Glucose-regulated Protein Research Articles

Characterization and Inhibition of the Chaperone Function of Plasmodium falciparum Glucose-Regulated Protein 94 kDa (PfGrp94).

Plasmodium falciparum expresses four heat shock protein 90 (Hsp90) members. Among these, one, glucose-regulated protein 94 (PfGrp94), is localized in the endoplasmic reticulum (ER). Both the cytosolic and ER-based Hsp90s are essential for parasite survival under all growth conditions. The cytosolic version has been extensively studied and has been targeted in several efforts through the repurposing of anticancer therapeutics as antimalarial drugs. However, PfGrp94 has not been fully characterized and some of its functions related to the ER stress response are not fully understood. Structural analysis of the recombinant full-length PfGrp94 protein showed a predominantly α-helical secondary structure and its thermal resilience was modulated by 5'-N-ethyl-carboxamide-adenosine (NECA) and nucleotides ATP/ADP. PfGrp94 exhibits ATPase activity and suppressed heat-induced aggregation of a model substrate, malate dehydrogenase, in a nucleotide-dependent manner. However, these PfGrp94 chaperone functions were abrogated by NECA. Molecular docking and molecular dynamics (MD) simulations showed that NECA interacted with unique residues on PfGrp94, which could be potentially exploited for selective drug design. Finally, using parasites maintained at the red blood stage, NECA exhibited moderate antiplasmodial activity (IC50 of 4.3, 7.4, and 10.0 μM) against three different P. falciparum strains. Findings from this study provide the first direct evidence for the correlation between in silico, biochemical, and invitro data toward utilizing the ER-based chaperone, PfGrp94, as a drug target against the malaria parasites.

Hypoxia Induced Lnc191 Upregulation Dictates the Progression of Esophageal Squamous Cell Carcinoma by Activating GRP78/ERK Pathway.

Hypoxia is a typical hallmark of solid tumors and plays a crucial role in the progression of esophageal squamous cell carcinogenesis (ESCC). Nevertheless, the precise mechanisms underlying the involvement of hypoxia in tumor development remain unclear. In the present study, a novel hypoxia-induced long noncoding RNA (lncRNA) is identified first, lnc191, which is highly expressed in clinical ESCC tissues and is positively correlated with poor prognosis of ESCC patients. These findings provide evidence that the hypoxia-inducible factor-1α (HIF-1α)-mediated transcriptional activation of lnc191 enhances the growth and metastasis of ESCC cells both in vitro and in vivo. Mechanistically, lnc191 interacts with GRP78 (78-kDa glucose-regulated protein), one of the endoplasmic reticulum chaperone proteins, leading to its translocation to the membrane, where GRP78 binds with EGFR and enhances its phosphorylation (Y845), further activates ERK/MAPK signaling pathway, and thereby in favor of the progression of ESCC. Overall, this data proposes lnc191 as a key driver during the development of ESCC and reveals the participation of the activated GRP78/ERK/MAPK axis in the ESCC progression mediated by lnc191. These findings indicate the potential of lnc191 as a promising diagnostic biomarker and therapeutic target in ESCC.

Taurine Protects Irinotecan-induced Muscle Dysfunction by Modulating Oxidative Stress and Endoplasmic Reticulum Stress in Human Skeletal Muscle Cells.

Irinotecan is a key component of standard first-line treatment for metastatic colorectal cancer. However, irinotecan-induced muscle dysfunction is a contributing factor to cancer cachexia. Here, we present the protective effect of taurine, a conditionally essential amino acid with great antioxidant properties, in attenuating muscle dysfunction induced by irinotecan. Irinotecan (20 μg/ml) was added to human skeletal muscle cells (HSkMCs) with or without pre-treatment of taurine (5 mM). The effects of taurine and irinotecan on the viability, cytotoxicity, and differentiation ability of HSkMC myoblasts were examined. The intracellular reactive oxygen species (ROS) and endoplasmic reticulum stress (ERS) were also monitored. Irinotecan caused cytotoxicity of HSkMCs, while taurine pretreatment increased cell viability and inhibited adenylate kinase release significantly in both myoblasts and myotubes. During differentiation, taurine increased ROS clearance and preserved the myotube differentiation ability impaired by irinotecan. Irinotecan exposure resulted in the up-regulation of CCAAT/enhancer-binding protein homologous protein (CHOP) and glucose-regulated protein 78 (GRP78). Taurine pretreatment could combat such irinotecan-induced ERS. The current in vitro study provides molecular evidence that taurine plays a beneficial role in protecting against irinotecan-induced muscle dysfunction by modulating oxidative stress and endoplasmic reticulum stress.

Serine protease inhibitor AEBSF(4-(2-aminoethyl)-benzenesulfonyl fluoride) decreased ischemic brain injury through inhibiting endoplasmic reticulum stress, oxidative stress, and autophagy in rats

4-(2-Aminoethyl)-benzenesulfonyl fluoride (AEBSF) is a serine protease inhibitor that may alleviate endoplasmic reticulum (ER) stress, a significant contributing factor to cerebral ischemia/reperfusion injury. The molecular crosstalk between ER stress, oxidative stress and autophagy represents a vicious cycle that can be pharmacologically targeted to minimize neuronal death after acute injuries to the central nervous system. However, the neuroprotective effects of AEBSF in the context of cerebral ischemia/reperfusion injury remain unknown. In this study,we reported the neuroprotective effect of AEBSF against cerebral ischemia/reperfusion injury and explored the mechanisms involved, particularly its role in reducing ER stress, oxidative stress and autophagy. Rats were pretreated with AEBSF or a vehicle before a 90-minute middle cerebral artery occlusion (MCAO) followed by 24 h of reperfusion. Our results demonstrate that AEBSF treatment reduced infarct volume and improved neurological function compared to vehicle treated rats after 24 h of reperfusion. Furthermore,AEBSF treatment decreased the expression of caspase-3, suggesting a decrease in neuronal apoptosis. Additionally, AEBSF treatment lowered levels of key ER stress biomarkers, including glucose-regulated protein 78 (GRP78), phosphorylated eukaryotic initiation factor 2α (p-eIF2α), and CCAAT-enhancer-binding protein homologous protein (CHOP), while the levels of inositol-requiring enzyme 1α (IRE1α) remained unchanged. AEBSF also decreased the oxidative stress biomarker neuronal nitric oxide synthase (nNOS) and its related molecule pro-MMP-9. Importantly, treatment with AEBSF reversed the trends of autophagy biomarker LC3B II/α-tubulin, Beclin1, and SQSTM1 at 24 h after reperfusion. In conclusion, AEBSF significantly mitigates ischemic brain damage and promotes neurological recovery by inhibiting ER stress, oxidative stress, and autophagy, highlighting its potential as a therapeutic option for ischemic stroke.

Function of PxGrp78 in the Reproduction of Plutella xylostella.

Glucose-regulated protein 78 (Grp78), a crucial molecular chaperone in the endoplasmic reticulum, has been extensively investigated in vertebrates. However, its functional exploration in insects remains limited. This study cloned the full-length cDNA sequence of Grp78 in Plutella xylostella (L.), which is 2583 bp long. The open reading frame (ORF) is 2004 bp in length and encodes a total of 667 amino acids, including three conserved characteristic sequences of the HSP70 family. PxGrp78 is expressed in various developmental stages of P. xylostella, with the highest expression observed in third instar larvae and higher expression in female adults compared to male adults. The interference with PxGrp78 in female adults was found to significantly reduce the quantity of egg laying and the hatching rate, as well as shorten the oviposition period and down-regulate the expression of the PxVg gene. These results suggested an important role for PxGrp78 in the reproduction of P. xylostella.

Sinapic acid alleviates glutamate-induced excitotoxicity by inhibiting neuroinflammation and endoplasmic reticulum stress pathway in C6 glioma cells

Sinapic acid (SA) is a polyphenol compound derived from hydroxycinnamic acid found in various foods such as cereals and vegetables and has antioxidant, anti-inflammatory and neuroprotective properties. However, its effects on glutamate-induced excitotoxicity, which is important in neurodegenerative diseases, have not been fully elucidated. This study aimed to investigate the effect of SA on glutamate excitotoxicity and the possible role of proinflammatory cytokines and the endoplasmic reticulum (ER) stress pathway. In the study, C6 rat glioma cell line was used and the cells were divided into 4 groups: control, glutamate, SA and glutamate+SA. Cells were treated with 10 mM glutamate for 24 h to induce excitotoxicity. Additionally, SA was applied to cells at concentrations of 12.5 to 100 μM to examine its effects on glutamate excitotoxicity. XTT test was used for cell viability, and apoptotic cells were determined by immunofluorescence and flow cytometry methods. Proinflammatory cytokines (tumor necrosis factor-alpha, TNF-α and interleukin-beta, IL-1β), ER stress markers (glucose regulatory protein 78, GRP78; C/EBP homologous protein, CHOP and activating transcription factor-4, ATF-4) and caspase-3 was used to measure ELISA method. Findings indicated that SA (50 μM) significantly increased cell viability against glutamate-induced excitotoxicity (p < 0.05). Also, SA caused a significant decrease in TNF-α, IL-1β, GRP78, CHOP, ATF-4 and caspase-3 levels in glutamate-treated cells (p < 0.05). Flow cytometry and immunofluorescence staining results showed that SA reduced apoptosis in C6 glioma cells. In conclusion, our findings suggested that SA attenuated glutamate-induced excitotoxicity by preventing apoptosis through reducing proinflammatory cytokines and ER stress protein levels.

Internal Quality Network Stress and Inflammation Reaction Indicators in the Expression and Significance of the Serum of Limited Pregnant Women's Serum.

Aims/Background Fetal growth restriction (FGR) is a common clinical disorder in pregnant women. Inflammation and endoplasmic reticulum stress play an important role in the occurrence and development of FGR. The purpose of this study was to explore the expression and significance of endoplasmic reticulum stress and inflammatory response indicators in the serum of pregnant women with FGR. Methods The data of pregnant women admitted to Changzhou Maternal and Child Health Care Hospital from January 2020 to June 2023 were collected and analyzed by propensity score matching (PSM). Pregnant women with FGR were included in the observation group (n = 65), whereas healthy pregnant women admitted to the hospital during the same period were included in the control group (n = 65). Enzyme-linked immunosorbent assay (ELISA) was used to detect the serum levels of inflammatory markers such as tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) in all the pregnant women recruited in this study. The serum levels of glucose regulatory protein 78 (GRP78) and C/EBP homologous protein (CHOP), which are characteristic indicators of endoplasmic reticulum stress, in the two groups of pregnant women, were also detected and analyzed. A range of parameters concerning fetal growth and development indicators, such as fetal head circumference, abdominal circumference and fetal development index, of the two groups of study subjects were recorded and compared. Analyze the correlation between inflammatory response indicators and endoplasmic reticulum stress factors in pregnant women with FGR and fetal growth and development indicators. Results The serum levels of TNF-α, IL-1β and IL-6 in the observation group were significantly higher than those in the control group (p < 0.001). The serum levels of GRP78 and CHOP in the observation group were significantly higher than those in the control group (p < 0.001). The levels of GRP78 and CHOP in pregnant women with FGR were negatively correlated with fetal head circumference, abdominal circumference, and fetal growth and development index (p < 0.05). Conclusion The serum levels of inflammatory markers such as TNF-α, IL-1β and IL-6 in pregnant women with FGR were abnormally elevated, indicating severe inflammatory response. In addition, endoplasmic reticulum stress was observed in pregnant women with FGR, marked by significantly elevated levels of GRP78 and CHOP. The levels of TNF-α, IL-1β, IL-6, GRP78 and CHOP were negatively correlated with fetal head circumference, abdominal circumference and fetal growth and development index, implying their impacts on the occurrence and development of FGR.

Predictive utility of nomogram based on serum glucose-regulated protein 78 and kidney function for long-term kidney graft survival

The estimated glomerular filtration rate (eGFR) at 1 year post-transplantation is a well-established predictor of long-term graft survival; however, its predictive accuracy needs improvement. We retrospectively analyzed data from 51 kidney transplant recipients at Kanazawa Medical University Hospital (January 2001–February 2015). Cox regression was used to identify risk factors for death-censored graft loss and create a nomogram to predict graft survival at 15 years post-transplantation. The predictive factors ultimately included in the nomogram included eGFR and serum glucose-regulated protein 78 (GRP78) at 1 year post-transplantation. In terms of discrimination, assessed by area under the receiver operating characteristic curve (AUC-ROC), no significant difference was noted between the eGFR model (AUC 0.84 [0.67–1.00]) and nomogram (AUC 0.92 [0.82–1.00]) (p = 0.38). However, calibration, evaluated by the calibration plot, indicated superiority of the nomogram over the eGFR model, confirmed in the internal validation cohort using the Bootstrap method. Regarding clinical value evaluated by decision curve analysis, the nomogram showed a greater net benefit than the eGFR model, especially at wider diagnostic thresholds (particularly important lower thresholds). Our findings suggest the added predictive value of serum GRP78 at 1 year post-transplantation for long-term graft survival prediction.

GRP78 mediates mitochondrial fusion and fission in cigarette smoke-induced inflammatory responses in airway epithelial cells

Objective Chronic obstructive pulmonary disease (COPD) is characterized by persistent airway inflammation, with cigarette smoke being a major contributor to epithelial injury. Recent studies have shown that abnormal mitochondrial function is closely linked to the onset and progression of airway inflammation. This study aims to explore the role and underlying molecular mechanisms of mitochondrial dynamics in cigarette smoke-induced airway inflammation. Materials and Methods Human bronchial epithelial (HBE) cells were exposed to cigarette smoke extract (CSE) to assess the expression of mitochondrial fusion markers MFN2 and OPA1, the fission marker DRP1, and the glucose-regulated protein GRP78. The siRNA and pharmaceutics targeting DRP1, MFN2, and GRP78 were employed. Both cells and supernatants were analyzed for inflammatory factor levels and the related signaling pathways. Results In this study, HBE cells exposed to CSE showed a significant decrease in the proteins MFN2 and OPA1 and an increase in DRP1. The inhibition of DRP1 expression mitigated inflammation while silencing MFN2 exacerbated it. This was similarly corroborated by the use of the DRP1 inhibitor mdivi-1 and the MFN2 activator leflunomide. Additionally, we proved that GRP78 played an important regulatory role as an essential endoplasmic reticulum protein, regulating the mitochondrial fusion/fission process and subsequently activating the NF-κB pathway to regulate airway inflammation. Discussion and Conclusion Taken together, these results suggested that the GRP78-mediated mitochondrial fusion and fission process played a vital role in cigarette smoke-induced airway inflammation and might be a potential therapeutic target in this regard.

AKAP1/PKA-mediated GRP75 phosphorylation at mitochondria-associated endoplasmic reticulum membranes protects cancer cells against ferroptosis.

Emerging evidence suggests that signaling pathways can be spatially regulated to ensure rapid and efficient responses to dynamically changing local cues. Ferroptosis is a recently defined form of lipid peroxidation-driven cell death. Although the molecular mechanisms underlying ferroptosis are emerging, spatial aspects of its signaling remain largely unexplored. By analyzing a public database, we found that a mitochondrial chaperone protein, glucose-regulated protein 75 (GRP75), may have a previously undefined role in regulating ferroptosis. This was subsequently validated. Interestingly, under ferroptotic conditions, GRP75 translocated from mitochondria to mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) and the cytosol. Further mechanistic studies revealed a highly spatial regulation of GRP75-mediated antiferroptotic signaling. Under ferroptotic conditions, lipid peroxidation predominantly accumulated at the ER, which activated protein kinase A (PKA) in a cAMP-dependent manner. In particular, a signaling microdomain, the outer mitochondrial membrane protein A-kinase anchor protein 1 (AKAP1)-anchored PKA, phosphorylated GRP75 at S148 in MAMs. This caused GRP75 to be sequestered outside the mitochondria, where it competed with Nrf2 for Keap1 binding through a conserved high-affinity RGD-binding motif, ETGE. Nrf2 was then stabilized and activated, leading to the transcriptional activation of a panel of antiferroptotic genes. Blockade of the PKA/GRP75 axis dramatically increased the responses of cancer cells to ferroptosis both in vivo and in vitro. Our identification a localized signaling cascade involved in protecting cancer cells from ferroptosis broadens our understanding of cellular defense mechanisms against ferroptosis and also provides a new target axis (AKAP1/PKA/GRP75) to improve the responses of cancer cells to ferroptosis.

ETS1 promotes cisplatin resistance of NSCLC cells by promoting GRP78 transcription.

Non-small cell lung cancer (NSCLC) is a common malignant tumor characterized by rapid growth and invasive power. Glucose regulatory protein 78 (GRP78) is important in cancer cell progression. Here, this study aimed to explore the effect and mechanism of GRP78 on cisplatin (DDP) resistance of NSCLC cells. qRT-PCR and Western blot detected the expression of genes and proteins. Flow cytometry was used to analyze endoplasmic reticulum stress (ERS) induced by DDP in NSCLC. Cell proliferation and apoptosis were examined using cell counting kit-8 (CCK8), cell cloning, and flow cytometry, respectively. Chromatin immunoprecipitation assay (CHIP) and dual-luciferase reporter assays were performed to determine the binding of ETS1 and GRP78 promoter. Mouse xenograft models were constructed for in vivo analysis. ERS was induced by DDP in NSCLC cells. GRP78 were upregulated in DDP-resistant NSCLC tissues, and knockdown of GRP78 suppressed DDP resistance, clone formation, promoted apoptosis, and inhibited ERS in DDP-resistant NSCLC cells. ETS1 knockdown repressed GRP78 expression and NSCLC tumor growth. Interestingly, ETS1 played a role in DDP-resistant NSCLC via GRP78. ETS1 inhibits cisplatin sensitivity of NSCLC cells by promoting GRP78 transcription.

Acetylcholinesterase – glucose-regulated protein 78 binding site prediction, a hope to cure neurological disorders such as Alzheimer’s disease

Cerebral amyloid plaques in the brain define the elderly neuralgic disorder, Alzheimer’s disease (AD). The enzyme Acetylcholinesterase (AChE) was reported to play a vital role in AD. It was shown that AChE induces amyloid fibril formation forming highly toxic AChE-Amyloid-β (Aβ) complexes. AChE can accelerate amyloid formation, and its inhibition could prevent such alterations to the enzyme. Understanding the proteostasis of AChE and its binding site to cellular chaperone GRP78 (Glucose-regulated protein 78) would help find a treatment for AD. In this study, the state of the art computational tools were utilized to predict the binding location of AChE that can stably associate with the cellular chaperone, GRP78. Sequence comparison along with molecular docking predicts two binding locations on AChE (C69–C96 and C257–C272) that could bind to GRP78 substrate binding domain β (SBDβ). The analysis of the docking data suggests that the former location has the best average binding affinity value (-12.16 kcal/mol) and average interaction pattern (13.9 ± 3.5 H-bonds, 5.5 ± 1.4 hydrophobic contacts, and 1.4 ± 1.2 salt bridges).

Short-term Uridine Treatment Alleviates Endoplasmic Reticulum Stress via Regulating Inflammation and Oxidative Stress in Lithium-Pilocarpine Model of Status Epilepticus

Status Epilepticus (SE) leads to the development of epilepsy with the contribution of Endoplasmic Reticulum (ER) stress. Uridine, a pyrimidine nucleoside, has been shown to have neuroprotective and antiepileptogenic effects in animal models. This study aimed to determine whether uridine ameliorates ER stress and apoptosis following epileptogenic insult. Secondly, this study aimed to establish the effect of uridine on inflammatory and oxidative stress parameters that contribute to ER stress. Status epilepticus was induced using lithium-pilocarpine in adult male Sprague-Dawley rats. Following SE termination, rats were treated with uridine, 4-phenylbutyric acid (4-PBA), or saline twice daily for 48 h. Expressions of hippocampal glucose-regulated protein 78 (GRP78), Inositol- Requiring Protein 1 (IRE1α), Protein kinase RNA-like Endoplasmic Reticulum Kinase (PERK), and C/EBP Homologous Protein (CHOP) were determined by western blotting 48 h after SE. Uridine's effects on apoptosis, inflammation or oxidation were evaluated by analyses of cleaved caspase-3 and poly(ADP-ribose) polymerase 1 (PARP1) protein expressions or pro-inflammatory cytokine levels or levels of oxidative stress markers, respectively. Expressions of all ER stress-related proteins significantly increased 48 h after SE. Uridine treatment markedly decreased GRP78, IRE1α, and CHOP levels. A decrease in the PERK level was observed following the administration of 4-PBA; however, uridine had no effect. Cleaved caspase-3 and PARP1 levels were increased in the SHAM group, while uridine and 4-PBA treatment effectively decreased their expressions. Treatment with uridine significantly reduced Myeloperoxidase (MPO) and Malondialdehyde (MDA) levels while tending to increase Catalase (CAT) and Glutathione Peroxidase (GPx) levels. Uridine treatment also significantly attenuated levels of TNF-α and IL-1β, the pro-inflammatory cytokines, which increased 48 h post-SE. Our data indicate that uridine alleviates ER stress after SE. This effect may be attributed to the regulation of inflammation and oxidative stress. Uridine shows promise as a potential preventive agent for epilepsy.

P-Coumaric Acid alleviates non-alcoholic fatty liver disease by promoting GLP-1 secretion via the GRP78-Ca2+ signaling axis

In individuals with obesity-related non-alcoholic fatty liver disease (NAFLD), endogenous Glucagon-like peptide-1 (GLP-1) secretion is compromised, and supplementation with GLP-1 receptor agonists has shown efficacy in ameliorating NAFLD. However, there is currently a scarcity of research focused on the direct promotion of endogenous GLP-1 secretion from L-cells by plant-derived phenolic acids. Hence, this study reveals that p-Coumaric Acid (p-CA), a widely occurring phenolic acid in the plant, holds potential as a therapeutic approach by stimulating the secretion of GLP-1 to improve glucose-lipid metabolism alterations and alleviate NAFLD symptoms. In diet-induced obese (DIO) mouse models, administration of high doses of p-CA led to a significant elevation in serum GLP-1 levels, as well as improvements in body weight gain, glucose level, insufficient insulin secretion, and biochemical abnormalities associated with NAFLD in mice fed with a high-fat diet. Moreover, the number of lipid droplets in the liver tissue of p-CA-treated mice was notably reduced, suggesting its effectiveness in mitigating liver fat accumulation and ameliorating NAFLD severity. Experimental evidence suggests that p-CA exerts its effects by influencing the transcriptional regulation of Glucose-regulated protein 78 kDa (GRP78) through interaction with Nucleophosmin 1 (NPM1), thereby downregulating GRP78 expression. This decrease in GRP78 expression subsequently leads to an increase in intracellular Calcium ions (Ca2+) concentration, which promotes the production and exocytosis of GLP-1-containing vesicles in L cells, consequently enhancing GLP-1 secretion. This research demonstrates the mechanistic role and therapeutic effects of p-CA in regulating GLP-1 secretion and its implications for improving NAFLD, underscoring the pharmacological significance of this natural compound as a dietary supplement.

Dapagliflozin mitigates cellular stress and inflammation through PI3K/AKT pathway modulation in cardiomyocytes, aortic endothelial cells, and stem cell-derived β cells

Dapagliflozin (DAPA), a sodium-glucose cotransporter 2 (SGLT2) inhibitor, is well-recognized for its therapeutic benefits in type 2 diabetes (T2D) and cardiovascular diseases. In this comprehensive in vitro study, we investigated DAPA’s effects on cardiomyocytes, aortic endothelial cells (AECs), and stem cell-derived beta cells (SC-β), focusing on its impact on hypertrophy, inflammation, and cellular stress. Our results demonstrate that DAPA effectively attenuates isoproterenol (ISO)-induced hypertrophy in cardiomyocytes, reducing cell size and improving cellular structure. Mechanistically, DAPA mitigates reactive oxygen species (ROS) production and inflammation by activating the AKT pathway, which influences downstream markers of fibrosis, hypertrophy, and inflammation. Additionally, DAPA’s modulation of SGLT2, the Na+/H + exchanger 1 (NHE1), and glucose transporter (GLUT 1) type 1 highlights its critical role in maintaining cellular ion balance and glucose metabolism, providing insights into its cardioprotective mechanisms. In aortic endothelial cells (AECs), DAPA exhibited notable anti-inflammatory properties by restoring AKT and phosphoinositide 3-kinase (PI3K) expression, enhancing mitogen-activated protein kinase (MAPK) activation, and downregulating inflammatory cytokines at both the gene and protein levels. Furthermore, DAPA alleviated tumor necrosis factor (TNFα)-induced inflammation and stress responses while enhancing endothelial nitric oxide synthase (eNOS) expression, suggesting its potential to preserve vascular function and improve endothelial health. Investigating SC-β cells, we found that DAPA enhances insulin functionality without altering cell identity, indicating potential benefits for diabetes management. DAPA also upregulated MAFA, PI3K, and NRF2 expression, positively influencing β-cell function and stress response. Additionally, it attenuated NLRP3 activation in inflammation and reduced NHE1 and glucose-regulated protein GRP78 expression, offering novel insights into its anti-inflammatory and stress-modulating effects. Overall, our findings elucidate the multifaceted therapeutic potential of DAPA across various cellular models, emphasizing its role in mitigating hypertrophy, inflammation, and cellular stress through the activation of the AKT pathway and other signaling cascades. These mechanisms may not only contribute to enhanced cardiac and endothelial function but also underscore DAPA’s potential to address metabolic dysregulation in T2D.Graphical abstract

L-arginine, aminoguanidine and mesenchymal stem cells reduce the level of endoplasmic reticulum stress markers and D-dimer in the lungs of mice with antiphospholipid syndrome

Antiphospholipid syndrome (APS) is an autoimmune disease characterized by damage to the intima of the microcirculatory blood vessels as a result of the formation of autoimmune antibodies to phospholipids of cell membranes. Recent data indicate a possible link between the occurrence of autoimmune diseases and endoplasmic reticulum stress, impaired nitric oxide availability, high plasma D-dimer level. The aim of the study was to estimate the effect of nitric oxide synthesis modulators L-arginine and aminoguanidine, and mesenchymal stem cells on the level of inositol-requiring enzyme-1a (IRE-1a), glucose-regulated protein 78 (GRP-78) as ER stress markers, and the level of D-dimer in the lung tissue of female BALB/c line mice with experimental APS induced with cardiolipin administration. 30 experimental animals were divided into five groups: 1 – control animals; 2 – mice with APS; 3 – mice with APS, injected intraperitoneally with L-arginine hydrochloride (25 mg/kg) and aminoguanidine (10 mg/kg); 4 – mice with APS, injected intraperitoneally with stem cells (5×106/kg); 5 – mice with APS, injected with L-arginine hydrochloride, aminoguanidine and stem cells in combination. After 10 days post APS formation animals were removed from the experiment, proteins were extracted from the lung tissue and their level was determined with Western blotting. It was established that in group with APS the levels of IRE-1, GRP-78 and D-dimer were substantially increased as compared to the control group. After separate administration of both arginine with aminoguanidine and MSC, as well as with their combined use, the level of IRE-1, GRP-78 and D-dimer decreased compared to the indices in animals with induced APS. The obtained data indicated that this effect is probably due to the reduction of ER stress through iNOS inhibition and the anti-inflammatory action of MSCs. Keywords: aminoguanidine, antiphospholipid syndrome, D-dimer, endoplasmic reticulume stress, GRP-78, IRE-1, L-arginine, lung, mesenchymal stem cells

Human Cripto-1 and Cripto-3 Protein Expression in Normal and Malignant Settings That Conflicts with Established Conventions.

Background/Objectives: Cripto-1 (CR1) is a plurifunctional embryonic protein required for implantation and re-expressed in the adult during wound repair, inflammation, and tumorigenesis. CR1 and its predicted CR1 pseudogene product Cripto-3/CR3 are highly homologous proteins, and given this physical attribute, commercially available antibodies cannot discriminate between CR1 and CR3. Methods: A series of mouse monoclonal antibodies [MoAbs] were developed with a high-affinity binding that can differentiate human CR1/CR3 proteins and showed no measurable cross-reactivity. Results: Using these reagents, we confirm that CR3 is a bona fide translated protein found in human tumor tissue, cancer cell lysates, and in normal/cancer patient donor sera. We also reveal that CR1 and CR3 compete for binding to signal transduction protein Nodal, glucose-regulated protein 78Da (GRP78), and activin receptor-like kinase 4 (Alk4). Our discriminatory MoAbs provide new reagents to help clarify current CR1/CR3 protein expression vagaries in the Cripto field of study, challenging established CR1 conventions. In addition, our data validate CR3 involvement in human carcinogenesis and cell signaling pathways, with potential clinical relevance in determining cancer patient prognosis and disease severity.

Hydrogen sulfide ameliorated endothelial dysfunction in hyperhomocysteinemia rats: Mechanism of IRE1α/JNK pathway-mediated autophagy

Previous studies showed that hyperhomocysteinemia (HHcy) induced endothelial dysfunction by endoplasmic reticulum (ER) stress induction and autophagy stimulation. This study aimed to determine the effect of hydrogen sulfide (H2S) in homocysteine (Hcy)-induced endothelial dysfunction and observe the possible mechanism involved. Male Wistar rats (160–180g) were used and randomly divided into four groups: Control group, HHcy group, HHcy+Sodium hydrosulfide (NaHS) group and NaHS group. Rats were fed with 2% high methionine diet for 8 weeks to set up HHcy model. Plasma concentration of Hcy was measured by ELISA. Endothelium-dependent and non-endothelium-dependent vasodilation of rat renal arteries were determined by myograph. The protein expression of cystathionine-γ-lyase (CSE), ER stress- and autophagy-related proteins in renal arteries or human umbilical vein endothelial cells (HUVECs) were analyzed by western blotting. The endothelial function was impaired in HHcy rats and HUVECs. NaHS supplementation could improve the ACh-induced vasodilation, however it was eliminated by ER stress inducer Tunicamycin (TM) or autophagy inducer Rapamycin. Western blotting in renal arteries showed that Glucose-regulated protein 78 (GRP78) and three branches of ER stress (p-IRE1α, p-PERK, ATF6) , p-JNK1+p-JNK2 were downregulated, simultaneously the autophagy marker Beclin1, LC3BII/LC3BI ratio were decreased and p62 was increased with NaHS treatment in HHcy rats. In HUVECs, IRE1α-JNK induced autophagy was involved in HHcy-induced endothelial dysfunction, while NaHS stimulation reversed the protein expression in IRE1α/JNK-autophagy pathway with Hcy incubation. This study might suggest that endothelial dysfunction induced by HHcy might be correlated with IRE1α-JNK-autophagy axis pathway, which was suppressed by exogenous supplementation of H2S donor, NaHS.

Protective Effects of Trimetazidine and Dexmedetomidine on Liver Injury in a Mesenteric Artery Ischemia-Reperfusion Rat Model via Endoplasmic Reticulum Stress.

Acute mesenteric ischemia can lead to severe liver damage due to ischemia-reperfusion (I/R) injury. This study investigated the protective effects of trimetazidine (TMZ) and dexmedetomidine (DEX) against liver damage induced by mesenteric artery I/R via endoplasmic reticulum stress (ERS) mechanisms. Twenty-four rats were divided into four groups: control, I/R, I/R+TMZ, and I/R+DEX. TMZ (20 mg/kg) was administered orally for seven days, and DEX (100 µg/kg) was given intraper-itoneally 30 min before I/R induction. Liver tissues were analyzed for creatinine, alanine ami-notransferase (ALT), aspartate aminotransferase (AST), thiobarbituric acid reactive substances (TBARS), and total thiol (TT) levels. Compared with the control group, the I/R group presented significantly increased AST, ALT, TBARS, and TT levels. TMZ notably reduced creatinine levels. I/R caused significant liver necrosis, inflammation, and congestion. TMZ and DEX treatments reduced this histopathological damage, with DEX resulting in a more significant reduction in infiltrative areas and vascular congestion. The increase in the expression of caspase-3, Bax, 8-OHdG, C/EBP homologous protein (CHOP), and glucose-regulated protein 78 (GRP78) decreased with the TMZ and DEX treatments. In addition, Bcl-2 positivity decreased both in the TMZ and DEX treatments. Both TMZ and DEX have protective effects against liver damage. These effects are likely mediated through the reduction in ERS and apoptosis, with DEX showing slightly superior protective effects compared with TMZ.

Endoplasmic reticulum stress and expression of nitric oxide synthases in heart failure with preserved and with reduced ejection fraction - pilot study.

Unfolded Protein Response (UPR), endoplasmic reticulum (ER) stress, and inducible nitric oxide synthase (iNOS) overexpression have been found to influence heart failure with preserved ejection fraction (HFpEF) pathogenesis. Their importance in heart failure with reduced ejection fraction (HFrEF) is not entirely established; there is little data involving a detailed comparison between HFpEF and HFrEF from this perspective. This pilot study aimed to compare circulating levels of Glucose-regulated protein 78kDa (GRP78) (ER - stress marker) and all NOS isoforms between both HFpEF and HFrEF and to analyze the correlation between these markers and the clinical characteristics of the patients. Forty-two patients with HFpEF and thirty-eight with HFrEF were involved in this study. Clinical characteristics and echocardiographic data were obtained. Basic laboratory tests were performed and ELISA tests for iNOS, endothelial NOS (eNOS), neuronal NOS (nNOS), and GRP78. Patients with HFpEF had lower circulating levels of GRP78 and higher iNOS concentrations when compared to HFrEF patients (P = 0.023, P < 0.0001, accordingly). The subgroup of the HFpEF population with eGFR < 60 mL/min/1.73m2 had higher nNOS and eNOS levels than HFpEF patients with normal GFR (P = 0.049, P = 0.035, respectively). In the HFrEF subgroup, patients with coexistent diabetes mellitus had elevated concentrations of nNOS compared to the subpopulation without diabetes mellitus (P = 0.041). There was a positive correlation between eNOS and nNOS concentrations (ρ = 0.86, P < 0.0001). In HFpEF, there is a more intensified iNOS overexpression, while in HFrEF, ER stress is more prominent.