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

Sec61β, a subunit of the Sec61 translocon complex, is not essential in yeast and commonly used as a marker of endoplasmic reticulum (ER). In higher eukaryotes, such as Drosophila, deletion of Sec61β causes lethality, but its physiological role is unclear. Here, we show that Sec61β interacts directly with microtubules. Overexpression of Sec61β containing small epitope tags, but not a RFP tag, induces dramatic bundling of the ER and microtubule. A basic region in the cytosolic domain of Sec61β is critical for microtubule association. Depletion of Sec61β induces ER stress in both mammalian cells and Caenorhabditis elegans, and subsequent restoration of ER homeostasis correlates with the microtubule binding ability of Sec61β. Loss of Sec61β causes increased mobility of translocon complexes and reduced level of membrane-bound ribosomes. These results suggest that Sec61β may stabilize protein translocation by linking translocon complex to microtubule and provide insight into the physiological function of ER-microtubule interaction.
Animals ; COS Cells ; Caenorhabditis elegans Proteins ; genetics ; metabolism ; Cell Line, Tumor ; Cercopithecus aethiops ; Endoplasmic Reticulum ; metabolism ; Homeostasis ; Humans ; Microtubules ; metabolism ; SEC Translocation Channels ; deficiency ; genetics ; metabolism

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

OBJECTIVEPERK/eIF2α/CHOP is a major signaling pathway mediating endoplasmic reticulum (ER) stress related with atherosclerosis. Oxidized LDL (ox-LDL) also induces endothelial apoptosis and plays a vital role in the initiation and progression of atherosclerosis. The present study was conducted to explore the regulatory effect of ox-LDL on PERK/eIF2α/CHOP signaling pathway in vascular endothelial cells.

METHODSThe effects of ox-LDL on PERK and p-eIF2α protein expression of primary human umbilical vein endothelial cells (HUVECs) were investigated by Western blot analysis. PERK gene silencing and selective eIF2α phosphatase inhibitor, salubrinal were used to inhibit the process of ox-LDL induced endothelial cell apoptosis, caspase-3 activity, and CHOP mRNA level.

RESULTSOx-LDL treatment significantly increased the expression of PERK, PERK-mediated inactivation of eIF2α phosphorylation, and the expression of CHOP, as well as the caspase-3 activity and apoptosis. The effects of ox-LDL were markedly decreased by knocking down PERK with stable transduction of lentiviral shRNA or by selective eIF2α phosphatase inhibitor, salubrinal.

CONCLUSIONThis study provides the first evidence that ox-LDL induces apoptosis in vascular endothelial cells mediated largely via the PERK/eIF2α/CHOP ER-stress pathway. It adds new insights into the molecular mechanisms underlying the pathogenesis and progression of atherosclerosis.

Apoptosis ; Endoplasmic Reticulum Stress ; Eukaryotic Initiation Factor-2 ; genetics ; metabolism ; Human Umbilical Vein Endothelial Cells ; metabolism ; Humans ; Lipoproteins, LDL ; genetics ; metabolism ; Signal Transduction ; Transcription Factor CHOP ; genetics ; metabolism ; eIF-2 Kinase ; genetics ; metabolism

Early brain injury (EBI) plays a key role in the pathogenesis of subarachnoid hemorrhage (SAH). This study investigated the role of glucose-regulated protein 78 (GRP78) in EBI after SAH. Male Sprague-Dawley rats (n=108) weighing 260±40 g were divided into control, sham-operated, and operated groups. Blood was injected into the prechiasmatic cistern of rats in the operated group. Neurological scores, ultrastructures of neurons, apoptosis, and GRP78 expression in the hippocampus were examined using Garcia scoring system, transmission electron microscopy, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling, and Western blotting at 1, 6, 12, 24, 48, and 72 h after SAH, respectively. The results showed that neurological scores were significantly decreased in the operated group as compared with those in control and sham-operated groups at 12, 24, 48, and 72 h. Metachromatin, chromatin pyknosis at the edge, endoplasmic reticulum swelling, and invagination of nuclear membrane were observed at 24 h in the operated group, indicating the early morphological changes of apoptosis. The number of apoptotic cells was significantly increased in the operated group as compared with that in control and sham-operated groups at 6, 12, 24, 48, and 72 h. The GRP78 protein expression levels in the operated group were significantly elevated at all time points and reached the peak at 12 h. GRP78 expression was positively associated with apoptosis cells and negatively with neurological scores. In conclusion, EBI was demonstrated to occur after SAH and GRP78 was involved in the development of EBI after SAH.
Animals ; Apoptosis ; Brain Injuries ; complications ; metabolism ; pathology ; Chromatin ; pathology ; Endoplasmic Reticulum Stress ; Heat-Shock Proteins ; genetics ; metabolism ; Male ; Rats ; Rats, Sprague-Dawley ; Subarachnoid Hemorrhage ; etiology ; metabolism ; pathology

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

Endoplasmic Reticulum ; genetics ; metabolism ; Plasmodium berghei ; genetics ; metabolism ; Plasmodium falciparum ; genetics ; metabolism ; Protozoan Proteins ; genetics ; metabolism

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

BACKGROUNDCalreticulin (CRT) is major Ca 2+ -binding chaperone mainly resident in the endoplasmic reticulum (ER) lumen. Recently, it has been shown that non-ER CRT regulates a wide array of cellular responses. We previously found that CRT was up-regulated during hypoxia/reoxygenation (H/R) and this study was aimed to investigate whether CRT nuclear translocation aggravates ER stress (ERS)-associated apoptosis during H/R injury in neonatal rat cardiomyocytes.

METHODSApoptosis rate and lactate dehydrogenase (LDH) leakage in culture medium were measured as indices of cell injury. Immunofluorescence staining showed the morphological changes of ER and intracellular translocation of CRT. Western blotting or reverse transcription polymerase chain reaction was used to detect the expression of target molecules.

RESULTSCompared with control, H/R increased apoptosis rate and LDH activity. The ER became condensed and bubbled, and CRT translocated to the nucleus. Western blotting showed up-regulation of CRT, Nrf2, activating transcription factor 4 (ATF4), CHOP and caspase-12 expression after H/R. Exogenous CRT overexpression induced by plasmid transfection before H/R increased cell apoptosis, LDH leakage, ER disorder, CRT nuclear translocation and the expression of ERS-associated molecules. However, administration of the ERS inhibitor, taurine, or CRT siRNA alleviated cell injury, ER disorder, and inhibited ERS-associated apoptosis.

CONCLUSIONSOur results indicated that during H/R stress, CRT translocation increases cell apoptosis and LDH leakage, aggravates ER disorder, up-regulates expression of nuclear transcription factors, Nrf2 and ATF4, and activates ERS-associated apoptosis.

Animals ; Apoptosis ; genetics ; physiology ; Calreticulin ; genetics ; metabolism ; Cell Hypoxia ; genetics ; physiology ; Cell Survival ; genetics ; physiology ; Cells, Cultured ; Endoplasmic Reticulum Stress ; physiology ; Myocytes, Cardiac ; cytology ; metabolism ; RNA Interference ; Rats