Expression Of Glucose-regulated Protein 78 Protein Research Articles

Effect of naringin on oxidative stress and endoplasmic reticulum stress in diabetic cardiomyopathy

To explore the protective effect of naringin(Nar) on the injury of myocardium tissues induced by streptozotocin(STZ) in diabetic rats and the relationship with oxidative stress and endoplasmic reticulum stress(ERS)， the male SD rats were intraperitoneally injected with streptozotocin(STZ， 60 mg·kg⁻¹) to establish the diabetic rat model and then randomly divided into the type 1 diabetic rat group(T1DR)， the low-dose Nar group(Nar25)， the middle-dose Nar group(Nar50) and the high-dose Nar group(Nar100). The normal rats were designed as control group(Con). Nar25， Nar50， Nar100 groups were orally administered with Nar at the doses of 25.0， 50.0， 100.0 mg·kg⁻¹ per day， respectively， while the normal group and the T1DR group were orally administered with saline. At the 8th week after treatment， fasting plasma glucose and heart mass index were measured. The pathological changes in myocardial tissues were observed by microscope. The cardiac malondialdehyde(MDA) level and superoxide dismutase(SOD) activities were measured. The gene and protein expressions of glucose-regulated protein 78(GRP78)， C/EBP homologous protein(CHOP)， cysteinyl aspartate-specific proteinase 12(caspase 12) were detected by qRT-PCR and Western blot. According to the results， compared with control group， the myocardial structure was damaged， the content of MDA was increased， while the activities of SOD were decreased(P<0.05) in T1DR group. GRP78， CHOP and caspase 12 mRNA and protein expressions were increased significantly in T1DR group(P<0.05， P<0.01). Compared with T1DR group， myocardial structure damage was alleviated in Nar treatment group. The content of MDA was decreased， while the activities of SOD were increased significantly. The mRNA and protein expressions of GRP78， CHOP and caspase 12 were increased， especially in middle and high-dose groups(P<0.05， P<0.01). After treatment with Nar for 8 weeks， myocardial structure damage was obviously alleviated in Nar treatment groups. The content of MDA was decreased， while the activities of SOD were increased significantly in myocardial tissues. The mRNA and protein expressions of GRP78， CHOP and caspase 12 were increased， especially in middle and high-dose groups(P<0.05， P<0.01). The findings suggest that Nar may protect myocardium in diabetic rats by reducing mitochondrial oxidative stress injuries and inhibiting the ERS-mediated cell apoptosis pathway.

Adaptive unfolded protein response promotes cell survival in rifampicin-treated L02 cells.

An important concept in drug-induced liver injury(DILI) is adaptation, which means the injury reverses with the continuation of the drug. The mechanism of adaption of drugs remains enigmatic, adaptive unfolded protein response (UPR) is possibly involved. We once observed adaptation phenomenon of rifampicin (RFP) in animal models, in this study, we investigate the effects of RFP on adaptive UPR in L02 cells, and after inhibiting UPR by using 4-phenylbutyrate(4-PBA), the change of cell viability and cell apoptosis in RFP-treated cells. We found that with the concentration of RFP increased and the treatment time was prolonged, the glucose-regulated protein78 (GRP78), a hallmark of the UPR, was upregulated, and was dose- and time-dependent. RFP also activates the p-eukaryotic initiation factor2α (eIF2α) protein expression. 4-PBA decreased GRP78 and p-eIF2α protein expression levels. Moreover, FCA showed that cell apoptosis rate obviously increased, and MTT assay showed that cell survival rate obviously decreased, this indicates that after inhibiting the UPR, the cell damage increased, which shows that the UPR is an adaptation mechanism to protect cells against injury induced by RFP. This also proves that when the degree of UPR induced by RFP is relatively mild, adaptive UPR is helpful for cell survival.

Effects of sevoflurane pretreatment on the apoptosis of rat H9c2 cardiomyocytes and the expression of GRP78.

The protective effect of sevoflurane on apoptosis of rat H9c2 cardiomyocytes induced by H2O2 and the effect on the expression of glucose-regulated protein 78 (GRP78) were investigated. H9c2 cells were routinely cultured and divided into the control, model and sevoflurane groups. Cells in the model group were treated with 400 µM H2O2, and cells in the sevoflurane group were pretreated with sevoflurane prior to treatment with 400 µM H2O2. MTT assay was used to assess cell viability. Annexin V-propidium iodide (AV-PI) double staining flow cytometry was used to detect apoptosis. The intracellular free Ca2+ concentration was measured by the fluorescence-based assay using Fluo-3 AM as a calcium ion fluorescence probe. The mRNA expression level of GRP78 and protein expression levels of GRP78, CHOP and caspase-12 were measured using reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot analysis, respectively. The assays showed that after sevoflurane pretreatment the H9c2 cell viability was significantly increased, whereas the H2O2-induced apoptosis, intracellular Ca2+ concentration, mRNA expression of GRP78, and the protein expression of GRP78, CHOP and caspase-12 were all reduced. The results show that pretreatment with sevoflurane inhibited H2O2-induced apoptosis in H9c2 cells. The mechanism may be related to inhibition of the stress-related protein GRP78 expression in endoplasmic reticulum, resulting in decreased intracellular Ca2+ concentration and the downregulation of CHOP and caspase-12 expression levels.

Grape seed proanthocyanidins protect cardiomyocytes against hypoxia/reoxygenation injury by attenuating endoplasmic reticulum stress through PERK/eIF2α pathway.

The aim of the present study was to observe the protective effect of grape seed proanthocyanidins (GSPs) against endoplasmic reticulum (ER) stress‑mediated apoptosis caused by hypoxia/reoxygenation (H/R) injury in H9C2 cardiomyocytes along with its potential mechanisms. H9C2 cardiomyocytes underwent hypoxia for 3h followed by reoxygenation for 3h. Different doses of GSPs (50, 100 and 200µg/ml) were administered 30min before hypoxia. Cell viability was assessed, as well as lactic dehydrogenase (LDH) activity, cell apoptosis rate, expression levels of glucose‑regulated protein 78 (GRP78), C/EBP‑homologous protein (CHOP), protein kinase RNA‑like ER kinase (PERK), and eukaryotic translation initiation factor‑2 (eIF2α) mRNA and protein. The results revealed that GSPs improved cell viability, reduced LDH activity and reduced the apoptosis rate in cells subjected to H/R, and that the protective effect was most significant when 100µg/ml in GSPs was administered. GSPs treatment also decreased mRNA and protein expression of GRP78, CHOP, eIF2α and the level of the phosphorylated form of PERK. Furthermore, GSPs displayed a similar protective effect to that of established ER stress inhibitor 4‑phenyl butyric acid. In conclusion, the findings of this study suggest that GSPs may protect H9C2 cardiomyocytes from H/R injury by decreasing ER stress‑mediated apoptosis through the suppression of the PERK/eIF2α signaling pathway.

Abstract 4506: UPR biomarker expression correlates with inhibition of migration of TNBC cells treated with low doses of chemotherapy

Abstract Much attention has been directed to the study of triple-negative breast cancer (TNBC) because of its short disease-free interval from diagnosis and more aggressive course in the metastatic setting. TNBC also lacks specific target receptors for treatment. Third-generation metronomic chemotherapy using available agents that target DNA repair complexes (i.e., taxanes), and cell proliferation (i.e., anthracycline) have shown promise results. However, specific regimens for TNBC at the different disease stages have not been clearly defined. Therefore, researchers are seeking molecular biomarkers to predict response to current chemotherapeutic agents. In a previous study of TNBC, we demonstrated that low doses of anthracyclines and taxanes, which are known to increase endoplasmic reticulum stress, increased tumor cell expression of glucose-regulated protein 78 (GRP78), a key regulator of the unfolding protein response (UPR), resulting in tumor cell apoptosis. GRP78 gene expression is upregulated via CREB2L1, an endoplasmic reticulum transmembrane transcription factor and member of the CREB/ATF family of transcription factors. CREB3L1 is a metastatic suppressor and functions as a transducer of UPR. The aim of the present study was to investigate the effect of low doses of doxorubicin and paclitaxel on UPR activation in metastatic TNBC cells by determining CREB3L1 protein expression in correlation to cell-surface GRP78 expression. Furthermore, we related CREB3L1 and cell-surface GRP78 expression with the migration potential of the cells in response to treatment. We found that metronomic doses of doxorubicin significantly induced CREB3L1 and cell surface GRP78 expression in TNBC cells. CREB3L1 increased by 2.5-fold in MDAMB231 and by 3-fold in MDAMB468 (p&amp;lt;0.04); GRP78 increased by 3.7-fold and 6.1-fold, respectively (p&amp;lt;0.01). Similar results were obtained with low doses of paclitaxel. A strong correlation was observed between CREB3L1 and cell-surface GRP78 protein expression. The increased expression of both proteins in MDAMB231 (highly metastatic) and MDAMB468 (moderated metastatic) was associated with significant inhibition of the migration capacity of the treated TNBC cells. In contrast, the migration capacity of treated estrogen-positive MCF7 cells (non-metastatic) and Her2- positive BT474 cells (highly metastatic) was poorly inhibited, coinciding with a non-significant increase in CREB3L1 and GRP78 expression. The results demonstrated that the drug effect via the UPR was specific for TNBC and unrelated to the metastatic profile of the cells. This study is the first stage in the identification of UPR-related biomarkers of beneficial outcome of metronomic chemotherapy for TNBC. Future mechanistic studies should focus on the possible use of cell surface GRP78 and CREB3L1 as targets for combination therapies. Citation Format: Annat Raiter, Julia Lipovetsky, Britta Hardy, Rinat Yerushalmi. UPR biomarker expression correlates with inhibition of migration of TNBC cells treated with low doses of chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4506. doi:10.1158/1538-7445.AM2017-4506

Effect of endoplasmic reticulum stress in trophocytes on the pathogenesis of intrahepatic cholestasis of pregnancy

Objective: To evaluate the effect of endoplasmic reticulum stress in trophocytes, in patients with intrahepatic cholestasis of pregnancy (ICP). Methods: Sixty-one pregnant women who were hospitalized in Women's Hospital, School of Medicine, Zhejiang University from January to December 2015 were recruited. Thirty-one women who were diagnosed as ICP were defined as the ICP group and 30 healthy pregnant women were defined as the control group. The localization and expression intensity of glucose regulated protein 78 (GRP-78) in placental tissues were detected by immunohistochemistry technique. Electronic microscope was used to observe ultra-microstructure change of the endoplasmic reticulum in trophocytes and cell line Swan71. Reverse transcription (RT)-PCR and western blot were used to investigate the expression of GRP-78 mRNA and protein in Swan 71 cell. Results: (1) GRP-78 protein was mainly expressed in the cytoplasm of cytotrophoblasts and syncytiotrophoblasts. The protein expression of GRP-78 in placentas of the ICP group (13.2±2.4) was significantly higher than that in the control group (7.8±1.3, P<0.01). (2) The volume of endoplasmie reticulum did not increase and the microvilli developed well, with no swelling and no expansion of endoplasmic reticulum in the control group.In the ICP group, microvilli injury, endoplasmic reticulum edema were found; the volume of endoplasmic reticulum increased, with dilation, vacuolation and significant degranulation. After treated with 100 μmol/L cholyglycine for 24 hours, universal dilatation of the endoplasmic reticulum were seen in the Swan71 cells. (3) In Swan71 cells, cholylglycine displayed a concentration-dependent up-regulation on the expression of GRP-78. The expressions of GRP-78 mRNA in 0, 25, 50, 100 μmol/L cholylglycine experimental group were 1.01±0.17, 2.17±0.16, 5.47±0.36, 5.65±0.82, respectively. The expression of GRP-78 protein in 0, 25, 50, 100 μmol/L cholylglycine experimental group were 1.01±0.04, 1.17±0.15, 1.33±0.13, 1.73±0.13, respectively. The expression of GRP-78 mRNA and protein in 100 and 50 μmol/L cholylglycine experimental group were significantly higher than 0 μmol/L (all P<0.01). Conclusion: The obvious expansion of endoplasmic reticulum and the increased expression of GRP-78 in trophocytes indicated that endoplasmic reticulum stress of trophocytes may be involved in the pathogenesis of ICP.

Therapeutic efficacy and mechanism of action of ginsenoside Rg1 in treating acute hepatic failure in mice

Objective: To examine the regulatory effect of ginsenoside Rg1 (G-Rg1) on endoplasmic reticulum stress and its effect on hepatocellular apoptosis in carbon tetrachloride (CCl(4))-induced acute liver failure (ALF). Methods: Forty healthy, adult male C57/BL mice were randomly divided into normal saline control (NS) group, G-Rg1 blank control (G-Rg1) group, CCl(4) model (CCl(4)) group, and G-Rg1 preventive treatment (CCl(4)+G-Rg1) group, and an ALF mouse model was established by CCl(4) induction. Blood and liver specimens were collected from all mice upon sacrifice at 12 hours post-intraperitoneal injection. Serum alanine aminotransferase (ALT), serum aspartate aminotransferase (AST) and total bilirubin (TBil) levels were determined using commercial test kits. The mRNA expression of glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP) was measured using real-time PCR. The protein expression of GRP78, CHOP, caspase12, and caspase3 were measured by Western blot. Histological changes in the liver were assessed by hematoxylin-eosin staining, and the expression of GRP78 and caspase3 was detected by immunohistochemistry. Hepatocyte apoptosis was determined using terminal transferase dUTP nick end labeling. Quantitative data were analyzed using one-way ANOVA, and subsequent pairwise comparisons were performed using the LSD-t method. Results: Serum ALT, AST, and TBil levels in the CCl(4)+G-Rg1 group were significantly reduced compared with those in the CCl(4) group (ALT: 691.30 ± 108.06 U/L vs 980.66 ± 110.29 U/L, F = 365.07, P < 0.05; AST: 195.40 ± 15.41 U/L vs 319.44 ± 89.32 U/L, F = 115.64, P < 0.05; TBil: 1.09 ± 0.11 mg/dl vs 1.56 ± 0.12 mg/dl, F = 211.29, P < 0.05). The relative mRNA expression of GRP78 and CHOP was significantly lower in the CCl(4) + G-Rg1 group than in the CCl(4) group (P < 0.05). The relative protein expression of caspase3, GRP78, caspase12, and CHOP was significantly reduced to different extents in the CCl(4)+G-Rg1 group compared with those in the CCl4 group (P < 0.05). The CCl(4) + G-Rg1 group showed reduced liver tissue degeneration and necrosis compared with the CCl(4) group. Furthermore, the CCl(4)+G-Rg1 group showed significantly fewer brown granules in the liver than the CCl4 group (P < 0.05), indicating that G-Rg1 preventive treatment reduced CCl(4)-induced hepatocyte apoptosis. Conclusion: G-Rg1 prophylaxis can inhibit inflammation and reduce hepatocyte necrosis and apoptosis during CCl(4)-induced ALF. Its mechanism may involve the suppression of endoplasmic reticulum stress-related signaling molecules to alleviate hepatocyte endoplasmic reticulum stress and apoptosis. The results of this study suggest that G-Rg1 may inhibit liver inflammation and hepatocyte apoptosis through multiple targets to protect liver function.

Protective effect of earthworm active ingredients against endoplasmic reticulum stress-induced acute liver injury in mice

To study the protective effect of earthworm active ingredients(EWAs) against endoplasmic reticulum stress(ERS)-induced acute liver injury in mice. The model of liver injury was induced through intraperitoneal injection of 10%CCl4. Serum glutamic-pyruvic transaminase(ALT), glutamic-oxaloacetic transaminase(AST), superoxide dismutase(SOD) and glutathione peroxidase(GSH-PX) activity and malondialdehyde(MDA) concentration were detected by colorimetric method. Histological examination was performed through hematoxylin-eosin staining and light microscopy, and apoptosis was detected using terminal transferase dUTP nick end labeling. The expressions of ERS related proteins, including glucose regulated protein 78(GRP78), protein kinase R-like ER kinase(PERK), eukaryotic transcription initiation factor 2α(eIF2α), active transcription factor-4(ATF4) and CCAAT/enhancer binding homologous protein(CHOP), were measured by immunohistochemistry and Western blot. According to the results, compared with the model group,serological indexes in the high, middle and low doses of EWAs were significantly improved (P<0.05 or P<0.01), the extent of liver lesion was decreased and the degree of injury was significantly reduced, and that the liver index and the spleen index of mice were significantly changed(P<0.05 or P<0.01). In liver tissue, the expressions of GRP78 and CHOP were significantly decreased(P<0.05 or P<0.01). The protein expressions of GRP78, CHOP and its upstream signaling pathway PERK-eIF2-ATF4 were significantly decreased in each dose group(P<0.05 or P<0.01). In summary, EWAs has a significant protective effect on ERS-induced acute liver injury, and its mechanism may be correlated with the inhibition of oxidative stress and ERS, and down-regulation of ERS marker protein CHOP expression, andinhibition of apoptosis.

Dexmedetomidine-induced cardioprotection in a mouse model of lung ischemia-reperfusion: the relationship with endoplasmic reticulum stress

Objective To evaluate dexmedetomidine-induced cardioprotection in a mouse model of lung ischemia-reperfusion(I/R)and the relationship with endoplasmic reticulum stress. Methods Forty healthy SPF male C57BL/6J mice, weighing 20-24 g, aged 8-10 weeks, were divided into 4 groups(n=10 each)using a random number table: sham operation group(Sham group), lung I/R group(I/R group), dexmedetomidine group(Dex group)and dexmedetomidine plus atipamezole(specific α2-adrenergic receptor antagonist)group(DA group). The model of lung I/R injury was established by clamping the left hilum of lung for 30 min followed by 180 min of reperfusion.In group Sham, only sternotomy was performed, the hilum of lung was not clamped, and the mice were mechanically ventilated for 210 min.In Dex and DA groups, dexmedetomidine 20 μg/kg and dexmedetomidine 20 μg/kg plus atipamezole 250 μg/kg were injected intraperitoneally, respectively, at 30 min before establishment of the model.At 180 min of reperfusion, blood samples were collected from the orbit for determination of creatine kinase-MB(CK-MB)and lactic dehydrogenase(LDH)activities in serum.The animals were then sacrificed, and hearts were removed for determination of apoptosis in cardiomyocytes(by TUNEL)and expression of phosphorylated c-Jun N-terminal kinase(p-JNK), caspase-12, CCAAT/enhancer-binding protein homologous protein(CHOP)and glucose-regulated protein 78(GRP78)in myocardial tissues(by Western blot), and expression of JNK, caspase-12, CHOP, GRP78 mRNA in myocardial tissues(by real-time polymerase chain reaction). Apoptosis index was calculated. Results Compared with Sham group, the serum CK-MB and LDH activities and apoptosis index were significantly increased, the expression of p-JNK, JNK mRNA, and caspase-12, CHOP and GRP78 protein and mRNA was up-regulated in I/R, Dex and DA groups(P 0.05). Compared with DEX group, the serum CK-MB and LDH activities and apoptosis index were significantly increased, the expression of p-JNK, JNK mRNA, and caspase-12 and CHOP protein and mRNA was up-regulated(P 0.05). Conclusion Dexmedetomidine can reduce myocardial injury induced by lung I/R, and the mechanism may be related to activation of α2-adrenergic receptors and inhibition of endoplasmic reticulum stress in myocardial cells of mice. Key words: Dexmedetomidine; Lung; Reperfusion injury; Myocardium; Endoplasmic reticulum; Stress

Astragalus polysaccharides attenuate PCV2 infection by inhibiting endoplasmic reticulum stress in vivo and in vitro

This study explored the effects of Astragalus polysaccharide (APS) on porcine circovirus type 2 (PCV2) infections and its mechanism in vivo and vitro. First, fifty 2-week-old mice were randomly divided into five groups: a group without PCV2 infection and groups with PCV2 infections at 0, 100, 200 or 400 mg/kg APS treatments. The trial lasted for 28 days. The results showed that APS treatments at 200 and 400 mg/kg reduced the pathological injury of tissues, inhibited PCV2 infection and decreased glucose-regulated protein 78 (GRP78) and GADD153/CHOP gene mRNA and protein expression significantly (P < 0.05). Second, a study on endoplasmic reticulum stress mechanism was carried out in PK15 cells. APS treatments at 15 and 45 μg/mL significantly reduced PCV2 infection and GRP78 mRNA and protein expression (P < 0.05). Tunicamycin supplementation increased GRP78 mRNA and protein expression and significantly attenuated the APS-induced inhibition of PCV2 infection (P < 0.05). Tauroursodeoxycholic acid supplementation decreased GRP78 mRNA and protein expression and significantly inhibited PCV2 infection (P < 0.05). In addition, fifty 2-week-old mice were randomly divided into five groups: Con, PCV2, APS + PCV2, TM + PCV2 and TM + APS + PCV2. The results were similar to those in PK15 cells. Taken together, it could be concluded that APS suppresses PCV2 infection by inhibiting endoplasmic reticulum stress.

Evaluation and Treatment of Endoplasmic Reticulum (ER) Stress in Right Ventricular Dysfunction during Monocrotaline-Induced Rat Pulmonary Arterial Hypertension.

Endoplasmic reticulum (ER) stress contributes to pulmonary artery hypertension (PAH). However, the exact roles of ER stress in right ventricular (RV) dysfunction, which is strongly associated with PAH, are largely unknown. Here, we aimed to explore how ER stress affects RV function in a rat PAH model and evaluated the effects of an ER stress inhibitor on RV dysfunction. We examined expression changes of an ER marker: chaperone glucose-regulated protein 78 (GRP78), three ER stress sensor proteins: activating transcription factor 6 (ATF6), inositol-requiring enzyme 1 (IRE1), and protein kinase RNA-like endoplasmic reticulum kinase (PERK), and a key ER stress-induced apoptosis indicator: CCAAT/enhancer-binding protein homologous protein (CHOP), with inflammation indicators: interleukin 6 (IL-6), tumor necrosis factor-α (TNF-α), and matrix metalloproteinases (MMPs) in RV at 3, 7, 14 and 28days following a single dose of monocrotaline (MCT) injection, with or without a preventive treatment [4-phenylbutyric acid (PBA)]. RV function was evaluated by histological, molecular and echocardiographic analysis. 1) GRP78 protein expression started to increase (1.5±0.06 fold change) at 3d post MCT injection, even before the formation of PAH. 2) ATF6, IRE1, and PERK showed distinctive expression patterns post MCT injection. 3) CHOP expression remained low at day 3 & 7, but significantly increased at day 14 (p<0.05), along with the peak of RV cardiomyocytes apoptosis. 4) PBA inhibited ER stress and alleviated remodeling and dysfunction in the RV. The early phase of ER stress might benefit RV function, whereas the extended phase led to RV cardiomyocyte apoptosis and dysfunction. Inhibition of ER stress by PBA during PAH directly improved RV function.

Selenite and ebselen supplementation attenuates D-galactose-induced oxidative stress and increases expression of SELR and SEP15 in rat lens.

Selenite and ebselen supplementation has been shown to possess anti-cataract potential in some experimental animal models of cataract, however, the underlying mechanisms remain unclear. The present study was designed to evaluate the anti-cataract effects and the underlying mechanisms of selenite and ebselen supplementation on galactose induced cataract in rats, a common animal model of sugar cataract. Transmission electron microscopy images of lens fiber cells (LFC) and lens epithelial cells (LEC) were observed in D-galactose-induced experimental cataractous rats treated with or without selenite and ebselen, also redox homeostasis and expression of proteins such as selenoprotein R (SELR), 15kD selenoprotein (SEP15), superoxide dismutase 1 (SOD1), catalase (CAT), β-crystallin protein, aldose reductase (AR) and glucose-regulated protein 78 (GRP78) were estimated in the lenses. The results showed that D-galactose injection injured rat lens and resulted in cataract formation; however, selenite and ebselen supplementation markedly alleviated ultrastructural injury of LFC and LEC. Moreover, selenite and ebselen supplementation could mitigate the oxidative damage in rat lens and increase the protein expressions of SELR, SEP15, SOD1, CAT and β-crystallin, as well as decrease the protein expressions of AR and GRP78. Taken together, these findings for the first time reveal the anti-cataract potential of selenite and ebselen in galactosemic cataract, and provide important new insights into the anti-cataract mechanisms of selenite and ebselen in sugar cataract.

Effects of electroacupuncture combined with dietary control on liver endoplasmic reticulum stress in rats with non-alcoholic fatty liver disease

To observe the effects of electroacupuncture (EA) combined with dietary control on liver endoplasmic reticulum stress (ERS) in rats with non-alcoholic fatty liver disease (NAFLD), and to explore the action mechanism of EA on NAFLD. Sixty male SD rats were randomly divided into a common diet group (15 rats) and a model group (45 rats, fed with high-fat diet to induce NAFLD model). After model establishment, 10 rats in the common diet group were randomly selected as a control group, and 40 rats in model group were randomly divided into a high-fat diet (HFD) group, a low-fat diet (LFD) group, an EA-1 group and an EA-2 group, 10 rats in each one. From the 2nd day of model establishment, rats in the HFD group and EA-1 group were fed with high-fat diet, while rats in the LFD group and EA-2 group were fed with common diet. Rats in the EA-1 group and EA-2 group were treated with acupuncture at unilateral "Zusanli" (ST 36), "Sanyinjiao" (SP 6) and "Taichong" (LR 3), besides,"Zusanli" (ST 36) and "Sanyinjiao" (SP 6) were connected with EA. The treatment was given once a day, and two sides of acupoints were selected alternatively. Each treatment last 20 min for totally 4 weeks. The rats in the remaining groups were treated with identical fixation method. After treatment, the liver tissues of rats in each group were collected and made into ultra-thin slice to perform transmission electron microscope (TEM) on the endoplasmic reticulum morphology; western blot and real-time PCR method were applied to observe the protein and gene expression of the specific ERS protein, glucose-regulated protein 78 (GRP78). Under TEM, plenty of rough endoplasmic reticulum in alignment was observed in the cytoplasm of rats in the control group, accompanied with indistinct adhering ribosome; the rough endoplasmic reticulum in the cytoplasm in the HFD group was in expansion and obvious breakage, presenting small vacuoles in irregular arrangement; the expansion of rough endoplasmic reticulum in the LFD group and EA-1 group was relieved without appa-rent breakage and disarrangement; the rough endoplasmic reticulum in the EA-2 group was in alignment without obvious expansion. The protein expression of GRP78 was little in the control group, while the protein and mRNA expression of GRP78 were obviously increased in the remaining groups (all P<0.01). The protein and mRNA expression of GRP78 in the LFD group and EA-1 group were significantly lower than those in the HFD group (all P<0.01); those in the EA-2 group were lower than those in the LFD group and EA-1 group (all P<0.01). EA effectively improves the liver ERS in NAFLD rats, which can be one of the action mechanisms of EA on NAFLD. In addition, the efficacy is more significant with diet control.

Effect of sevoflurane preconditioning on lung injury induced by limb ischemia-reperfusion in rats and the relationship with endoplasmic reticulum stress

Objective To investigate the effect of sevoflurane preconditioning on lung injury induced by limb ischemia-reperfusion in rats and the relationship with endoplasmic reticulum stress. Methods Forty-five healthy male Sprague-Dawley rats, aged 6-8 weeks, weighing 250-300 g, were randomly allocated into 3 groups(n=15 each)using a random number table: sham operation group(group S), limb ischemia-reperfusion group(group LIR), and sevoflurane preconditioning group(group SP). Limb ischemia-reperfusion was induced by clamping bilateral femoral arteries for 3 h followed by 3 h reperfusion in LIR and SP groups.In group SP, 2.5% sevoflurane was inhaled for 30 min, and 15 min later the model was established.Before ischemia and at 3 h of reperfusion(at the corresponding time point in group S), arterial blood samples were collected for blood gas analysis, and arterial oxygen partial pressure(PaO2)and arterial carbon dioxide partial pressure(PaCO2)were recorded.The rats were sacrificed at 3 h of reperfusion, and lung specimens were obtained for determination of wet to dry lung weight ratio(W/D ratio)and total lung water content(TLW)and for microscopic examination of the pathological changes, and index of quantitative evaluation for alveolar damage(IQA)was calculated.The expression of glucose-regulated protein 78(GRP78)and CCAAT/enhancer-binding protein homologous protein(CHOP)protein and mRNA in lung tissues was detected by Western blot and real-time polymerase chain reaction, respectively. Results Compared to group S, the PaO2 was significantly decreased at 3 h of reperfusion, the W/D ratio, TLW and IQA were significantly increased, the expression of GRP78 and CHOP protein and mRNA was significantly up-regulated(P 0.05). Compared to group LIR, the PaO2 was significantly increased at 3 h of reperfusion, the W/D ratio, TLW and IQA were significantly decreased, the expression of GRP78 and CHOP protein and mRNA was significantly down-regulated(P 0.05). Conclusion Sevoflurane preconditioning can ameliorate lung injury induced by limb ischemia-reperfusion in the rats, and the mechanism is related to inhibition of endoplasmic reticulum stress. Key words: Anesthetics, inhalation; Ischemic preconditioning; Extremities; Reperfusion injury; Respiratory distress syndrome, adult; Endoplasmic reticulum; Stress

ER stress contributes to alpha-naphthyl isothiocyanate-induced liver injury with cholestasis in mice

Endoplasmic reticulum (ER) stress is involved in the development of several liver diseases and tumors. This study investigated the underlying mechanisms of α-naphthyl isothiocyanate (ANIT)-induced liver injury with cholestasis in mice and found ER stress contributes to the injury. All animals were randomly divided into three groups. In the ANIT-intoxicated group, mice were intragastrically given 100mg/kg ANIT (dissolved in corn oil), while the other groups received an equal volume of vehicle as control. After 24 and 48h of ANIT administration, blood samples and liver tissues of all animals were collected for serum biochemistry and hepatic histopathological examinations to evaluate liver injuries with cholestasis. Hepatocellular apoptosis was assessed by the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. The expression of hepatic ER stress-related markers was determined by real-time PCR, immunohistochemical assay and Western blot. ANIT was found to significantly induce liver injury with cholestasis compared with control mice as evidenced by the increase of serum transaminases and total bilirubin (TBil), and histopathological changes in mice. ANIT remarkably induced hepatocellular apoptosis, upregulated the expression of caspase-9 and cytochrome c, and inhibited the gene and protein expression of proliferating cell nuclear antigen (PCNA). The gene expression of ER stress–related markers, including glucose-regulated protein 78 (GRP78), protein kinase R–like ER kinase (PERK), eukaryotic initiation factor 2α (eIF2α), inositol requiring enzyme-1α (IRE-1α) and activating transcription factor 6 (ATF6) was upregulated by ANIT in mice. ANIT also upregulated the protein expression of GRP78 and activated the phosphorylation of IRE1. These results suggested that ANIT induced liver injury with cholestasis partly due to its ability to activate the ER stress pathway.

Role of glucose-regulated protein 78 in early brain injury after experimental subarachnoid hemorrhage in rats.

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.

Effect of Taurine on the expression of glucose regulated protein 78 and growth arrest and DNA damage-inducible protein 34 in zebrafish larvae brain after hypoxia/reoxygenation brain injury

Objective To investigate the expression of endoplasmic reticulum stress-related factors, glucose-regulated protein 78 (GRP78)and growth arrest and DNA damage-inducible protein 34 (GADD34)and neuronal apoptosis in the brain of zebrafish larvae after hypoxia/reoxygenation brain injury, and the neuroprotective role of Taurine. Methods The 5 day post-fertilization zebrafish larvae were randomly assigned to 3 groups: control group, hypoxia/reoxygenation model group (model group) and Taurine treatment group(Taurine group). According to the different time points for observation, each group was subdivided into 5 subgroups (1 h, 3 h, 6 h, 24 h, 48 h)with 100 zebrafish larvae each.The pathological changes in the brain tissues and cell apoptosis were detected by fluorescence terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling(TUNEL). The expression of GRP78 and GADD34 mRNA in the brain of zebrafish larvae were detected by real-time quantitative reverse transcription PCR(qRT-PCR). The changes in GRP78 and GADD34 protein were detected by Western blot. Results (1)TUNEL: apoptosis index(AI)was increased after hypoxia/reoxygenation, and reached the peak at 3 h in model group, the AI in Taurine group was decreased compared with that in the model group at the same time point (1 h: 22.83±1.80 vs 30.18±1.81, 3 h: 23.22±2.46 vs 42.97±4.01, 6 h: 16.80±1.69 vs 22.97±1.91, all P<0.05). (2) qRT-PCR: the expression of GRP78 and GADD34 mRNA was increased at 1 h after hypoxia/reoxygenation, and reached the peak at 3 h in the model group, the expression in Taurine group was decreased compared with that in the model group at the same time point (GRP78 mRNA: 1 h: 2.35±0.13 vs 5.36±0.35, 3 h: 3.08±0.33 vs 4.27±0.52, 6 h: 1.57±0.12 vs 3.00±0.13, all P<0.05; GADD34 mRNA: 1 h: 5.14±0.55 vs 7.45±0.67, 3 h: 2.79±0.58 vs 5.83±0.51, 6 h: 1.79±0.22 vs 3.67±0.30, all P<0.05). (3) Western blot: the expression of GRP78 and GADD34 protein was increased at 1 h, reached the peak at 6 h, but it was decreased in Taurine group (GRP78 protein: 1 h: 1.12±0.11 vs 1.37±0.13, 3 h: 0.79±0.11 vs 1.25±0.10, 6 h: 0.55±0.10 vs 1.52±0.14, all P<0.05; GADD34 protein: 1 h: 0.92±0.11 vs 1.11±0.13, 3 h: 0.96±0.11 vs 1.52±0.09, 6 h: 0.76±0.05 vs 1.89±0.06, all P<0.05). (4) The expression of GRP78 and GADD34 protein was positively correlated with AI in the model group(r=0.53, 0.56 respectively, all P<0.05). Conclusion One of neuroprotective mechanisms of Taurine against hypoxia/reoxygenation brain injury may down-regulate GRP78 and GADD34 expression. Key words: Endoplasmic reticulum stress; Hypoxic-ischemic brain damage; Glucose-regulated protein 78; Growth arrest and DNA damage-inducible protein 34; Apoptosis

Effect of glucose regulated protein 78 on autophagy and apoptosis in ovarian epithelial carcinoma

To explore the effect and mechanism of glucose regulated protein 78 (GRP78) on autophagy and apoptosis in ovarian carcinoma, and to investigate the influence on the growth and sensitivity to cisplatin on the ovarian cancer cells. The human ovarian cancer cell line SKOV3 were treated by the GRP78 regulator BAPTA-AM and A23187, which were used to decrease or increase the expression levels of GRP78, respectively. The experiment were divided into three groups. Cells in the group of BAPTA-AM were treated by BAPTA-AM at the final concerntration of 40 µmol/L for 1 hour. Cells in the group of A23187 were treated by A23187 at the final concerntration of 4 µmol/L for 24 hours. While, cells in the control group were treated by culture medium without any GRP78 regulator for 24 hours. The expressions of GRP78, beclin1, Bcl-2 and CHOP mRNA and protein were detected by reverse transcription (RT)-PCR and western blot. The autophagy levels was observed by green fluorescent protein-microtubule-associated protein 1 light chain 3-II (GFP-LC3-II) fluorescence staining. The flow cytometry was used to analyse the apoptosis rates of cells. The effect on cell growth and the sensitivity to cisplatin of SKOV3 were accessed by methyl thiazolyl tetrazolium (MTT). (1) The mRNA expressions of GRP78, beclin1, Bcl-2 and CHOP in the group of BAPTA-AM were 0.583±0.025, 0.860±0.055, 0.714±0.032 and 0.811±0.004, respectively. The mRNA expressions of GRP78, beclin1, Bcl-2 and CHOP in the group of A23187 were 0.840±0.044, 0.654±0.065, 0.908±0.047 and 0.620±0.062, respectively. The mRNA expressions of GRP78, beclin1, Bcl-2 and CHOP in the control group were 0.687±0.032, 0.772±0.029, 0.845±0.018, 0.712±0.077, respectively. While the protein expressions of GRP78, beclin1, Bcl-2 and CHOP in the group of BAPTA-AM were 0.423±0.035, 0.952±0.022, 0.385±0.032, 0.681±0.095, respectively. The protein expressions of GRP78, beclin1, Bcl-2 and CHOP in the group of A23187 were 0.743±0.032, 0.638±0.025, 0.596±0.029, 0.431±0.095, respectively. The protein expressions of GRP78, beclin1, Bcl-2 and CHOP in the control group were 0.617±0.031, 0.789±0.083, 0.492±0.036, 0.531±0.003, respectively. The mRNA and protein expressions of beclin and CHOP in the group of BAPTA-AM were both higher than those in the control group (P<0.05). While, the mRNA and protein expressions of beclin and CHOP in the group of A23187 were both lower than those in the control group (P< 0.05). (2) The autophagy fluorescence of SKOV3 in the group of BAPTA-AM, A23187 and the control group were 706±117, 473±128, 595±126, respectively, in which there were significant differences among three groups (P<0.05). (3) The apoptosis rate of SKOV3 in the group of BAPTA-AM was (27.4±2.2)%, which was higher than that in the control group [(19.6±1.4)%, P<0.05]. The apoptosis rate of SKOV3 in the group of A23187 was (12.2±1.9)%, which was lower than that in the control group (P<0.05). (4) The comparison of the sensitivity to cisplatin in 3 groups of SKOV3. The 50% inhibition concentration (IC50) of SKOV3 to cisplatin was (3.02±0.62) mg/L. After treated by BAPTA-AM + cisplatin, the IC50 was (2.00±0.17) mg/L and the sensitivity of SKOV3 to cisplatin was increased by 33.8%, and there was significant difference (P<0.05), compared with the control group. And after treated by A23187 + cisplatin, the IC50 was (4.91±2.52) mg/L and the sensitivity of SKOV3 to cisplatin was decreasd by 62.6%; and there was significant difference (P<0.05), compared with the control group. GRP78 could regulate autophagy and apoptosis of ovarian cancer cells by regulating the expressions of beclin1, Bcl-2 and CHOP, thereby affecting the sensitivity to cisplatin in ovarian carcinoma, which may be a new method for the treatment and improvement of the sensitivity to cisplatin in ovarian carcinoma.

Expression of glucose regulated protein 78 and the pathogenesis of gestational diabetes mellitus

To investigate the relationship between the expression of glucose regulated protein 78 (GRP78) in the placental trophoblast cells and the pathogenesis of gestational diabetes mellitus (GDM). All the patients were recruited from Qingdao Municipal Hospital from May 2013 to May 2014. Among them, fifty women with GDM were assigned to the GDM group, and fifty healthy women were defined as the control group. All of them received cesarean section because of breech presentation, contracted pelvis, scarred uterus or on mother's demand. Real-time PCR was conducted to analyze the expression of GRP78 mRNA in the trophoblasts. Immunohistochemistry was performed to detect the localization of GRP78 protein in the placentasl trophoblast cells. (1) GRP78 mRNA expressed in the cytoplasm of trophoblasts of both the GDM group and the control group. The GRP78 mRNA levels in the GDM group and the control group were 15.6±0.4 and 6.0±0.7, respectively. The relative expression level of GRP78 mRNA in the GDM group was 2.6 times of that in the control group, with statistically significant difference (P<0.01). (2) The expression of GRP78 protein was found in the cytoplasm of the trophoblasts of the GDM group. It showed in deep, light brown or yellow after staining, according to the expression degree. The expression of GRP78 protein was also found in the cytoplasm of the trophoblasts of the control group, but it mainly showed yellow color (38/50). The strong positive rate of GRP78 protein in the GDM group (96%, 48/50) was higher than that in the control group (22%, 11/50; P<0.01). The expression of GRP78 increased in the placental trophoblast cells of GDM patients. It might suggest that GRP78 had some effect on the pathogenesis of GDM.

Involvement of GRP78 in the Resistance of Ovarian Carcinoma Cells to Paclitaxel.

Glucose regulated protein 78 (GRP78) is a type of molecular chaperone. It is a possible candidate protein that contributes to development of drug resistance. We first examined the involvement of GRP78 in chemotherapy-resistance in human ovarian cancer cell. The expression of GRP78 mRNA and protein were examined by RT-PCR and western blotting, respectively, in human ovarian cancer cells line (HO-8910). Sensitivity of HO-8910 to paclitaxel was determined with methyl thiazolyl tetrazolium (MTT). Suppression of GRP78 expression was performed using specific small-interfering RNA (siRNA) in HO-8910 cells, and cell apoptosis was assessed by flow cytometry. Statistical analysis was performed using the SPSS 15.0 statistical package. HO-8910 cells, with high basal levels of GRP78, exhibited low sensitivity to paclitaxel. The mRNA and protein levels of GRP78 were dramatically decreased at 24h, 48h and 72h after transfection and the sensitivity to paclitaxel was increased when the GRP78 gene was disturbed by specific siRNA transfection. The results suggested that high GRP78 expression might be one of the molecular mechanisms causing resistance to paclitaxel, and therefore siRNA of GRP78 may be useful in tumor-specific gene therapy for ovarian cancer.