C/EBP Homologous 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.

Ursolic acid alleviates paclitaxel-induced peripheral neuropathy through PPARγ activation

BackgroundChemotherapy-induced peripheral neuropathy (CIPN) reduces the overall quality of life and leads to interruption of chemotherapy. Ursolic acid, a triterpenoid naturally which presents in fruit peels and in many herbs and spices, can function as a peroxisome proliferator-activated receptor γ (PPARγ) agonist, and has been widely used as an herbal medicine with a wide spectrum of pharmacological activities, including anti-cancer, anti-inflammatory and neuroprotective effect. MethodsWe used a phenotypic drug screening approach to identify ursolic acid as a potential neuroprotective drug in vitro and in vivo and carried out additional biochemical experiments to identify its mechanism of action. ResultsOur study demonstrated that ursolic acid reduced neurotoxicity and cell apoptosis induced by pacilitaxel, resulting in an improvement of CIPN. Moreover, we explored the potential mechanisms of ursolic acid on CIPN. As a result, ursolic acid inhibited CHOP (C/EBP Homologous Protein) expression, indicating the endoplasmic reticulum (ER) stress suppression, and regulating CHOP related apoptosis regulator (the Bcl2 family) to reverse pacilitaxel induced apoptosis. Moreover, we showed that the therapeutic effect of ursolic acid on the pacilitaxel-induced peripheral neuropathy is PPARγ dependent. ConclusionsTaken together, the present study suggests ursolic acid has potential as a new PPARγ agonist targeting ER stress-related apoptotic pathways to ameliorate pacilitaxel-induced peripheral neuropathic pain and nerve injury, providing new clinical therapeutic method for CIPN.

Mechanism of Qili Qiangxin Capsule for Heart Failure Based on miR133a-Endoplasmic Reticulum Stress.

To investigate the pharmacological mechanism of Qili Qiangxin Capsule (QLQX) improvement of heart failure (HF) based on miR133a-endoplasmic reticulum stress (ERS) pathway. A left coronary artery ligation-induced HF after myocardial infarction model was used in this study. Rats were randomly assigned to the sham group, the model group, the QLQX group [0.32 g/(kg·d)], and the captopril group [2.25 mg/(kg·d)], 15 rats per group, followed by 4 weeks of medication. Cardiac function such as left ventricular ejection fraction (EF), fractional shortening (FS), left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), the maximal rate of increase of left ventricular pressure (+dp/dt max), and the maximal rate of decrease of left ventricular pressure (-dp/dt max) were monitored by echocardiography and hemodynamics. Hematoxylin and eosin (HE) and Masson stainings were used to visualize pathological changes in myocardial tissue. The mRNA expression of miR133a, glucose-regulated protein78 (GRP78), inositol-requiring enzyme 1 (IRE1), activating transcription factor 6 (ATF6), X-box binding protein1 (XBP1), C/EBP homologous protein (CHOP) and Caspase 12 were detected by RT-PCR. The protein expression of GRP78, p-IRE1/IRE1 ratio, cleaved-ATF6, XBP1-s (the spliced form of XBP1), CHOP and Caspase 12 were detected by Western blot. TdT-mediated dUTP nick-end labeling (TUNEL) staining was used to detect the rate of apoptosis. QLQX significantly improved cardiac function as evidenced by increased EF, FS, LVSP, +dp/dt max, -dp/dt max, and decreased LVEDP (P<0.05, P<0.01). HE staining showed that QLQX ameliorated cardiac pathologic damage to some extent. Masson staining indicated that QLQX significantly reduced collagen volume fraction in myocardial tissue (P<0.01). Results from RT-PCR and Western blot showed that QLQX significantly increased the expression of miR133a and inhibited the mRNA expressions of GRP78, IRE1, ATF6 and XBP1, as well as decreased the protein expressions of GRP78, cleaved-ATF6 and XBP1-s and decreased p-IRE1/IRE1 ratio (P<0.05, P<0.01). Further studies showed that QLQX significantly reduced the expression of CHOP and Caspase12, resulting in a significant reduction in apoptosis rate (P<0.05, P<0.01). The pharmacological mechanism of QLQX in improving HF is partly attributed to its regulatory effect on the miR133a-IRE1/XBP1 pathway.

The ameliorative effects of cannabidiol on methotrexate-induced neuroinflammation and neuronal apoptosis via inhibiting endoplasmic reticulum and mitochondrial stress.

Methotrexate (MTX) is an antineoplastic agent and has neurotoxic effects. It exerts its toxic effect on the brain by triggering inflammation and apoptosis. Cannabidiol (CBD) is an agent known for its antioxidant, anti-inflammatory effects in various tissues. The aim of this study is to examine the protective effects of CBD treatment in various brain structures from MTX damage and to evaluate the effect of intracellular pathways involved in apoptosis. Thirty-two adult Wistar Albino female rats were divided into four groups as control, MTX (20 mg/kg intraperitoneally [i.p.]), MTX + CBD (0.1 mL of 5 mg/kg i.p.), and CBD (for 7 days, i.p.). At the end of the experiment, brain tissues collected for biochemical analyses as total oxidant status (TOS), total antioxidant status, oxidative stress index (OSI), histopathological and immunohistochemical analyses as tumor necrosis factor-α (TNF-α), serotonin, mammalian target of rapamycin (mTOR) staining, genetic analyses as caspase-9 (Cas-9), caspase-12 (Cas-12), C/EBP homologous protein (CHOP), and cytochrome-c (Cyt-c) gene expressions. In the histopathological and immunohistochemical evaluation, hyperemia, microhemorrhage, neuronal loss, and significant decreasing expressions of seratonin were observed in the cortex, hippocampus, and cerebellum regions in the MTX group. mTOR, TNF-α, Cas-9, Cas-12, CHOP, and Cyt-c expressions with TOS and OSI levels were increased in the cortex. It was observed that these findings were reversed after CBD application in all regions. MTX triggers neuronal apoptosis via endoplasmic reticulum and mitochondrial stress while destroying serotonergic neurons. The reversal of the pathological changes with CBD treatment proves that it has anti-inflammatory and antiapoptotic activity in brain.

The Preventive Effects of Salubrinal against Pyrethroid-Induced Disruption of Adult Hippocampal Neurogenesis in Mice.

Environmental factors, including pesticide exposure, have been identified as substantial contributors to neurodegeneration and cognitive impairments. Previously, we demonstrated that repeated exposure to deltamethrin induces endoplasmic reticulum (ER) stress, reduces hippocampal neurogenesis, and impairs cognition in adult mice. Here, we investigated the potential relationship between ER stress and hippocampal neurogenesis following exposure to deltamethrin, utilizing both pharmacological and genetic approaches. To investigate whether ER stress is associated with inhibition of neurogenesis, mice were given two intraperitoneal injections of eIf2α inhibitor salubrinal (1 mg/kg) at 24 h and 30 min prior to the oral administration of deltamethrin (3 mg/kg). Salubrinal prevented hippocampal ER stress, as indicated by decreased levels of C/EBP-homologous protein (CHOP) and transcription factor 4 (ATF4) and attenuated deltamethrin-induced reductions in BrdU-, Ki-67-, and DCX-positive cells in the dentate gyrus (DG) of the hippocampus. To further explore the relationship between ER stress and adult neurogenesis, we used caspase-12 knockout (KO) mice. The caspase-12 KO mice exhibited significant protection against deltamethrin-induced reduction of BrdU-, Ki-67-, and DCX-positive cells in the hippocampus. In addition, deltamethrin exposure led to a notable upregulation of CHOP and caspase-12 expression in a significant portion of BrdU- and Ki-67-positive cells in WT mice. Conversely, both salubrinal-treated mice and caspase-12 KO mice exhibited a considerably lower number of CHOP-positive cells in the hippocampus. Together, these findings suggest that exposure to the insecticide deltamethrin triggers ER stress-mediated suppression of adult hippocampal neurogenesis, which may subsequently contribute to learning and memory deficits in mice.

Fasudil inhibits the expression of C/EBP homologous protein to protect against liver injury in acetaminophen-overdosed mice

Acetaminophen (APAP) overdoses can cause severe liver injury. In this study, the protective effect of fasudil against APAP-induced liver injury was investigated. APAP (400 mg/kg) was administered to male C57BL/6J mice to induce liver injury, and fasudil (20 or 40 mg/kg) was injected 30 min before APAP administration. Fasudil markedly suppressed APAP-induced elevation in serum transaminase activity and hepatic necrosis and significantly reduced an increase in nitrotyrosine and DNA fragmentation. However, fasudil did not affect cytochrome P450 2E1 expression, N-acetyl-p-benzoquinone imine production or c-jun N-terminal kinase activation. In contrast, fasudil significantly inhibited an APAP-induced increase in expression of the transcription factor C/EBP homologous protein (CHOP) in the liver, accompanied by transcriptional suppression of ER stress-related molecules such as Ero1α, Atf4 and Grp78. These findings indicate that suppression of CHOP expression by fasudil exhibits a remarkable protective effect against APAP liver injury by regulating ER stress. We suggest that fasudil is a promising therapeutic candidate for treating APAP-induced liver injury.

Punicalagin relieves lipotoxic injuries on pancreatic β-cells via regulating the oxidative stress and endoplasmic reticulum stress-mediated apoptosis.

Cellular toxicity of hyperlipidemia has been long considered a major cause of various intractable disease such as diabetes. Discovering lipotoxicity antagonist with high efficiency and low side effects is of importance to develop therapeutics for relevant diseases. In the current study, we evaluate the anti-lipotoxic potential of punicalagin (PU) on pancreatic cells and investigate its underpinning mechanism involved. The administration of PU effectively improved cell viability, quenched intracellular reactive oxygen species, alleviated lipid peroxidation, and enhanced cellular antioxidative capacity in RINm5F cells stimulated by sodium palmitate. Besides that, PU treatment significantly inhibited the overload of mitochondrial calcium ions; alleviated the activation of endoplasmic reticulum (ER) stress mediators including glucose-regulated protein 78, protein kinase RNA-like ER kinase, eukaryotic initiation factor 2α, activating transcription factor 6, caspase 12, and C/EBP homologous protein (CHOP); and attenuated the expression of cleaved caspase 3 and poly ADP-ribose polymerase in test cells. Further RNA interference experiment results and miR211-5p expression analysis revealed that PU may directly mitigate CHOP expression and upregulate the expression of miR211-5p to reduce ER stress-induced pancreatic cell death. The efficacy of PU in maintaining redox equilibrium and diminishing ER stress on pancreatic cells stressed by hyperlipidemia suggests that PU can be used as a promising dietary natural product to safeguard the pancreatic health against lipotoxicity.

UPR-Induced miR-616 Inhibits Human Breast Cancer Cell Growth and Migration by Targeting c-MYC.

C/EBP homologous protein (CHOP), also known as growth arrest and DNA damage-inducible protein 153 (GADD153), belongs to the CCAAT/enhancer-binding protein (C/EBP) family. CHOP expression is induced by unfolded protein response (UPR), and sustained CHOP activation acts as a pivotal trigger for ER stress-induced apoptosis. MicroRNA-616 is located within an intron of the CHOP gene. However, the regulation of miR-616 expression during UPR and its function in breast cancer is not clearly understood. Here we show that the expression of miR-616 and CHOP (host gene of miR-616) is downregulated in human breast cancer. Both miR-5p/-3p arms of miR-616 are expressed with levels of the 5p arm higher than the 3p arm. During conditions of ER stress, the expression of miR-616-5p and miR-616-3p arms was concordantly increased primarily through the PERK pathway. Our results show that ectopic expression of miR-616 significantly suppressed cell proliferation and colony formation, whereas knockout of miR-616 increased it. We found that miR-616 represses c-MYC expression via binding sites located in its protein coding region. Furthermore, we show that miR-616 exerted growth inhibitory effects on cells by suppressing c-MYC expression. Our results establish a new role for the CHOP locus by providing evidence that miR-616 can inhibit cell proliferation by targeting c-MYC. In summary, our results suggest a dual function for the CHOP locus, where CHOP protein and miR-616 can cooperate to inhibit cancer progression.

Empagliflozin reduces endoplasmic reticulum stress associated TXNIP/NLRP3 activation in tunicamycin-stimulated aortic endothelial cells.

Sodium-glucose cotransporter 2 inhibitors (SGLT2i) have proven to be of therapeutic significance for cardiovascular diseases beyond the treatment of type 2 diabetes. Recent studies have demonstrated the beneficial effects of SGLT2i on endothelial cell (EC) dysfunction, but the underlying cellular mechanisms remain to be clarified. In this study, we sought to understand the effect of empagliflozin (EMPA; Jardiance®) on cell homeostasis and endoplasmic reticulum (ER) stress signaling. ER stress was induced by tunicamycin (Tm) in human abdominal aortic ECs treated with EMPA over 24 h. Tm-induced ER stress caused increases in the protein expression of thioredoxin interacting protein (TXNIP), NLR-family pyrin domain-containing protein 3 (NLRP3), C/EBP homologous protein (CHOP), and in the ratio of phospho-eIF2α/eIF2α. EMPA (50-100 µM) resulted in a dampened downstream activation of ER stress as seen by the reduced expression of CHOP and TXNIP/NLRP3 in a dose-dependent manner. Nuclear factor erythroid 2-related factor 2 (nrf2) translocation was also attenuated in EMPA-treated ECs. These results suggest that EMPA improves redox signaling under ER stress which in turn attenuates the activation of TXNIP/NLRP3.

MicroRNA-211 Regulates Proliferation, Expansion, and Immune Inhibitory Function of Myeloid-Derived Suppressor Cells via Mediation of CHOP Expression

ABSTRACT Background Myeloid-derived suppressor cells (MDSCs) are capable of effectively repressing immune responses against tumors and orchestrating the tumor microenvironment, which can promote tumor angiogenesis and metastasis. The pathway networks used to modulate tumor-expanded MDSC accumulation and function remain unclear. This study identified microRNA-211 (miR-211), whose expression was significantly decreased by factors derived from tumors. Methods miR-211 was assumed to be critical in modulating the accumulation and activity of MDSCs isolated from ovarian cancer (OC)-bearing mice by targeting C/EBP homologous protein (CHOP). Results The upregulation of miR-211 repressed MDSC proliferation, inhibited MDSC immunosuppressive functions, and increased the number of co-incubated CD4+ and CD8+ cells. Furthermore, overexpression of miR-211 led to decreased activities of the NF-κB, PI3K/Akt, and STAT3 pathways and the subsequent downregulation of matrix metalloproteinases to promote tumor cell invasion and metastasis. CHOP overexpression counteracted the effects of miR-211 elevation on these phenotypic changes. Upregulation of miR-211 also dramatically impaired the activity of MDSCs and suppressed OC tumor growth in vivo. Conclusion These results indicated that the miR-211-CHOP axis in MDSCs plays an essential role in the metastasis and proliferation of tumor-expanded MDSCs and might represent a promising cancer treatment target.

Gene deletion of Interleukin-1α reduces ER stress-induced CHOP expression in macrophages and attenuates the progression of atherosclerosis in apoE-deficient mice

The pathophysiology of atherosclerosis initiation and progression involves many inflammatory cytokines, one of them is interleukin (IL)-1α that has been shown to be secreted by activated macrophages. We have previously shown that IL-1α from bone marrow-derived cells is critical for early atherosclerosis development in mice. It is known that endoplasmic reticulum (ER) stress in macrophages is involved in progression to more advanced atherosclerosis, but it is still unknown whether this effect is mediated through cytokine activation or secretion. We previously demonstrated that IL-1α is required in ER stress-induced activation of inflammatory cytokines in hepatocytes and in the associated induction of steatohepatitis. In the current study, we aimed to examine the potential role of IL-1α in ER stress-induced activation of macrophages, which is relevant to progression of atherosclerosis. First, we demonstrated that IL-1α is required for atherosclerosis development and progression in the apoE knockout (KO) mouse model of atherosclerosis. Next, we showed that ER stress in mouse macrophages results in the protein production and secretion of IL-1α in a dose-dependent manner, and that IL-1α is required in ER stress-induced production of the C/EBP homologous protein (CHOP), a critical step in ER stress-mediated apoptosis. We further demonstrated that IL-1α-dependent CHOP production in macrophages is specifically mediated through the PERK-ATF4 signaling pathway. Altogether, these findings highlight IL-1α as a potential target for prevention and treatment of atherosclerotic cardiovascular disease.

Activating Transcription Factor 6 (ATF6) Modulates The Proteostatic Response To Mutant Surfactant Protein C Conformers In Alveolar Epithelial Cells

Alveolar Type II (AT2) epithelial cell dysfunction is a hallmark of the pathogenesis of Idiopathic Pulmonary Fibrosis (IPF), a progressive interstitial lung disease. Mutations in the surfactant protein C (SP-C) gene (SFTPC) are a high effect size etiological cause of PF in a subset of these patients. Prior in vitro studies from our lab showed that SFTPC mutations located within the BRICHOS domain of the SP-C proprotein result in production of ER retained and aggregation prone conformers. A major aspect of the proteostasis network is the unfolded protein response (UPR), which is initiated when misfolded proteins activate one or more of 3 ER transmembrane sensing proteins (Protein kinase R-like ER kinase (PERK), the Inositol-Requiring kinase 1 (IRE1) and Activating Transcription Factor 6 (ATF6). Consistent with this, we published that clinical IPF associated BRICHOS SFTPC mutations activate all 3 arms of UPR/ER stress signaling in mice in vivo. The goal of this study was to define AT2 proteostatic responses to SFTPC expression and the contribution of ATF6 to the UPR. For this, we generated stable doxycycline-inducible Mouse Lung Epithelial (MLE-12) cell lines expressing either Wild Type (SP-CWT) or an aggregating mutant (SP-CC121G) which were interrogated using cycloheximide chase and / or treatments with activators or inhibitors of ATF6, IRE1, and PERK as well as ERAD (vp97) or autophagy. Readouts included qPCR, luciferase reporter assays, and immunoblotting to assess UPR signaling and apoptosis, coupled with fluorescence microscopy to localize SP-C expression. Using this model system we found that SP-CC121G was ER retained and activated the UPR within 8 hours of Dox induction expression as measured by BIP expression. Commensurate with this both IRE1a (as spliced XBP1 (sXBP1)) and ATF6 were activated by SP-CC121G. The clearance of both mutant SP-CC121G as well as a portion of proSP-CWT was dependent on ERAD as its disruption using the vp97 inhibitor Eeyarestatin I induced formation of higher molecular weight aggregates while bafilomycin failed to induce SP-CC121G aggregates or block their clearance. ATF6 and IRE1 activation during ERAD inhibition accelerated aggregate protein degradation. When SP-CC121G MLE12 cells were treated with either an ATF6 inhibitor or an IRE1 activator, marked increases in levels of C/EBP Homologous Protein (CHOP) occur while either ATF6 agonism or IRE1 inhibition decreased CHOP expression. Collectively, these our data demonstrate a primary role for ERAD in the proteostatic response to SP-C expression while ATF6 agonism appears to provide protection from SFTPC mutant expression in part by accelerating degradation of SP-C aggregates in the face of IRE1 UPR signaling. NIH R01 HL145408 (MFB), VA Merit Review 2I01BX001176 (MFB) and NIH F32HL160011 (LRR) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

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.

Apigenin Alleviates Endoplasmic Reticulum Stress-Mediated Apoptosis in INS-1 β-Cells.

The improvement of type 2 diabetes mellitus induced by naturally occurring polyphenols, known as flavonoids, has received considerable attention. However, there is a dearth of information regarding the effect of the trihydroxyflavone apigenin on pancreatic β-cell function. In the present study, the anti-diabetic effect of apigenin on pancreatic β-cell insulin secretion, apoptosis, and the mechanism underlying its anti-diabetic effects, were investigated in the INS-ID β-cell line. The results showed that apigenin concentration-dependently facilitated 11.1-mM glucose-induced insulin secretion, which peaked at 30 µM. Apigenin also concentration-dependently inhibited the expression of endoplasmic reticulum (ER) stress signaling proteins, CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP) and cleaved caspase-3, which was elevated by thapsigargin in INS-1D cells, with peak suppression at 30 µM. This was strongly correlated with the results of flow cytometric analysis of annexin V/propidium iodide (PI) staining and DNA fragmentation analysis. Moreover, the increased expression of thioredoxin-interacting protein (TXNIP) induced by thapsigargin was remarkably reduced by apigenin in a concentration-dependent manner. These results suggest that apigenin is an attractive candidate with remarkable and potent anti-diabetic effects on β-cells, which are mediated by facilitating glucose-stimulated insulin secretion and preventing ER stress-mediated β-cell apoptosis, the latter of which may be possibly mediated by reduced expression of CHOP and TXNIP, thereby promoting β-cell survival and function.

Some Macrophages With High Expression of CHOP Undergo Necroptosis in Chronic Rhinosinusitis.

Endoplasmic reticulum stress (ER stress) is activated in chronic sinusitis with nasal polyps (CRSwNP) and leads to increased expression of C/EBP homologous protein (CHOP). However, the role of CHOP in the pathogenesis of CRSwNP remains unclear. CHOP expression was detected by immunohistochemistry staining in nasal mucosa of control and CRSwNP patients. Co-localization of CHOP and cleaved caspase3, p-MLKL, and CD68 was detected by immunofluorescence staining in CRSwNP patients. TNFα, IFNγ, IL1β, LPS, and tunicamycin were added to primary dispersed nasal polyp cells (DNPCs) to explore their roles in cell death. Western blot, CCK8 assay, and flow cytometry were employed to detect cell death. CHOP was specifically activated in CRSwNP compared to controls. It was mainly macrophages that highly expressed CHOP, some of which underwent apoptosis and the other underwent necroptosis. IL1β induced increased CHOP and apoptosis, and a slight p-MLKL. In addition, ER stress could also promote p-MLKL expression. Whereas classical TNFα plus IFNγ and LPS did not induce increased necroptosis in DNPCs. IL1β induced the apoptotic pathway and minor necroptosis. And ER stress also plays a role in the occurrence of necroptosis in CRSwNP.

DHA Induces Cell Death through the Production of ROS and the Upregulation of CHOP in Fibroblast-like Synovial Cells from Human Rheumatoid Arthritis Patients.

Rheumatoid arthritis (RA) is an inflammatory disease marked by a massive proliferation of synovial cells in the joints. In this study, we investigated the pro-apoptotic effects of docosahexaenoic acid (DHA) in human fibroblast-like synovial cells from RA patients (RA-FLS). An in vitro study using MH7A cells showed that DHA treatment induced caspase-8-dependent apoptosis in a dose-dependent manner and reduced the TNF-α-mediated induction of MMP-9 and IL-1β. DHA also induced the phosphorylation of eIF2α, the expression of the ER stress markers ATF4 and C/EBP homologous protein (CHOP), and death receptor 5 (DR5). The knockdown of CHOP or DR5 increased cell viability and reduced apoptosis in DHA-treated cells. Furthermore, the knockdown of CHOP reduced DHA-mediated DR5 expression, while the overexpression of CHOP increased DR5 expression. We also found that DHA treatment induced the accumulation of reactive oxygen species (ROS), and pretreatment with the anti-oxidant Tiron effectively abrogated not only the expression of CHOP and DR5, but also DHA-induced apoptosis. Under this condition, cell viability was increased, while PARP-1 cleavage and caspase-8 activation were reduced. All the findings were reproduced in human primary synovial cells obtained from RA patients. These results suggest that the DHA-mediated induction of ROS and CHOP induced apoptosis through the upregulation of DR5 in RA-FLSs, and that CHOP could be used as a therapy for RA.

Proflactic effects of rosmarinic acid on spinal cord injury in rats.

Spinal cord injury (SCI) causes various neurological consequences that disrupt the structure of axons. The C/EBP Homologous Protein (CHOP) acts in neuronal death by apoptosis has been demonstrated in experimental models. Rosmarinic acid (RA) is a phenolic compound used for therapeutic purposes in many diseases. In this study, we investigated the therapeutic effect of Rosmarinic acid application on inflammation and apoptotic development after spinal cord injury. Male Wistar albino rats (n: 24) were assigned to three group: control, SCI and SCI+ RA. All rats were fixed on the operating table after anesthesia, the skin of the thoracic region was opened with a midline incision and the paravertebral muscles were dissected and T10-T11 laminas were exposed. A cylindrical tube of 10 cm length was fixed to the area to be laminectomy. A metal weight of 15 grams was left down the tube. Spinal damage was created, skin incisions were sutured. 50 mg/kg rosmarinic acid was given orally for 7 days after the spinal injury. Spinal tissues were fixed in formaldehyde solution and processed for paraffin wax tissue protocol and 4-5 μm sections were taken with microtome for further immunohistochemical examination. Caspase-12 and CHOP antibodies were applied to sections. Remaining tissues were carried out in glutaraldehyde for the first fixation then in osmium tetroxide for the second. Tissues were kept in pure araldite and thin sections were taken for transmission electron microscope. Values of malondialdehyde (MDA), myeloperoxidase (MPO), glutathione peroxidase (GSH), neuronal degeneration, vascular dilation, inflammation, CHOP and Caspase-12 expression were increased in SCI group compared to control group. Only glutathione peroxidase content was decreased in SCI group. In SCI group, disruption of basement membrane structure in canalis ependymalis, degeneration in structures of unipolar bipolar and multipolar neurons, and apoptotic changes were seen with increased inflammation in the piamater region and positive CHOP expression in vascular endothelial cells. In SCI+RA group, reorganization of basement membrane pill in canalis ependymalis were observed with mild Caspase-12 activity in some canalis ependymal and glial cells. Also, moderate CHOP expression in multipolar and bipolar neurons and glia cells were observed. The application of RA has a significant effect on preventing damage in SCI. It was thought that CHOP and Caspase-12 mediated oxidative stress could be a guide in showing the potential and therapeutic target to stop the apoptotic course after SCI injury.

[Role of IRE1 in autophagy induced by vitamin E succinate in human gastric cancer cell].

To investigate the role of inositol-requiring enzyme 1(IRE1) in autophagy of human gastric cancer cells induced by vitamin E succinate(VES). Human gastric cancer SGC-7901 cells were cultured in vitro and divided into solvent control group(0.1% ethanol absolute), different doses(5, 10, 15 and 20 μg/mL) VES group, 4μ8C group, and VES + 4μ8C group. The endoplasmic reticulum stress-related molecules glucose regulated protein 78(GRP78) and C/EBP homologous protein(CHOP), autophagy marker microtubule associated Protein1 light chain 3(LC3), Beclin-1, unfolded protein response branching pathway Inositol-requiring enzyme 1(IRE1), X box-binding protein 1(XBP1), c-Jun n-terminal kinase(JNK) and p-JNK were detected by Western blot in the solvent control group and different doses of VES group. IRE1 was inhibited by 4μ8C. The expressions of IRE1, XBP1, JNK, p-JNK, GRP78 and CHOP were detected by Western blot, and the expressions of LC3 and Beclin-1 were detected. The expression of GRP78(1.16±0.06) and CHOP(1.36±0.11) in 20 μg/mL VES group were significantly higher than those in solvent control group GRP78(0.36±0.10) and CHOP(0.48±0.05)(P&lt;0.001). The expression of Beclin-1(1.09±0.20) and LC3-Ⅱ/LC3-Ⅰ(1.29±0.03) in 20 μg/mL VES group were significantly higher than those in solvent control group(0.27±0.07) and LC3-Ⅱ/LC3-Ⅰ(0.43±0.06)(P&lt;0.001). The expression levels of IRE1(1.07±0.20), XBP1(1.33±0.07) and p-JNK/JNK(1.19±0.31) in 20 μg/mL VES group were significantly higher than those in the solvent control group(P&lt;0.01). After IRE1 is inhibited: The expression level of IRE1(0.63±0.27), XBP1(0.74±0.09), p-JNK/JNK(0.35±0.04), GRP78(0.66±0.02), CHOP(0.51±0.02), LC3-Ⅱ/LC3-Ⅰ(0.72±0.01), Beclin-1(0.70±0.15) was significantly lower than that of VES group(P&lt;0.05). VES may participate in the regulation of autophagy in gastric cancer cells by upregulating IRE1 pathway.

Hydrogen sulfide alleviates ischemia induced liver injury by repressing the SPHK1/S1P pathway.

Ischemia/reperfusion (I/R) induced liver injury is a severe pathological process which frequently occurs during clinical hepatic operations. The current study investigated the protective function and underlying mechanisms of hydrogen sulfide (H2S) in I/R induced liver injury. The effects of H2S were examined using the fibroblast-like rat liver cell line BRL-3A (the name of normal hepatocytes in rats) cultured under hypoxic conditions and an I/R rat model. The viability of BRL-3A cells was assessed using the methylthiazolyldiphenyl-tetrazolium (MTT) assay and Hoechst analysis. The expression of C/EBP homologous protein (CHOP), sphingosine kinase 1 (SPHK1), and sphingosine 1-phosphate (S1P) were determined in hypoxic BRL-3A cells with or without H2S treatment. CHOP was overexpressed in hypoxic BRL-3A cells to further evaluate whether H2S protected the liver against I/R injury by decreasing endoplasmic reticulum (ER) stress. Finally, the inflammation levels in the serum and the histopathological changes of liver were examined in the I/R rat model to evaluate the therapeutic function of H2S on I/R induced liver injury in vivo. H2S alleviated hypoxic damage in BRL-3A cells. In addition, hypoxia increased the expression of CHOP, SPHK1, and S1P in BRL-3A cells, and this was abolished by H2S pretreatment. Notably, overexpression of CHOP significantly inhibited the effect of H2S on the viability of BRL-3A cells during hypoxia. Overall, H2S effectively protected against I/R induced liver injury, decreased the inflammatory responses, and attenuated apoptosis of hepatocyte via inhibiting the ER stress response. These findings demonstrated that pre-treatment of H2S protected against I/R induced liver injury by repressing the SPHK1/S1P pathway via inhibition of ER stress, suggesting an effective therapeutic method for the treatment of I/R induced liver injury.

Effects of Simvastatin on Endoplasmic Reticulum Stress-Mediated Apoptosis in Atherosclerotic Calcification

Objective: The effectiveness of statins in reducing atherosclerotic calcification remains controversial. The aim of this study was to confirm that simvastatin reduces atherosclerotic calcification and stabilizes plaque by restricting endoplasmic reticulum stress (ERS)-mediated apoptosis. Methods: Twenty-four 8-week-old male apolipoprotein E (ApoE)-/- mice (C57BL/6J genetic background) were selected and randomly divided into model (n = 12) and simvastatin (n = 12) groups. Twelve male C57BL/6J mice were selected as control group (n = 12). The mice were adaptively fed for 2 weeks and were put on a high-fat diet thereafter. After 9 weeks, they were treated with simvastatin (20 mg/kg) or phosphate-buffered saline daily for 8 weeks. Aortic sinus samples were obtained from ApoE-/- and C57BL/6J mice for hematoxylin and eosin, von Kossa, alizarin Red S, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, and immunohistochemical staining after in vivo treatment with simvastatin. In addition, mouse vascular smooth muscle cells were analyzed after exposure to simvastatin in vitro. Results: Administration of simvastatin in vivo drastically attenuated the atherosclerosis, calcification, and apoptosis, and decreased the serum levels of triglycerides, total cholesterol, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol. The expression levels of glucose-regulated protein, 78 kDa (GRP78), C/EBP homologous protein (CHOP), and caspase 12 (CASP12) in the aortic sinus decreased in the simvastatin group compared with the model group. In vitro, simvastatin or simvastatin plus ERS inhibitor (taurine) attenuated calcification and apoptosis, and reduced the expression of ERS-related proteins GRP78, CHOP, and CASP12. Conclusion: Treatment with simvastatin suppressed atherosclerotic calcification. This effect may be mediated through the inhibition of ERS-related apoptosis.