The aim of this study was to investigate the role of selenoprotein M (SelM) in endoplasmic reticulum stress and apoptosis in nickel-exposed mouse hearts and to explore the detoxifying effects of melatonin. At 21 d after intraperitoneal injection of nickel chloride (NiCl₂) and/or melatonin into male wild-type (WT) and SelM knockout (KO) C57BL/6J mice, NiCl₂ was found to induce changes in the microstructure and ultrastructure of the hearts of both WT and SelM KO mice, which were caused by oxidative stress, endoplasmic reticulum stress, and apoptosis, as evidenced by decreases in malondialdehyde (MDA) content and total antioxidant capacity (T-AOC) activity. Changes in the messenger RNA (mRNA) and protein expression of genes related to endoplasmic reticulum stress (activating transcription factor 4 (ATF4), inositol-requiring protein 1 (IRE1), c-Jun N-terminal kinase (JNK), and C/EBP homologous protein (CHOP)) and apoptosis (B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), Caspase-3, Caspase-9, and Caspase-12) were also observed. Notably, the observed damage was worse in SelM KO mice. Furthermore, melatonin alleviated the heart injury caused by NiCl₂ in WT mice but could not exert a good protective effect in the heart of SelM KO mice. Overall, the findings suggested that the antioxidant capacity of SelM, as well as its modulation of endoplasmic reticulum stress and apoptosis, plays important roles in nickel-induced heart injury.
Animals ; Male ; Mice ; Antioxidants/pharmacology* ; Apoptosis ; Endoplasmic Reticulum Stress ; Melatonin/pharmacology* ; Mice, Inbred C57BL ; Nickel/adverse effects* ; Selenoproteins/genetics* ; Heart/drug effects*

BACKGROUND: Pilea umbrosa (Urticaceae) is used by local communities (district Abbotabad) for liver disorders, as anticancer, in rheumatism and in skin disorders. METHODS: Methanol extract of P. umbrosa (PUM) was investigated for the presence of polyphenolic constituents by HPLC-DAD analysis. PUM (150 mg/kg and 300 mg/kg) was administered on alternate days for eight weeks in rats exposed with carbon tetrachloride (CCl). Serum analysis was performed for liver function tests while in liver tissues level of antioxidant enzymes and biochemical markers were also studied. In addition, semi quantitative estimation of antioxidant genes, endoplasmic reticulum (ER) induced stress markers, pro-inflammatory cytokines and fibrosis related genes were carried out on liver tissues by RT-PCR analysis. Liver tissues were also studied for histopathological injuries. RESULTS: Level of antioxidant enzymes such as catalase (CAT), superoxide dismutase (SOD), peroxidase (POD) and glutathione (GSH) decreased (p < 0.05) whereas level of thiobarbituric acid reactive substance (TBARS), HO and nitrite increased in liver tissues of CCl treated rat. Likewise increase in the level of serum markers; alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) and total bilirubin was observed. Moreover, CCl caused many fold increase in expression of ER stress markers; glucose regulated protein (GRP-78), x-box binding protein1-total (XBP-1 t), x-box binding protein1-unspliced (XBP-1 u) and x-box binding protein1-spliced (XBP-1 s). The level of inflammatory mediators such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) was aggregated whereas suppressed the level of antioxidant enzymes; γ-glutamylcysteine ligase (GCLC), protein disulfide isomerase (PDI) and nuclear erythroid 2 p45-related factor 2 (Nrf-2). Additionally, level of fibrosis markers; transforming growth factor-β (TGF-β), Smad-3 and collagen type 1 (Col1-α) increased with CCl induced liver toxicity. Histopathological scrutiny depicted damaged liver cells, neutrophils infiltration and dilated sinusoids in CCl intoxicated rats. PUM was enriched with rutin, catechin, caffeic acid and apigenin as evidenced by HPLC analysis. Simultaneous administration of PUM and CCl in rats retrieved the normal expression of these markers and prevented hepatic injuries. CONCLUSION Collectively these results suggest that PUM constituted of strong antioxidant chemicals and could be a potential therapeutic agent for stress related liver disorders.
Animals ; Carbon Tetrachloride ; adverse effects ; Chemical and Drug Induced Liver Injury ; drug therapy ; etiology ; pathology ; Endoplasmic Reticulum Stress ; drug effects ; Fibrosis ; drug therapy ; genetics ; Inflammation ; drug therapy ; genetics ; Liver ; drug effects ; enzymology ; metabolism ; Male ; Protective Agents ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Urticaceae ; chemistry

OBJECTIVE: This study was conducted to investigate the regulation of endoplasmic reticulum stress on Nrf2 signaling pathway in the kidneys of rats. METHODS: Rats were divided into twelve groups of six animals each. Some groups were pre-administered with bacitracin or tauroursodeoxycholic acid (TUDCA), and all of them were treated with 5-20 μmol/kg cadmium (Cd) for 48 h. The oxidative stress levels were analyzed using kits. The mRNA and protein expression levels of endoplasmic reticulum stress-related factors and Nrf2 signaling pathway-related factors were determined using RT-PCR and western blot. RESULTS: Cd exposure resulted in oxidative stress in the kidneys of rats and upregulated the expression of endoplasmic reticulum stress (ERS)-related factors and Nrf2 signaling pathway-related factors, especially at doses of 10 and 20 μmol/kg Cd, and the expression changes were particularly obvious. Moreover, after pretreatment with bacitracin, Cd upregulated the expression of ERS-related factors to a certain extent and, at higher doses, increased the mRNA expression of Nrf2. After pretreatment with TUDCA, Cd reduced the level of ERS to a certain extent; however, at these doses, there were no significant changes in the expression of Nrf2. CONCLUSION Cadmium can result in ERS and oxidative stress in the kidneys of rats, activate Nrf2, and upregulate the transcriptional expression of phase II detoxification enzymes under these experimental conditions. ERS has a positive regulation effect on Nrf2 signaling pathway but has little effect on the negative regulation of Nrf2 signaling pathway in cadmium toxicity.
Animals ; Cadmium ; toxicity ; Endoplasmic Reticulum Stress ; drug effects ; Environmental Pollutants ; toxicity ; Female ; Kidney ; drug effects ; metabolism ; Male ; NF-E2-Related Factor 2 ; genetics ; metabolism ; Oxidative Stress ; drug effects ; Rats, Sprague-Dawley ; Signal Transduction ; drug effects ; Taurochenodeoxycholic Acid ; pharmacology

As the chemotherapeutic resistance of extranodal NK/T-cell lymphoma (ENKTL) rises year by year, searching for novel chemoprevention compounds has become imminent. Gambogic acid (GA) has recently been shown to have anti-tumor effects, but its role and underling mechanism in ENKTL are rather elusive. In the present study, we showed that GA inhibited the cell growth and potently induced the apoptosis of ENKTL cells in vitro in a time- and concentration-dependent manner. Furthermore, GA induced cell death through endoplasmic reticulum stress (ERS) mediated suppression of Akt signaling pathways and finally the release of the caspase-3 proteases. Overall, our data provided evidences supporting GA as a potential therapeutic agent for ENKTL, which may facilitate further preclinical development of anti-tumor drugs.
Apoptosis ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Endoplasmic Reticulum Stress ; drug effects ; Humans ; Lymphoma, Extranodal NK-T-Cell ; drug therapy ; genetics ; metabolism ; physiopathology ; Proto-Oncogene Proteins c-akt ; genetics ; metabolism ; Signal Transduction ; drug effects ; Xanthones ; pharmacology

PURPOSE: To investigate the production of long pentraxin 3 (PTX3) in response to tunicamycin-induced endoplasmic reticulum (ER) stress and its role in ER stress-associated cell death, PTX3 expression was evaluated in the human retinal pigment epithelial cell line, ARPE-19. METHODS: PTX3 production in ARPE-19 cells was analyzed in the absence or presence of tunicamycin treatment by enzyme-linked immunosorbent assay. PTX3 protein and mRNA levels were estimated using western blot analysis and real-time reverse transcription-polymerase chain reaction, respectively. Protein and mRNA levels of CCAAT-enhancer-binding protein homologous protein (CHOP) and ARPE-19 cell viability were measured in the presence of tunicamycin-induced ER stress in control or PTX3 small hairpin RNA (shRNA)-transfected ARPE-19 cells. RESULTS: The protein and mRNA levels of PTX3 were found to be significantly increased by tunicamycin treatment. PTX3 production was significantly decreased in inositol-requiring enzyme 1α shRNA-transfected ARPE-19 cells compared to control shRNA-transfected cells. Furthermore, pretreatment with the NF-κB inhibitor abolished tunicamycin-induced PTX3 production. Decreased cell viability and prolonged protein and mRNA expression of CHOP were observed under tunicamycin-induced ER stress in PTX3 shRNA transfected ARPE-19 cells. CONCLUSIONS: These results suggest that PTX3 production increased in the presence of tunicamycin-induced ER stress. Therefore, PTX3 could be an important protector of ER stress-induced cell death in human retinal pigment epithelial cells. Inositol-requiring enzyme 1α and the NF-κB signaling pathway may serve as potential targets for regulation of PTX3 expression in the retina. Therefore, their role in PTX3 expression needs to be further investigated.
Anti-Bacterial Agents/pharmacology ; Apoptosis ; Blotting, Western ; C-Reactive Protein/biosynthesis/*genetics ; Cells, Cultured ; Endoplasmic Reticulum Stress/*drug effects/genetics ; Enzyme-Linked Immunosorbent Assay ; *Gene Expression Regulation ; Humans ; Polymerase Chain Reaction ; RNA, Messenger/*genetics ; Retinal Pigment Epithelium/*metabolism/pathology ; Serum Amyloid P-Component/biosynthesis/*genetics ; Tunicamycin/*pharmacology

Arisaema heterophyllum Blume is one of the three medicinal plants known as traditional Chinese medicine Rhizoma Arisaematis (RA). RA has been popularly used to treat patients with convulsions, inflammation, and cancer for a long time. However, the underlying mechanisms for RA effects are still unclear. The present study was designed to determine the cytotoxicity of agglutinin isolated from Arisema heterophyllum Blume (AHA) and explore the possible mechanisms in human non-small-cell lung cancer A549 cells. AHA with purity up to 95% was isolated and purified from Arisaema heterophyllum Blume using hydrophobic interaction chromatography. AHA dose-dependently inhibited the proliferation of A549 cells and induced G phase cell cycle arrest. AHA induced apoptosis by up-regulating pro-apoptotic Bax, decreasing anti-apoptotic Bcl-2, and activating caspase-9 and caspase-3. In A549 cells treated with AHA, the PI3K/Akt pathway was inhibited. Furthermore, AHA induced increase in the levels of ER stress markers such as phosphorylated eukaryotic initiation factor 2α (p-eIF2α), C/EBP-homologous protein (CHOP), inositol-requiring enzyme 1α (IRE1α), and phosphorylated c-Jun NH-terminal kinase (p-JNK). AHA also induced autophagy in A549 cells. Staining of acidic vesicular organelles (AVOs) and increase in the levels of LC3II and ATG7 were observed in AHA-treated cells. These findings suggested that AHA might be one of the active components with anti-cancer effects in Arisaema heterophyllum Blume. In conclusion, cytotoxicity of AHA on cancer cells might be related to its effects on apoptosis and autophagy through inhibition of PI3K/Akt pathway and induction of ER stress.
A549 Cells ; Agglutinins ; pharmacology ; Apoptosis ; drug effects ; Arisaema ; chemistry ; Autophagy ; drug effects ; Carcinoma, Non-Small-Cell Lung ; drug therapy ; enzymology ; metabolism ; physiopathology ; Cell Line, Tumor ; Drugs, Chinese Herbal ; pharmacology ; Endoplasmic Reticulum Stress ; drug effects ; Humans ; MAP Kinase Signaling System ; drug effects ; Phosphatidylinositol 3-Kinases ; genetics ; metabolism ; Phosphorylation ; drug effects ; Proto-Oncogene Proteins c-akt ; genetics ; metabolism

BACKGROUNDPrevious studies have indicated that endoplasmic reticulum stress participates in and mediates liver injury and apoptosis in brain-dead (BD) rats. In this study, we observed the effect of salubrinal (Sal, Sigma, USA) on liver cells in BD rats and explored its relevant mechanisms.

METHODSThirty Sprague-Dawley rats were equally randomized into three groups: BD group, Sal group, and DMSO group. The BD models were established by increasing intracranial pressure in a modified, slow, and intermittent way. In the drug groups, Sal was administered 1 h before the induction of BD. After modeling was completed, the blood and liver samples were harvested. CHOP and Caspase-12 mRNA expression was detected using quantitative polymerase chain reaction. PKR-like ER kinase (PERK), P-eukaryotic translation initiation factor 2α (eIF2α), eIF2α, CHOP and caspase-12 expression was detected using western blotting (WB). CHOP and caspase-12 distribution and expression in liver tissues were determined using immunohistochemistry (IHC). Alanine aminotransferase and aspartate aminotransferase level were detected using an automatic biochemical analyzer. Hepatic cell apoptosis was detected using TUNEL. The results were analyzed using Quantity-one v4.62 software (Bio-Rad, USA).

RESULTSCHOP and caspase-12 expression and PERK, eIF2α, and P-eIF2α protein expression showed no significant difference between BD group and DMSO group. Compared with BD group, Sal group had a significantly higher P-eIF2C level and a lower P-PERK level 2 h and 6 h after BD (P < 0.05). However, eIF2α expression showed no significant difference (P > 0.05). After the Sal treatment, CHOP and caspase-12 mRNA expression significantly decreased 4 h after BD (P < 0.05). WB and IHC indicated that CHOP and caspase-12 expression also significantly decreased after Sal treatment. Sal was associated with improved liver function and decreased hepatic cell apoptosis.

CONCLUSIONSSal can significantly reduce apoptosis in hepatic cells of BD rats. This protective effect may be achieved via the PERK-eIF2α signaling pathway.

Animals ; Apoptosis ; drug effects ; Blotting, Western ; Brain Death ; metabolism ; Caspase 12 ; genetics ; metabolism ; Cinnamates ; Disease Models, Animal ; Endoplasmic Reticulum Stress ; drug effects ; Eukaryotic Initiation Factor-2 ; genetics ; metabolism ; Immunohistochemistry ; Liver ; drug effects ; injuries ; Male ; Rats ; Rats, Sprague-Dawley ; Real-Time Polymerase Chain Reaction ; Thiourea ; analogs & derivatives ; Transcription Factor CHOP ; genetics ; metabolism

Serum palmitic acid (PA), a type of saturated fatty acid, causes lipid accumulation and induces toxicity in hepatocytes. Ethanol (EtOH) is metabolized by the liver and induces hepatic injury and inflammation. Herein, we analyzed the effects of EtOH on PA-induced lipotoxicity in the liver. Our results indicated that EtOH aggravated PA-induced apoptosis and lipid accumulation in primary rat hepatocytes in dose-dependent manner. EtOH intensified PA-caused endoplasmic reticulum (ER) stress response in vitro and in vivo, and the expressions of CHOP, ATF4, and XBP-1 in nucleus were significantly increased. EtOH also increased PA-caused cleaved caspase-3 in cytoplasm. In wild type and CHOP(-/-) mice treated with EtOH and high fat diet (HFD), EtOH worsened the HFD-induced liver injury and dyslipidemia, while CHOP knockout blocked toxic effects of EtOH and PA. Our study suggested that targeting UPR-signaling pathways is a promising, novel approach to reducing EtOH and saturated fatty acid-induced metabolic complications.
Activating Transcription Factor 4 ; drug effects ; metabolism ; Animals ; Apoptosis ; drug effects ; Caspase 3 ; drug effects ; Chemical and Drug Induced Liver Injury ; metabolism ; DNA-Binding Proteins ; drug effects ; metabolism ; Diet, High-Fat ; adverse effects ; Dose-Response Relationship, Drug ; Dyslipidemias ; chemically induced ; metabolism ; Endoplasmic Reticulum Stress ; drug effects ; Ethanol ; metabolism ; toxicity ; Fatty Liver ; chemically induced ; metabolism ; Gene Knockout Techniques ; Hepatocytes ; drug effects ; metabolism ; Lipid Metabolism ; drug effects ; Liver ; metabolism ; Male ; Mice ; Palmitic Acid ; toxicity ; Rats ; Rats, Sprague-Dawley ; Regulatory Factor X Transcription Factors ; Signal Transduction ; drug effects ; Transcription Factor CHOP ; drug effects ; genetics ; metabolism ; Transcription Factors ; drug effects ; metabolism ; Unfolded Protein Response ; drug effects ; X-Box Binding Protein 1

5-Hydroxymethylfurfural (5-HMF), a water-soluble compound extracted from wine-processed Fructus corni, is a novel hepatic protectant for treating acute liver injury. The present study was designed to investigate the protective effect of 5-HMF in human L02 hepatocytes injured by D-galactosamine (GalN) and tumor necrosis factor-α (TNF-α) in vitro and to explore the underlying mechanisms of action. Our results showed that 5-HMF caused significant increase in the viability of L02 cells injured by GalN/TNF-α, in accordance with a dose-dependent decrease in apoptotic cell death confirmed by morphological and flow cytometric analyses. Based on immunofluorescence and Western blot assays, we found that GalN/TNF-α induced ER stress in the cells, as indicated by the disturbance of intracellular Ca(2+) concentration, the activation of protein kinase RNA (PKR)-like ER kinase (PERK), phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α), and expression of ATF4 and CHOP proteins, which was reversed by 5-HMF pre-treatment in a dose-dependent manner. The anti-apoptotic effect of 5-HMF was further evidenced by balancing the expression of Bcl-2 family members. In addition, the knockdown of PERK suppressed the expression of phospho-PERK, phospho-eIF2α, ATF4, and CHOP, resulting in a significant decrease in cell apoptosis after the treatment with GalN/TNF-α. 5-HMF could enhance the effects of PERK knockdown, protecting the cells against the GalN/TNF-α insult. In conclusion, these findings demonstrate that 5-HMF can effectively protect GalN/TNF-α-injured L02 hepatocytes against ER stress-induced apoptosis through the regulation of the PERK-eIF2α signaling pathway, suggesting that it is a possible candidate for liver disease therapy.
Apoptosis ; drug effects ; Cornus ; chemistry ; Endoplasmic Reticulum Stress ; drug effects ; Eukaryotic Initiation Factor-2 ; genetics ; metabolism ; Furaldehyde ; analogs & derivatives ; pharmacology ; Galactosamine ; metabolism ; Hepatocytes ; cytology ; drug effects ; metabolism ; Humans ; Liver ; cytology ; drug effects ; metabolism ; Plant Extracts ; pharmacology ; Protective Agents ; pharmacology ; Signal Transduction ; drug effects ; Tumor Necrosis Factor-alpha ; genetics ; metabolism ; eIF-2 Kinase ; genetics ; metabolism

OBJECTIVETo explore the possible mechanism of curcumin in inducing the apoptosis of breast cancer cell MDA-MB-231.

METHODCurcumin of different concentrations at 0, 10 25, 50, 100, 150, 200 micromol x L(-1) were used to intervene breast cancer cells MDA-MB-231 for 24 hours. MTT was used to observe its effect on the proliferation of breast cancer cells. The flow cytometry was used to detect its effect on the cell apoptosis. The real-time quantitative PCR and Western blot was used to assess the expression levels of GRP78 and CHOP in breast cancer cells.

RESULTCurcumin could inhibit the proliferative ability of breast cancer cells by inducing them in a concentration-dependent manner. Curcumin could significantly increase the expression levels of GRP78 and CHOP in breast cancer cells.

CONCLUSIONCurcumin could induce the apoptosis of breast cancer cells MDA-MB-231 by activating endoplasmic reticulum stress.

Apoptosis ; drug effects ; Breast Neoplasms ; drug therapy ; genetics ; metabolism ; physiopathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Curcumin ; pharmacology ; Drugs, Chinese Herbal ; pharmacology ; Endoplasmic Reticulum Stress ; drug effects ; Female ; Heat-Shock Proteins ; genetics ; metabolism ; Humans ; Transcription Factor CHOP ; genetics ; metabolism