Levels Of C/EBP Homologous Protein Research Articles

Borax affects cellular viability by inducing ER stress in hepatocellular carcinoma cells by targeting SLC12A5.

Hepatocellular carcinoma (HCC) presents a persistent challenge to conventional therapeutic approaches. SLC12A5 is implicated in an oncogenic capacity and facilitates the progression of cancer. The objective of this investigation is to scrutinize the inhibitory effects of borax on endoplasmic reticulum (ER)-stress and apoptosis mediated by SLC12A5 in HepG2 cells. Initially, we evaluated the cytotoxic impact of borax on both HL-7702 and HepG2 cell lines. Subsequently, the effects of borax on cellular morphology and the cell cycle of these lines were examined. Following this, we explored the impact of borax treatment on the mRNA and protein expression levels of SLC12A5, C/EBP homologous protein (CHOP), glucose-regulated protein-78 (GRP78), activating transcription factor-6 (ATF6), caspase-3 (CASP3), and cytochrome c (CYC) in these cellular populations. The determined IC50 value of borax for HL-7702 cells was 40.8 mM, whereas for HepG2 cells, this value was 22.6 mM. The concentrations of IC50 (22.6 mM) and IC75 (45.7 mM) of borax in HepG2 cells did not manifest morphological aberrations in HL-7702 cells. Conversely, these concentrations in HepG2 cells induced observable morphological and nuclear abnormalities, resulting in cell cycle arrest in the G1/G0 phase. Additionally, the levels of SLC12A5, ATF6, CHOP, GRP78, CASP3, and CYC were elevated in HepG2 cells in comparison to HL-7702 cells. Moreover, SLC12A5 levels decreased following borax treatment in HepG2 cells, whereas ATF6, CHOP, GRP78, CASP3, and CYC levels exhibited a significant increase. In conclusion, our data highlight the potential therapeutic effects of borax through the regulation of ER stress in HCC by targeting SLC12A5.

SERCA1 Overexpression in Skeletal Muscle Attenuates Muscle Atrophy and Improves Motor Function in a Mouse Model of ALS.

Amyotrophic lateral sclerosis (ALS) is characterized by progressive loss of muscle mass and muscle function. Previous work from our lab demonstrated that skeletal muscles from a mouse model of ALS show elevated intracellular calcium (Ca2+) levels and heightened endoplasmic reticulum (ER) stress. To investigate whether overexpression of sarcoplasmic reticulum (SR) Ca2+ ATPase 1 (SERCA1) in skeletal muscle would improve intracellular Ca2+ handling, attenuate ER stress, and improve motor function ALS transgenic mice. B6SJL-Tg (SOD1*G93A)1Gur/J (ALS-Tg) mice were bred with skeletal muscle α-actinin SERCA1 overexpressing mice to generate wild type (WT), SERCA1 overexpression (WT/+SERCA1), ALS-Tg, and SERCA1 overexpressing ALS-Tg (ALS-Tg/+SERCA1) mice. Motor function (grip test) was assessed weekly and skeletal muscles were harvested at 16 weeks of age to evaluate muscle mass, SR-Ca2+ ATPase activity, levels of SERCA1 and ER stress proteins - protein disulfide isomerase (PDI), Grp78/BiP, and C/EBP homologous protein (CHOP). Single muscle fibers were also isolated from the flexor digitorum brevis muscle to assess changes in resting and peak Fura-2 ratios. ALS-Tg/+SERCA1 mice showed improved motor function, delayed onset of disease, and improved muscle mass compared to ALS-Tg. Further, ALS-Tg/+SERCA1 mice returned levels of SERCA1 protein and SR-Ca2+ ATPase activity back to levels in WT mice. Unexpectedly, SERCA-1 overexpression increased levels of the ER stress maker Grp78/BiP in both WT and ALS-Tg mice, while not altering protein levels of PDI or CHOP. Lastly, single muscle fibers from ALS-Tg/+SERCA1 had similar resting but lower peak Fura-2 levels (at 30 Hz and 100 Hz) compared to ALS-Tg mice. These data indicate that SERCA1 overexpression attenuates the progressive loss of muscle mass and maintains motor function in ALS-Tg mice while not lowering resting Ca2+ levels or ER stress.

ERp29 Attenuates Nicotine-Induced Endoplasmic Reticulum Stress and Inhibits Choroidal Neovascularization.

Nicotine-induced endoplasmic reticulum (ER) stress in retinal pigment epithelium (RPE) cells is thought to be one pathological mechanism underlying age-related macular degeneration (AMD). ERp29 attenuates tobacco extract-induced ER stress and mitigates tight junction damage in RPE cells. Herein, we aimed to further investigate the role of ERp29 in nicotine-induced ER stress and choroidal neovascularization (CNV). We found that the expression of ERp29 and GRP78 in ARPE-19 cells was increased in response to nicotine exposure. Overexpression of ERp29 decreased the levels of GRP78 and the C/EBP homologous protein (CHOP). Knockdown of ERp29 increased the levels of GRP78 and CHOP while reducing the viability of ARPE-19 cells under nicotine exposure conditions. In the ARPE-19 cell/macrophage coculture system, overexpression of ERp29 decreased the levels of M2 markers and increased the levels of M1 markers. The viability, migration and tube formation of human umbilical vein endothelial cells (HUVECs) were inhibited by conditioned medium from the ERp29-overexpressing group. Moreover, overexpression of ERp29 inhibits the activity and growth of CNV in mice exposed to nicotine in vivo. Taken together, our results revealed that ERp29 attenuated nicotine-induced ER stress, regulated macrophage polarization and inhibited CNV.

Effect and mechanism of 1, 25(OH)2D3 on myocardial inflammation induced by Coxsackie virus B3 in mice

Objective: To explore the effect and mechanism of 1, 25(OH)2D3 on myocardial inflammation induced by Coxsackie virus B3 (CVB3) in mice. Methods: Wild type (WT) and 1α-hydroxylase knockout [1(OH)ase-/-] male mice were divided into four groups: WT group, WT+CVB3 group, 1(OH)ase-/-+CVB3 group and 1(OH)ase-/-+CVB3+VD3 group, with 8 mice in each group. The indicators for evaluating myocardial cell injury were examined by different methods. The mRNA levels of pro-inflammatory cytokines [interlenkin (IL)-1β, IL-6, interferon γ (IFN-γ) and tumor necrosis factor α (TNF-α)] were determined by quantitative real-time PCR. Hematoxylin-eosin (HE) staining was used to observe the myocardial histopathological changes. The apoptosis of myocardial cells was detected by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining and flow cytometry. Fluo-4/AM fluorescence probe was used to detect intracellular calcium ion content. Meanwhile, the expression levels of Ca2+/Calmodulin-dependent protein kinase Ⅱ (CaMKⅡ) protein as well as endoplasmic reticulum stress-related proteins like glucose-related protein 78 (GRP78) and C/EBP homologous protein (CHOP) in the myocardial tissues were detected by Western blot. Results: Compared with WT group, the mRNA levels of pro-inflammatory factors increased in the cardiomyocytes of mice in WT+CVB3 group, including IL-1β (14.88±3.32 vs 1.03±0.02, P=0.009), IL-6 (7.00±1.09 vs 1.81±0.18, P=0.005), IFN-γ (4.70±1.11 vs 1.34±0.34, P=0.006) and TNF-α (17.20±3.22 vs 1.02±0.12, P<0.001). Similarly, the infiltration of inflammatory cells, and the apoptosis rate of cardiomyocytes elevated (16.66%±1.09% vs 7.85%±1.12%, P=0.012). The level of calcium ions in myocardial cytoplasm was significantly higher in WT+CVB3 group than that in the WT group (2.98±1.05 vs 0.96±0.10, P=0.006). Likewise, the expression levels of pCaMKⅡ(1.97±0.34 vs 1.00±0, P<0.001), GRP78 (1.78±0.19 vs 1.00±0, P=0.005) and CHOP (1.62±0.09 vs 1.00±0, P=0.002) in WT+CVB3 group up-regulated. The above myocardial cell injury markers were more significant in the 1(OH)ase-/-+CVB3 group. In the 1(OH)ase-/-+CVB3+VD3 group, 1, 25(OH)2D3 supplementation significantly improved myocardial cell injury indicators. Meanwhile, the specific inhibitors of CaMKⅡ can also reduce the myocardial injury and apoptosis rate of CVB3-infected mice. Conclusion: 1, 25(OH)2D3 deficiency can aggravate myocardial inflammation through over activation of CaMKⅡ.

Sivelestat sodium alleviated lipopolysaccharide-induced acute lung injury by improving endoplasmic reticulum stress

Acute lung injury (ALI) is a common inflammatory respiratory disorder characterized by a high incidence and mortality rate. This study aimed to investigate the potential therapeutic effects of the neutrophil elastase inhibitor Sivelestat sodium (SIV) in improving endoplasmic reticulum stress (ERS) while treating lipopolysaccharide (LPS)-induced ALI. An ALI model was established using LPS induction. The effects of SIV on ALI were observed both in vivo and in vitro, along with its impact on ERS. Lung tissue damage was assessed using Hematoxylin-eosin (H&E) staining. Lung edema was measured by the lung wet/dry weight ratio. The expression levels of protein kinase R-like ER kinase (PERK), Phospho-protein kinase R-like ER kinase (p-PERK), activating transcription factor 4 (ATF4), eukaryotic translation initiation factor 2α (EIF2a), phosphorylated α subunit of eukaryotic initiation factor 2α (P-EIF2a), and C/EBP homologous protein (CHOP) were analyzed by Western blotting in vivo and in vitro. The levels of tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) in Lung tissue samples supernatants were measured by ELISA. Oxidative stress markers were measured by ELISA. Apoptosis was measured using the TUNEL assay. Apoptosis-associated proteins B-cell lymphoma-2 (Bcl-2)、Bcl2-associated × (Bax)、caspase-3 were evaluated through Western blotting in vivo and in vitro. The expression levels of ERS-related proteins, including p-PERK, ATF4, P-EIF2a, and CHOP, were significantly increased in the LPS-induced ALI model. However, SIV markedly reduced the expression levels of these proteins, suppressing the LPS-induced ERS response. Further investigations revealed that SIV exerted a protective effect on ALI by alleviating lung tissue damage and apoptosis, improving lung function, and reducing inflammation and oxidative stress levels. However, when SIV was co-administered with Tunicamycin (TUN), TUN blocked the beneficial effects of SIV on ERS and reversed the protective effects of SIV on ALI. In conclusion, SIV alleviated lung tissue damage and apoptosis, improving lung function, and reducing inflammation and oxidative stress in LPS-induced ALI by improving ERS.

Effects of Brimonidine, Omidenepag Isopropyl, and Ripasudil Ophthalmic Solutions to Protect against H2O2-Induced Oxidative Stress in Human Trabecular Meshwork Cells

Purpose We investigated whether hydrogen peroxide (H2O2)-induced oxidative stress causes human trabecular meshwork (HTM) cell dysfunction observed in open angle glaucoma (OAG) in vitro, and the effects of topical glaucoma medications on oxidative stress in HTM cells. Methods We used commercially available ophthalmic solutions of brimonidine, omidenepag isopropyl, and ripasudil in the study. HTM cells were exposed to H2O2 for 1 h, with or without glaucoma medications. We assessed cell viability and senescence via WST-1 and senescence-associated-β-galactosidase (SA-β-Gal) activity assays. After exposure to H2O2 and glaucoma medications, we evaluated changes in markers of fibrosis and stress by using real-time quantitative polymerase chain reaction (qPCR) to measure the mRNA levels of collagen type I alpha 1 chain (COL1A1), fibronectin, alpha-smooth muscle actin (α-SMA), matrix metalloproteinase-2 (MMP-2), endoplasmic reticulum stress markers of C/EBP homologous protein (CHOP), 78-kDa glucose-regulated protein (GRP78), and splicing X-box binding protein-1 (sXBP-1). Results HTM cell viability decreased and SA-β-Gal activity increased significantly after exposure to H2O2. Treatment with three ophthalmic solutions attenuated these changes. Real-time qPCR revealed that H2O2 upregulated the mRNA levels of COL1A1, fibronectin, α-SMA, CHOP, GRP78, and sXBP-1, whereas it downregulated MMP-2 mRNA expression significantly. Brimonidine suppressed the upregulation of stress markers CHOP and GRP78. Additionally, omidenepag isopropyl and ripasudil decreased the upregulation of COL1A1 and sXBP-1. Furthermore, ripasudil significantly suppressed fibrotic markers fibronectin and α-SMA, compared with the other two medications. Conclusion In vitro, H2O2 treatment of HTM cells induced characteristic changes of OAG, such as fibrosis changes and the upregulation of stress markers. These glaucomatous changes were attenuated by additional treatments with brimonidine, omidenepag isopropyl, and ripasudil ophthalmic solutions.

Inhibition of glycolysis represses the growth and alleviates the endoplasmic reticulum stress of breast cancer cells by regulating TMTC3.

Considering the role of glycolysis inhibition as a novel therapeutic strategy for cancer, including breast cancer (BC), we wondered whether glycolysis could affect BC progression by regulating transmembrane O-mannosyltransferase-targeting cadherins 3 (TMTC3). Following the intervention, lactic acid production in BC cells was monitored, and viability, proliferation, and apoptosis assays were performed. The expressions of TMTC3 and endoplasmic reticulum (ER) stress- and apoptosis-related factors Caspase-12, C/EBP homologous protein (CHOP), glucose-regulated protein 78 (GRP78), B-cell lymphoma-2 (Bcl-2), and Bcl-2 associated X (Bax) were quantified. TMTC3 was lowly expressed in BC tissue and cell. The promotion of glycolysis via glucose represses TMTC3 expression and apoptosis yet enhances lactic acid production and growth of BC cell, along with promoted levels of Caspase-12, CHOP, GRP78, and Bcl-2 yet repressed level of Bax, while the contrary results were evidenced after 2-deoxyglycouse intervention. Overexpressed TMTC3 additionally abrogated the effects of glycolysis on increasing the viability and proliferation yet inhibiting the apoptosis of BC cells, with the increased expressions of Caspase-12, CHOP, and GRP78, and Bcl-2 yet decreased level of Bax. Collectively, inhibiting glycolysis restrained the growth and attenuated the ER stress of BC cell by regulating TMTC3.

AGK2 pre-treatment protects against thioacetamide-induced acute liver failure via regulating the MFN2-PERK axis and ferroptosis signaling pathway

BackgroundAcute liver failure (ALF) is an unpredictable and life-threatening critical illness. The pathological characteristic of ALF is massive necrosis of hepatocytes and lots of inflammatory cells infiltration which may lead to multiple organ failure. MethodsAnimals were divided into 3 groups, normal, thioacetamide (TAA, ALF model) and TAA + AGK2. Cultured L02 cells were divided into 5 groups, normal, TAA, TAA + mitofusin 2 (MFN2)-siRNA, TAA + AGK2, and TAA + AGK2 + MFN2-siRNA groups. The liver histology was evaluated with hematoxylin and eosin staining, inositol-requiring enzyme 1 (IRE1), activating transcription factor 6β (ATF6β), protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) and phosphorylated-PERK (p-PERK). C/EBP homologous protein (CHOP), reactive oxygen species (ROS), MFN2 and glutathione peroxidase 4 (GPX4) were measured with Western blotting, and cell viability and liver chemistry were also measured. Mitochondria-associated endoplasmic reticulum membranes (MAMs) were measured by immunofluorescence. ResultsThe liver tissue in the ALF group had massive inflammatory cell infiltration and hepatocytes necrosis, which were reduced by AGK2 pre-treatment. In comparison to the normal group, apoptosis rate and levels of IRE1, ATF6β, p-PERK, CHOP, ROS and Fe2+ in the TAA-induced ALF model group were significantly increased, which were decreased by AGK2 pre-treatment. The levels of MFN2 and GPX4 were decreased in TAA-induced mice compared with the normal group, which were enhanced by AGK2 pre-treatment. Compared with the TAA-induced L02 cell, apoptosis rate and levels of IRE1, ATF6β, p-PERK, CHOP, ROS and Fe2+ were further increased and levels of MFN2 and GPX4 were decreased in the MFN2-siRNA group. AGK2 pre-treatment decreased the apoptosis rate and levels of IRE1, ATF6β, p-PERK, CHOP, ROS and Fe2+ and enhanced the protein expression of MFN2 and GPX4 in MFN2-siRNA treated L02 cell. Immunofluorescence observation showed that level of MAMs was promoted in the AGK2 pre-treatment group when compared with the TAA-induced group in both mice and L02 cells. ConclusionsThe data suggested that AGK2 pre-treatment had hepatoprotective role in TAA-induced ALF via upregulating the expression of MFN2 and then inhibiting PERK and ferroptosis pathway in ALF.

Effect of palmitoylethanolamide on degeneration of a human-derived retinal pigment epithelial cell induced by all-trans retinal.

To study the effect of palmitoylethanolamide (PEA) on apoptosis of retinal pigment epithelial (RPE) cells induced by all-trans retinal (atRAL) and to explore the possible molecular mechanism. CellTiter 96® Aqueous One Solution Cell Proliferation Assay (MTS) was used to detect the effect of PEA on human-derived retinal epithelial cells (ARPE-19) viability induced by atRAL. A Leica DMi8 inverted microscope was used to observe cell morphology. Reactive oxygen species (ROS) production was evaluated with 2',7'-dichlorodihydrof-luorescein diacetate (H2DCFDA) staining and fluorescence microscopy. Expression of c-Jun N-terminal kinase (JNK), phosphorylated JNK (p-JNK), c-Jun, phosphorylated c-Jun (p-c-Jun), Bak, cleaved caspase-3, C/EBP homologous protein (CHOP), and binding (Bip) protein levels were tested by Western blot. Abca4 -/- Rdh8 -/- mice, mouse models of atRAL clearance defects which displays some symbolic characteristics of dry age-related macular degeneration (AMD) and Stargardt disease (STGD1). In the animal models, PEA was injected intraperitoneally. The full-field electroretinogram was used to detect visual function under scotopic conditions traced from mice. Optical coherence tomography showed reconstitution or thickening of the retinal pigment epithelium layer. Effect of PEA on fundus injury induced by light in Abca4-/-Rdh8-/- mice was observed by fundus photography. PEA ameliorated ARPE-19 cells apoptosis and inhibited ROS (including mitochondrial ROS) production induced by atRAL. PEA improved the retinal functional, prohibited both RPE and photoreceptor from death, ameliorates light-induced fundus impairment in Abca4 -/- Rdh8 -/- mice. In vitro and in vivo, PEA inhibited JNK, p-JNK, c-Jun, p-c-Jun, Bak, cleaved caspase-3, CHOP, and Bip protein levels induced by all-trans retinal in ARPE-19 cells. PEA has effect on treating RPE cells apoptosis in retinopathy caused by atRAL accumulation. PEA is a potential treatment strategy for dry AMD and STGD1. The molecular mechanism is affecting the ROS-JNK-CHOP signaling pathway partly.

The clinical significance of circulating glucose-regulated protein 78, Caspase-3, and C/EBP homologous protein levels in patients with heart failure

Background and aimsThe destruction of endoplasmic reticulum (ER) homeostasis leads to heart failure (HF), which further aggravates ER stress. Limited data are available on the levels of ER stress markers in HF patients in clinical practice. This study aimed to determine the clinical significance of the ER stress markers, glucose-regulated protein 78 (GRP78), Caspase-3, and C/EBP homologous protein (CHOP), in predicting HF and its severity. Materials and methodsA total of 62 patients with HF and 44 healthy controls were enrolled in the study, and all participants were followed-up for 2 years. ResultsSerum GRP78, Caspase-3, and CHOP levels were significantly higher in patients with HF than those in healthy controls. The level of GRP78 increased with the severity of HF. GRP78 levels were negatively correlated with left ventricular ejection fraction, and positively correlated with N-terminal B-type natriuretic peptide, D-dimer, and lactic acid. Serum GRP78 and Caspase-3 levels showed moderate predictive values for HF patients. ConclusionER stress markers, GRP78 and Caspase-3, had a certain predictive value in HF and can be used as serum biomarkers for the diagnosis of HF. Additionally, GRP78 showed a certain predictive value in HF severity.

Genipin Attenuates Sepsis-Induced Splenocyte Apoptosis via the Inhibition of Endoplasmic Reticulum Stress.

Endoplasmic reticulum (ER) dysfunction is characterized by ER stress, which can be triggered by sepsis. Recent studies have reported that lessening ER stress is a promising therapeutic approach to improving the outcome of sepsis. Genipin is derived from gardenia fruit, which is a traditional Chinese medicinal herb for anti-inflammation. Here, mice were treated with genipin (2.5 mg/kg) intravenously to assess its biological effects and underlying mechanism against polymicrobial sepsis. Furthermore, the present study focused on detecting the levels of ER stress-related proteins, including protein kinase R-like ER kinase (PERK), glucose-regulated protein of 78 kDa (GRP78), phosphorylated-eukaryotic initiation factor 2α (p-eIF2α), and CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP). The results demonstrated that genipin significantly decreased the serum concentrations of tumor necrosis factor-α and interleukin-6, alleviated histopathological damage to the lungs, livers and spleens, and even improved the survival rates of septic mice. Moreover, sepsis significantly upregulated the protein expression levels of splenic GRP78, PERK, p-eIF2α and CHOP, but their levels were significantly suppressed by genipin. Furthermore, genipin also significantly downregulated cleaved caspase-3 expression levels and reduced sepsis-induced splenocyte apoptosis. In conclusion, genipin potentially improved the survival rate of sepsis and attenuated sepsis-induced organ injury and an excessive inflammatory response in mice. The effects of genipin against sepsis were potentially associated with decreased splenocyte apoptosis via the attenuation of sepsis-induced ER stress to further inhibit ER stress-induced apoptosis.

The Clinical Significance of iNOS/NO Signaling Pathway in Traumatic Shock and the Mechanism under the Promotion on the Development of Traumatic Shock via Endoplasmic Reticulum Stress.

This study aims to clarify the clinical significance of the inducible nitric oxide synthase (iNOs)/nitric oxide (NO) signaling pathway and endoplasmic reticulum stress (ERS) in traumatic shock (TS) and the mechanism of action, so as to offer a novel direction for the emergency treatment of TS in the future. The clinical data of 90 patients with TS treated in our hospital between June 2019 and January 2021 were retrospectively analyzed. Patients were divided into mild (n = 30), moderate (n = 30), and severe group (n = 30) based on their disease severity. Furthermore, patients were assigned into Groups A and B for fluid resuscitation based on a pulse index continuous cardiac output (PICCO) monitor and fluid resuscitation based on monitoring results of central venous pressure (CVP) and mean arterial pressure (MAP), respectively. Additionally, the 18 purchased Sprague Dawley (SD) rats were randomized into model (TS model), control (normal rats) and intervention (TS model injected with iNOS inhibitor) groups, with 6 rats each. iNOs and NO levels were measured by colorimetry, and the concentrations of inflammatory factors were quantified using enzyme-linked immunosorbent assays (ELISAs). Polymerase chain reaction (PCR) and western blot were adopted for the quantification of ERS markers (glucose-related protein 78 (GRP78), GRP94 and C/EBP homologous protein (CHOP)), and hematoxylin-eosin (HE) staining of rat cardiac tissue was carried out to observe the pathological state of myocardial tissue. The moderate group showed higher levels of iNOS, NO, GRP78, GRP94 and CHOP than the mild group and lower levels of them than the severe group (all p < 0.05). MAP, extravascular lung water index (EVLWI), pulmonary vascular permeability index (PVPI), and locus control region (LCR) increased in both Groups A and B after resuscitation, with more significant increases of these parameters in Group A. The application of PICCO technique lowered the levels of iNOS, NO, inflammatory factors, GRP78, GRP94 and CHOP in TS patients. In addition, the intervention group had lower levels of iNOS, NO, inflammatory factors, GRP78, GRP94, and CHOP than the model group and higher levels of them than the control group. According to the results of HE staining of myocardial tissue, the intervention group had significantly alleviated myocardial necrosis than the model group, with slightly stained cytoplasm, visible contraction bands in most myocardium, and significantly reduced neutrophil infiltration. iNOS/NO and ERS increase with the severity of TS, and PICCO can effectively lower their levels. The results of animal experiments suggest that the inhibition of iNOS/NO can relieve inflammation and ERS intensification, thus alleviating the progression of TS.

Endoplasmic Reticulum Stress in the Pathogenesis of Hyperglycemia Induced by Thiamine-Responsive Megaloblastic Anemia.

Thiamine responsive megaloblastic anemia (TRMA) is a genetic disease caused by SLC19A2 gene mutation. This study aimed to preliminarily explore the relationship between endoplasmic reticulum stress (ERS)-PERK signaling pathway and the pathogenesis of hyperglycemia induced by TRMA. Islet β (INS.1 and β-TC-6) and HEK293T cell line models with stable overexpression of SLC19A2 and SLC19A2 (c.1409insT) were established. The cells were divided into empty virus group (control), wild-type group (overexpressed SLC19A2), and mutation group (overexpressed SLC19A2 (c.1409insT)). Quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and western blotting were used to detect the expression levels of ERS-PERK signaling pathway-related proteins, including glucose-regulated protein 78 (GRP78), protein kinase R-like ER kinase (PERK), and eukaryotic initiation factor 2 (eIF2α), activating transcription factor 4 (ATF4), and C/EBP homologous protein (CHOP) in islet β cells. Protein localization was assessed by immunofluorescence staining. Compared with the control group, the mRNA expression levels of SLC19A2 in wild-type and mutant islet β cells (INS.1 and β-TC-6) and HEK293T cells were significantly upregulated (all p < 0.05). Compared with the control group and the wild-type group, the mRNA expression levels of GRP78, PERK, eIF2α, ATF4, and CHOP were increased (all p < 0.05) in the mutant islet β cells; the protein expression levels of PERK, GRP78, and eIF2α were elevated (all p < 0.05). In addition, the results of immunofluorescence staining showed that SLC19A2 (c.1409insT) mutation changed the localization of the proteins in the cells. Thus, they were not located on the cell surface, but in the cytoplasm and nuclei, and protein aggregation occurred in the cytoplasm. 1. Islet β and HEK293Tcell lines, stably overexpressing SLC19A2 and SLC19A2 (c.1409insT) mutations, were successfully constructed. 2. SLC19A2 (c.1409insT) mutation could raise the expression levels of ERS-PERK signaling pathway-related proteins (GRP78, PERK, eIF2α, ATF4, and CHOP), and activate apoptosis pathway. 3. SLC19A2 (c.1409insT) mutation could change the localization of proteins and produce protein aggregation in cells. It could lead to protein misfolding and ERS, which would participate in the pathological mechanism of hyperglycemia induced by TRMA.

Cholesterol accumulation in hepatocytes mediates IRE1/p38 branch of endoplasmic reticulum stress to promote nonalcoholic steatohepatitis.

Non-alcoholic fatty liver disease (NAFLD), based on the elevating obesity incidence, is one of the major health issue worldwide. Transition from NAFLD to non-alcoholic steatohepatitis (NASH) is driven by increased apoptosis and is relevant to higher morbidity rates. In regard to limited understanding on cholesterol mediated hepatocyte alterations in NALFD/NASH transition, we investigated endoplasmic reticulum (ER) stress and related apoptosis. Our findings suggest that cholesterol upregulates ER stress and enhances C/EBP homologous protein (CHOP) either in hypercholesterolemic rabbits or in hepatocytes treated with liposome-cholesterol complex. Mechanistically, cholesterol accumulation in hepatocytes activates IRE1/p38 branch of ER stress, stimulating CHOP levels. In liver tissues of cholesterol fed rabbits, α-tocopherol supplementation decreased IRE1/p38/CHOP activation and prevented NASH development. Thus, our study provides a critical role of hepatocyte cholesterol in inducing IRE1/p38/CHOP pathway and suggests novel candidates for therapeutic targets against NASH.

Roles of C/EBP-homologous protein and histone H3 lysine 4 methylation in arsenic-induced mitochondrial apoptosis in hepatocytes.

C/EBP-homologous protein (CHOP) and histone H3 lysine 4 (H3K4) methylation have been verified to be correlated with apoptosis, whereas their biological function in arsenic-induced hepatocyte apoptosis through the mitochondrial pathway is still unclear. This study aimed to explore the specific regulatory mechanism of CHOP and H3K4me1/2 in arsenic-induced mitochondrial apoptosis in hepatocytes. Apoptosis and proliferation results showed arsenic promoted apoptosis and inhibited cell growth in BRL-3A cells. Meanwhile, arsenic treatment significantly upregulated the 78-kDa glucose-regulated protein (GRP78), CHOP, su(var)-3-9,enhancer-of-zeste,trithorax (SET) domain containing 7/9 (SET7/9), H3K4me1/2, BIM and BAX expression, while markedly downregulated lysine-specific histone demethylase 1 (LSD1) and BCL2 expression. After down-regulating CHOP, LSD1, and (su(var)-3-9,enhancer-of-zeste,trithorax) domain-containing protein 7/9 (SET7/9) in BRL-3A cells by siRNA, silencing CHOP and SET7/9 notably attenuated the pro-apoptotic and anti-proliferative effects of arsenic treatment on BRL-3A cells, which was reversed after inhibiting LSD1. In addition, our results suggested that knockdown of CHOP altered the expression of mitochondrial-associated proteins BCL2 and BIM, whereas knockdown of LSD1 and SET7/8 regulated the level of H3K4me1/2 modification and BAX protein. Coupled with chromatin immunoprecipitation results, we found that the level of CHOP in the promoter regions of BCL2 and BIM was significantly increased in BRL-3A cells exposed to 30µmol/L NaAsO2 for 24h, whereas the levels of H3K4me1/2 in the promoter regions of BAX were unchanged. Collectively, these data indicated that arsenic triggered the mitochondrial pathway to induce hepatocyte apoptosis by up-regulating the levels of CHOP and H3K4me1/2.

Oxidative and endoplasmic reticulum stress develop adverse metabolic effects due to the high-fat high-fructose diet consumption from birth to young adulthood

AimsThe early postnatal dietary intake has been considered a crucial factor affecting the offspring later life metabolic status. Consistently, this study investigated the oxidative and endoplasmic reticulum (ER) stress interventions in the induction of adverse metabolic effects due to the high-fat high-fructose diet (HFHFD) consumption from birth to young adulthood in rat offspring. Materials and methodsAfter delivery, the dams with their pups were randomly allocated into the normal diet (ND) and HFHFD groups. At weaning, the male offspring were divided into ND-None, ND-DMSO, ND-4-phenyl butyric acid (4-PBA), HFHFD-None, HFHFD-DMSO, and HFHFD-4-PBA groups and fed on their respected diets for five weeks. Then, the drug was injected for ten days. Subsequently, glucose and lipid metabolism parameters, oxidative and ER stress markers, and Wolfram syndrome1 (Wfs1) expression were assessed. Key findingsIn the HFHFD group, anthropometrical parameters, plasma high-density lipoprotein (HDL), and glucose-stimulated insulin secretion and content were decreased. Whereas, the levels of plasma leptin, low-density lipoprotein (LDL) and glucose, hypothalamic leptin, pancreatic catalase activity and glutathione (GSH), pancreatic and hypothalamic malondialdehyde (MDA), binding immunoglobulin protein (BIP) and C/EBP homologous protein (CHOP), and pancreatic WFS1 protein were increased. 4-PBA administration in the HFHFD group, decreased the hypothalamic and pancreatic MDA, BIP and CHOP levels, while, increased the Insulin mRNA and glucose-stimulated insulin secretion and content. SignificanceHFHFD intake from birth to young adulthood through the development of pancreatic and hypothalamic oxidative and ER stress, increased the pancreatic WFS1 protein and impaired glucose and lipid homeostasis in male rat offspring.

RBM10 Regulates Embryonic Trophoblast Injury via Endoplasmic Reticulum Stress

Background: Previous studies shown that RNA binding motif proteins (RBM) participate in regulating various physiological processes such as cell autophagy, proliferation, and apoptosis, and are abnormally highly expressed in placental trophoblast cells intervened by hypoxia in vitro, but their molecular mechanisms regulating placental trophoblast damage remain unclear. This study aims to investigate the role and molecular mechanism of RBM10 in regulating hypoxia-induced placental trophoblast injury through endoplasmic reticulum stress. Methods: CCK-8 cell proliferation assay and Transwell cell invasion assay were applied to detect the proliferation and invasion ability of normal, hypoxic and RBM10 up-regulated plus hypoxic embryonic trophoblast cells, respectively. The expression of endoplasmic reticulum stress-related proteins (ERN-1) and C/EBP homologous protein (CHOP), apoptosis-related proteins B cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax) and Caspase-3, and autophagy-related proteins including microtubule-associated protein 1 light chain 3 (LC-3), Beclin-1 and P62 were also detected by western blot assays. The effects of hypoxia and overexpression of RBM10 on placental trophoblast apoptosis were examined using flow cytometry. Results: We found that the growth and invasion ability of placental trophoblast cells treated with hypoxia were significantly decreased (p &lt; 0.05), and the upregulation of RBM10 further led to the decrease of the growth and invasion ability of hypoxic placental trophoblast cells. In addition, hypoxia promoted the expression of endoplasmic reticulum stress-related proteins (p &lt; 0.05), which triggered apoptosis and autophagy of embryonic trophoblast cells. The data showed that embryonic trophoblast cells regulated cell injury by stimulating endoplasmic reticulum stress after hypoxia. after upregulation of RBM10 expression, the expression levels of endoplasmic reticulum stress-related proteins ERN-1 and CHOP were further increased (p &lt; 0.05), and the apoptosis rate of embryonic trophoblast cells was further increased (p &lt; 0.05). Conclusions: Overall, our findings suggest that post-hypoxia mediates autophagy in embryonic trophoblast cells through stimulation of endoplasmic reticulum stress, thereby promoting apoptosis. Overexpression of RBM10 levels regulates the proliferative, apoptotic capacity of trophoblast cells by affecting cellular endoplasmic reticulum stress. RBM10 plays an important role in regulating hypoxia-induced autophagy and apoptosis in trophoblast cells, and RBM10 upregulation can further stimulate endoplasmic reticulum stress-mediated autophagy and apoptosis in trophoblast cells.

Protective Effects of Shen-Yuan-Dan Capsule against Ischemia/Reperfusion Injury in Cardiomyocytes by Alleviating Endoplasmic Reticulum Stress.

Objective Endoplasmic reticulum (ER) stress leads to the accumulation of misfolded proteins and an active unfolded protein response (UPR). If the ER stress is not resolved, the UPR triggers activation of the apoptotic cell death program. It has been shown that ischemia/reperfusion (I/R) injury can induce apoptosis via the ER stress pathway. We previously found that Shen-Yuan-Dan capsule (SYDC), a widely used traditional Chinese medicine, reduces I/R injury. Here, we investigated whether SYDC protects against cardiomyocyte apoptosis by reducing ER stress during I/R injury and. if so, explored its mechanism of action. Methods We use forty male Wistar rats to prepare the SYDC pharmacological serum. An I/R injury model was established using cultures of neonatal rat ventricular myocytes where cells were exposed to 2 h of reduced oxygenation followed by 4 h of normal oxygenation. After treatment of cultured cells with serum containing SYDC for 4 h, reverse transcription polymerase chain reaction and western blotting were performed to assess the expression levels of target molecules. Results Ischemia/reperfusion (I/R) clearly decreased cell viability. Treatment of cells with SYDC in serum (5% and 10%) increased cell viability compared with control serum-treated I/R cardiomyocytes. The mRNA levels of glucose-regulated protein 78 (Grp78), C/EBP homologous protein (CHOP), and caspase-12 were significantly upregulated in the I/R group. The mRNA levels of Grp78, CHOP, and caspase-12 were significantly decreased in the 5% and 10% SYDC groups compared to the I/R group. The protein expression levels of Grp78, CHOP, and caspase-12 were significantly upregulated in the I/R group. Treatment of I/R cardiomyocytes with 5% or 10% SYDC reduced the expression levels of CHOP and caspase-12, while the control serum did not show this effect. Conclusions These findings demonstrate that SYDC alleviates ER stress and prevents ER stress-induced apoptosis via the CHOP-dependent pathway.

Tauroursodeoxycholic acid inhibits TGF-β1-induced renal fibrosis markers in cultured renal mesangial cells by regulating endoplasmic reticulum stress.

Chronic kidney disease (CKD) has a worldwide prevalence of higher than 10% with an increasing mortality rate. As it involves the deterioration of renal function, it represents a serious risk to human health and, if left untreated, significantly lowers the quality of the patient's life. CKD is characterized by renal fibrosis. Studies have shown that transforming growth factor β1 (TGF-β1), a key driving factor of renal fibrosis, is closely related to the activation of renal fibrosis pathways such as endoplasmic reticulum stress (ERS). Tauroursodeoxycholic acid (TUDCA), an endogenous bile acid derivative, can effectively inhibit endogenous ERS. Here, we explored the effects and actions of TUDCA on renal fibrosis by establishing a renal mesangial cell (RMC) model. The RMC was stimulated with TGF-β1, and PCR and western blotting were used to detect the expression of ERS-related chaperone proteins and fibrotic indicators. The expression of glucose-regulated protein 78 (GRP78) was silenced in RMC cells to investigate the role of GRP78 in renal fibrosis. Finally, PCR and western blotting were used to detect the effects of TUDCA on the expression of GRP78, C/EBP homologous protein (CHOP), α-smooth muscle actin (α-SMA), and fibronectin (FN) in the TGF-β1-stimulated RMCs. The results showed that TUDCA significantly downregulated TGF-β1-induced levels of GRP78, CHOP, α-SMA and FN in RMCs. In addition, downregulation of GRP78 inhibited the expression of FN and α-SMA in the RMCs. In conclusion, downregulation of GRP78 and CHOP expression is one of the mechanisms by which TUDCA inhibits TGF-β1-induced renal mesangial cell fibrosis.

The diagnostic value of endoplasmic reticulum stress-related specific proteins GRP78 and CHOP in patients with sepsis: a diagnostic cohort study.

BackgroundSepsis is a life-threatening disease with high mortality. Early diagnosis is critical as early treatment improves outcomes. The protein levels of glucose regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP), markers of endoplasmic reticulum stress (ERS) activation, were reported increasing rapidly and continuously in the serum of patients with sepsis. Therefore, they might serve as a potential biomarker for sepsis diagnosis. This study aimed to analyze the role of GRP78 and CHOP in the diagnosis of patients with sepsis.MethodsThis study enrolled a total of 92 infected patients with or without sepsis who were admitted to the intensive care unit (ICU) from February 1, 2018 to September 30, 2018. According to 2016 SCCM/ESICM Sepsis 3.0 diagnostic criteria, patients with sepsis were allocated into group I (sepsis infected group) and patients without sepsis were allocated into group II (non-sepsis infected group). Serum samples were collected on days 1, 2, 3, and 7 after admission to ICU, and the concentrations of GRP78 and CHOP in the serum were analyzed by enzyme-linked immunosorbent assay (ELISA). The diagnostic ability of GRP78, CHOP, and other traditional inflammatory markers was assessed with receiver operating characteristic (ROC)/area under the ROC curves (AUC) analysis. Patients were shortly follow-up for the 28-day mortality.ResultsSerum GRP78 and CHOP levels in group I patients were higher than that in group II patients (P=0.021, P=0.00, respectively). When GRP78 was used to diagnose sepsis, the maximum area under the ROC curve (AUC) was 0.771 (95% CI: 0.662–0.880) and the optimal threshold was 157.29 ng/L (sensitivity, 75.0%; specificity, 73.1%) on day 2. When CHOP was used for the diagnosis of sepsis, the maximum AUC was 0.813(95% CI: 0.721–0.906) and the optimal threshold was 4.915 ng/L (sensitivity, 57.7%; specificity, 96.2%) on day 2.ConclusionsCompared with traditional inflammatory markers, ERS-related specific proteins GRP78 and CHOP have better sensitivity and specificity in the diagnosis of sepsis, which is helpful for clinicians in the diagnosis of sepsis.