C/EBP Homologous Protein Protein Expression Research Articles

Role of C/EBP Homologous Protein in Vascular Stenosis After Carotid Artery Injury.

The study aims to explore the fluctuating expression of C/EBP Homologous Protein (CHOP) following rat carotid artery injury and its central role in vascular stenosis. Using in vivo rat carotid artery injury models and in vitro ischemia and hypoxia cell models employing human aortic endothelial cells (HAECs) and vascular smooth muscle cells (T/G HA-VSMCs), a comprehensive investigative framework was established. Histological analysis confirmed intimal hyperplasia in rat models. CHOP expression in vascular tissues was assessed using Western blot and immunohistochemical staining, and its presence in HAECs and T/G HA-VSMCs was determined through RT-PCR and Western blot. The study evaluated HAEC apoptosis, inflammatory cytokine secretion, cell proliferation, and T/G HA-VSMCs migration through Western blot, ELISA, CCK8, and Transwell migration assays. The rat carotid artery injury model revealed substantial fibrous plaque formation and vascular stenosis, resulting in an increased intimal area and plaque-to-lumen area ratio. Notably, CHOP is markedly elevated in vessels of the carotid artery injury model compared to normal vessels. Atorvastatin effectively mitigated vascular stenosis and suppresses CHOP protein expression. In HAECs, ischemia and hypoxia-induced CHOP upregulation, along with heightened TNFα, IL-6, caspase3, and caspase8 levels, while reducing cell proliferation. Atorvastatin demonstrated a dose-dependent suppression of CHOP expression in HAECs. Downregulation of CHOP or atorvastatin treatment led to reduced IL-6 and TNFα secretion, coupled with augmented cell proliferation. Similarly, ischemia and hypoxia conditions increased CHOP expression in T/G HA-VSMCs, which was concentration-dependently inhibited by atorvastatin. Furthermore, significantly increased MMP-9 and MMP-2 concentrations in the cell culture supernatant correlated with enhanced T/G HA-VSMCs migration. However, interventions targeting CHOP downregulation and atorvastatin usage curtailed MMP-9 and MMP-2 secretion and suppressed cell migration. In conclusion, CHOP plays a crucial role in endothelial injury, proliferation, and VSMCs migration during carotid artery injury, serving as a pivotal regulator in post-injury fibrous plaque formation and vascular remodeling. Statins emerge as protectors of endothelial cells, restraining VSMCs migration by modulating CHOP expression.

Relaxin-3 Ameliorates Diabetic Cardiomyopathy by Inhibiting Endoplasmic Reticulum Stress

Background This study is aimed at investigating whether relaxin-3 exhibits protective effects against cardiomyopathy in diabetic rats by suppressing ERS. Methods Eighty male SD rats were randomly divided into two groups: controls (n = 20) and diabetes (n = 60). The streptozotocin-treated rats were randomly divided into three groups: diabetic group (DM), low-dose relaxin-3 group (0.2 μg/kg/d), and high-dose relaxin-3 group (2 μg/kg/d). The myocardial tissues and collagen fiber were observed by hematoxylin and eosin (H&E) and Masson staining. Serum brain natriuretic peptide (BNP), troponin (TNI), myoglobin, interleukin (IL-17), interleukin (IL)-1α, and tumor necrosis factor (TNF)-α were determined by ELISA. The protein expression of glucose regulatory protein 78 (GRP78) and C/EBP homologous protein (CHOP) in the heart tissue of each group was detected by Western blot analysis. Results (1) HE and Masson staining indicated that relaxin-3 could attenuate myocardial lesions and myocardial collagen volume fraction. (2) BNP, TnI, and myoglobin in the DM group at four and eight weeks were significantly higher than in the controls (P < 0.01). The relaxin-3-treated groups showed significantly reduced serum BNP, TnI, and myoglobin levels compared with the DM group (P < 0.05). (3) IL-17, IL-1α, and TNF-α levels in the DM rats at 4 weeks were higher than in the controls (P < 0.05). Low or high dose of relaxin-3-treated groups showed reduced serum IL-17 and TNF-α levels compared with the DM group at four and eight weeks (P < 0.05). (4) CHOP and GRP78 protein expression was increased in the DM group at four and eight weeks compared with the controls (P < 0.01), and small and large doses of relaxin-3 significantly reduced GRP78 and CHOP protein expression. Conclusions Exogenous relaxin-3 ameliorates diabetic cardiomyopathy by inhibiting ERS in diabetic rats.

Inhibitory Effect of Trihydroxy Isoflavone on Neuronal Apoptosis in Natural Aging Rats.

Objective To explore the impact of genistein (Gen) on the apoptosis of neuronal cells in naturally aged rats and its mechanism. Methods Fifty SD male rats were allocated into five groups at random, including youth group (3M group), natural aging group (24M group), and Gen low-, medium-, and high-dose groups. Starting from 18 months of age, Gen 10, 30, and 60 mg-kg−1 were administered via gavage to the Gen low-, medium-, and high-dose groups, respectively, while the rats in the natural aging group was given saline by gavage until 24 months of age, and the drug was stopped for 1 d per week for 6 months. The protein expression of target genes was examined using western blotting. Results In contrast to the 3M group, the 24M group rats showed disturbed neuronal cell arrangement and massive cell degeneration. After 6 months of Gen intervention, in contrast to the 24M group, the neural cell pathology in the CA3 area of the hippocampus improved and cell apoptotic decreased observably. In contrast to the 3M group, the protein expression of c-Jun amino-terminal kinase (p-JNK), C/EBP homologous protein (CHOP), inflammatory vesicle 3-associated factor (NLRP3), cysteine protease-1 (Caspase-1), and apoptosis-related punctate protein (ASC) and downstream inflammatory factors in the hippocampus was obviously increased in the 24M group. In contrast to the 24M group, the protein expression of p-JNK, CHOP, NLRP3, Caspase-1, and ASC and downstream inflammatory factors in the hippocampus was observably declined in Gen groups. Conclusion Gen has a protective effect on hippocampal neurons in aging rat brain tissue via the inhibition of the ERS apoptotic signaling pathway and NLRP3 inflammatory vesicle activation.

Methylmercury Induces Mitochondria- and Endoplasmic Reticulum Stress-Dependent Pancreatic β-Cell Apoptosis via an Oxidative Stress-Mediated JNK Signaling Pathway.

Methylmercury (MeHg), a long-lasting organic pollutant, is known to induce cytotoxic effects in mammalian cells. Epidemiological studies have suggested that environmental exposure to MeHg is linked to the development of diabetes mellitus (DM). The exact molecular mechanism of MeHg-induced pancreatic β-cell cytotoxicity is still unclear. Here, we found that MeHg (1-4 μM) significantly decreased insulin secretion and cell viability in pancreatic β-cell-derived RIN-m5F cells. A concomitant elevation of mitochondrial-dependent apoptotic events was observed, including decreased mitochondrial membrane potential and increased proapoptotic (Bax, Bak, p53)/antiapoptotic (Bcl-2) mRNA ratio, cytochrome c release, annexin V-Cy3 binding, caspase-3 activity, and caspase-3/-7/-9 activation. Exposure of RIN-m5F cells to MeHg (2 μM) also induced protein expression of endoplasmic reticulum (ER) stress-related signaling molecules, including C/EBP homologous protein (CHOP), X-box binding protein (XBP-1), and caspase-12. Pretreatment with 4-phenylbutyric acid (4-PBA; an ER stress inhibitor) and specific siRNAs for CHOP and XBP-1 significantly inhibited their expression and caspase-3/-12 activation in MeHg-exposed RIN-mF cells. MeHg could also evoke c-Jun N-terminal kinase (JNK) activation and reactive oxygen species (ROS) generation. Antioxidant N-acetylcysteine (NAC; 1mM) or 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox; 100 μM) markedly prevented MeH-induced ROS generation and decreased cell viability in RIN-m5F cells. Furthermore, pretreatment of cells with SP600125 (JNK inhibitor; 10 μM) or NAC (1 mM) or transfection with JNK-specific siRNA obviously attenuated the MeHg-induced JNK phosphorylation, CHOP and XBP-1 protein expression, apoptotic events, and insulin secretion dysfunction. NAC significantly inhibited MeHg-activated JNK signaling, but SP600125 could not effectively reduce MeHg-induced ROS generation. Collectively, these findings demonstrate that the induction of ROS-activated JNK signaling is a crucial mechanism underlying MeHg-induced mitochondria- and ER stress-dependent apoptosis, ultimately leading to β-cell death.

Effects of endoplasmic reticulum stress-mediated CREB3L1 on apoptosis of glioma cells.

The association between endoplasmic reticulum stress (ERS) and apoptosis has been extensively studied. Cyclic adenosine monophosphate responsive element binding protein 3 like 1 (CREB3L1) has an important role in the development of glioma. In the present study, the potential association between ERS-induced apoptosis and CREB3L1 and its clinical implications were investigated. From a total of 30 cases, brain gliomas with different pathological grades surgically resected at the Department of Neurosurgery of the Affiliated Hospital of Guizhou Medical University (Guiyang, China) between January 2018 and January 2019 were collected. The expression of CREB3L1 and ERS-related proteins in gliomas with different degrees of malignancy was detected by immunohistochemistry. Furthermore, U87-MG glioma cells were cultured in vitro and treated with different concentrations of ERS inducer thapsigargin (TG). The Cell Counting Kit-8 (CCK-8) assay was performed to detect changes in cell activity at different incubation times and drug concentrations. Cell apoptosis was detected by Annexin Ⅴ-FITC/propidium iodide double staining and the protein expression levels of CREB3L1 and ERS were detected by western blot analysis. Immunohistochemical analysis suggested that the expression levels of CREB3L1, glucose-regulated protein, 78 kDa (GRP78) and C/EBP-homologous protein (CHOP) in World Health Organization (WHO) grade I glioma were higher than those in WHO grade Ⅱ-Ⅳ (all P<0.01). The results of the CCK-8 assay suggested that the activity of U87-MG glioma cells was significantly decreased after treatment with TG (all P<0.05), and this effect was time- and drug concentration-dependent. Flow cytometric analysis indicated that the apoptotic rate of the cells was increased, which was significant when the concentration of TG was 0.1 µmol/l (P<0.01). Furthermore, the protein expression of CREB3L1, GRP78 and CHOP in glioma cells treated with TG was increased (P<0.05). However, the expression level of Bcl-2 decreased (P<0.05). In conclusion, ERS may reduce the cell proliferative activity and promote apoptosis through mediating CREB3L1 expression. CREB3L1 may be a novel potential target for glioma therapy.

Upregulation of Zip14 correlates with induction of endoplasmic reticulum stress (ERS) in hypertrophied hearts of Dahl saltsensitive rats

Zinc is a trace element involved in maintaining cellular structure and function. Although zinc is associated with left ventricular hypertrophy (LVH), there have been few reports on this association. This study aimed to evaluate the correlation between Zip14 and expression of endoplasmic reticulum stress (ERS) associated molecules in hypertrophied hearts of rats. Dahl salt-sensitive rats were fed a high salt diet to establish a left ventricular hypertrophy (LVH) rat model. RT-PCR was used to determine Zip14, activating transcription factor (ATF4), ATF6, x-box-binding protein 1 (xBP1), C/EBP homologous protein (CHOP), immunoglobulin-binding protein (BiP) mRNA expression. Western blotting was used to evaluate Zip14, BiP, CHOP, GAPDH expression. Zinc levels were measured by Inductively Coupled Plasma Optical Emission Spectroscopy. The results indicated that compared with the Control group, Zip14 mRNA and protein expression in LVH rat hearts were markedly increased (P < 0.01). Zinc content in rat heart tissue was significantly increased in the LVH group compared with the Control group (P < 0.05). ATF4, ATF6, xBP1 mRNA expressions were increased in LVH rat hearts compared with Control hearts (P < 0.001). Compared with the Control group, CHOP and BiP mRNA and protein expression were markedly increased in LVH rat hearts (P < 0.05, P < 0.01). Linear regression models showed that Zip14 mRNA expressions were positively correlated with zinc concentration, ATF4 and ATF6 mRNA expressions in Control hearts (P = 0.0005, P = 0.0052, P = 0.0026, respectively) and LVH rat hearts (P < 0.0001, P = 0.0119, P = 0.0033, respectively). In conclusion, upregulation of Zip14 in LVH rat hearts correlated with zinc accumulation and induction of ERS.

Upregulation of Zip14 correlates with induction of endoplasmic reticulum stress (ERS) in hypertrophied hearts of Dahl saltsensitive rats

Zinc is a trace element involved in maintaining cellular structure and function. Although zinc is associated with left ventricular hypertrophy (LVH), there have been few reports on this association. This study aimed to evaluate the correlation between Zip14 and expression of endoplasmic reticulum stress (ERS) associated molecules in hypertrophied hearts of rats. Dahl salt-sensitive rats were fed a high salt diet to establish a left ventricular hypertrophy (LVH) rat model. RT-PCR was used to determine Zip14, activating transcription factor (ATF4), ATF6, x-box-binding protein 1 (xBP1), C/EBP homologous protein (CHOP), immunoglobulin-binding protein (BiP) mRNA expression. Western blotting was used to evaluate Zip14, BiP, CHOP, GAPDH expression. Zinc levels were measured by Inductively Coupled Plasma Optical Emission Spectroscopy. The results indicated that compared with the Control group, Zip14 mRNA and protein expression in LVH rat hearts were markedly increased (P < 0.01). Zinc content in rat heart tissue was significantly increased in the LVH group compared with the Control group (P < 0.05). ATF4, ATF6, xBP1 mRNA expressions were increased in LVH rat hearts compared with Control hearts (P < 0.001). Compared with the Control group, CHOP and BiP mRNA and protein expression were markedly increased in LVH rat hearts (P < 0.05, P < 0.01). Linear regression models showed that Zip14 mRNA expressions were positively correlated with zinc concentration, ATF4 and ATF6 mRNA expressions in Control hearts (P = 0.0005, P = 0.0052, P = 0.0026, respectively) and LVH rat hearts (P < 0.0001, P = 0.0119, P = 0.0033, respectively). In conclusion, upregulation of Zip14 in LVH rat hearts correlated with zinc accumulation and induction of ERS.

Endoplasmic reticulum stress promotes local immunoglobulin E production in allergic rhinitis.

BackgroundThe role of endoplasmic reticulum (ER) stress in the pathogenesis of allergic rhinitis (AR) remains elusive.MethodsReal‐time polymerase chain reaction (RT‐PCR), immunohistochemistry, and western blotting analyses were performed to detect the expression of ER stress and unfolded protein response markers: 78‐kDa glucose‐regulated protein (GRP78), C/EBP homologous protein (CHOP), activating transcription factor 6 (ATF6α), spliced X‐box binding protein 1 (sXBP‐1), and phosphorylated eukaryotic initiation factor 2α (p‐eIF2α), in inferior turbinate tissue samples from patients with AR and non‐AR controls. Nasal tissues from patients with AR were cultured ex vivo and treated with 4‐phenylbutyric acid (4‐PBA), an ER stress inhibitor.ResultsCompared to those in non‐AR controls, the mRNA and protein levels of GRP78, CHOP, ATF6α, sXBP‐1, and p‐eIF2α were significantly increased in nasal tissues from patients with AR. GRP78 and CHOP were mainly expressed in CD138+ plasma cells in nasal tissues from patients with AR. The frequency of IgE+CD138+ plasma cells was significantly higher in nasal tissues from patients with AR than that in non‐AR controls. IgE levels in nasal secretions and tissues were positively correlated with GRP78 and CHOP mRNA levels in the nasal tissues. After 4‐PBA treatment, the protein expression of GRP78, CHOP, ATF6α, sXBP‐1, and p‐eIF2α was significantly reduced in cultured AR‐derived nasal tissues, and IgE levels were simultaneously decreased in cultured supernatants.ConclusionsER stress may be involved in the regulation of local IgE production in patients with AR. Inhibition of ER stress potentially provides a therapeutic avenue in AR by reducing local IgE production.Level of EvidenceNA

The role of C/EBP homologous protein (CHOP) in regulating macrophage polarization in RAW264.7 cells.

Schistosomiasis is a zoonotic parasitic disease that is endemic in Asia. Macrophages are mainly involved in the inflammatory response of late schistosoma infection. Our previous study found that C/EBP homologous protein (CHOP) expression is significantly increased, and M2 macrophages are activated in schistosome-induced liver fibrosis mice. However, the role of CHOP in the regulation of macrophage polarization remains to be further studied. Western blotting or quantitative PCR revealed that IL-4 increased the expression of arginase-1, macrophage mannose receptor 1, phosphorylation signal transducer and activator of transcription 6 (p-STAT6), Krüppel-like factor 4 (KLF4), CHOP, and IL-13 receptor alpha (IL-13Rα) and induced M2 polarization in RAW264.7 as measured by flow cytometry. Inhibiting STAT6 phosphorylation (AS1517499) reduced the IL-4-induced expression of KLF4, CHOP, and IL-13Rα and also the number of M2 macrophages. The overexpression of CHOP stimulated M2 polarization, but AS1517499 inhibited this effect. CHOP increased the protein expression of KLF4 but did not change the expression of p-STAT6. Soluble egg antigen (SEA) could promote the IL-4-induced protein expression of p-STAT6, CHOP, and KLF4. Overall, the findings show that SEA can promote the activation of M2 macrophages by causing increased CHOP-induced KLF4 levels and activation of STAT6 phosphorylation.

P0015SIRT2 REGULATES CISPLATIN-INDUCED ENDOPLASMIC RETICULUM STRESS THROUGH HEAT SHOCK FACTOR 1 DEACETYLATION

Abstract Background and Aims Nephrotoxicity is an important cisplatin-induced adverse reaction and restricts the use of cisplatin to treat malignant tumors. Endoplasmic reticulum (ER) stress is caused by the accumulation of misfolded proteins, and is induced by cisplatin in kidneys. SIRT2 nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase is a member of the sirtuin family, but its role in cisplatin-induced ER stress remains unclear. Method To investigate the effect of SIRT2 on cisplatin-induced ER stress using SIRT2 knockout mice and human proximal tubular epithelial cells (HK-2 cells). We treated cisplatin (20 µg/mL) or induced by intraperitoneal injection of cisplatin (20 mg/kg) and evaluated the changes of ER stress and its signal mechanism. Results Cisplatin administration was found to significantly increase the expressions of PRKR-like ER kinase (PERK), phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), and the C/EBP homologous protein (CHOP) and caspase-12 in the kidneys of SIRT2-wild type mice. However, cisplatin-induced increases in the expressions of p-PERK, p-eIF2α, CHOP and, caspase-12 were diminished in kidneys of SIRT2 knockout mice. In vitro, cisplatin significantly increased the expressions of p-PERK, p-eIF2α, CHOP, and caspase-12 in HK-2 cells. When the effect of SIRT2 on cisplatin-induced ER stress was evaluated using SIRT2-siRNA (ON-TARGET plus human SIRT2 siRNA) or the SIRT2 inhibitors, AGK2 and AK1, knockdown or inhibition of SIRT2 significantly attenuated the cisplatin-induced protein expression of p-PERK, p-eIF2α, CHOP, and caspase-12. Immunoprecipitation studies showed SIRT2 bound physically to heat shock factor (HSF)1 and that HSF1 acetylation was significantly increased by cisplatin. In addition, knockdown of SIRT2 increased cisplatin-induced HSF1 acetylation and increased the expression of heat shock protein (HSP)70. Conclusion These observations suggest that suppression of SIRT2 ameliorates cisplatin-induced ER stress by increasing HSF1 acetylation and HSP expression.

ΜicroRNA‑421 participates in vitiligo development through regulating human melanocyte survival by targeting receptor‑interacting serine/threonine kinase 1.

Vitiligo is a common localized or generalized skin pigmentation disorder. Endoplasmic reticulum (ER) stress may be implicated in the development of vitiligo. microRNA-421 (miR-421) has been reported to be dysregulated in various human tumors. However, there is no report to date on the role of miR-421 in vitiligo development. The present study demonstrated that 3 µM tunicamycin (TM) increased the expression of the ER stress-related proteins protein kinase RNA-like endoplasmic reticulum kinase (PERK), α subunit of eukaryotic translation initiation factor 2 (eIF2α) and C/EBP homologous protein (CHOP) in human primary epidermal melanocytes. Moreover, TM suppressed melanocyte viability and induced apoptosis. Reverse transcription-quantitative PCR analysis demonstrated that TM promoted miR-421 expression in human melanocytes. Next, TargetScan and dual luciferase reporter gene assay indicated that receptor-interacting serine/threonine kinase 1 (RIPK1) was a direct target of miR-421. RIPK1 expression was significantly downregulated in TM-induced human melanocytes. Subsequently, the effect of miR-421 downregulation on the damage of human melanocytes induced by ER stress was investigated. Human melanocytes were transfected with inhibitor control, miR-421 inhibitor, miR-421 inhibitor + control-short hairpin (sh)RNA, or miR-421 inhibitor + RIPK1-shRNA for 24 h and then treated with TM (3 µM) for 48 h. TM was found to upregulate PERK, eIF2α and CHOP protein expression in human melanocytes, which was reduced by an miR-421 inhibitor. In addition, the miR-421 inhibitor increased viability and reduced apoptosis in TM-treated melanocytes. Furthermore, all these effects of the miR-421 inhibitor on TM-induced human melanocytes were reversed by RIPK1-shRNA. Further analyses revealed that the miR-421 inhibitor activated the phosphoinositide 3 kinase/protein kinase B/mammalian target of rapamycin signaling pathway in TM-induced human melanocytes. These data collectively suggest that miR-421 may serve as a new treatment target in vitiligo development.

Cadmium exposure induces pancreatic β-cell death via a Ca2+-triggered JNK/CHOP-related apoptotic signaling pathway

Cadmium (Cd) is known to be ranked the 7th hazardous substance in the Substance Priority List by Agency for Toxic Substances and Disease Registry. The experimental and epidemiological data have suggested that Cd is linked to the development of diabetes mellitus (DM). The molecular mechanism of Cd on the pancreatic β-cell cytotoxicity still remains unclear. Evidence has pointed toward that Ca2+ is an important regulator of toxic insult-induced β-cell cytotoxicity. The role of Ca2+ in the Cd-induced β-cell cytotoxicity is still unknown. In this study, we found that Cd exposure significantly inhibited insulin secretion and cell viability in the pancreatic β-cell-derived RIN-m5F cells. Cd exposure induced apoptotic events, including the increased populations of apoptotic cells and sub-G1 hypodiploid cells, and caspase-3/-7/-9 and poly (ADP-ribose) polymerase (PARP) activation, which largely depended on the activation of c-Jun N-terminal kinase (JNK) and C/EBP homologous protein (CHOP). Transfection with siRNAs for JNK and CHOP or pretreatment with specific pharmacological inhibitor of JNK (SP600125) in β-cells effectively prevented the Cd-induced insulin secretion dysfunction and apoptosis. JNK-specific siRNA dramatically suppressed Cd-induced JNK phosphorylation and CHOP protein expression, but JNK phosphorylation could not be inhibited by CHOP-specific siRNA. Furthermore, Cd exposure significantly increased the intracellular calcium ([Ca2+]i) levels. Buffering the Ca2+ response with BAPTA/AM effectively abrogated the Cd-induced [Ca2+]i elevation, insulin secretion dysfunction, apoptosis, and protein expression of JNK phosphorylation and CHOP activation. Taken together, these findings demonstrated that Cd exposure exerts β-cell death via a [Ca2+]i-dependent JNK activation-activated downstream CHOP-related apoptotic signaling pathway.

MON-201 ER Stress Activated by Androgen Enhances Apoptosis of Granulosa Cells via Induction of Death Receptor 5 in PCOS

PCOS is associated with hyperandrogenism and growth arrest of antral follicles. Excess androgens work as a stage-specific inhibitor of a follicular growth suppressing the growth of late-stage follicles. Previously, we found that endoplasmic reticulum (ER) stress is activated in granulosa cells of antral follicles in PCOS (1). This is evidenced by the activation of various unfolded protein response (UPR) genes, including a pro-apoptotic UPR transcription factor, C/EBP homologous protein (CHOP). Death receptor (DR) 5, which harbors a CHOP-binding site in its promoter region, plays a crucial role in ER stress-induced apoptosis by increasing autocrine death-ligand signal, independent of its extracellular ligand TNF-related apoptosis-inducing ligand (TRAIL) (2). Based on these observations, we hypothesized that ER stress is activated by androgen in granulosa cells of antral follicles in PCOS and activated ER stress promotes apoptosis via induction of CHOP and subsequent activation of DR5. Using RT-PCR (qPCR), we showed that testosterone induced the mRNA expression of various UPR factors including XBP1(S), HSPA5, ATF4 and CHOP along with DR5 in cultured human granulosa-lutein cells (GLCs). Treatment with an ER stress inhibitor, tauroursodeoxycholic acid (TUDCA), inhibited testosterone-induced mRNA and protein expression of DR5 and CHOP, with a concomitant reduction in apoptosis examined by qPCR, western blotting and flow cytometry respectively. Furthermore, knockdown of CHOP or DR5 by RNA interference inhibited testosterone-induced apoptosis and knockdown of CHOP reduced testosterone-induced DR5 mRNA expression. Treatment with an androgen receptor (AR) antagonist, flutamide, as well as the knockdown of AR, decreased testosterone-induced DR5 and CHOP mRNA expression and apoptosis. In addition, expression of DR5 and CHOP was upregulated in GLCs of PCOS patients and granulosa cells of antral follicles in ovarian sections obtained from both PCOS patients and dehydroepiandrosterone (DHEA)-induced PCOS mice, proven by qPCR and immunohistochemistry (IHC). Treatment of PCOS mice with TUDCA decreased apoptosis and DR5 expression in granulosa cells of antral follicles, with a concomitant reduction in CHOP expression, shown by TUNEL and IHC staining. Our findings indicate that ER stress activated by hyperandrogenism in PCOS promotes apoptosis of granulosa cells of antral follicles via induction of DR5. Reference: (1) Takahashi N, Harada M, et al. Sci Rep 2017, 7:10824 (2) Lu M et al. Science 2014;345:98-101.

Kaempferol protects mice from d-GalN/LPS-induced acute liver failure by regulating the ER stress-Grp78-CHOP signaling pathway

Kaempferol is a flavonoid compound that has many functions, such as anti-inflammation and antioxidation. Acute liver failure (ALF) is a life-threatening illness accompanied by serious inflammation and extensive hepatocyte apoptosis. The aim of this study was to examine the therapeutic potential of kaempferol and its mechanism in ALF. In a murine ALF model induced by d-galactosamine (d-GalN, 700 mg/kg) / lipopolysaccharide (LPS, 10 μg/kg), mice were pretreated with kaempferol at 2 h before d-GalN/LPS administration and then sacrificed 6 h after d-GalN/LPS injection. Lethality, liver damage, endoplasmic reticulum(ER) stress, hepatocyte viability and apoptosis were evaluated. Whether pretreatment of kaempferol protected hepatocytes from ER stress-induced apoptosis was detected in vitro. Pretreatment of kaempferol decreased lethality, prolonged the survival time and significantly protected against liver injury, which was indicated by decreased transaminase levels and the well-preserved liver structure. The protective effect of kaempferol on the ALF mouse model was achieved by inhibiting hepatocyte apoptosis. Moreover, pretreatment of kaempferol increased the expression of glucose-regulated/binding immunoglobulin protein 78 (Grp78), decreased the expression of C/EBP-homologous protein (CHOP), and protected hepatocytes from ER stress-induced apoptosis in vitro. Our results showed that pretreatment of Grp78 siRNA partially negated the hepatic protection from kaempferol and reversed the inhibition of CHOP protein expression in d-GalN/LPS-induced ALF mice. In conclusion, kaempferol inhibits hepatocyte apoptosis to protect mice from liver failure by regulating the ER stress-Grp78-CHOP signaling pathway. Therefore, kaempferol may be used to treat ALF.

Baicalein stimulates fibroblast growth factor 21 expression by up-regulating retinoic acid receptor-related orphan receptor α in C2C12 myotubes

Retinoic acid receptor-related orphan receptor (ROR) α has been implicated in various physiological functions, including the immune system, inflammation, and circadian rhythms. In the present study, the synthetic RORα/γ agonist SR1078 stimulated the production and gene expression of fibroblast growth factor 21 (FGF21) in C2C12 myotubes. FGF21, a member of the FGF family, plays important roles in the regulation of peripheral glucose tolerance and lipid metabolism and improves metabolic health. The mRNA expression and secretion of FGF21 was significantly weaker in Rora-silenced cells than in cells transfected with non-targeting control siRNA. SR1078 significantly up-regulated C/EBP homologous protein (CHOP), an established marker of ER stress, in a dose-dependent manner in C2C12 myotubes, while CHOP expression was decreased in Rora-silenced C2C12 cells, suggesting that RORα is involved in the regulation of FGF21 expression and stimulates ER stress in C2C12 myotubes. The naturally occurring compound baicalein up-regulated FGF21 expression and secretion in C2C12 myotubes. Additionally, the up-regulation of CHOP mRNA and protein expression was observed in C2C12 myotubes after the baicalein treatment. Furthermore, the knockdown of RORα prevented the augmentation of FGF21 and up-regulation of CHOP in response to baicalein in C2C12 cells. Collectively, these results suggest that baicalein stimulates the ER stress response and FGF21 expression through an RORα-dependent mechanism in C2C12 myotubes, and indicate the potential of baicalein as an effective anti-obesity therapy via its ability to enhance FGF21 production.

Exercise Training Attenuates Non-Alcoholic Fatty Liver Disease in rats with Diabetes via Endoplasmic Reticulum Stress

Non-alcoholic fatty liver disease (NAFLD) is highly prevalent in type 2 diabetes (T2D). Recent evidence supports that endoplasmic reticulum (ER) stress plays a critical role in the development of NAFLD. Although exercise training has been commonly prescribed for the treatment of NAFLD, the effects of different types of exercise on ER stress in NAFLD under type 2 diabetic condition are largely unknown. PURPOSE: The purpose of this study was to determine the effects of aerobic and resistance exercises on hepatic ER stress response and NAFLD in rats with T2D. METHODS: Male Sprague-Dawley rats were randomly assigned to four groups (n=10/group): Control (CON), T2D, T2D with aerobic exercise (T2D+AE; treadmill walking at 30 m/min, 0° incline, 60 mins/day, 5 day/week, for 8 weeks) and T2D with resistance exercise (T2D+RE; climbing a 80° incline vertical ladder with weights progressively increased from 50% to 100% of maximal carrying capacity on the tail, 3 times/day, 5 days/ week, for 8 weeks). Liver tissue samples were collected for histopathological analysis of the density of lipid droplets, and immunoblot analysis of expression levels of ER stress proteins, including glucose-regulated protein (GRP78), C/EBP homologous protein (CHOP), caspase 12, and c-Jun N-terminal kinase (JNK). One-way ANOVAs and Tukey’s -test were used for data analysis. RESULTS: The density of lipid droplets in the liver was significantly higher in the T2D group than in the CON group (p<0.01), but was significantly lower in the T2D+AE and T2D+RE groups when compared to the T2D group (both p<0.01). In addition, the T2D group had significantly higher levels of protein expression of GRP78, CHOP, caspase 12 and JNK when compared to the CON group (all p<0.01). Both exercise groups had significantly lower levels of protein expression of GRP 78, CHOP, Caspase 12 and JNK when compared to the T2D group (p<0.05 to p<0.01). CONCLUSION: Our findings suggest that both aerobic and resistance exercises are protective against NAFLD in rats with T2D by potentially regulating proteins involved in ER stress response.

WITHDRAWN: Inhibition of CHOP weakens the promotion of chronic intermittent hypoxia on epithelial cells apoptosis via inhibiting PI3K-Akt and NF-κB signaling pathways

// Yi-Bin Wang 1, * , Yin Ni 2, * , Ren-Hua Sun 2 , Xiao-Zhou Mou 3 , Fang Han 2 , Qian Li 2 , Hai-Jun Huang 4 , Jing-Quan Liu 2 and Yue-Xing Tu 2 1 Department of Cardiology, Chun’an First People’s Hospital, Zhejiang Provincial People’s Hospital Chun’an Branch, Hangzhou, Zhejiang 311700, China 2 Department of Critical Care Medicine, Zhejiang Provincial People’s Hospital, People’s Hospital of Hangzhou Medical College, Hangzhou, Zhejiang 310014, China 3 Clinical Research Institute, Zhejiang Provincial People’s Hospital, People’s Hospital of Hangzhou Medicine College, Hangzhou, Zhejiang 310014, China 4 Department of Infectious Diseases, Zhejiang Provincial People’s Hospital, People’s Hospital of Hangzhou Medical College, Hangzhou, Zhejiang 310014, China * These authors contributed equally to this work Correspondence to: Yue-Xing Tu, email: tuyuexing_12@126.com Keywords: chronic intermittent hypoxia; CHOP; PI3K/Akt; NF-κB Received: September 01, 2017 Accepted: November 23, 2017 Published: January 02, 2018 ABSTRACT Objective: To explore the role of transcription factor C/EBP homologous protein (CHOP) on chronic intermittent hypoxia (CIH) epithelial cells and the related molecular mechanism. Results: Expression of CHOP in CIH epithelial cells was significantly higher than the control. CIH promoted the apoptosis of epithelial cells, while transfection of siRNA-CHOP reduced the promotion by CIH. Meanwhile, CIH increased the protein expression cleaved-caspase-3, cleaved-caspase-12 and Bax/Bcl-2. The protein expression of P-PI3K, P-AKT and NF-κB were also markedly increased by CIH, while knockdown of CHOP showed inhibition effect against the increase of P-PI3K, P-AKT and NF-κB by CIH. Conclusion: Inhibition of CHOP could inhibit the promotion of CIH on the apoptotic rate of epithelial cells via inhibition of PI3K/Akt and NF-κB signaling pathway. Methods: In the present study, we used Western blotting was to determine the protein expression of CHOP, interleukin (IL)-1β, monocyte chemoattractant protein (MCP)-1, tumor necrosis factor (TNF)-α, and cell apoptosis-related factors (cleaved-caspase-3, cleaved-caspase-12, Bcl-2 and Bax) in CIH cells and the control cells. Western blotting and RT-PCR were applied to verify the transfection effect of siRNA-CHOP on epithelial cells. Then the protein expression of activating transcription factor 6 (ATF6), P-PI3K, PI3K, P-AKT, P-AKT and nuclear factor-kappaB (NF-κB) in control or CIH cells was determined after transfection with siRNA-CHOP. Apoptpsis of epithelial cells was determined by flow cytometry assay (FCM) after transfection with siRNA-CHOP.

Necrostatin-1 Mitigates Endoplasmic Reticulum Stress After Spinal Cord Injury

Necrostatin-1 (Nec-1) has been shown to inhibit necroptosis and convey a significant protective effect after spinal cord injury (SCI). This small molecule inhibitor may reduce tissue damage and restore neurological function by lessening mitochondrial injury after SCI and preserving energy homeostasis. However, the effects of Nec-1 on endoplasmic reticulum stress (ERS)-an important pathological consequence of SCI-are still not clear. The present study investigates the relationship between necroptosis and ERS in a rat model of SCI. Electron microscopy was employed to observe ultra-structural changes in the endoplasmic reticulum and mitochondria after lesioning. Real-time quantitative PCR was used to measure the mRNA levels of ERS-related pro-apoptotic molecules such as C/EBP homologous protein (CHOP), immunoglobulin-binding protein (BiP/GRP78) and X box-binding protein-1 (XBP-1). Western blot and immunofluorescence were conducted to analyze CHOP, GRP78 and XBP-1 protein expression after lesioning. Results demonstrated that applying Nec-1 in SCI reduces ultra-structural damage to the endoplasmic reticulum and mitochondria and inhibits expression of ERS-related genes and proteins after lesioning. Immunofluorescence also shows ERS-related proteins mainly expressed in the cytoplasm of nerve cells. Taken together, these results demonstrate that Nec-1 has protective effect on the endoplasmic reticulum and mitochondria and alleviates ERS after SCI.

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

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

Mechanism of pingyangmycin-induced apoptosis of cultured human umbilical vein endothelial cells.

In this study, we investigated the effects of pingyangmycin (PYM) on the growth inhibition and apoptosis of human umbilical vein endothelial cells (HUVEC). In this study, we aimed to explore the optimal concentration of PYM to induce the apoptosis of HUVEC and to determine its mechanism of action. After treatment of HUVEC with different concentrations of PYM for 24 h, cell counting kit-8 (CCK-8) was used to detect growth inhibiting effects. Annexin V-FITC/propidium iodide stain was used to detect apoptosis, and western blot was used to detect the expression of glucose-related protein 78 (GPR78) and C/EBP homologous protein (CHOP) endoplasmic reticulum stress proteins. With increasing PYM concentration, the growth inhibition of HUVEC increased (P < 0.05), the apoptotic numbers of HUVEC increased (P < 0.05), with higher PYM concentrations inducing necrosis, and the protein expression of GRP78 and CHOP increased (P < 0.05). PYM could obviously inhibit the proliferation and promote the apoptosis of HUVEC. Necrotic cells were more prevalent than apoptotic cells at high PYM concentrations. This study helped to determine the proper concentration of PYM to induce more apoptosis than necrosis, which is critical to minimize inflammation, enhance the healing of the skin, and maintain safety for the patient. PYM might induce HUVEC apoptosis through the endoplasmic reticulum stress pathway.