxicity in BRL-3A cells.

Gap junctions (GJs), which are important plasma membrane channels for the transfer of signaling molecules between adjacent cells, have been implicated in drug-induced liver injury. However, the influence and the underlying mechanisms of GJs in propylthiouracil (PTU)-induced hepatotoxicity are unclear. In the present study, distinct manipulations were performed to regulate GJ function in the BRL-3A rat liver cell line. The results indicated that the toxic effect of PTU in BRL-3A cells was mediated by GJ intercellular communication, as cell death was significantly attenuated in the absence of functional GJ channels. Furthermore, the specific knockdown of connexin-32 (Cx32; a major GJ component protein in hepatocytes) using small interfering RNA was observed to decrease necrosis, intracellular PTU content and the level of reactive oxygen species (ROS) following PTU exposure. These observations demonstrated that suppressing GJ Cx32 could confer protection against PTU-induced cytotoxicity through decreasing the accumulation of PTU and ROS. To the best of our knowledge, the present study is the first to demonstrate the role and possible underlying mechanisms of GJs in the regulation of PTU-induced toxicity in BRL-3A rat liver cells.

Curcumin and allopurinol ameliorate fructose-induced hepatic inflammation in rats via miR-200a-mediated TXNIP/NLRP3 inflammasome inhibition.

Excess fructose consumption causes high prevalence of metabolic syndrome and inflammatory liver diseases. The aim of the current study was to investigate the therapeutic effects and underlying molecular mechanisms of curcumin and allopurinol in high fructose-induced hepatic inflammation. Male Sprague-Dawley rats were supplied with standard rat chow and drinking water containing 10% (w/v) fructose for consecutive 12 weeks. Curcumin (15, 30 and 60 mg/kg) and allopurinol (5 mg/kg) were administered to rats via oral gavage daily from Week 7 to 12. For in vitro experiments, curcumin (2.5 μM) and allopurinol (100 μM) were treated to 5 mM fructose-exposed Buffalo rat liver cell line (BRL-3 A) and human hepatoblastoma cell line (HepG2), respectively. The data from these animal and hepatocyte models showed that curcumin and allopurinol ameliorated fructose-induced metabolic symptom, especially hepatic inflammation in rats. Interestingly, down-regulation of microRNA-200a (miR-200a) was screened out in livers of fructose-fed rats and then validated in fructose-exposed BRL-3 A and HepG2 cells. Fructose-induced miR-200a low-expression was identified as a negative mediator of thioredoxin interacting protein (TXNIP) by direct targeting of 3'UTR-rTXNIP, subsequently activating the NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome in BRL-3 A cells. Curcumin, as well as allopurinol, notably up-regulated miR-200a expression, accordingly, down-regulated TXNIP and inhibited NLRP3 inflammasome activation in fructose-fed rat livers and fructose-exposed BRL-3 A and HepG2 cells. Taken together, this study firstly identified miR-200a as a biomarker of fructose-induced hepatic inflammation, and revealed the hepatoprotection of curcumin and allopurinol via up-regulating miR-200a-mediated TXNIP/NLRP3 inflammasome pathway.

RNA interference against stromal interacting molecule-1 (STIM1) ameliorates ethanol-induced hepatotoxicity

Previously we have demonstrated that stromal interacting molecule-1 (STIM1) was involved in ethanol induced liver injury. However, the exact pathogenic mechanism of STIM1 in alcoholic liver disease (ALD) is still unknown. We constructed plasmid vectors encoding short-hairpin RNA against STIM1 to investigate its role in ALD in the rat liver cell line BRL and in Sprague-Dawley rats. The results showed that STIM1 targeted sh-RNA (Sh-STIM1) significantly ameliorated ethanol-induced BRL cells injury and liver injury in rats with 20 weeks-induced alcoholic liver disease. Inhibition of STIM1 also reduced intracellular calcium ion concentration, reactive oxygen species (ROS) production, lipid peroxidation, NF-kappa B activation and TNF-α production under ethanol exposure. STIM1 may play an important role in the pathogenesis of alcoholic liver disease. Silencing STIM1 may be effective in preventing alcoholic liver disease.