Glucose-regulated Protein 78 mRNA Research Articles

Biomarker potential of the GRP78 cell-free RNA in endometrial cancer

BackgroundCirculating tumor cells represent an opportunity for the assessment of early recurrent disease or for real-time tracing of cancer. Glucose Regulated Protein 78 (GRP78) is known in the literature as a stress factor in endometrial cancer. We aimed to investigate the importance of the gene by targeting tumor traces circulating in the cell fluids of patients with Type 1 endometrial cancer, examining cell-free RNAs in patients’ samples and performing ROC analysis.MethodologyIn this study, 32 endometrial cancer patients and 20 controls were included. This in vitro study evaluated, the GRP78 cell-free mRNA expression levels in endometrial cancer patients, by quantitative real-time polymerase chain reaction qRT–PCR Light Cycler. Receiver operating characteristic (ROC) analysis is a tool used to identify the precision of a diagnostic test or prediction model. In our study, we investigated whether the expression levels of cell-free GRP78 mRNA could be used as a diagnostic criterion.ResultsThe ROC curve results for endometrial cancer diagnostic criterion of cfRNA GRP78 mRNA indicated quite a significant value (p < 0.001).ConclusionCurrent findings show that cell-free mRNA GRP78 is now a criterion that can be used together with smear mRNA GRP78 without the need for invasive methods in endometrial cancer studies.

Mechanistic Research into the Effects of the Jianpi Xiaozhi Formula on Liver Injury in Diabetic Rats.

Objective The purpose of this study was to explore the mechanism of Jianpi Xiaozhi Formula (JPXZF) action in attenuating liver injury in a rat model of type 2 diabetes mellitus (T2DM). Methods A rat model of T2DM was established. Forty-eight male Sprague–Dawley (SD) rats were randomly allocated to six groups: healthy untreated rats (normal control (NC)), rats with diabetes mellitus (DM), diabetic rats treated with low-dosage JPXZF (DM + JL), diabetic rats treated with an intermediate JPXZF dosage (DM + JM), diabetic rats treated with high-dosage JPXZF (DM + JH), and diabetic rats treated with 4-phenylbutyric acid (PBA) (DM + PBA). The rats in each group were given the indicated drugs for 8 weeks, and pathological changes in the liver tissues of each rat group were observed by haematoxylin-eosin (HE) staining. Reverse-transcription polymerase chain reaction (RT-PCR) and Western blotting (WB) were performed to determine the expression of glucose-regulated protein 78 (GRP78), activating transcription factor 6 (ATF6), family with sequence similarity 134, member B (FAM134B), P62, Beclin-1, and light chain 3II/I (LC3II/I) genes and proteins in the liver tissues of the rats in each group. Immunofluorescence was used to observe changes in FAM134B expression. Results After successfully establishing the rat model, RT-PCR assays revealed that, compared with those in the NC group rats, the expression levels of GRP78, ATF6, and P62 mRNA in the livers of the DM group rats were significantly increased, and the relative expression levels of FAM134B and Beclin-1 mRNA were significantly decreased. Compared with that in the DM group, the relative expression of GRP78, ATF6, and P62 mRNA in the liver of the rats in each JPXZF intervention group was decreased in a dosage-dependent manner, and the relative expression of FAM134B and Beclin-1 mRNA was increased significantly (p < 0.05). WB indicated that, compared with that in the NC group rats, the LC3II/I protein expression ratio in the liver of the DM group rats was significantly reduced, and the LC3II/I protein expression ratio in the liver of the rats in each JPXZF intervention group was significantly increased. In addition, the expression of the other measured proteins was consistent with that of the corresponding mRNA measured by RT-PCR (p < 0.05). The immunofluorescence assay results showed that FAM134B changes were consistent with the results obtained by RT-PCR and WB (p < 0.05). Conclusion Jianpi Xiaozhi Formula may be effective in treating liver injury in diabetic rats by regulating autophagy induced by endoplasmic reticulum stress (ERS).

IDF21-0230 Anti-inflammatory activity of glyoxalase-1 inducer supports further evaluation for prevention of COVID-19

Background: Pre-clinical and clinical studies suggest an essential role of proteases, plasma membrane-associated transmembrane protease serine 2 (TMPRSS2) and furin, in virion fusion with lung alveolar epithelial cells for viral infectivity of coronavirus SARS-CoV-2. Angiotensin converting enzyme-2 (ACE2) and glucose regulated protein-78 (GRP78) are considered to be receptors for cell surface binding of the spike protein of SARS-CoV-2. Risk of progression of associated COVID-19 disease to severe symptoms and risk of mortality is linked to increased levels of inflammatory mediators, including monocyte chemoattract protein-1 (MCP-1) and interleukin-8 (IL8), which are also likely risk factors for severe, life-threatening disease. Increased MCP-1 and IL8 in obese subjects and patients with diabetes may contribute to poor clinical outcomes when infected with coronavirus. In a double blind placebo-controlled clinical crossover study in overweight and obese subjects, treatment with Glo1 inducer, trans-resveratrol and hesperetin combination (tRES-HESP), once daily by oral capsule dietary supplement for 8 weeks, decreased expression of MCP-1, IL8, the receptor for advanced glycation endproducts (RAGE) and cyclo-oxygenase-2 (COX-2) in peripheral blood mononuclear cells (PBMCs). Placebo had no effect. We hypothesized that tRES-HESP may decrease protease and inflammatory gene expression linked to SARS-CoV-2 infection and COVID-19 progression. If so, the anti-inflammatory activity of Glo1 inducer in PBMCs found in clinical trial may also be evidenced in human small airway alveolar epithelial cells in primary culture. Aim: The aim of this project was to assess the anti-inflammatory effect of tRES-HESP on human alveolar epithelial cells in primary culture, assessing effects on protease and inflammatory gene expression considered essential for SARS-CoV-2 infectivity and progression of COVID-19 severity. Method: Human small airway epithelial cells (SAECs) were purchased from Lonza. SAECs were cultured in Small Airway Growth Media with supplements, according to the supplier’s instructions. SAECs (10,000 cells/cm2) were cultured with and without 5 µM tRES-HESP and the effect of basal gene expression studied after 72 h for mRNA (qRT-PCR) and protein (Western blotting); and significance of effect assessed by Student’s t-test, n = 3 or 4. The concentration of glucose in the medium was 6 mM. Results: In SAEC primary cultures, tRES-HESP decreased basal protein levels of TMPRSS2 (- 58%; P<0.001) and furin protease (- 43%; P<0.01) after 72 h. tRES-HESP also decreased mRNA of TMPRSS2 (- 62%, P<0.01), GRP78 (- 67%, P<0.001), COX-2 (-34%, P<0.001) and RAGE mRNA (- 56%, P<.001) and decreased basal secretion of MCP-1 and IL8 (-29% and - 25%, P<0.001). Discussion: tRES-HESP decreased basal expression of proteases essential for SARS-CoV-2 infection, GRP78 and secretion of inflammatory mediator risk predictors of severe COVID-19. These effects are likely due to activation of Nrf2 by tRES-HESP and decrease of unfolded protein response activation by suppression of dicarbonyl stress. These outcomes suggest previous anti-inflammatory response of tRES-HESP in overweight and obese subjects found in PBMCs may also occur concomitantly in the lung. TMPRSS2 is an antioxidant response element-linked gene down regulated by activation of Nrf2. These anti-proteolytic and anti-inflammatory responses of tRES-HESP suggest it is suitable for further evaluation for prevention of COVID-19, particularly in obesity and diabetes.

Mechanistic Insights into Ameliorating Effect of Geraniol on d-Galactose Induced Memory Impairment in Rats

Geraniol (GE), an important ingredient in several essential oils, displayed pleiotropic biological activities through targeting multiple signaling cascades. In the current study, we aimed to examine the protective effect of GE on d-galactose (d-gal) induced cognitive impairment and explore the underlying mechanisms. Forty male Wistar rats (8 weeks old) were randomly categorized into 4 groups; Group I (saline + vehicle [edible oil]), group II (saline + geraniol) (100 mg/kg/day orally), group III (d-galactose) (100 mg/kg/day subcutaneously injected), and group IV (d-galactose + geraniol). Behavioral impairments were evaluated. Brain levels of malondialdehyde (MDA) and reduced glutathione (GSH) as well as superoxide dismutase (SOD) and acetylcholinesterase (AchE) activities were estimated. The levels of inflammatory markers [tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, IL-6, and nuclear factor kappa beta (NF-kβ)], endoplasmic reticulum stress sensors [inositol requiring protein 1(IRE1) and protein kinase RNA–like endoplasmic reticulum kinase (PERK)], brain-derived neurotrophic factor (BDNF), and mitogen-activated protein kinases (MAPK) pathway were measured by ELISA. Also, hippocampal histopathological assessment and immunohistochemical analysis of glial fibrillary acidic protein (GFAP) and caspase-3 were performed. Glucose regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP) mRNA expression and protein levels were assessed. GE effectively ameliorated aging-related memory impairment through increasing GSH, BDNF, Ach levels, and SOD activity. Additionally, GE treatment caused a decrease in the levels of MDA, inflammatory mediators, and ER stress sensors as well as the AchE activity together with concomitant down-regulation of GRP78 and CHOP mRNA expression. Moreover, GE improved neuronal architecture and rat's spatial memory; this is evidenced by the shortened escape latency and increased platform crossing number. Therefore, GE offers a unique pharmacological approach for aging-associated neurodegenerative disorders.Graphical

Enhanced GRP78 protein expression via the IRE1α/ASK1/p38 MAPK pathway during As2O3-induced endoplasmic reticulum stress in BEAS-2B cells

Inorganic arsenic is widely present in the environment. Exposure to moderate to high concentrations of arsenic from drinking water or air can cause various cancers and multisystem dysfunction. Glucose-regulated protein 78 (GRP78) is an endoplasmic reticulum (ER) stress sensor of unfolded protein response (UPR) under stress conditions and it enhances cell survival. The aim of this study is to investigate molecular mechanisms of arsenic-induced GRP78 expression in BEAS-2B cells model. We found that GRP78 protein expression was enhanced, while the level of GRP78 mRNA expression did not change under arsenic trioxide (As2O3)-induced ER stress condition in BEAS-2B cells. Cycloheximide, a protein synthesis inhibitor, completely inhibited As2O3-induced GRP78 protein expression. GRP78 mRNA expression was inhibited by actinomycin-D (Act-D). However, GRP78 protein expression was upregulated in the presence of Act-D under As2O3-induced ER stress condition. These data indicated that the upregulation of GRP78 protein under As2O3-induced UPR condition was possibly due to the increased biosynthesis of GRP78 protein. Moreover, both inositol-requiring enzyme 1α (IRE1α) RNase and kinase inhibitor KIRA6 and IRE1α kinase inhibitor APY29 completely inhibited As2O3-induced GRP78 protein expression and phosphorylation of JNK, ERK and p38 MAPK. Activation of apoptotic signaling kinase 1 (ASK1) is a downstream effector of IRE1α kinase. ASK1 inhibitor selonsertib and p38 MAPK inhibitor SB203580 partially inhibited As2O3-induced GRP78 protein expression, respectively. Our results suggested that As2O3 enhanced GRP78 protein expression in BEAS-2B cells via IRE1α kinase/ASK1/p38 MAPK signaling pathway. To our knowledge, this is the first report on illuminating the related mechanisms of increased GRP78 protein expression in As2O3-induced ER stress condition through a novel IRE1α pathway.

6-Formylindolo[3,2-b]carbazole alleviates lipopolysaccharide-induced acute lung injury via suppressing endoplasmic reticulum stress

To investigate the effect and mechanism of 6-formylindolo[3,2-b]carbazole (FICZ) on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. Male C57BL/6J mice aged 8-12 weeks were divided into 4 groups with 8 mice in each group, according to the method of simple random sampling. Sepsis-induced ALI mice model was established by intraperitoneal injection of LPS 5 mg/kg (LPS group), and phosphate buffer saline (PBS) control group (PBS group) was injected with equal volume of PBS. The LPS+FICZ group was intervened by intraperitoneal injection of 1 μg FICZ 1 hour after LPS stimuli, while the FICZ control group (FICZ group) was given the same amount of FICZ 1 hour after intraperitoneal injection of PBS. Serum and lung tissue were collected 24 hours after LPS stimuli, and the pathological changes of lung tissue were analyzed by hematoxylin-eosin (HE) staining and wet/dry weight (W/D) ratio of lung tissue. The concentrations of inflammatory factors in serum and lung tissue were detected by enzyme linked immunosorbent assay (ELISA). The expression levels of endoplasmic reticulum stress signaling pathway related molecules were detected by real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) and Western blotting. Compared with PBS group, inflammatory cell infiltration, alveolar collapse and obvious alveolar exudative lesions had increased, lung tissue W/D ratio was significantly increased, serum interleukin-6 (IL-6) level, lung tissue IL-6 mRNA expression, and the mRNA expressions of glucose-regulated protein 78 (GRP78), protein kinase R-like endoplasmic reticulum kinase (PERK), CCAAT/EBP homologous protein (CHOP), and the protein expressions of GRP78, PERK, activating transcription factor 6 (ATF6), CHOP in lung tissue were significantly increased in LPS group. However, the indexes of FICZ group were not affected. Compared with LPS group, LPS+FICZ group had less inflammatory cell infiltration, relatively intact alveolar structure. Lung W/D weight ratio in LPS+FICZ group was significantly decreased (5.38±0.10 vs. 6.60±0.30, P < 0.01), so as serum IL-6 (ng/L: 15.55±3.77 vs. 32.22±3.84) and lung IL-6 mRNA expression (2-ΔΔCt: 0.79±0.21 vs. 6.89±0.92, both P < 0.01). The mRNA expressions of GRP78, PERK and CHOP were also significantly decreased [GRP78 mRNA (2-ΔΔCt): 1.90±0.16 vs. 7.55±1.29, PERK mRNA (2-ΔΔCt): 1.68±0.20 vs. 4.54±0.89, CHOP mRNA (2-ΔΔCt): 1.13±0.24 vs. 4.44±1.13, all P < 0.05], and the protein expressions of GRP78, PERK, ATF6 and CHOP were significantly decreased (GRP78/GAPDH: 0.59±0.02 vs. 0.77±0.01, PERK/GAPDH: 0.48±0.03 vs. 1.04±0.05, ATF6/GAPDH: 0.51±0.03 vs. 0.65±0.01, CHOP/GAPDH: 0.91±0.05 vs. 1.11±0.07, all P < 0.05). FICZ protects LPS-induced ALI possibly via suppressing endoplasmic reticulum stress and reducing IL-6 expression in blood and lung tissue.

Kuanoniamine C stimulates bortezomib-induced cell death via suppression of glucose-regulated protein 78 in osteosarcoma

Osteosarcoma is the most frequent and intractable malignancy of the bone in children and young adults. Surgical operation requires extensive excision of the cancer tissue and neighboring normal tissues. In addition, anticancer drugs and radiation therapy are thought to be almost ineffective. Glucose-regulated protein 78 (GRP78), a cell-protective endoplasmic reticulum (ER) chaperone protein, is one of the most promising anticancer targets for osteosarcoma. Here, by analyzing the molecular mechanisms of kuanoniamine C, we report that kuanoniamine C suppresses GRP78 expression via GRP78 mRNA degradation in an ER stress response-independent manner. Interestingly, kuanoniamine C-induced cell death and downregulation of GRP78 expression was regulated by p53 signaling. Moreover, co-treatment with bortezomib, which is a newly identified anticancer drug for osteosarcoma, and kuanoniamine C suppressed GRP78 protein expression, which is essential for the stimulation of bortezomib-induced cell death. These results suggest that co-treatment with bortezomib and kuanoniamine C is a novel therapeutic strategy for the treatment of osteosarcoma that enhances bortezomib-dependent cell death by the downregulation of GRP78, and this combination selectively targets the major cell population of osteosarcoma, which expresses wild-type p53.

GRP78 Level Is Altered in the Brain, but Not in Plasma or Cerebrospinal Fluid in Parkinson's Disease Patients.

Accumulation of misfolded proteins results in cellular stress, and is detected by specific sensors in the endoplasmic reticulum, collectively known as the unfolded protein response (UPR). It has been prominently proposed that the UPR is involved in the pathophysiology of Parkinson’s disease (PD). In the present study, the levels of the UPR proteins and mRNA transcripts were quantified in post mortem brain tissue from PD patients and matched controls. The level of a key mediator of the UPR pathway, glucose-regulated protein 78 (GRP78), was significantly decreased in temporal cortex and cingulate gyrus, whereas there were no significant changes in the caudate nucleus, prefrontal, or parietal cortex regions. On the other hand, GRP78 mRNA level was significantly increased in caudate nucleus, cingulate gyrus, prefrontal, and parietal cortex regions. GRP78 protein level was also measured in plasma and cerebrospinal fluid, but there were no differences in these levels between PD patients and control subjects. Furthermore, immunofluorescence labeling of the CD4+ T cells from PD patients showed that GRP78 protein is found in the cytoplasm. However, GRP78 level in PD patients was not significantly different from control subjects. Unlike the previous Lewy body dementia study, the present investigation reports reduced cortical protein, but increased transcript levels of GPR78 in PD. In summary, these data provide further evidence that GRP78 regulation is dysfunctional in the brains of PD patients.

Protective effect of melatonin on brain injury of cerebral ischemia/reperfusion and its mechanism

Objective To investigate the protective effect of melatonin on brain injury in rats with cerebral ischemia/reperfusion and its mechanism. Methods Ninety rats were divided into control group, model group and melatonin group accorded to random number method, thirty rats in each group. The cerebral infarction volume and neurobehavioral score of rats were determined. The C/EBP homologous protein (CHOP), phosphorylated pancreatic endoplasmic reticulase (p-PERK), glucose-regulated protein 78 (GRP78) and cleaved caspase-3 protein levels were determined by Western blotting analysis. The levels of CHOP, PERK and GRP78 mRNA in rat brain tissue were determined by reverse transcription-polymerase chain reaction (RT-PCR). Used t test and analysis of variance. Results The cerebral infarction volume [(17.83±1.46)%] and neurological deficit scores [(1.34±0.22) scores] of the melatonin group were lower than those of the model group [(44.25±2.92)%, (3.12±0.46) scores](t=30.280, 18.472, P<0.05). Compared with the control group, the levels of CHOP, p-PERK, GRP78 and cleaved caspase-3 protein in the brain tissue of the model group and the melatonin group were increased (F=22.879, 47.469, 25.788, P<0.05). Compared with the model group, the levels of CHOP, p-PERK, GRP78 and cleaved caspase-3 protein in the brain tissue of the melatonin group were decreased (t=5.134, 6.274, 5.618、4.382, P<0.05). Compared with the control group, the levels of CHOP, PERK and GRP78 mRNA in the brain tissue of the model group and melatonin group were increased (F=65.940, 23.848, 16.277, P<0.05). Compared with the model group, the levels of CHOP, PERK and GRP78 mRNA in the brain tissue of melatonin group were decreased (t=7.463, 5.164, 4.026, P<0.05). Conclusion Melatonin has a protective effect on brain tissue of rats with cerebral ischemia/reperfusion, and its mechanism may be that melatonin exerts brain protection by inhibiting endoplasmic reticulum stress in brain tissue. Key words: Melatonin; Brain; Ischemia/reperfusion; Rat; C/EBP homologous protein; Phosphorylated pancreatic endoplasmic reticulum kinase; Glucose regulatory protein 78

Effect of penehyclidine hydrochloride on hyperoxia-induced acute lung injury and the relationship with endoplasmic reticulum stress-dependent apoptosis in infantile rats

Objective To evaluate the effect of penehyclidine hydrochloride on hyperoxia-induced acute lung injury and the relationship with endoplasmic reticulum stress-dependent apoptosis in infantile rats. Methods Ninety-six clean-grade male infantile Sprague-Dawley rats, weighing 40-50 g, aged 14 days, were allocated into 4 groups (n=24 each) using a random number table method: control group (C group), penehyclidine hydrochloride group (P group), hyperoxia group (HO group) and hyperoxia plus penehyclidine hydrochloride group (HP group). Infantile rats were intravenously injected with penehyclidine hydrochloride (0.3 mg/kg) at the same time point every day for 3 consecutive days in P and HP groups.Infantile rats were injected with the equal volume of normal saline instead of penehyclidine hydrochloride in C and H groups.Acute lung injury was induced by inhaling oxygen at concentration greater than 90% for 72 h starting from 4th day after administration.Infantile rats were sacrificed at the end of inhaling, and lungs were removed for examination of the pathological changes and for determination of weight to dry weight ratio (W/D ratio), index of quantitative evaluation for alveolar damage (IQA), pneumonocyte apoptosis (using TUNEL), expression of glucose-regulated protein 78 (GRP78) and CCAAT/enhancer-binding protein homologous protein(CHOP) protein and mRNA in lung tissues (by Western blot or using reverse transcription-polymerase chain reaction). The mitochondrial injury score was assessed, and apoptotic index (AI) was determined. Results Compared with C group, the W/D ratio, IQA, AI and mitochondrial injury score were significantly increased, and the expression of GRP78 and CHOP protein and mRNA was up-regulated in HO and HP groups(P 0.05). Compared with HO group, the W/D ratio, IQA, AI and mitochondrial injury score were significantly decreased, the expression of GRP78 and CHOP protein and mRNA was down-regulated (P<0.05), and the pathological changes of lung tissues were significantly attenuated in HP group. Conclusion Penehyclidine hydrochloride can mitigate hyperoxia-induced acute lung injury, and the mechanism may be related to inhibiting endoplasmic reticulum stress-dependent apoptosis in infantile rats. Key words: Cholinergic antagonist; Acute lung injury; Hyperoxia; Endoplasmic reticulum stress; Apoptosis

MiR‑495 enhances the efficacy of radiotherapy by targeting GRP78 to regulate EMT in nasopharyngeal carcinoma cells.

Glucose-regulated protein 78 (GRP78) was revealed to be associated with the radioresistance of nasopharyngeal carcinoma (NPC) in our previous study. GRP78 is a highly expressed cell surface protein, and holds great promise as a cancer specific target. Its expression may be impacted by the regulation of miRNAs, which may be involved in the radioresistance of NPC. A better understanding of the mechanisms of radioresistance may generate new targets of therapy for NPC patients. The present study was designed to investigate the effect of microRNA targeting GRP78 on the radiosensitivity of NPC. First, we used miRWalk software to predict miRNAs that may interact with GRP78. Subsequently, analysis of miR-495 and GRP78 expression was performed in the primary tissues of 92 NPC tissues and cell lines by immunohistochemistry and real-time PCR and the results revealed that miR-495 expression was lower in radioresistant NPC tissues in comparison to chronic rhinitis tissues, and also lower in radioresistant 5-8F cells (5-8F-IR) in comparison to its parental 5-8F cells. Notably, we observed an inverse association between the expression miR-495 and GRP78. Our bioinformatics analysis led to the identification of miR-495 as the optimal miRNA interacting with GRP78 mRNA. Furthermore, miR-495 targeting the 3′untranslated region (UTR) of GRP78 was detected by a Dual-Glo Luciferase Assay system. Finally, we observed that miR-495 inhibition led to a significant increase in the radioresistance of 5-8F cells and higher GRP78 expression, which may be involved in epithelial-mesenchymal transition (EMT) phenotype. miR-495 targeted the 3′UTR of GRP78 and contributed to the efficacy of radiation therapy in NPC.

The effect of L-carnitine on the apoptosis of human lens epithelial cells through endoplasmic reticulum stress

Objective: To investigate the effect of L-carnitine on the apoptosis of human lens epithelial cells through endoplasmic reticulum (ER) stress pathway. Methods: HLE-B3 cell lines were used to set up an oxidative stress model with H(2)O(2) treatment for 12 h, and lead to ER stress. Cells were divided into four groups: H(2)O(2) group, L-carnitine (100 μmol/L) with H(2)O(2) group, phosphate buffered saline (PBS) group and L-carnitine (100 μmol/L) group. Cell counting kit-8 was used to detect the cell viability under the treatment of different concentrations (200, 400, 600 and 800 μmol/L) of H(2)O(2). The apoptosis ratio of HLE-B3 treated by 400 μmol/L H(2)O(2) was tested by flow cytometry. When HLE-B3 was treated by 400 μmol/L H(2)O(2), the expression levels of cysteinyl aspartate specific proteinase 3 (caspase-3) gene and glucose-regulated protein 78 (GRP78) gene were measured by RT-PCR, and the expression levels of caspase-3 protein and GRP78 protein were assayed by Western blotting. Data from groups was analyzed by the one-way analysis of variance, and the LSD-t test was used for the comparison of groups. Results: Cell counting kit-8 assay showed that when H(2)O(2) concentration was 200, 400, 600 and 800 μmol/L, there was significant difference in the H(2)O(2) group(77.6%±0.8%,58.1%±3.1%,39.2%±1.5%,28.1%±2.2%), L-carnitine with H(2)O(2) group(83.3%±4.2%,74.5%±3.1%,46.4%±1.7%,32.4%±1.2%), PBS group(97.6%±2.1%,98.3%±0.2%,96.3%±2.2%,98.5%±1.1%) and L-carnitine group(98.5%±1.3%, 96.1%±2.1%, 98.1%±0.2%, 97.3%±1.4%) (all P<0.05). There was no significant difference between groups (PBS group compared to L-carnitine group, all P>0.05). When the concentration of H(2)O(2) was 400 μmol/L, the survival rate of the L-carnitine with H(2)O(2) group was higher than the H(2)O(2) group. The difference was statistically significant (t=18.14, P=0.020). With increasing of the H(2)O(2) concentration, cell necrosis was increased. The cell survival rate had no significant difference between the L-carnitine with H(2)O(2) group and H(2)O(2) group (both P>0.05). Flow cytometry results of the H(2)O(2) group, L-carnitine with H(2)O(2) group, PBS group and L-carnitine group were 31.4%±4.5%, 16.5%±2.8%, 2.1%±0.2% and 1.9%±1.8%, respectively (F=126.784, P=0.024) . The rate of apoptosis in the L-carnitine with H(2)O(2) group was lower than that in the H(2)O(2) group (t=24.67, P=0.013). There was no significant difference between the PBS group and L-carnitine group (P>0.05). The results of RT-PCR showed that the expression of caspase-3 mRNA in the L-carnitine with H(2)O(2) group was lower than the H(2)O(2) group (0.424±0.041 vs. 0.752±0.203), and the expression of GRP78 mRNA in the L-carnitine with H(2)O(2) group was lower than the H(2)O(2) group (0.521±0.223 vs. 0.821±0.103). The difference was statistically significant (caspase-3: t=27.92, P=0.018;GRP78: t=16.31, P=0.019). Western blotting showed that the protein expression of caspase-3 in the L-carnitine with H(2)O(2) group was lower than the H(2)O(2) group (0.712±0.212 vs. 1.126±0.251), and the GRP78 protein expression in the L-carnitine with H(2)O(2) group was lower than the H(2)O(2) group (0.512±0.012 vs. 0.735±0.051). The difference was statistically significant (caspase-3: t=15.43, P=0.010;GRP78: t=20.62, P=0.018). Conclusion: L-carnitine can reduce the apoptosis rate of HLE-B3 during oxidative stress through ER stress pathway. (Chin J Ophthalmol, 2018, 54: 363-368).

Effects of excessive endoplasmic reticulum stress on lung ischemia/reperfusion induced myocardial injury in mice

To investigate the effects of excessive endoplasmic reticulum stress on lung ischemia/reperfusion (I/R) induced myocardial injury in mice. Forty healthy SPF male C57BL/6J mice were divided into 4 groups randomly (n=10):sham operation group (Sham group), lung I/R group (I/R group), endoplasmic reticulum stress (ERS) pathway agonist Tunicamycin group (TM) and ERS inhibitor 4-phenyl butyric acid group (4-PBA). 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 sham group, only sternotomy was performed, the hilum of lung was not clamped, and the mice were mechanically ventilated for 210 min. In TM and 4-PBA groups, TM 1mg/kg and 4-PBA 400 mg/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 myocardial enzyme. The animals were then sacrificed, and hearts were removed for determination of light microscope, TUNEL, Caspase 3 enzymatic activity, real-time polymerase chain reaction and Western blot. Compared with sham group, the cardiomyocytes had obvious damage under light microscope, and the serum creatine kinase-MB (CK-MB) and lactic dehydrogenase (LDH) activities, apoptosis index and Caspase 3 enzymatic activity were increased significantly, the expressions of p-Jun N-terminalkinase(p-JNK), Caspase 12, CCAAT/enhancer-binding protein homologous protein (CHOP) and glucose regulated protein 78(GRP78) protein and mRNA were up-regulated in I/R, TM and 4-PBA groups (P<0.01). Compared with I/R group, the cardiomyocytes damage was obvious under light microscope, and the serum CK-MB and LDH activities, apoptosis index and Caspase 3 enzymatic activity were increased significantly, the expressions of p-JNK, Caspase 12, CHOP and GRP78 protein and mRNA were up-regulated in group TM; while all above changes were relieved in group 4-PBA (P<0.01). Compared with TM group, the cardiomyocytes damage was relieved under light microscope, and the serum CK-MB and LDH activities, apoptosis index and Caspase 3 enzymatic activity were decreased significantly, the expressions of p-JNK, Caspase 12,CHOP and GRP78 protein and mRNA were down-regulated in group 4-PBA. The excessive endoplasmic reticulum stress participates in myocardial injury induced by lung ischemia/reperfusion (I/R) and inhibit excessive endoplasmic reticulum stress response can relieved myocardial injury.

Effect of Huoxue Jiedu Jiangtang Formulation on Endoplasmic Reticulum Stress in Diabetic Atherosclerosis

Objecive: To study the effects of Huoxue Jiedu Jiangtang formulation (HJJF) on endoplasmic reticulum stress (ERS) coupled inflammatory response From the molecular mechanism in diabetic vascular disease. Methods: Type 2 diabetes were established in male SD rats by feeding high lipid diet and injection of streptozotocin. after T2DM models established successfully, the rats were divided into model group, low-dose HJJF group (HJJF1), high-dose HJJF group (HJJF2), and Western medicine group (Gliquidone+Benazepril), and accepted corresponding drugs for 8 weeks respectively. Another normal group were used as control group, fed with normal diet, no drug intervention. The levels of fasting blood glucose (FBG), serum superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), IL-6 and TNF-α were tested. The ERS signaling molecules glucose-regulated protein 78 (GRP78) and c-Jun N-terminal kinases (JNK) mRNA transcription level in thoracic aorta vessel were determined by reverse-transcription polymerase chain reaction (RT-PCR). Results: Compared with model group, After drugs intervention, all administered groups could significantly decrease FPG (P<0.05), elevate SOD and GSH-Px (P<0.05); all could reduce levels of TC, TG, LDL-C, IL-6 and TNF-α (P<0.05), and raise HDL-C (P<0.05); all could depress the transcription levels of GRP78 and JNK mRNA (P<0.05). With increasing dose, the HJJF effect is more significant (P<0.05). Conclusion: HJJF could improve insulin resistance, correct lipid metabolism disorders, and enhance the antioxidative ability, inhibit the response of ERS coupled inflammatory, showing the multi-target treatment characteristics of Traditional Chinese Medicine.

Experimental study of inhibitory effects of diallyl trisulfide on the growth of human osteosarcoma Saos-2 cells by downregulating expression of glucose-regulated protein 78.

BackgroundDiallyl trisulfide (DATS) is a natural organic sulfur compound isolated from garlic that has good anticancer activity according to many previous reports. There are many studies pointing out that DATS can downregulate expression of the glucose-regulated protein 78 (GRP78), which is associated with poor prognosis and drug resistance in various types of human cancers. However, it remains unknown whether DATS has the same effect on human osteosarcoma cells. This study attempted to clarify the potential molecular mechanisms of the action of DATS in human osteosarcoma Saos-2 cells.MethodsWe used an inverted phase microscope and immunofluorescent staining to observe the morphological changes of Saos-2 cells after being cultured in different concentrations of DATS (0, 25, 50, and 100 μM) for 24 h, or for four time periods (24, 48, 72, and 96 h) in the same DATS concentration (50 μM). Quantitative real-time polymerase chain reaction and Western blot were used to detect the expression level of GRP78 mRNA and proteins in Saos-2 cells. GRP78 expression was suppressed in Saos-2 cells by utilizing small-interfering RNA, and the cells were subsequently used to study the anti-proliferative effects of DATS treatment.ResultsThe expression level of GRP78 mRNA and proteins was significantly downregulated due to the increased concentration and effective times of DATS (P<0.05). In addition, there were significant associations between GRP78 silencing and cell proliferation (P<0.05) of DATS treatment.ConclusionThese results indicate that DATS inhibits the growth of human osteosarcoma Saos-2 cells by downregulating the expression of GRP78.

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.

Effect of oxidative stress on myocardial apoptosis, endoplasmic reticulum stress and apoptosis factor in suckling mouse atria myocardium

To investigate the relationship between oxidative stress and myocardial apoptosis, endoplasmic reticulum stress, apoptosis-inducing factors in the process of by researching the effect of oxidative stress on myocardial apoptosis, endoplasmic reticulum stress and apoptosis factor--chop, bax, bcl-2 in suckling mouse atria myocardium. The primary cultured suckling mouse myocardium were randomly divided into control group and oxidative stress group. Firstly, the suckling mouse atria cardiomyocytes were treated with H2O2 at the concentration of 100 m μmol/L for 2 hours. Then, the index of oxidative stress and anti-oxidative stress superoxide dismutase (SOD), the con-tents of malondialdehyde (MDA) and glutathione (GSH) of this two groups were detected by ELISA. Myocardial apoptosis of the two groups was detected by TUNEL. The expressions of glucose-regulated protein 78 (GRP78), glucose-regulated protein 94 (GRP94), C/EBP homolo-gous protein (chop), Bcl-2 associated X protein (bax), B-cell leukemia 2 protein (bcl-2) mRNA were detected by real time PCR. Compared with the control group, the viability of SOD and the contents of MDA in oxidative stress group were reduced, the contents of MDA was increased (P < 0.01). Compared with the control group, the expression of myocardial apoptosis in oxidative stress group was increased(P < 0.01); the expressions of GRP78, GRP94, chop and bax mRNA were increased, while the expression of bcl-2 mRNA was reduced in ox-idative stress group. Oxidative stress may induce the endoplasmic reticulum stress, activate the expressions of apoptosis factors, and finally increase the myocardial apoptosis of atria cardiomyocytes. This may connected to the incident of atrial fibrillation.

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.

The relationship between miRNA378, calumenin and endoplasmic reticulum stress in suckling mouse myocardium with myocarditis caused by adriamycin

To investigate the relationship between miRNA378, calumenin and endoplasmic reticulum stress in suckling mouse myocardium with myocarditis caused by adriamycin. The suckling mouse myocardium of primary culture were randomly divided into control group, adriamycin group, lentivirus infection miRNA378 over expression control group, lentivirus infection miRNA378 over expression group, lentivirus infection miRNA378 silence control group and lentivirus infection miRNA378 silence group. Firstly to identify the suckling mouse myocardium, α-SMA was monitored by immunohistochemical method, and secondly the ventricular myocytes were transfered by lentivirus plasmids. Then the expression change of miRNA378, calumenin and glucose regulated protein 78(GRP78) mRNA were detected by Quantitative Real-time PCR. Compared with the adriamycin infection group, the expression of calumenin mRNA in lentivirus infection miRNA378 over expression group was increased(P<0.01), while the expression of GRP78 mRNA was reduced(P<0.01); Compared with the adriamycin infection group, the expressions of calumenin and GRP78 mRNA in lentivirus infection miRNA378 silence group did not change insignificantly. Adriamycin injection may cause expression of calumenin in suckling mouse myocardium with myocarditis reduced, which may lead to the endoplasmic reticulum stress. This effect is related with miRNA378.