Zearalenone induces apoptosis in bovine mammary epithelial cells by activating endoplasmic reticulum stress

Zearalenone (ZEA) is a common mycotoxin produced by fungi within the genus Fusarium. However, few studies have examined the direct effects of the toxin on the mammary glands. In the present study, the effects of ZEA treatment on bovine mammary epithelial cells (MAC-T) from dairy cows were investigated. The cells were treated with different concentrations of ZEA to evaluate the effect of the toxin on cell viability, intracellular reactive oxygen species (ROS) concentrations, mitochondrial membrane potential, endoplasmic reticulum (ER) stress, and the expression of apoptosis-related genes. The results indicated that different concentrations (5, 10, 15, 20, 25, 30, 50, 60, or 100 μM) of ZEA were able to inhibit growth of MAC-T cells. After exposing the MAC-T cells to 30 μM ZEA, compared with the control group, ROS levels increased, mitochondrial membrane potential decreased, and mRNA expression of the ER-specific stress-related genes GRP78, HSP70, ATF6, EIF2A, ASK1, and CHOP was upregulated in the ZEA-treated group. Further, we analyzed the increase in apoptotic rate by flow cytometry. At the mRNA level, compared with the control group, the expression of the apoptosis-promoting gene BAX was increased in the ZEA-treated group, the expression of the inhibitory gene BCL2 decreased, and the expression of the gene CASP3 increased. We observed a significant increase in caspase-3 activity in ZEA-treated MAC-T cells. Furthermore, the apoptotic rate of the cells in the ZEA group treated with 4-phenylbutyric acid (ER stress inhibitor) decreased and the mRNA expression levels of ER stress markers GRP78 and CHOP decreased. Compared with the ZEA treatment group, the mRNA expression level of the apoptosis-related gene BAX was decreased and the expression level of BCL2 was increased in the ZEA + 4-phenylbutyric acid cotreatment group. These findings indicate that ZEA-induced ER stress increases apoptosis in MAC-T cells. The treatment of MAC-T cells with ZEA reduced cell viability, increased ROS content, decreased mitochondrial membrane potential, increased ER stress marker expression, and induced apoptosis.     

Long-chain fatty acid effects on peroxisome proliferator-activated receptor-α-regulated genes in Madin-Darby bovine kidney cells: Optimization of culture conditions using palmitate

Studying long-chain fatty acid (LCFA) effects on gene network expression in bovine cells could provide useful information for future practical applications. An optimized in vitro system that does not require tissue collection or cell isolation could fill a niche in the study of PPARα activity in ruminants. Specific aims were to optimize culture conditions in Madin-Darby bovine kidney (MDBK) cells to achieve maximal mRNA expression of known peroxisome proliferator-activated receptor-α (PPARα) target genes using palmitate (16:0) as a representative LCFA. Variables included length of incubation time, use of albumin-bound (4:1 molar proportion) 16:0 (A16:0), or addition of insulin. A first time-course experiment tested culturing cells in Dulbecco's modified Eagle's medium with 150 μM PPAR ligand Wy-14643 (WY) and A16:0. A second experiment tested the effects of albumin and insulin using 150 μM of 16:0 without albumin or insulin (−Alb/−Ins), 16:0 without albumin plus 5 mg/L of bovine insulin (−Alb/+Ins), A16:0 without insulin (+Alb/−Ins), or a control. A third experiment was a preliminary metabolic characterization of cells and assessed intracellular lipid droplet formation after treatment with 150 μM of 16:0 or an ethanol control. For all experiments, cells were harvested at 0, 6, 12, 18, and 24 h posttreatment. In experiments 1 and 2, mRNA expression was assessed by quantitative PCR of selected PPARα target genes as well as PPARα coactivators (ACOX1, CPT1A, ACADVL, ACSL1, PPARA, PPARGC1A, LPIN1). In experiment 1, there was a linear increase in mRNA expression of CPT1A (∼500%) and ACSL1 (50 to 200%) by 6 h of incubation with both WY and A16:0. The LPIN1 mRNA increased by >100% by 6 h only with A16:0. Further, there was a linear increase in expression of PPARA (∼100%) with A16:0 through 24 h of incubation. In experiment 2, insulin increased, and coupling LCFA with albumin tended to delay the response in expression of CPT1A and ACSL1 to 16:0. Data indicated a toxic effect of 150 μM free 16:0 as assessed by cell counts after 12 h of incubation. In experiment 3, MDBK cells appeared to use glucose and AA as energy sources and were able to secrete triglycerides. In addition, MDBK cells cultured with 150 μM of 16:0 had a substantial uptake of LCFA and synthesized intracellular lipid droplets. Overall, results indicated that a 6-h incubation with free LCFA and addition of insulin was suitable to detect marked effects on mRNA expression of PPARα target genes in MDBK cells.     

AMPK-endoplasmic reticulum stress axis contributes to lipopolysaccharide-caused mitochondrial dysfunction by regulating mitochondria-associated membrane function in bovine hepatocytes

Mitochondrial homeostasis is closely associated with cellular homeostasis process, whereas mitochondrial dysfunction contributes to apoptosis and mitophagy. Hence, analyzing the mechanism of lipopolysaccharide (LPS)-caused mitochondrial damage is necessary to understand how cellular homeostasis is maintained in bovine hepatocytes. Mitochondria-associated membranes (MAM), a connection between endoplasmic reticulum (ER) and mitochondria, is important to control mitochondrial function. To investigate the underlying mechanisms of the LPS-caused mitochondrial dysfunction, hepatocytes isolated from dairy cows at ∼160 d in milk (DIM) were pretreated with the specific inhibitors of adenosine 5′-monophosphate-activated protein kinase (AMPK), ER stress, RNA-activated protein kinase-like ER kinase (PERK), inositol-requiring enzyme 1α (IRE1α), c-Jun N-terminal kinase, and autophagy followed by a 12 I1/4g/mL LPS treatment. The results showed that inhibiting ER stress with 4-phenylbutyric acid decreased the levels of autophagy and mitochondrial damage with AMPK inactivation in LPS-treated hepatocytes. The AMPK inhibitor compound C pretreatment alleviated LPS-induced ER stress, autophagy and mitochondrial dysfunction by regulating the expression of MAM-related genes, such as mitofusin 2 (MFN2), PERK, and IRE1α. Moreover, inhibiting PERK and IRE1α mitigated autophagy and mitochondrial dynamic disruption by regulating the MAM function. Additionally, blocking c-Jun N-terminal kinase, the downstream sensor of IRE1α, could reduce the levels of autophagy and apoptosis and restore the balance of mitochondrial fusion and fission by modulating the B cell leukemia 2 (BCL-2)/BCL-2 interacting protein 1 (BECLIN1) complex in the LPS-treated bovine hepatocytes. Furthermore, autophagy blockage with chloroquine could intervene in LPS-caused apoptosis to restore mitochondrial function. Collectively, these findings suggest that the AMPK-ER stress axis is involved in the LPS-caused mitochondrial dysfunction by mediating the MAM activity in bovine hepatocytes.     

n-3多不饱和脂肪酸、内质网应激与非酒精性脂肪肝病的研究进展

非酒精性脂肪性肝病(nonalcoholic fatty liver disease, NAFLD)已逐渐成为一个全球性健康问题, 但其发病的具体机制不甚明朗。内质网是细胞内蛋白质加工、脂质合成和钙储存的主要场所, 内质网结构和功能的失常所致的内质网应激(endoplasmic reticulum stress, ERS)对脂代谢和细胞功能具有重要调控作用, 可能是NAFLD的发生和发展的重要机制。近来大量研究显示, 脂类中特定的脂质负荷(饱和脂肪酸、胆固醇)可能是诱导ERS导致NAFLD重要原因, 而另一方面, n-3多不饱和脂肪酸(n-3 polyunsaturated fatty acids, n-3 PUFA)的摄入量却与NAFLD患病率呈负相关。本文就脂肪酸与内质网应激及其与NAFLD的联系作一综述, 进一步探讨n-3多不饱和脂肪酸防治NAFLD的机制。     

Influence of passage number on glycosylation of alkyl lactosides by Madin-Darby canine kidney (MDCK) cells

The cell function on saccharide biosynthesis can be evaluated by employing the saccharide primer method. This study demonstrated that the characteristics of Madin-Darby canine kidney (MDCK) cells changed in relation with passage number when 12-azidododecyl β-lactoside (Lac-12N(3) primer) was incorporated into MDCK cells and afforded GM3-, GD3-, sialylparagloboside (SPG), and NeuAc-Gal-GlcNAc-Gal-GlcNAc-Lac-type oligosaccharides. By measuring the amount of glycosylated products from relatively early to late passage numbers, results showed that there was an appropriate passage number that optimized oligosaccharide production and that the higher passage number resulted to a decrease in oligosaccharide production. Moreover, results suggested that aside from sialyltransferase, the activity of several kinds of enzymes that control the amount of saccharide production was presumably affected depending upon the biological senescence.     

Structure of the yeast endoplasmic reticulum: localization of ER proteins using immunofluorescence and immunoelectron microscopy.

The endoplasmic reticulum (ER) and other secretory compartments of Saccharomyces cerevisiae have biochemical functions that closely parallel those described in higher eukaryotic cells, yet the morphology of the yeast organelles is quite distinct. In order to associate ER functions with the corresponding cellular structures, we localized several proteins, each of which is expected to be associated with the ER on the basis of enzymatic activity, biological function, or oligosaccharide content. These marker proteins were visualized by immunofluorescence or immunoelectron microscopy, allowing definition of the S. cerevisiae ER structure, both in intact cells and at the ultrastructural level. Each marker protein was most abundant within the membranes that envelop the nucleus and several were also found in extensions of the ER that frequently juxtapose the plasma membrane. Double-labeling experiments were entirely consistent with the idea that the marker proteins reside within the same compartment. This analysis has permitted, for the first time, a detailed characterization of the ER morphology as yeast cells proceed through their growth and division cycles.     

Bovine milk phospholipid fraction protects Neuro2a cells from endoplasmic reticulum stress via PKC activation and autophagy

Endoplasmic reticulum stress commonly causes neuronal damage in a lot of neurodegenerative diseases. In this study, we examined neuroprotective effect of bovine milk phospholipid fraction (mPL) on mouse neuroblastoma Neuro2a cells from endoplasmic reticulum (ER) stress induced cell death. Neuro2a cells were induced cell death by ER stressor tunicamycin (TM) or thapsigargin (TG), and studied whether mPL could attenuate the toxicity. By preincubation with mPL, the cell viabilities were significantly increased in TM or TG treated cells, and caspase-12 activated cells induced by TM or TG treatment were significantly decreased. Protein kinase C inhibitor GF109203x significantly reduced the protective effect on TM induced cell death, and autophagy inhibitor 3-methyladenine reduced the protective effect on TM or TG induced cell death. Moreover, preincubation with mPL significantly stimulated autophagosomes formation observed by dansylcadaverine staining. Our data suggest that mPL will be applicable to prevent neurodegenerative diseases caused by ER stress.     

Kaempferol induced apoptosis via endoplasmic reticulum stress and mitochondria‐dependent pathway in human osteosarcoma U‐2 OS cells

Kaempferol is a natural flavonoid. Previous studies have reported that kaempferol has anti‐proliferation activities and induces apoptosis in many cancer cell lines. However, there are no reports on human osteosarcoma. In this study, we investigate the anti‐cancer effects and molecular mechanisms of kaempferol in human osteosarcoma cells. Our results demonstrate that kaempferol significantly reduces cell viabilities of U‐2 OS, HOB and 143B cells, especially U‐2 OS cells in a dose‐dependent manner, but exerts low cytotoxicity on human fetal osteoblast progenitor hFOB cells. Comet assay, DAPI staining and DNA gel electrophoresis confirm the effects of DNA damage and apoptosis in U‐2 OS cells. Flow cytometry detects the increase of cytoplasmic Ca²⁺ levels and the decrease of mitochondria membrane potential. Western blotting and fluorogenic enzymatic assay show that kaempferol treatment influences the time‐dependent expression of proteins involved in the endoplasmic reticulum stress pathway and mitochondrial signaling pathway. In addition, pretreating cells with caspase inhibitors, BAPTA or calpeptin before exposure to kaempferol increases cell viabilities. The anti‐cancer effects of kaempferol in vivo are evaluated in BALB/c^nu/nu mice inoculated with U‐2 OS cells, and the results indicate inhibition of tumor growth. In conclusion, kaempferol inhibits human osteosarcoma cells in vivo and in vitro.     

Effects of body condition,monensin, and essential oils on ruminal lipopolysaccharide concentration,inflammatory markers,and endoplasmatic reticulum stress of transition dairy cows

Evidence exists that dairy cows experience inflammatory-like phenomena in the transition period. Rumen health and alterations in metabolic processes and gene networks in the liver as the central metabolic organ might be key factors for cows’ health and productivity in early lactation. This study made use of an animal model to generate experimental groups with different manifestations of postpartal fat mobilization and ketogenesis. In total, 60 German Holstein cows were allocated 6 wk antepartum to 3 high-body condition score (BCS) groups (BCS 3.95) and 1 low-BCS group (LC; BCS 2.77). High-BCS cows were fed an antepartal forage-to-concentrate ratio of 40:60 on dry matter basis, in contrast to 80:20 in the LC group, and received a monensin controlled-release capsule (HC/MO), a blend of essential oils (HC/EO), or formed a control group (HC). We evaluated serum haptoglobin, kynurenine, tryptophan, ruminal lipopolysaccharide concentration and mRNA abundance of nuclear factor kappa B (NF-κB), nuclear factor E2-related factor 2 (Nrf2), and endoplasmatic reticulum stress-induced unfolded protein response (UPR) target genes in liver biopsy samples from d ?42 until +56 relative to calving. Nearly all parameters were highly dependent on time, with greatest variation near calving. The ruminal lipopolysaccharide concentration and evaluated target genes were not generally influenced by antepartal BCS and feeding management. The kynurenine-to-tryptophan ratio was higher in LC than in HC/MO treatment on d 7. Ruminal lipopolysaccharide concentration was higher in HC/MO than in the HC group, but not increased in HC/EO group. Abundance of UPR target gene X-box binding protein 1 was higher in HC/MO than in HC/EO group on d 7. Hepatic mRNA abundance of Nrf2 target gene glutathione peroxidase 3 was higher, whereas expression of NF-κB target gene haptoglobin tended to be higher in LC than in HC/EO cows. The HC/MO cows showed the most prominent increase in the abundance of glutathione peroxidase 3 and haptoglobin after calving in comparison to antepartal values. Results indicate the presence of inflammatory-like phenomena near calving. Simultaneously, alterations in UPR and Nrf2 target genes with antioxidative properties and haptoglobin occurred, being most prominent in LC and HC/MO group.     

Effect of frying oils on the postprandial endoplasmic reticulum stress in obese people

The addition of antioxidants to frying oil reduces postprandial oxidative stress and the inflammatory response. ER stress may trigger both inflammation and oxidative stress processes. We aimed to determine the biological effects of the intake of four models of frying oils on postprandial ER stress in peripheral blood mononuclear cells. Twenty obese people received four breakfasts following a randomized crossover design, consisting of muffins made with different oils (virgin olive oil (VOO), sunflower oil (SFO), and a mixture of seed oils (SFO/canola oil) with either dimethylpolysiloxane (SOD) or natural antioxidants from olives (SOP) added), which were previously subjected to 20 heating cycles. ER stress was assessed by measuring the mRNA levels of sXBP1, BiP, CRT, and CNX in peripheral blood mononuclear cells. Our study showed that the intake of the muffins made with SFO induced the postprandial increase of the mRNA levels of the ER stress‐sensor sXBP1, and the ER stress related chaperones BiP and CRT (all p‐values <0.05). The harmful effects associated with the use of SFO as frying oil, in terms of inflammatory response and postprandial oxidative stress, may be partially mediated by the induction of postprandial ER stress.     

Scp160p,a new yeast protein associated with the nuclear membrane and the endoplasmic reticulum,is necessary for maintenance of exact ploidy

We have cloned a new gene, SCP160, from Saccharomyces cerevisiae, the deduced amino acid sequence of which does not exhibit overall similarity to any known yeast protein. A weak resemblance between the C-terminal part of the Scp160 protein and regulatory subunits of cAMP-dependent protein kinases from eukaryotes as well as the pstB protein of Escherichia coli was observed. The SCP160 gene resides on the left arm of chromosome X and codes for a polypeptide of molecular weight around 160 kDa. By immunofluorescence microscopy the Scp160 protein appears to be localized to the nuclear envelope and to the endoplasmic reticulum (ER). However, no signal sequence or membrane-spanning region exists, suggesting that the Scp160 protein is attached to the cytoplasmic surface of the ER–nuclear envelope membranes. Disruption of the SCP160 gene is not lethal but results in cells of decreased viability, abnormal morphology and increased DNA content. This phenotype is not reversible by transformation with a plasmid carrying the wild-type gene. Crosses of SCP160 deletion mutant strains among each other or with unrelated strains lead to irregular segregation of genetic markers. Taken together the data suggest that the Scp160 protein is required during cell division for faithful partitioning of the ER–nuclear envelope membranes which in S. cerevisiae enclose the duplicated chromosomes.     

Dietary Monascus adlay supplements facilitate suppression of cigarette smoke-induced pulmonary endoplasmic reticulum stress,autophagy, apoptosis and emphysema-related PLGF in the rat

Cigarette smoke (CS) exposure may cause oxidative stress in the lung, leading to cell death and long-term injury. Monascus adlay (MA) with antioxidant components produced by inoculating adlay (Cois lachrymal-jobi L. var. ma-yuen Stapf) with Monascus purpureus may protect lung against CS-induced lung injuries in rats. MA and lovastatin had higher antioxidant activities than either M. purpureus or adlay. CS exposure caused significant lung damage, as evidenced by higher levels of reactive oxygen species (ROS), neutrophil infiltration, dityrosine and 4-HNE, as well as lower levels of Mn-superoxide dismutase and catalase expression. Lung tissues with CS exposure had higher levels of ER stress, apoptosis, autophagy and emphysema-related placenta growth factor (PlGF) expressions. All CS-induced injuries were significantly suppressed by MA supplements. MA would be a beneficial nutritional therapy to ameliorate CS-induced lung injury via preserving antioxidant defense mechanisms, decreasing oxidative stress and inhibiting ER stress, autophagy, apoptosis and emphysema-related risk factor.     

Roles of stress response-related signaling and its contribution to the toxicity of zearalenone in mammals

Zearalenone (ZEA) is a mycotoxin frequently found in cereal crops and cereal-derived foodstuffs worldwide. It affects plant productivity, and is also a serious hazard to humans and animals if being exposed to food/feed contaminated by ZEA. Studies over the last decade have shown that the toxicity of ZEA in animals is mainly mediated by the various stress responses, such as endoplasmic reticulum (ER) stress, oxidative stress, and others. Accumulating evidence shows that oxidative stress and ER stress signaling are actively implicated in and contributes to the pathophysiology of various diseases. Biochemically, the deleterious effects of ZEA are associated with apoptosis, DNA damage, and lipid peroxidation by regulating the expression of genes implicated in these biological processes. Despite these findings, the underlying mechanisms responsible for these alterations remain unclear. This review summarized the characteristics, metabolism, toxicity and the deleterious effects of ZEA exposure in various tissues of animals. Stress response signaling implicated in the toxicity as well as potential therapeutic options with the ability to reduce the deleterious effects of ZEA in animals were highlighted and discussed.