GRP78 Gene Expression Research Articles

ACE2-independent SARS-CoV-2 virus entry through cell surface GRP78 on monocytes – evidence from a translational clinical and experimental approach

SARS-CoV-2 infects host cells via an ACE2/TMPRSS2 entry mechanism. Monocytes and macrophages, which play a key role during severe COVID-19 express only low or no ACE2, suggesting alternative entry mechanisms in these cells. In silico analyses predicted GRP78, which is constitutively expressed on monocytes and macrophages, to be a potential candidate receptor for SARS-CoV-2 virus entry. Hospitalized COVID-19 patients were characterized regarding their pro-inflammatory state and cell surface GRP78 (csGRP78) expression in comparison to healthy controls. RNA from CD14+ monocytes of patients and controls were subjected to transcriptome analysis that was specifically complemented by bioinformatic re-analyses of bronchoalveolar lavage fluid (BALF) datasets of COVID-19 patients with a focus on monocyte/macrophage subsets, SARS-CoV-2 infection state as well as GRP78 gene expression. Monocyte and macrophage immunohistocytochemistry on GRP78 was conducted in post-mortem lung tissues. SARS-CoV-2 spike and GRP78 protein interaction was analyzed by surface plasmon resonance, GST Pull-down and Co-Immunoprecipitation. SARS-CoV-2 pseudovirus or single spike protein uptake was quantified in csGRP78high THP-1cells. Cytokine patterns, monocyte activation markers and transcriptomic changes indicated typical COVID-19 associated inflammation accompanied by upregulated csGRP78 expression on peripheral blood and lung monocytes/macrophages. Subsequent cell culture experiments confirmed an association between elevated pro-inflammatory cytokine levels and upregulation of csGRP78. Interaction of csGRP78 and SARS-CoV-2 spike protein with a dissociation constant of KD=55.2nM was validated invitro. Infection rate analyses in ACE2low and GRP78high THP-1cells showed increased uptake of pseudovirus expressing SARS-CoV-2 spike protein. Our results demonstrate that csGRP78 acts as a receptor for SARS-CoV-2 spike protein to mediate ACE2-independent virus entry into monocytes. Funded by the Sino-German-Center for Science Promotion (C-0040) and the Germany Ministry BMWi/K [DLR-grant 50WB1931 and RP1920 to AC, DM, TW].

Redox profile and mediators of the unfolded protein response (UPR) in the placenta of rats during pregnancy.

Proliferation, differentiation, migration and apoptosis of trophoblastic cells are influenced by hypoxia, as well as adequate modulation of oxidative stress and the unfolded protein response (UPR) pathway. We aimed to evaluate the expression profile of redox and UPR mediators in the placenta of rats throughout pregnancy. Placental expression of hypoxia-inducible factor 1α (HIF1α), 8-Hydroxy-2'-deoxyguanosine (8-OHdG), superoxide dismutase 1 (SOD1), glutathione peroxidase (GPX), catalase (Cat), activating transcription factor 6 (ATF6), protein kinase RNA-like endoplasmic reticulum kinase (PERK), 78 kD glucose-regulated protein (GRP78) and C/EBP-homologous protein (CHOP), as well as reactive oxygen species (ROS) and peroxynitrite production, were evaluated in Wistar rats on the 10th, 12th, 14th, 16th and 18th day of pregnancy (DP). Increased immunostaining of HIF1α was observed on the 16th and 18th DP, while 8-OHdG and ROS production were greater on the 14th DP. SOD1 and Cat had increased immunostaining on the 14th and 18th DP, while staining of GPX1/2, GRP78 and CHOP was greater on the 18th DP. With regard to gene expression, Hif1α and Sod1 showed increased mRNA expression on the 12th and 16th DP, while Gpx1 had increased expression on the 10th and 16th DP. Cat, Perk and Grp78 gene expression was greater on the 14th DP, unlike Atf6, which showed greater expression on the 12th DP. In contrast, Chop maintained increased expression from the 12th to the 18th DP. The placental expression of redox and UPR mediators in rats is influenced by gestational age, with greater expression in periods of greater HIF1α and 8-OHdG expression and at the end of the pregnancy. This study provides data on the physiological modulation of redox and UPR mediators during placental development in rats.

The involvement of oxidative stress mediated endoplasmic reticulum pathway in apoptosis of Golden Pompano (Trachinotus blochii) liver under PS-MPs stress

Globally, microplastics (MPs) are highly prevalent, especially in coastal areas. Unfortunately, golden pompano as a major marine fish in China is typically raised in floating marine cages near coasts, facing these MPs sources. However, toxicological studies on Golden Pompano which farm in coastal areas and face actual microplastic exposure are rare. Therefore, golden pompano were exposed to 10.0 μg/L, 100.0 μg/L, and 1000.0 μg/L polystyrene MPs (PS-MPs) for 14 days to study the potential impact of the microplastics on the Golden Pompano. Fish show slowed growth after 14 days of exposure. Histopathology shows irregular shaped nuclei and nuclear and cytoplasmic vacuolation traits in liver. Oxidative stress-related enzyme activity and gene expression data show that oxidative damage occurs in the high-concentrations (100.0 μg/L and 1000.0 μg/L) of PS-MPs exposures. Up-regulation of Grp78, Xbp-1, Eif-2α and chop gene expression indicates the occurrence of endoplasmic reticulum stress, and the western blot results also confirmed this. Severe oxidative stress also caused ERS, which ultimately increased BAX/Bcl-2 ratios and induces apoptosis. Furthermore, up-regulated anaerobic respiration, altered lipid metabolism, and immune disturbance were exhibited during PS-MPs stress. Therefore, oxidative stress appeared to be the main toxicity issue caused by MPs, while ERS-mediated apoptosis, metabolic alterations, and immune responses were induced by this stress. Notably, endoplasmic reticulum stress and apoptosis are a self-protective mechanism, which may be an intermediate link in the toxicity of microplastics. This study highlights the role of endoplasmic reticulum stress in MPs toxicology and assesses the adverse effects of microplastics on Golden Pompano.

The effects of overwintering temperature on the survival of female adult mud crab, Scylla paramamosain, under recirculating aquaculture systems as examined by histological analysis of the hepatopancreas and expression of apoptosis-related genes

The effects of the overwintering temperature (8, 12, 16, and 20 °C) on survival, antioxidant capacity, and heat shock protein (HSP) gene expression in Scylla paramamosain were examined during an experimental period of eight weeks. The effects of the temperature on survival were parabolic, with the highest and lowest survival rates occurring at 16 °C and 8 °C, respectively. Histological examination of the hepatopancreas revealed that the hepatopancreas tissue structure remained intact and that cell boundaries were clearly observed at 16 °C and 20 °C. However, hepatopancreas cells in the 12 °C and 8 °C groups appeared to be vacuolated and indistinct, respectively. Furthermore, high levels of reactive oxygen species (ROS) and malondialdehyde (MDA) and low levels of total antioxidant capacity (T-AOC) and superoxide dismutase (SOD) in the hepatopancreas were observed in the 8 °C group. Hsp60 and hsp70 gene expression were dramatically upregulated, but hsp10, hsp40, hsp90, and glucose regulatory protein 78 (grp78) gene expression were downregulated at 8 °C. The terminal deoxynucleotidyl transferase deoxy-UTP nick end labeling (TUNEL) method showed that there was more DNA damage at 8 °C and 12 °C than at 16 °C. Moreover, apoptosis was significantly induced with decreasing temperature, and the 8 °C group had the highest apoptosis index. Overall, these results suggest that the overwintering temperature greatly impacts the survival of mud crabs and that these effects may be mediated through antioxidant, HSP response, and apoptosis pathways. Significantly, the optimal overwintering temperature for S. paramamosain was extrapolated to be 14.2–16.1 °C. This suggests that temperature control should be considered in the process of mud crab overwintering.

Effect of endoplasmic reticulum stress on human trophoblast cells: Survival triggering or catastrophe resulting in death

Endoplasmic reticulum (ER) stress has been reported to play a role in the pathogenesis of intrauterine growth retardation and preeclampsia, especially implantation failure. Although in vitro ER stress studies in human trophoblast cell line have been conducted in recent years, the influence of Thapsigargin on intracellular dynamics on calcium homeostasis has not been proven. Here, the effects of ER stress and impaired calcium homeostasis on apoptosis, autophagy, cytoskeleton, hypoxia, and adhesion molecules in 2D and spheroid cultures of human trophectoderm cells were investigated at gene expression and protein levels. Thapsigargin caused ER stress by increasing GRP78 gene expression and protein levels. Human trophectoderm cells displayed different characterization properties in 2D and spheroids. While it moves in the pathway of EIF2A and IRE1A mechanisms in 2D, it proceeds in the pathway of EIF2A and ATF6 mechanisms in spheroids and triggers different responses in survival and programmed cell death mechanisms such as apoptosis and autophagy. This led to changes in the cytoskeleton, cell adhesion molecules and cell-cell interactions by affecting the hypoxia mechanism.

7,8-Dihydroxyflavone alleviates Endoplasmic Reticulum Stress in cafeteria diet-induced metabolic syndrome

AimsProlonged Endoplasmic Reticulum Stress (ERS) is involved in the pathogenesis of metabolic syndrome, including type-2 diabetes mellitus, cardiovascular diseases, atherosclerosis, obesity, and fatty liver disease. There have been significant efforts to discover molecules to treat ERS and/or to ameliorate associate symptoms. In this study, we investigated the effect of 7,8-Dihydroxyflavone (7,8-DHF) on ERS in liver and pancreas tissues in a cafeteria (CAF) diet induced metabolic syndrome model. Main methodsMale C57BL/6 mice were fed CAF diet for 16 weeks and 7,8-DHF was administered intraperitoneally (5 mg/kg/day) for last four weeks. 78-kDa glucose-regulated protein (GRP78) and C/EBP homologous protein (CHOP) in liver and pancreas tissues, insulin and interleukin-1β (IL-1β) in serum were analyzed by ELISA method and serum biochemistry parameters were analyzed with autoanalyzer. GRP78 and CHOP gene expression levels were determined by qRT-PCR. In addition, histopathological analyzes were performed on liver and pancreas tissues. Key findingsFindings revealed that CAF diet caused metabolic abnormalities, insulin resistance and inflammation in serum and triggered ERS in pancreas and liver tissues. 7,8-DHF treatment significantly reduced metabolic abnormalities by reducing serum biochemical parameters, HOMO-IR and IL-1β levels. qRT-PCR and ELISA results indicated that 7,8-DHF treatment down-regulated GRP78 and CHOP expression and protein levels in the liver and GRP78 expression in pancreas. Efficiency of 7,8-DHF in these tissues was also demonstrated by histopathological tests. SignificanceIn conclusion, CAF diet-induced metabolic syndrome model, 7,8-DHF suppressed ERS and ERS-induced metabolic disorders in both liver and pancreas. Therefore, 7,8-DHF may potentially be a novel therapeutic compound to ameliorate ERS and related metabolic symptoms.

Possible Involvement of Adipose Tissue in Patients With Older Age, Obesity, and Diabetes With SARS-CoV-2 Infection (COVID-19) via GRP78 (BIP/HSPA5): Significance of Hyperinsulinemia Management in COVID-19.

Aging, obesity, and diabetes are major risk factors for the severe progression and outcome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection (coronavirus disease 2019 [COVID-19]), but the underlying mechanism is not yet fully understood. In this study, we found that the SARS-CoV-2 spike protein physically interacts with cell surface GRP78, which promotes the binding to and accumulation in ACE2-expressing cells. GRP78 was highly expressed in adipose tissue and increased in humans and mice with older age, obesity, and diabetes. The overexpression of GRP78 was attributed to hyperinsulinemia in adipocytes, which was in part mediated by the stress-responsive transcription factor XBP-1s. Management of hyperinsulinemia by pharmacological approaches, including metformin, sodium–glucose cotransporter 2 inhibitor, or β3-adrenergic receptor agonist, decreased GRP78 gene expression in adipose tissue. Environmental interventions, including exercise, calorie restriction, fasting, or cold exposure, reduced the gene expression of GRP78 in adipose tissue. This study provides scientific evidence for the role of GRP78 as a binding partner of the SARS-CoV-2 spike protein and ACE2, which might be related to the severe progression and outcome of COVID-19 in patients with older age, obesity, and diabetes. The management of hyperinsulinemia and the related GRP78 expression could be a therapeutic or preventative target.

Baking of methionine-choline deficient diet aggravates testis injury in mice

Dietary pattern and cooking methods are important factors to determine the nutrients supplementation for male reproduction. Methionine and choline are two methyl donors in daily diet, which could mediate the lipid metabolism, but their effects on the sperms are not clear. In this study, we fed the mice with methionine-choline deficient (MCD) diet or the baked MCD diet for 6 weeks to evaluate this dietary pattern and the appended high temperature cooking on the spermatogenesis. The results have shown that MCD diet induced testis degradation and the damage of spermatocytes, reduced sperm vitality, motility, but elevated sperm deformity. Additionally, baking of MCD diet aggravated the testis injury, further reduced sperm density, sperm motility, and decreased normal sperm morphology dramatically. These changes were not related to the blood-testis barrier nor the Leydig cells dysfunction, but related to spermatocytes lost and apoptosis. The spermatocyte apoptosis was mediated by reticulum stress, including GRP78, XBP-1 and CHOP gene expression. Our study has shown the importance of methionine and choline in diet, and emphasized the crucial role of cooking condition, which are dietary factors to influence the quality of sperms.

A Weak Response to Endoplasmic Reticulum Stress Is Associated With Postoperative Organ Failure in Patients Undergoing Cardiac Surgery With Cardiopulmonary Bypass.

Introduction: Endoplasmic reticulum stress (ERS) is involved in inflammatory organ failure. Our objective was to describe ERS, its unfolded protein response (UPR) expression/kinetics during cardiac surgery with cardiopulmonary bypass (CPB) and its association with postoperative organ failure (OF).Methods: Prospective study conducted on patients undergoing cardiac surgery with CPB. Blood samples were taken before (Pre-CPB), 2 h (H2-CPB) and 24 h (H24-CPB) after CPB. Plasma levels of 78 kDa Glucose- Regulated Protein (GRP78, final effector of UPR) were evaluated by ELISA. The expression of genes coding for key elements of UPR (ATF6, ATF4, sXBP1, CHOP) was evaluated by quantitative PCR performed on total blood. OF was defined as invasive mechanical ventilation and/or acute kidney injury and/or hemodynamic failure requiring catecholamines.Results: We included 46 patients, GRP78 was decreased at H2-CPB [1,328 (878–1,730) ng/ml vs. 2,348 (1,655–3,730) ng/ml Pre-CPB; p < 0.001] but returned to basal levels at H24-CPB [2,068 (1,436–3,005) ng/ml]. The genes involved in UPR had increased expression at H2 and H24. GRP78 plasma levels in patients with OF at H24-CPB (n = 10) remained below Pre-CPB levels [−27.6 (−51.5; −24.2)%] compared to patients without OF (n = 36) in whom GRP78 levels returned to basal levels [0.6 (−28.1; 26.6)%; p < 0.01]. H24-CPB ATF6 and CHOP expressions were lower in patients with OF than in patients without OF [2.3 (1.3–3.1) vs. 3.0 (2.7–3.7), p < 0.05 and 1.3 (0.9–2.0) vs. 2.2 (1.7–2.9), p < 0.05, respectively].Conclusions: Low relative levels of GRP78 and weak UPR gene expression appeared associated with postoperative OF. Further studies are needed to understand ERS implication during acute organ failure in humans.

Preliminary Virtual Screening Studies to Identify GRP78 Inhibitors Which May Interfere with SARS-CoV-2 Infection.

SARS-CoV-2 Spike protein was predicted by molecular docking to bind the host cell surface GRP78, which was suggested as a putative good molecular target to inhibit Covid-19. We aimed to confirm that GRP78 gene expression was increased in blood of SARS-CoV-2 (+) versus SARS-CoV-2 (−) pneumonia patients. In addition, we aimed to identify drugs that could be repurposed to inhibit GRP78, thus with potential anti-SARS-CoV-2 activity. Gene expression studies were performed in 10 SARS-CoV-2 (−) and 24 SARS-CoV-2 (+) pneumonia patients. A structure-based virtual screen was performed with 10,761 small molecules retrieved from DrugBank, using the GRP78 nucleotide binding domain and substrate binding domain as molecular targets. Results indicated that GRP78 mRNA levels were approximately four times higher in the blood of SARS-CoV-2 (+) versus SARS-CoV-2 (−) pneumonia patients, further suggesting that GRP78 might be a good molecular target to treat Covid-19. In addition, a total of 409 compounds were identified with potential as GRP78 inhibitors. In conclusion, we found preliminary evidence that further proposes GRP78 as a possible molecular target to treat Covid-19 and that many clinically approved drugs bind GRP78 as an off-target effect. We suggest that further work should be urgently carried out to confirm if GRP78 is indeed a good molecular target and if some of those drugs have potential to be repurposed for SARS-CoV-2 antiviral activity.

The Mitochondrial Antioxidant SS-31 Modulates Oxidative Stress, Endoplasmic Reticulum Stress, and Autophagy in Type 2 Diabetes.

Mitochondrial dysfunction has been shown to play a central role in the pathophysiology of type 2 diabetes (T2D), and mitochondria-targeted agents such as SS-31 are emerging as a promising strategy for its treatment. We aimed to study the effects of SS-31 on leukocytes from T2D patients by evaluating oxidative stress, endoplasmic reticulum (ER) stress and autophagy. Sixty-one T2D patients and 53 controls were included. Anthropometric and analytical measurements were performed. We also assessed reactive oxygen species (ROS) production, calcium content, the expression of ER stress markers GRP78, CHOP, P-eIF2α, and autophagy-related proteins Beclin1, LC3 II/I, and p62 in leukocytes from T2D and control subjects treated or not with SS-31. Furthermore, we have evaluated the action of SS-31 on leukocyte-endothelium interactions. T2D patients exhibited elevated ROS concentration, calcium levels and presence of ER markers (GRP78 and CHOP gene expression, and GRP78 and P-eIF2α protein expression), all of which were reduced by SS-31 treatment. SS-31 also led to a drop in BECN1 gene expression, and Beclin1 and LC3 II/I protein expression in T2D patients. In contrast, the T2D group displayed reduced p62 protein levels that were restored by SS-31. SS-20 (with non-antioxidant activity) did not change any analyzed parameter. In addition, SS-31 decreased rolling flux and leukocyte adhesion, and increased rolling velocity in T2D patients. Our findings suggest that SS-31 exerts potentially beneficial effects on leukocytes of T2D patients modulating oxidative stress and autophagy, and ameliorating ER stress.

Efficacy of systemic temozolomide‐activated phage‐targeted gene therapy in human glioblastoma

Glioblastoma multiforme (GBM) is the most lethal primary intracranial malignant neoplasm in adults and most resistant to treatment. Integration of gene therapy and chemotherapy, chemovirotherapy, has the potential to improve treatment. We have introduced an intravenous bacteriophage (phage) vector for dual targeting of therapeutic genes to glioblastoma. It is a hybrid AAV/phage, AAVP, designed to deliver a recombinant adeno‐associated virus genome (rAAV) by the capsid of M13 phage. In this vector, dual tumor targeting is first achieved by phage capsid display of the RGD4C ligand that binds the αvβ3 integrin receptor. Second, genes are expressed from a tumor‐activated and temozolomide (TMZ)‐induced promoter of the glucose‐regulated protein, Grp78. Here, we investigated systemic combination therapy using TMZ and targeted suicide gene therapy by the RGD4C/AAVP‐Grp78. Firstly, in vitro we showed that TMZ increases endogenous Grp78 gene expression and boosts transgene expression from the RGD4C/AAVP‐Grp78 in human GBM cells. Next, RGD4C/AAVP‐Grp78 targets intracranial tumors in mice following intravenous administration. Finally, combination of TMZ and RGD4C/AAVP‐Grp78 targeted gene therapy exerts a synergistic effect to suppress growth of orthotopic glioblastoma.

Icariin Regulates Cellular Functions and Gene Expression of Osteoarthritis Patient-Derived Human Fibroblast-Like Synoviocytes.

Synovial inflammation plays an important role in the pathogenesis and progress of osteoarthritis (OA). There is an urgent need to find safe and effective drugs that can reduce the inflammation and regulate the pathogenesis of cytokines of the OA disease. Here, we investigated the effect of icariin, the major pharmacological active component of herb Epimedium on human osteoarthritis fibroblast-like synoviocytes (OA–FLSs). The OA–FLSs were isolated from patients with osteoarthritis and cultured in vitro with different concentrations of icariin. Then, cell viability, proliferation, and migration were investigated; MMP14, GRP78, and IL-1β gene expression levels were detected via qRT-PCR. Icariin showed low cytotoxicity to OA–FLSs at a concentration of under 10 μM and decreased the proliferation of the cells at concentrations of 1 and 10 μM. Icariin inhibited cell migration with concentrations ranging from 0.1 to 1 μM. Also, the expression of three cytokines for the pathogenesis of OA which include IL-1β, MMP14 and GRP78 was decreased by the various concentrations of icariin. These preliminary results imply that icariin might be an effective compound for the treatment of OA disease.

Oxidative and endoplasmic reticulum stress is impaired in leukocytes from metabolically unhealthy vs healthy obese individuals.

Oxidative stress and inflammation are related to obesity, but the influence of metabolic disturbances on these parameters and their relationship with endoplasmic reticulum (ER) stress is unknown. Therefore, this study was performed to evaluate whether metabolic profile influences ER and oxidative stress in an obese population with/without comorbidities. A total of 113 obese patients were enrolled in the study; 29 were metabolically healthy (MHO), 53 were metabolically abnormal (MAO) and 31 had type 2 diabetes (MADO). We assessed metabolic parameters, proinflammatory cytokines (TNFα and IL-6), mitochondrial and total reactive oxygen species (ROS) production, glutathione levels, antioxidant enzymes activity, total antioxidant status, mitochondrial membrane potential and ER stress marker expression levels (glucose-regulated protein (GRP78), spliced X-box binding protein 1 (XBP1), P-subunit 1 alpha (P-eIF2α) and activating transcription factor 6 (ATF6). The MAO and MADO groups showed higher blood pressure, atherogenic dyslipidemia, insulin resistance and inflammatory profile than that of MHO subjects. Total and mitochondrial ROS production was enhanced in MAO and MADO patients, and mitochondrial membrane potential and catalase activity differed significantly between the MADO and MHO groups. In addition, decreases in glutathione levels and superoxide dismutase activity were observed in the MADO vs MAO and MHO groups. GRP78 and CHOP protein and gene expression were higher in the MAO and MADO groups with respect to MHO subjects, and sXBP1 gene expression was associated with the presence of diabetes. Furthermore, MAO patients exhibited higher levels of ATF6 than their MHO counterparts. Waist circumference was positively correlated with ATF6 and GRP78, and A1c was positively correlated with P-Eif2α. Interestingly, CHOP was positively correlated with TNFα and total ROS production and GRP78 was negatively correlated with glutathione levels. Our findings support the hypothesis that both inflammation and oxidative stress are involved in the induction of ER stress signaling pathways in the leukocytes of metabolically unhealthy obese vs healthy obese subjects.

Downregulation of microRNA‑199a‑5p protects cardiomyocytes in cyanotic congenital heart disease by attenuating endoplasmic reticulum stress.

Chronic hypoxia is a key pathological change in patients with cyanotic congenital heart defect (CCHD). It has been demonstrated that enhanced myocardial unfolded protein response (UPR) increases the capacity to buffer endoplasmic reticulum (ER) stress and to avoid subsequent apoptosis caused by the hypoxia that underlies CCHD. The present study was performed to determine the regulatory role of microRNAs (miRNAs) in this cytoprotective UPR process. The results revealed that miR‑199a‑5p was markedly downregulated in the cardiac tissue of patients with CCHD and in human myocardial cells cultured in hypoxic conditions. The two major UPR modulators, 78 kDa glucose‑regulated protein (GRP78) and activating transcription factor 6 (ATF6), were potential target genes of miR‑199a‑5p in CCHD myocardial specimens. In addition, the miR‑199a‑5p mimic and inhibitor were evidently able to change GRP78 and ATF6 gene expression and ER stress‑associated apoptosis in hypoxia‑treated cardiomyocytes. The interaction between miR‑199a‑5p and the ATF6 and GRP78 3'‑UTR binding sites in myocardial cells was also confirmed by luciferase assay. Thus, it is concluded that myocardial downregulation of miR‑199a‑5p favors the UPR against hypoxia‑induced ER stress in CCHD, which contributes to myocardial protection.

Hepatocyte growth factor secreted by bone marrow stem cell reduce ER stress and improves repair in alveolar epithelial II cells

Idiopathic Pulmonary Fibrosis (IPF) is a progressive, irreversible lung disease with complex pathophysiology. Evidence of endoplasmic reticulum (ER) stress has been reported in alveolar epithelial cells (AEC) in IPF patients. Secreted mediators from bone marrow stem cells (BMSC-cm) have regenerative properties. In this study we investigate the beneficial effects of BMSC-cm on ER stress response in primary AEC and ER stressed A549 cells. We hypothesize that BMSC-cm reduces ER stress. Primary AEC isolated from IPF patients were treated with BMSC-cm. To induce ER stress A549 cells were incubated with Tunicamycin or Thapsigargin and treated with BMSC-cm, or control media. Primary IPF-AEC had high Grp78 and CHOP gene expression, which was lowered after BMSC-cm treatment. Similar results were observed in ER stressed A549 cells. Alveolar epithelial repair increased in presence of BMSC-cm in ER stressed A549 cells. Hepatocyte growth factor (HGF) was detected in biologically relevant levels in BMSC-cm. Neutralization of HGF in BMSC-cm attenuated the beneficial effects of BMSC-cm including synthesis of surfactant protein C (SP-C) in primary AEC, indicating a crucial role of HGF in ER homeostasis and alveolar epithelial repair. Our data suggest that BMSC-cm may be a potential therapeutic option for treating pulmonary fibrosis.

Naltrexone changes the expression of lipid metabolism-related proteins in the endoplasmic reticulum stress induced hepatic steatosis in mice.

Endoplasmic reticulum (ER) stress is closely associated with several chronic diseases such as obesity, atherosclerosis, type 2 diabetes, and hepatic steatosis. Steatosis in hepatocytes may also lead to disorders such as nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH), fibrosis, and possibly cirrhosis. Opioid peptides are involved in triglyceride and cholesterol dysregulation. Naltrexone also attenuates ER stress induced hepatic steatosis in mice. In this study, we evaluated the effects of naltrexone on the expression of lipid metabolism-related nuclear factors and enzymes in the ER stress induced hepatic steatosis. C57/BL6 mice received saline, DMSO and naltrexone as control groups. In a fourth group, ER stress was induced by tunicamycin (TM) injection and in the last group, naltrexone was given before TM administration. Histopathological evaluations, real-time RT-PCR and western blot were performed. We found that GRP78, IRE1α, PERK and ATF6 gene expression and steatosis significantly reduced in naltrexone treated animals. Naltrexone alleviated the gene and protein expression of SREBP1c. Expression of ACAT1, apolipoprotein B (ApoB) and PPARα also increased after naltrexone treatment. In conclusion, this study, for the first time, shows that naltrexone has a considerable role in attenuation of ER stress-induced liver injury.

Altered expression of Chop and Grp78 in neural tube of rat with spina bifida

Chop and Grp78 genes are key genes in endoplasmic reticulum stress(ERS). To explore the gene alteration of Chop and Grp78 in embryo with spina bifida, we studied Chop and Grp78 gene expression by qRT-PCR in neural tube of rat with spina bifida in E12 and primary cultured neuroepithelial stem cells(NEP). We found higher expression of both Chop and Grp78 in neural tube of rat with spina bifida than normal rats.While Chop and Grp78 genes were up-regulated after NEP differentiated into neuron.The up-regulation of Chop and Grp78 genes in neural tube of rat and NEP might be involved in the abnormal apoptosis in spina bifida related with ERS.And we found the primary culture of NEP provided a good model for embryo early development research. Key words: spina bifida; Chop; Grp78; Neuroepithelial stem cell

Glucose-regulated protein Grp78 affects characteristics of sleep and thermoregulation in rats

It was shown previously that sleep deprivation in representatives of warm-blooded animals evokes an elevation of the glucose-regulated protein 78 (Grp78) gene expression in the brain structures involved in the control of sleep and thermoregulation. However it is currently unknown what role the increased Grp78 expression plays in the mechanisms that maintain homeostasis of sleep and thermoregulation. Using electrophysiological methods, it has been shown in this study that microinjections of Grp78 into the third brain ventricle of Wistar rats evokes an increase mostly in deep non-rapid-eye-movement (NREM) sleep (due to a lengthening of the NREM sleep episodes) and a decrease in the amount of rapid-eye-movement sleep. The period of deep NREM sleep was accompanied by a reduced muscle contractile activity. Our results provide evidence that Grp78 is implicated in the molecular mechanisms of maintenance of deep NREM sleep typically accompanied by a decrease in muscle contractile activity.

Electro-acupuncture on Nei Guan and Bai Hui: How Does It Affect GRP78 and Caspase-12 Gene Expression in Rats with Ischemia-Reperfusion Injury

Abstract Objective: To observe the effects of electro-acupuncture (EA) on GRP78 and Caspase-12 gene expression in rats with ischemia- reperfusion injury (IRI) by stimulation on Nei Guan (PC6) and Bai Hui (GV20) points, so that to understand whether or not the protective effects of acupuncture is related to endocytoplasmic reticulum (ER) stressapoptosis passage. Methods: 50 rats were randomly assigned to five groups (10 in each group): normal control(A), pseudo-operation(B), operation(C), Edaravone(D) and EA(E). The ischemia/reperfusion model of middle cerebral artery occlusion (MCAO) was established by suture embolic method. TUNEL staining method was employed to measure the apoptosis index of nerve cells in rats. Real-time polymerase chain reaction (RT-PCR) was employed to measure the mRNA expression of GRP78 and Caspase-12. Results: Compared with normal group and pseudo-operation group, the apoptosis indexes and mRNA expression of GRP78 and Caspase-12 in operation group, Edaravone group and EA group were increased, with statistical significance（P＜0.05 or P＜0.01）; compared with operation group, the apoptosis indexes and Caspase-12 mRNA expression in Edaravone group and EA group were decreased（P＜0.05 or P＜0.01）, but GRP78 mRNA expression were increased（P＜0.01）; there were no significant difference between Edaravone group and EA group on the above indexes（P＞0.05). Conclusion: Acupuncture on Nei Guan and Bai Hui points could effectively suppress the nerve cell apoptosis in cerebral ischemia. The underlying mechanism might be related to upregulation of the ERS-protective GRP78 expression and downregulation of apoptosis-promotion Caspase-12 expression.