Changes In Endoplasmic Reticulum Stress Research Articles

Seasonal changes in endoplasmic reticulum stress and steroidogenesis in the ovary of the wild ground squirrels (Citellus dauricus Brandt)

The development of the follicle is accompanied by steroidogenesis and secretion, the endoplasmic reticulum (ER) requires significant synthesis of relevant proteins to support changes in the follicular microenvironment. The aim of this study was to investigate whether seasonal changes in gonadotropins and ovarian steroid hormones in the wild ground squirrels induce endoplasmic reticulum stress (ERS) and changes in ERS-mediated unfolded protein response (UPR) signaling. There were significant seasonal differences in ovarian mass, with values higher in the breeding season and relatively low in the non-breeding season. Histological observations revealed that ovaries in the breeding season had germ cells including primordial follicles, primary follicles, secondary follicles, tertiary follicles, and the corpus luteal, whereas ovaries consisted mainly of primary and secondary follicles in the non-breeding season. Analysis of ovarian transcriptome data showed that 1298 genes were up-regulated in expression and 1432 genes were down-regulated in expression during both periods. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated that these genes were mainly enriched in estrogen signaling pathways, ovarian steroidogenesis and endoplasmic reticulum protein processing pathways. The expression levels of steroidogenic enzymes (P450scc, P450c17, 3β-HSD, and P450arom) and gonadotropin receptor (FSHR and LHR) were significantly increased during the breeding season compared to the non-breeding season. GRP78 and UPR signaling factors (ATF4, ATF6, XBP1s) associated with ERS were expressed in both seasons. The mRNA expressions of Atf6 and Xbp1s were higher in the breeding season than those of the non-breeding season. Conversely, Atf4 and its downstream homologous protein (Chop) exhibited higher expression during the non-breeding season. In addition, follicle stimulating hormone (FSH), luteinizing hormone (LH), estradiol-17β, and progesterone of serum were significantly higher in the breeding season than those of the non-breeding season. These results suggested that UPR signaling, associated with seasonal changes in ovarian steroidogenesis, was activated during the breeding season and that ERS might be involved in regulating seasonal changes in ovarian steroidogenesis in the wild ground squirrels.

Umbelliferone Ameliorates Hepatic Steatosis and Lipid-Induced ER Stress in High-Fat Diet-Induced Obese Mice.

Among the characteristics of non-alcoholic fatty liver disease (NAFLD), hepatic steatosis is due to excessive fat accumulation and causes liver damage and lipotoxicity, which are associated with insulin resistance, endoplasmic reticulum (ER) stress, and apoptosis. Umbelliferone (UMB) has various powerful pharmacological properties, such as antioxidant, anti-hyperglycemic, anti-viral, and anti-inflammatory effects. However, the mechanism of action in hepatic steatosis and lipid-induced ER stress is still unclear. Thus, the efficacy of UMB in hepatic steatosis and palmitate (PA)-induced hepatocellular lipotoxicity was evaluated in the present study. Male C57BL/6J mice (n=40) were divided into four groups: regular diet (RD), UMB-supplemented RD, high-fat diet (HFD), and UMB-supplemented HFD. All mice were fed orally for 12 weeks. In addition, the effects of UMB on lipotoxicity were investigated in AML12 cells treated with PA (250 µM) for 24 h; Western blot analysis was used to evaluate the changes in ER stress and apoptotic-associated proteins. Administration with UMB in HFD-fed mice reduced lipid accumulation and hepatic triglyceride (TG) as well as serum insulin and glucose levels. In AML12 cells, UMB treatment reduced lipid accumulation as indicated by decreases in the levels of lipogenesis markers, such as SREBP1, FAS, PPAR-γ, and ADRP. Furthermore, UMB reduced both oxidative stress and ER stress-related cellular apoptosis. UMB supplementation ameliorated hepatic steatosis and improved insulin resistance by inhibiting lipid accumulation and regulating ER stress. These findings strongly suggest that UMB may be a potential therapeutic compound against NAFLD.

Parathyroid hormone-induced vascular smooth muscle cells calcification by endoplasmic reticulum stress.

Vascular smooth muscle cells (VSMCs) play a key role in vascular calcification, which is associated with enhanced mortality in chronic kidney disease. To ascertain the concentration of parathyroid hormone (PTH) that induces apoptosis of VSMCs and to explore whether the mechanism of endoplasmic reticulum (ER) stress is involved in PTH-induced calcification of VSMCs. The appropriate concentration and intervention time of PTH-inducing apoptosis of VSMCs were screened by flow cytometry. To investigate the effects of PTH on ER stress-related and apoptotic proteins in VSMCs, they were divided into four groups. These were the control group, the PTH group, the siC/EBP homologous protein (CHOP) + PTH group (in which siCHOP was used to knockdown the expression of CHOP at first), and the sp600125 + PTH group (in which the cells were pretreated with sp600125 at a concentration of 10 ng/ml for 24 h at first). Then, all groups except the control group were given 1x10-6 mol/L PTH to stimulate VSMCs. The changes in ER stress and apoptosis-related proteins in each group were detected, and the cell calcification was tested by Alizarin Red staining. Flow cytometry showed that the concentration of 1x10-6 mol/L PTH induced apoptosis most significantly. The apoptosis rate of the cells increased with the extension of stimulation time. The apoptosis of VSMCs pretreated with the Jun-N-terminal kinase (JNK) antagonist sp600125 was significantly reduced. The expression of cleaved caspase-3, PERK, IRE1, and CHOP was detected by Western blot analysis when cells were stimulated with 10-6 mol/L PTH for 14 days. After the knockdown of the CHOP expression by siCHOP, cleaved caspase-3 expression was significantly reduced. Alizarin Red staining showed siCHOP and sp600125 inhibits the VSMCs' calcification induced by PTH. In conclusion: the ER stress mechanism is involved in VSMCs' calcification induced by PTH.

Endoplasmic reticulum stress promoted acinar cell necroptosis in acute pancreatitis through cathepsinB-mediated AP-1 activation.

Acinar cell death and inflammatory response are two important events which determine the severity of acute pancreatitis (AP). Endoplasmic reticulum (ER) stress and necroptosis are involved in this process, but the relationships between them remain unknown. Here, we analyzed the interaction between ER stress and necroptosis and the underlying mechanisms during AP. Experimental pancreatitis was induced in Balb/C mice by caerulein (Cae) and lipopolysaccharide (LPS) or L-arginine (L-Arg) in vivo, and pancreatic acinar cells were also used to follow cellular mechanisms during cholecystokinin (CCK) stimulation in vitro. AP severity was assessed by serum amylase, lipase levels and histological examination. Changes in ER stress, trypsinogen activation and necroptosis levels were analyzed by western blotting, enzyme-linked immunosorbent assay (ELISA), adenosine triphosphate (ATP) analysis or lactate dehydrogenase (LDH) assay. The protein kinase C (PKC)α -mitogen-activated protein kinase (MAPK) -cJun pathway and cathepsin B (CTSB) activation were evaluated by western blotting. Activating protein 1 (AP-1) binding activity was detected by electrophoretic mobility shift assay (EMSA). We found that ER stress is initiated before necroptosis in CCK-stimulated acinar cells in vitro. Inhibition of ER stress by 4-phenylbutyrate (4-PBA) can significantly alleviate AP severity both in two AP models in vivo. 4-PBA markedly inhibited ER stress and necroptosis of pancreatic acinar cells both in vitro and in vivo. Mechanistically, we found that 4-PBA significantly reduced CTSB maturation and PKCα-JNK-cJun pathway -mediated AP-1 activation during AP. Besides, CTSB inhibitor CA074Me markedly blocked PKCα-JNK-cJun pathway -mediated AP-1 activation and necroptosis in AP. However, pharmacologic inhibition of trypsin activity with benzamidine hydrochloride had no effect on PKCα-JNK-cJun pathway and necroptosis in CCK-stimulated pancreatic acinar cells. Furthermore, SR11302, the inhibitor of AP-1, significantly lowered tumor necrosis factor (TNF) α levels, and its subsequent receptor interacting protein kinases (RIP)3 and phosphorylated mixed lineagekinase domain-like (pMLKL) levels, ATP depletion and LDH release rate in CCK-stimulated pancreatic acinar cells. To sum up, all the results indicated that during AP, ER stress promoted pancreatic acinar cell necroptosis through CTSB maturation, thus induced AP-1 activation and TNFα secretion via PKCα-JNK-cJun pathway, not related with trypsin activity. These findings provided potential therapeutic target and treatment strategies for AP or other cell death-related diseases.

4-Hydroxyisoleucine inhibits tumor growth by triggering endoplasmic reticulum stress and autophagy

4-Hydroxyisoleucine（4-HIL）is a non-protein amino acid that is able to reduce obesity and improve insulin sensitivity in mice, and recently emerged as a drug candidate against hypoglycemia. For the first time, we found that 4-HIL exhibits a potent anti-tumor activity in various cancer cell lines in vitro and in vivo. Most importantly, 4-HIL has no cytotoxic effect on normal or non-malignant cells. Proteomic data analysis revealed changes in endoplasmic reticulum stress（ERS）related protein and autophagy related protein. Western blot revealed that molecular components of the ERS pathway were activated, including phosphorylation of perk and EIF2a increased, while levels of GRP78 reduced, the cellular process of ERS potentially contributed to the activation of autophagy, Transmission electron microscopy revealed the formation of autophagic vesicles under 4-HIL treatment, and LC3B was increased. Meanwhile, activation of ERS inhibits intracellular protein synthesis rate, our results suggest that 4-HIL exhibits anti-tumor activity in various cancer cell lines by increasing ERS and triggering autophagy responses without causing damage to normal cells.

Endoplasmic Reticulum Stress Is Associated with the Mesencephalic Dopaminergic Neuron Injury in Stressed Rats.

An increasing number of people are in a state of stress due to social and psychological pressures, which may result in mental disorders. Previous studies indicated that mesencephalic dopaminergic neurons are associated with not only reward-related behaviors but also with stress-induced mental disorders. To explore the effect of stress on dopaminergic neuron and potential mechanism, we established stressed rat models of different time durations and observed pathological changes in dopaminergic neurons of the ventral tegmental area (VTA) through HE and thionine staining. Immunohistochemistry coupled with microscopy-based multicolor tissue cytometry (MMTC) was employed to investigate the number changes of dopaminergic neurons. Double immunofluorescence labelling was used to investigate expression changes of endoplasmic reticulum stress (ERS) protein GRP78 and CHOP in dopaminergic neurons. Our results showed that prolonged stress led to pathological alteration in dopaminergic neurons of VTA, such as missing of Nissl bodies and pyknosis in dopaminergic neurons. Immunohistochemistry with MMTC indicated that chronic stress exposure resulted in a significant decrease in dopaminergic neurons. Double immunofluorescence labelling showed that the endoplasmic reticulum stress protein took part in the injury of dopaminergic neurons. Taken together, these results indicated the involvement of ERS in mesencephalic dopaminergic neuron injury induced by stress exposure.

Pulmonary Hypertension in Obese Mice Is Accompanied by a Reduction in PPAR-γ Expression in Pulmonary Artery.

Obesity and insulin resistance (IR) are well-studied risk factors for systemic cardiovascular disease, but their impact on pulmonary hypertension (PH) is not well clarified. This study aims to investigate if diet-induced obesity induces PH and if peroxisome-proliferator-activated receptor (PPAR-γ) and/or endoplasmic reticulum (ER) stress are involved in this process. Mice were maintained on a high-fat diet (HFD) for 4 months, and IR and PH were confirmed. In a separate group, after 4 months of HFD, mice were treated with pioglitazone (PIO) or 4-phenylbutyric acid for the last month. The results demonstrated that HFD for at least 4 months is able to increase pulmonary artery pressure, which is maintained, and this animal model can be used to investigate the link between IR and PH, without changes in ER stress in the pulmonary artery. There was also a reduction in circulating adiponectin and in perivascular adiponectin expression in the pulmonary artery, associated with a reduction in PPAR-γ expression. Treatment with PIO improved IR and PH and reversed the lower expression of adiponectin and PPAR-γ in the pulmonary artery, highlighting this drug as potential benefit for this poorly recognized complication of obesity.

P–424 Evaluating the relationship between endoplasmic reticulum stress and recurrent pregnancy loss in a young population cohort

Abstract Study question Does endoplasmic reticulum (ER) stress evaluated by X-box binding protein 1 (XBP–1) among patients aged 18–30 years play a role in recurrent pregnancy loss (RPL)? Summary answer High levels of XBP–1 observed in patients with known RPL indicate that ER stress plays an important role in RPL. What is known already Female reproductive tract undergoes dynamic changes during oogenesis which require protein synthesis, folding, maturation, which take place in the ER. ER is also responsible for destruction of unfolded/misfolded proteins. Excess accumulation of these faulty proteins leads to ER stress, which actives unfolded protein response (UPR). XBP–1 is a transcription factor involved in UPR and regulates ER stress-mediated apoptosis. Regulation of ER homeostasis is important in folliculogenesis, oocyte maturation and embryogenesis. It is also known that ER stress has a positive correlation with age and it is associated with age-related diseases. Study design, size, duration This prospective case-controlled study was conducted at University of Health Sciences Turkey, Istanbul Kanuni Sultan Suleyman Training and Research Hospital Department of Obstetrics and Gynecology between March 2020 – September 2020. A total of 70 subjects were included in the study. All patients gave their written informed consent before their enrollment in the study. Participants/materials, setting, methods 38 patients aged 18–30 years with a history of RPL were included in the study. Patients who had miscarriages due to fetal abnormalities, patients with infections, endocrine or genetic disorders, smokers, alcohol and/or drug abusers, with acute/chronic inflammatory diseases, patients using steroids, anti-inflammatory and antioxidant medications were excluded from the study. Age-matched 32 healthy subjects without RPL were included in the control group. XBP–1 levels were determined using Human XBP–1 ELISA Kit (Elabscience Co., USA). Main results and the role of chance The mean age in the control group and in the study group were 25.21±3.3 and 25.26±2.6, respectively and they were statistically similar (p = 0.324). When groups were compared according to thyroid stimulating hormone (TSH) levels and body mass index (BMI), which are additional risk factors of RPL both groups were statistically similar (p = 0.642 and 0.942, respectively). As expected gravidity and abortus numbers were significantly higher in the study group (p < 0.001). A mean XBP–1 level of 1233.41± 3902.97 was determined in the control group. The mean value of the study group was calculated to be 2251.49± 9621.12. Mean XBP–1 level in the study group was significantly high (p < 0.001). A receiver operating curve (ROC) analysis was conducted in the study group. The area under the curve was found to be 87% (95% CI: 79% - 95%). The specificity was 75%, sensitivity was 89%, positive LR was 3.5, negative LR was 0.15, positive predictive value was 80% and negative predictive value was 87% for the cut-off XBP–1 level at 1364.68 pg/mL. Limitations, reasons for caution Small sample size is an important limitation of this study. In addition, evaluating XBP–1 only in serum samples does not let us drive any conclusions on the local changes of ER stress. Studies with larger samples sizes and studying XBP–1 levels in tissue samples of endometrial material is needed. Wider implications of the findings: The significantly high levels of XBP–1 in RPL patients younger than 30 years, indicate higher ER stress in this group even when age dependent increase in ER stress is calculated out of the equation. XBP–1 can be a promising marker in evaluating patients with a fertility wish for RPL risk. Trial registration number NCT04455256

Hydrogen-Rich Medium Regulates Cr(VI)-Induced ER Stress and Autophagy Signaling in DF-1 Cells.

Related studies have shown that chromium (Cr) is toxic to cells, and hydrogen can protect cells by regulating endoplasmic reticulum (ER) stress and autophagy. However, there are few reports on the protective effects of hydrogen on heavy metal-induced cell damage. The objective of this study was to investigate the protection of hydrogen-rich medium (HRM) on Cr(VI)-induced ER stress and autophagy in DF-1 cells. Therefore, HRM were pretreated for 30min before Cr(VI) treatment, and detected the autophagy and ER stress-related indicators to determine the role of HRM. The results showed that HRM could reduce the cell damage caused by Cr(VI), and 3-methyladenine (3-MA) could protect cells by inhibiting over autophagy. HRM can reverse the changes of ER stress- and autophagy-related indexes caused by Cr(VI), and inhibit the excessive autophagy caused by Cr(VI). In conclusion, HRM can protect cells from damage induced by Cr(VI), and play a role by inhibiting ER stress-mediated autophagy.

Developmental programming: Metabolic tissue-specific changes in endoplasmic reticulum stress, mitochondrial oxidative and telomere length status induced by prenatal testosterone excess in the female sheep

Prenatal testosterone (T) excess-induced metabolic dysfunctions involve tissue specific changes in insulin sensitivity with insulin resistant, oxidative and lipotoxic state in liver/muscle and insulin sensitive but inflammatory and oxidative state in visceral adipose tissues (VAT). We hypothesized that mitochondrial dysfunction, endoplasmic reticulum (ER) stress and premature cellular senescence are contributors to the tissue-specific changes in insulin sensitivity. Markers of mitochondrial number, function, and oxidative phosphorylation (OxPhos), ER stress and cellular senescence (telomere length) were assessed in liver, muscle and 4 adipose (VAT, subcutaneous [SAT], epicardiac [ECAT] and perirenal [PRAT]) depots collected from control and prenatal T-treated female sheep at 21 months of age. Prenatal T treatment led to: (a) reduction in mitochondrial number and OxPhos complexes and increase in ER stress markers in muscle; (b) increase in fibrosis with trend towards increase in short telomere fragments in liver (c) depot-specific mitochondrial changes with OxPhos complexes namely increase in SAT and reduction in PRAT and increase in mitochondrial number in ECAT; (d) depot-specific ER stress marker changes with increase in VAT, reduction in SAT, contrasting changes in ECAT and no changes in PRAT; and (d) reduced shorter telomere fragments in SAT, ECAT and PRAT. These changes indicate insulin resistance may be driven by mitochondrial and ER dysfunction in muscle, fibrosis and premature senescence in liver, and depot-specific changes in mitochondrial function and ER stress without involving cellular senescence in adipose tissue. These findings provide mechanistic insights into pathophysiology of metabolic dysfunction among female offspring from hyperandrogenic pregnancies.

Inhibition of CACNA1H can alleviate endoplasmic reticulum stress and reduce myocardial cell apoptosis caused by myocardial infarction.

In recent years, coronary heart disease (CHD) has become a disease that cannot be ignored by residents of our country, because CHD will not only endanger people's quality of life, but also threaten their lives. Therefore, this research mainly explores the correlation between myocardial infarction (MI) with endoplasmic reticulum (ER) stress and apoptosis. First, we constructed a model of myocardial ischemia and hypoxia (I/H) in vivo and in vitro, and examined the change of CACNA1H expression. At the same time, in order to research the role of CACNA1H, we chose CACNA1H-specific inhibitor ABT-639 to next research and detect changes in heart injury by detecting changes in creatine kinase (CK) content and lactate dehydrogenase (LDH) activity. Next, we used TUNEL staining and immunofluorescence staining to detect changes in apoptosis and ER stress, and analyzed changes in ER stress and apoptotic pathway expression by Western blotting and quantitative Real-Time Polymerase Chain Reaction (qRT-PCR). At 28 days after MI, the cardiac function of the mice was significantly reduced, the myocardial cell apoptosis rate was dramatically increased, and CACNA1H expression was dramatically increased in vivo and in vitro. In addition, we treated the model group with the ABT-639, and found that ABT-639 can partially protect myocardial function and relieve myocardial cell apoptosis. At the same time, ABT-639 may reduce H9c2 injury after I/H by reducing the degree of ER stress, because we found that the use of ABT-639 can dramatically reduce ER stress-related factors expression, and can inhibit the expression of apoptosis-related factors Caspase-3 and Caspase-9. The CACNA1H inhibitor ABT-639 can alleviate myocardial cell apoptosis caused by MI by reducing the ER stress response.

Maternal hypercortisolemia alters placental metabolism: a multiomics view.

Previous studies have suggested that increases in maternal cortisol or maternal stress in late pregnancy increase the risk of stillbirth at term. In an ovine model with increased maternal cortisol over the last 0.20 of gestation, we have previously found evidence of disruption of fetal serum and cardiac metabolomics and altered expression of genes related to mitochondrial function and metabolism in biceps femoris, diaphragm, and cardiac muscle. The present studies were designed to test for effects of chronically increased maternal cortisol on gene expression and metabolomics in placentomes near term. We hypothesized that changes in placenta might underlie or contribute to the alterations in fetal serum metabolomics and thereby contribute to changes in striated muscle metabolism. Placentomes were collected from pregnancies in early labor (143 ± 1 days gestation) of control ewes (n = 7) or ewes treated with cortisol (1 mg·kg-1·day-1 iv; n = 5) starting at day 115 of gestation. Transcriptomics and metabolomics were performed using an ovine gene expression microarray (Agilent 019921) and HR-MAS NMR, respectively. Multiomic analysis indicates that amino acid metabolism, particularly of branched-chain amino acids and glutamate, occur in placenta; changes in amino acid metabolism, degradation, or biosynthesis in placenta were consistent with changes in valine, isoleucine, leucine, and glycine in fetal serum. The analysis also indicates changes in glycerophospholipid metabolism and suggests changes in endoplasmic reticulum stress and antioxidant status in the placenta. These findings suggest that changes in placental function occurring with excess maternal cortisol in late gestation may contribute to metabolic dysfunction at birth.

Oxymatrine attenuates arsenic-induced endoplasmic reticulum stress and calcium dyshomeostasis in hepatic stellate cells.

BackgroundOxymatrine is the main bioactive component of Sophora flavescens. It exhibits various biological activities and has been used in various liver diseases, including hepatic fibrosis (HF). Hepatic stellate cells (HSCs) are the primary cell type involved during HF progression. Oxymatrine treatment could suppress the proliferation of HSCs and degrade the extracellular cell matrix (ECM), presumed to be associated with HF. However, the mechanism is still unknown.MethodsNaAsO2 induces HF in LX2 cells. Oxymatrine was used to treat NaAsO2- induced LX2 cells. Then, the LX2 cell proliferation, apoptosis, ECM secretion protein, oxidative stress index, and intracellular calcium concentration were respectively measured. Furthermore, after knocking down GRP78 [endoplasmic reticulum (ER) chaperone BiP] or overexpressing of SERCA2 (ATPase sarcoplasmic/ER Ca2+ transporting 2) in NaAsO2-induced LX2 cells, we detected the changes in ER stress and calcium homeostasis in LX2 cells.ResultsNaAsO2 exposure promoted apoptosis, increased ECM secretion, produced ER stress, and disrupted calcium homeostasis, which could be attenuated by oxymatrine treatment. Furthermore, knockdown of GRP78 to alleviate ER stress, or overexpression of SERCA2 to restore intracellular calcium homeostasis can inhibit the NaAsO2 effect.ConclusionsOxymatrine treatment could improve calcium homeostasis and attenuate ER stress to reverse NaAsO2-induced HSC activation and ECM secretion, which are the significant phenotypes of HF. The ER stress and calcium homeostasis may be the therapeutic targets for HF.

P0015SIRT2 REGULATES CISPLATIN-INDUCED ENDOPLASMIC RETICULUM STRESS THROUGH HEAT SHOCK FACTOR 1 DEACETYLATION

Abstract Background and Aims Nephrotoxicity is an important cisplatin-induced adverse reaction and restricts the use of cisplatin to treat malignant tumors. Endoplasmic reticulum (ER) stress is caused by the accumulation of misfolded proteins, and is induced by cisplatin in kidneys. SIRT2 nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase is a member of the sirtuin family, but its role in cisplatin-induced ER stress remains unclear. Method To investigate the effect of SIRT2 on cisplatin-induced ER stress using SIRT2 knockout mice and human proximal tubular epithelial cells (HK-2 cells). We treated cisplatin (20 µg/mL) or induced by intraperitoneal injection of cisplatin (20 mg/kg) and evaluated the changes of ER stress and its signal mechanism. Results Cisplatin administration was found to significantly increase the expressions of PRKR-like ER kinase (PERK), phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), and the C/EBP homologous protein (CHOP) and caspase-12 in the kidneys of SIRT2-wild type mice. However, cisplatin-induced increases in the expressions of p-PERK, p-eIF2α, CHOP and, caspase-12 were diminished in kidneys of SIRT2 knockout mice. In vitro, cisplatin significantly increased the expressions of p-PERK, p-eIF2α, CHOP, and caspase-12 in HK-2 cells. When the effect of SIRT2 on cisplatin-induced ER stress was evaluated using SIRT2-siRNA (ON-TARGET plus human SIRT2 siRNA) or the SIRT2 inhibitors, AGK2 and AK1, knockdown or inhibition of SIRT2 significantly attenuated the cisplatin-induced protein expression of p-PERK, p-eIF2α, CHOP, and caspase-12. Immunoprecipitation studies showed SIRT2 bound physically to heat shock factor (HSF)1 and that HSF1 acetylation was significantly increased by cisplatin. In addition, knockdown of SIRT2 increased cisplatin-induced HSF1 acetylation and increased the expression of heat shock protein (HSP)70. Conclusion These observations suggest that suppression of SIRT2 ameliorates cisplatin-induced ER stress by increasing HSF1 acetylation and HSP expression.

Changes in endoplasmic reticulum stress in spinal cord of rats with diabetic neuropathic pain

Objective To evaluate the changes in the endoplasmic reticulum stress in the spinal cord of rats with diabetic neuropathic pain (DNP). Methods Clean-grade healthy male Sprague-Dawley rats, aged 8 weeks, weighing 120-160 g, were fed a high-fat and high-glucose diet for 8 weeks, then diabetes mellitus was induced by intraperitoneal streptozotocin 35 mg/kg and confirmed by blood glucose level >16.7 mmol/L 3 days later.The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured at day 14 after injection.The establishment of DNP model was considered successful when MWT and TWL were lower than 85% of the baseline value.Fifteen rats in which the DNP model was successfully established served as DNP group, 15 rats in which the DNP model was not successfully established served as non-NDNP group (NDNP), and another 15 normal rats were selected and served as control group (group C). The MWT and TWL were measured at 3, 7 and 14 days after successful establishment of the model.The rats were then sacrificed, and the lumbar enlargement segments (L4-6) of the spinal cord were harvested to detect the expression of inositol-requiring enzyme-1α, phosphorylated JNK (p-JNK) and Beclin1 by Western blot. Results Compared with C and NDNP groups, the MWT was significantly decreased and the TWL was shortened at 3, 7 and 14 days after successful establishment of the model, and the expression of inositol-requiring enzyme-1α, p-JNK and Beclin1 was up-regulated in DNP group (P<0.05). Conclusion The mechanism of development and maintenance of DNP may be related to aggravated endoplasmic reticulum stress in the spinal cord and to autophagy induced by up-regulated expression of p-JNK in rats. Key words: Diabetic neuropathies; Endoplasmic reticulum stress; Spinal cord

In vitro culture expansion impairs chondrogenic differentiation and the therapeutic effect of mesenchymal stem cells by regulating the unfolded protein response

In vitro expansion of mesenchymal stem cells (MSCs) has been implicated in loss of multipotency, leading to impaired chondrogenic potential and an eventual therapeutic effect, as reported in our previous study. However, the precise regulatory mechanism is still unclear. Here, we demonstrate that endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) were involved in transformation of MSCs induced by in vitro culture based on the comparative profiling of in vitro cultured bone marrow MSCs at passage 3 (P3 BMSCs) vs. fresh P0 BMSCs by microarray analysis. Indeed, RT-PCR and Western blot analysis showed significantly lower expression levels of three key UPR-related molecules, ATF4, ATF6 and XBP1, in P3 BMSCs than P0 BMSCs. Further, we found that UPR suppression by 4-phenylbutyrate (4-PBA) reduced the chondrogenic potential of P0 BMSCs and further cartilage regeneration. Conversely, UPR induction by tunicamycin (TM) enhanced the chondrogenic differentiation of P3 BMSCs and the therapeutic effect on cartilage repair. Thus, the decline in the chondrogenic potential of stem cells after in vitro culture and expansion may be due to changes in ER stress and the UPR pathway.

Effects of insulin-like growth factor-1 on endoplasmic reticulum stress and autophagy in rat gastric smooth muscle cells cultured at different glucose concentrations in vitro.

The purpose of the study was to observe changes in endoplasmic reticulum stress (ERS)- and autophagy-related proteins in gastric smooth muscle tissues of diabetic rats with gastroparesis, investigate the effect of insulin-like growth factor 1 (IGF-1) on ERS and autophagy in rat gastric smooth muscle cells cultured under different glucose concentrations, and explore the influence of IGF-1 on development of diabetic gastroparesis (DGP). After establishing a rat model of DGP, rats were divided into normal control (NC) and 6-week diabetic model (DM6W) groups. Expression of ERS-related and autophagy-related proteins was detected by western blot analysis and immunofluorescence assay in rat gastric smooth muscle tissue and in vitro-cultured rat gastric smooth muscle cells exposed to different glucose concentrations and treatment with IGF-1 for 24 or 48h. Changes in glucose-regulated-protein-78 (GRP78), growth arrest and DNA damage-inducible gene 153 (CHOP), and microtubule-associated protein 1A/1B light chain 3B (LC3) expression levels were detected by western blot analysis, and GRP78 and LC3 expression were examined by confocal laser-scanning microscopy. In vivo expression levels of GRP78, CHOP, and LC3 were significantly higher in the DM6W group compared with the NC group (p < 0.001). Twenty-four hours after cells were cultured at different glucose concentrations in vitro, expression of GRP78, CHOP, and LC3II/Iwas significantly higher in the high glucose-treated group compared with the normal glucose group (p < 0.05). After IGF-1 intervention, CHOP and GRP78 expression were significantly higher in the normal glucose + IGF-1 group compared with the normal glucose group (p < 0.01), while no significant difference was found between high glucose and high glucose + IGF-1 groups. LC3II/Iexpression was significantly lower in the normal glucose + IGF-1 group compared with the normal glucose group, and was significantly lower in the high glucose and high glucose + IGF-1 groups (p < 0.05). After 48h of culture, CHOP expression was significantly higher and LC3II/I expression was significantly lower in the high glucose group compared with the normal glucose group (p < 0.05), but no significant change in GRP78 expression was observed between these two groups. After IGF-1 intervention, there was no difference in CHOP or GRP78 expression between normal glucose + IGF-1 and normal glucose groups. However, CHOP and GRP78 expression were significantly lower in the high glucose + IGF-1 group compared with the high glucose group (p < 0.05). There was no significant difference in LC3II/I expression between normal glucose + IGF-1 and normal glucose groups, or high glucose + IGF-1 and high glucose groups. Results of confocal laser-scanning microscopy showed significantly lower expression of LC3II/Iin the high glucose + IGF-1 group compared with the high glucose group (p < 0.05). ERS and autophagy were involved in the occurrence of DGP. IGF-1 exerted an inhibitory effect on ERS in rat gastric smooth muscle cells cultured under high glucose conditions, and this inhibitory effect increased with time. IGF-1 inhibited the level of autophagy in rat gastric smooth muscle cells cultured under high glucose conditions at early stages, which may be achieved through inhibition of ERS.

Hepatic topographical changes of endoplasmic reticulum stress and unfolded protein response signaling after hemorrhagic shock and reperfusion

BackgroundEndoplasmic reticulum (ER) stress plays a crucial role in cell death decisions in context of various diseases. Although it is known that ER stress occurs in livers subjected to hemorrhagic shock and reperfusion (HS/R), there is no understanding about the influence of the liver architecture on ER stress and the activation of the unfolded protein response (UPR). Materials and methodsMice were subjected to a pressure-controlled HS (30 ± 5 mmHg) for 90 min. Mice were sacrificed 2, 4, 6, 8, 10, 14, 18, and 24 h after shock induction. Plasma levels of inflammatory cytokines (IL-6, CXCL1, CXCL9, CXCL10, CCL2, CCL3) and transaminases were measured. Hematoxylin and eosin stains of paraffin-embedded liver tissue sections were evaluated for liver damage. Immunohistochemistry was used to analyze the hepatic topography of ER stress marker binding immunoglobulin protein and the activation of the three major pathways of the UPR. ResultsCompared with sham-operated mice, HS/R led to profound liver damage and an elevation of inflammatory cytokines. We found time-dependent topographical changes of ER stress in the livers. Furthermore, the three major pathways of the UPR represented by protein kinase RNA-like ER kinase, activating transcription factor 6, and inositol-requiring enzyme 1 were activated in differing ways dependent on the zonation within the liver acinus. ConclusionsThese findings show that the liver architecture must be taken into account when investigating the role of ER stress and the UPR in ischemia-reperfusion injury after HS/R.

Sequential changes of endoplasmic reticulum stress and apoptosis in myocardial fibrosis of diabetes mellitus-induced rats.

The endoplasmic reticulum (ER) is an organelle in which proteins form their appropriate structures. However, several of these proteins become unfolded or misfolded when exposed to stimuli, including hyperglycemia, oxidative stress, ischemia, disturbance of calcium homeostasis and overexpression of abnormal proteins, which activates ER stress and the unfolded protein response (UPR). To date, investigations have demonstrated that ER stress is important in diabetic myocardial fibrosis by inducing cardiac cell apoptosis. Therefore, in the present study, the polymerase chain reaction, western blotting analysis and tissue staining were performed to identify the changes in UPR signaling proteins and apoptotic proteins in diabetic rats at different time points, and to determine whether the myocardial fibrosis is associated with ER-stress-mediated apoptosis using a diabetes mellitus (DM) rat model. It was found that the upregulation of ER stress markers and apoptotic molecules developed over time. It was also demonstrated that anti-apoptotic markers and proapoptotic markers were activated early following model establishment, and then decreased in months 4 and 5. The changes in myocardial fibrosis were found to accelerate in a time-dependent manner with apoptosis in the DM rats.

India ink induces apoptosis in the yellow clam Mesodesma mactroides (Deshayes, 1854). Optical and ultrastructural study.

This paper reports on the acute inflammatory and cellular process in the yellow clam, Mesodesma mactroides, induced by injection of India ink into the muscular foot. Histological observations with optical and electronic microscopy were made at 24 and 48 h after injection. The induced cellular inflammatory response consisted of a general hemocyte infiltration without necrosis and apoptotic activity. Migration of ink-laden phagocytes across the intestinal epithelium was recorded. It appeared that the yellow clam "excreted" ink particles through the gill and kidney. The positive staining for apoptosis was observed in the digestive gland. Electronic microscopy revealed ultrastructural changes of endoplasmic reticulum stress and apoptotic bodies in the digestive gland. The mechanism by which the India ink particles induce apoptosis remains unknown, but might possibly be associated with the endoplasmic reticulum stress. This work has highlighted features that require further discussion in the restricted field the inflammatory responses of mollusks.