Sevoflurane postconditioning alleviates hypoxic-ischemic brain damage in rats by inhibiting the endoplasmic reticulum stress PERK/ATF4/CHOP pathway.

Hypoxic-ischemic brain damage (HIBD) frequently induces cognitive impairments. Investigating the role of sevoflurane postconditioning (SPC) in HIBD, we conducted experiments involving HIBD modeling, SPC treatment, and interventions with the PERK inhibitor GSK2656157 or the PERK activator CCT020312, administered 30 min before modeling, followed by SPC treatment. Behavioral testing using the Morris water maze test and Neurological Deficiency Scale (NDS) was conducted. Additionally, Nissl staining assessed hippocampal CA1 area neuronal density, TUNEL staining evaluated hippocampal CA1 area neuronal apoptosis, and Western blot determined hippocampal CA1 area protein levels, including Bax, Bcl-2, p-PERK/PERK, p-eIF2/eIF2, ATF4, CHOP, GRP78, Bax, and Bcl-2 protein levels. Following SPC treatment, HIBD rats exhibited improved spatial learning and memory abilities, reduced neuronal apoptosis, increased neuronal density in the hippocampal CA1 area, elevated Bcl-2 protein level, decreased Bax protein levels, and decreased levels of endoplasmic reticulum stress pathway related proteins (p-PERK/PERK, p-eIF2/eIF2, ATF4, CHOP and GRP78). Pre-modeling treatment with the PERK inhibitor treatment improved outcomes in HIBD rats. However, pre-modeling treatment with the PERK activator CCT020312 counteracted the protective effects of SPC against HIBD in rats. In conclusion, SPC alleviates neuronal apoptosis in the hippocampus CA1 area of HIBD rats by inhibiting the endoplasmic reticulum stress pathway PERK/ATF4/CHOP, thereby mitigating HIBD in rats.

Astragaloside IV protects cardiomyocytes from hypoxic injury by regulating endoplasmic reticulum stress via eIF2α/CHOP signaling pathway.

Endoplasmic reticulum stress (ER stress) is suggested to promote cardiomyocyte apoptosis and ultimately lead to ischemic injury. Inhibition of ER stress-induced apoptosis may be a therapeutic strategy for MI injury. Astragaloside-IV (AST) from Astragalus membranaceus (Fisch) Bge, was reported to have cardioprotective properties. In this study, we investigated the protective effect of AST on cardiomyocytes against hypoxia injury by regulating ER stress and inhibiting apoptosis. H9c2 cardiomyocytes were divided into three groups, normal group, hypoxia group and AST group. Cell viability was determined by CCK-8 assay. Intracellular reactive oxygen species (ROS) production was detected by DCFH-DA (2,7- dichloro-dihydrofluorescein diacetate) florescent staining. The study showed that AST treatment could significantly increase the cell viability of H9c2 cells exposed to hypoxia. Furthermore, AST could restrain cell apoptosis and decrease the production of ROS. Compared with normal group, the protein levels of Bax, caspase-3, caspase-9, GRP78, p-eIF2α, and CHOP were enhanced in the hypoxia group, whereas the protein level of Bcl-2 was dramatically reduced. Compared with hypoxia group, AST markedly inhibited the phosphorylation of eIF2α and the expression of caspase-3, caspase-9 and CHOP, and promoted the protein expression of Bcl-2. Thus, AST can inhibit the ER stress-mediated apoptosis, partly through the eIF2α/CHOP pathway suppression to inhibit ER stress.

Oridonin Promotes Apoptosis in Rheumatoid Arthritis Fibroblast-like Synoviocytes Through PERK/eIF2α/CHOP of Endoplasmic Reticulum Stress Pathway.

Oridonin (ORI), derived from Chinese herbs Rabdosia rubescens, has anti-inflammatory, proapoptotic, anticancer effects. Previous studies have found that ORI induces apoptosis in rheumatoid arthritis fibroblast synovial cells (RA-FLSs), but this mechanism is not clear. We will investigate the apoptosis mechanism of ORI on RA-FLSs. RA-FLSs were treated with various concentrations of ORI (0, 5, 10, 15, 20, 25, and 30 µM) for 24 h. CCK8, LDH, and hochest/PI assay determined the viability, cytotoxicity, and death of ORI on RA-FLSs. The endoplasmic reticulum probe was used to observe structural changes of endoplasmic reticulum in RA-FLSs. RNA expression was detected with RNA sequencing analysis and quantitative real-time PCR. The PERK/eIF2α/CHOP pathway protein of the endoplasmic reticulum was verified with Western Blot. Our results show that ORI induced the apoptosis of RA-FLSs from CCK8, LDH, and Hochest/PI. The endoplasmic reticulum distribution was altered in RA-FLSs after being treated with ORI. Bioinformatics analysis of RNA sequencing data found that 1453 genes were elevated. The PERK/eIF2α/CHOP pathway of the endoplasmic reticulum was regulated from the Gene ontology and KEGG analysis. The results of quantitative real-time PCR and Western blot analysis verified the regulation of PERK/eIF2α/CHOP pathway in RA-FLSs. Our data imply that the endoplasmic reticulum's PERK/eIF2α/CHOP signaling pathway is certainly implicated in the induction of RA-FLS apoptosis by ORI. This study has important implications for the pharmacological effects of ORI and the treatment of RA.

C5a-C5aR1 induces endoplasmic reticulum stress to accelerate vascular calcification via PERK-eIF2α-ATF4-CREB3L1 pathway.

AIMS: Vascular calcification (VC) predicts the morbidity and mortality in cardiovascular diseases. Vascular smooth muscle cells (VSMCs) osteogenic transdifferentiation is the crucial pathological basis for VC. To date, the molecular pathogenesis is still largely unclear. Notably, C5a-C5aR1 contributes to the development of cardiovascular diseases, and its closely related to physiological bone mineralization which is similar to VSMCs osteogenic transdifferentiation. However, the role and underlying mechanisms of C5a-C5aR1 in VC remain unexplored. METHODS AND RESULTS: A cross-sectional clinical study was utilized to examine the association between C5a and VC. Chronic kidney diseases mice and calcifying VSMCs models were established to investigate the effect of C5a-C5aR1 in VC, evaluated by changes in calcium deposition and osteogenic markers. The cross-sectional study identified that high level of C5a was associated with increased risk of VC. C5a dose-responsively accelerated VSMCs osteogenic transdifferentiation accompanying with increased the expression of C5aR1. Meanwhile, the antagonists of C5aR1, PMX 53, reduced calcium deposition, and osteogenic transdifferentiation both in vivo and in vitro. Mechanistically, C5a-C5aR1 induced endoplasmic reticulum (ER) stress and then activated PERK-eIF2α-ATF4 pathway to accelerated VSMCs osteogenic transdifferentiation. In addition, cAMP-response element-binding protein 3-like 1 (CREB3L1) was a key downstream mediator of PERK-eIF2α-ATF4 pathway which accelerated VSMCs osteogenic transdifferentiation by promoting the expression of COL1α1. CONCLUSIONS: High level of C5a was associated with increased risk of VC, and it accelerated VC by activating the receptor C5aR1. PERK-eIF2α-ATF4-CREB3L1 pathway of ER stress was activated by C5a-C5aR1, hence promoting VSMCs osteogenic transdifferentiation. Targeting C5 or C5aR1 may be an appealing therapeutic target for VC.

Interaction of Bmal1 and eIF2α/ATF4 pathway was involved in Shuxie compound alleviation of circadian rhythm disturbance-induced hepatic endoplasmic reticulum stress.

ETHNOPHARMACOLOGICAL RELEVANCE: Shuxie Compound (SX) combines the composition and efficacy of Suanzaoren decoction and Huanglian Wendan decoction. It can soothe the liver, regulate the qi, nourish the blood and calm the mind. It is used in the clinical treatment of sleep disorder with liver stagnation. Modern studies have proved that circadian rhythm disorder (CRD) can cause sleep deprivation and liver damage, which can be effectively ameliorated by traditional Chinese medicine to soothe the liver stagnation. However, the mechanism of SX is unclear. AIM OF THE STUDY: This study was designed to demonstrate the impact of SX on CRD in vivo, and confirm the molecular mechanisms of SX in vitro. MATERIALS AND METHODS: The quality of SX and drug-containing serum was controlled by UPLC-Q-TOF/MS, which were used in vivo and in vitro experiments, respectively. In vivo, a light deprivation mouse model was used. In vitro, a stable knockdown Bmal1 cell line was used to explore SX mechanism. RESULTS: Low-dose SX (SXL) could restore (1) circadian activity pattern, (2) 24-h basal metabolic pattern, (3) liver injury, and (4) Endoplasmic reticulum (ER) stress in CRD mice. CRD decreased the liver Bmal1 protein at ZT15, which was reversed by SXL treatment. Besides, SXL decreased the mRNA expression of Grp78/ATF4/Chop and the protein expression of ATF4/Chop at ZT11. In vitro experiments, SX reduced the protein expression of thapsigargin (tg)-induced p-eIF2α/ATF4 pathway and increase the viability of AML12 cells by increasing the expression of Bmal1 protein. CONCLUSIONS: SXL relieved CRD-induced ER stress and improve cell viability by up-regulating the expression of Bmal1 protein in the liver and then inhibiting the protein expression of p-eIF2α/ATF4.

Pre-treatment with IL-6 potentiates ß-cell death induced by pro-inflammatory cytokines.

BACKGROUND: Type I Diabetes mellitus (T1D) is characterized by a specific destruction of ß-cells by the immune system. During this process pro-inflammatory cytokines are released in the pancreatic islets and contribute for ß-cells demise. Cytokine-induced iNOS activation, via NF-κB, is implicated in induction of ß-cells death, which includes ER stress activation. Physical exercise has been used as an adjunct for better glycemic control in patients with T1D, since it is able to increase glucose uptake independent of insulin. Recently, it was observed that the release of IL-6 by skeletal muscle, during physical exercise, could prevent ß-cells death induced by pro-inflammatory cytokines. However, the molecular mechanisms involved in this beneficial effect on ß-cells are not yet completely elucidated. Our aim was to evaluate the effect of IL-6 on ß-cells exposed to pro-inflammatory cytokines. RESULTS: Pre-treatment with IL-6 sensitized INS-1E cells to cytokine-induced cell death, increasing cytokine-induced iNOS and Caspase-3 expression. Under these conditions, however, there was a decrease in cytokines-induced p-eIF2-α but not p-IRE1expression, proteins related to ER stress. To address if this prevention of adequate UPR response is involved in the increase in ß-cells death markers induced by IL-6 pre-treatment, we used a chemical chaperone (TUDCA), which improves ER folding capacity. Use of TUDCA increased cytokines-induced Caspase-3 expression and Bax/Bcl-2 ratio in the presence of IL-6 pre-treatment. However, there is no modulation of p-eIF2-α expression by TUDCA in this condition, with increase of CHOP expression. CONCLUSION: Treatment with IL-6 alone is not beneficial for ß-cells, leading to increased cell death markers and impaired UPR activation. In addition, TUDCA has not been able to restore ER homeostasis or improve ß-cells viability under this condition, suggesting that other mechanisms may be involved.

Protective role of PERK-eIF2α-ATF4 pathway in chronic renal failure induced injury of rat hippocampal neurons.

PURPOSE: Chronic renal failure (CRF) is associated with impairment of hippocampal neurons. This study investigated the effect of PERK-eIF2α-ATF4 pathway in CRF. METHODS: Rat CRF model was established and rat hippocampal neurons were separated. Xanthine Oxidase method, fluorescence spectrophotometry and flow cytometry were applied to detect superoxide dismutase (SOD) content, reactive oxygen species (ROS) level and apoptosis in hippocampal neurons, respectively. The levels of phosphorylated (p)-PERK, phosphorylated (p)-eIF2α, CHOP, Bax, C-Caspase-3 and Bcl-2 in rats were measured using Western blot. Then, the neurotoxicity of serum from CRF rats was assessed in rat hippocampal neurons after treatment with rat CRF serum and transfection with or without PERK overexpression or knockdown plasmid. RESULTS: SOD activity was reduced, while ROS level and apoptosis rate were increased in hippocampal tissues of CRF rats. PERK-eIF2α-ATF4 and apoptosis pathways were activated in CRF rats. Cells treated with serum from CRF rats showed increases in apoptosis rate and LDH and ROS levels, and decreases in cell viability and SOD activity. However, overexpressed PERK could reverse the cytotoxic effect of serum from CRF rats. PERK overexpression could enhance the activation of PERK-eIF2α-ATF4 pathway in hippocampal neurons induced by serum from CRF rats. Furthermore, PERK overexpression could alleviate the increases in CHOP, Bax, C-Caspase-3 expressions and the reduction of Bcl-2 expression in hippocampal neurons induced by serum from CRF rats. CONCLUSION: PERK-eIF2α-ATF4 pathway induced by increased endoplasmic reticulum stress may alleviate CRF-induced hippocampal neuronal damage.

Gardenia jasminoides Extract GJ-4 Alleviates Memory Deficiency of Vascular Dementia in Rats through PERK-Mediated Endoplasmic Reticulum Stress Pathway.

Endoplasmic reticulum stress (ERS) is involved in the pathological process of vascular dementia (VD). GJ-4 is extracted from Gardenia jasminoides J. Ellis and has been reported to have protective roles in ischemia-related brain damage. However, the role of GJ-4 in ERS has not been elucidated. We established a VD rat model through bilateral common carotid arteries occlusion (2-VO). The rats were intragastrically administrated with GJ-4 (10, 25, and 50[Formula: see text]mg/kg) and nimodipine (10[Formula: see text]mg/kg). Data from a Morris water maze test showed that GJ-4 could significantly alleviate learning and memory deficits in VD rats. Nissl and cleaved caspase-3 staining revealed that GJ-4 can inhibit apoptosis and thus exert a protective role in the brain of 2-VO rats. Western blot results suggested that GJ-4 significantly reduced ERS-related protein expression and inhibited apoptosis through suppression of the PERK/eIF2[Formula: see text]/ATF4/CHOP signaling pathway. For in vitro studies, the oxygen-glucose deprivation (OGD) SH-SY5Y model was employed. Western blot and Hoechst 33342/PI double staining were utilized to explore the effects of crocetin, the main active metabolite of GJ-4. Like GJ-4 in vivo, crocetin in vitro also decreased ERS-related protein expression and inhibited the activation of the PERK/eIF2[Formula: see text]/ATF4/CHOP signaling pathway. Thus, crocetin exerted similar protective roles on OGD challenged SH-SY5Y cells in vitro. In summary, GJ-4 and crocetin reduce the ERS in the brain of VD rats and SY5Y cells subjected to OGD and inhibit neuronal apoptosis through suppression of the PERK/eIF2[Formula: see text]/ATF4/CHOP pathway, suggesting that GJ-4 may be useful for the treatment of VD.

CPNE1 regulates myogenesis through the PERK-eIF2α pathway mediated by endoplasmic reticulum stress.

Sarcopenia is characterized by a progressive reduction in muscle mass or muscle physiological function associated with aging, but the relevant molecular mechanisms are not clear. Here, we identify the role of the myogenesis modifier CPNE1 in sarcopenia. CPNE1 is upregulated in aged skeletal muscles and young skeletal muscle satellite cells with palmitate-induced atrophy. The overexpression of CPNE1 hinders proliferation and differentiation and increases muscle atrophy characteristics in young skeletal muscle-derived satellite cells. In addition, CPNE1 overexpression disrupts the balance of mitochondrial fusion and division and causes endoplasmic reticulum stress. We found that the effects of CPNE1 on mitochondrial function are dependent on the PERK/eIF2α/ATF4 pathway. The overexpression of CPNE1 in young muscles alters membrane lipid composition, reduces skeletal muscle fibrosis regeneration, and exercise capacity in mice. These effects were reversed by PERK inhibitor GSK2606414. Moreover, immunoprecipitation indicates that CPNE1 overexpression greatly increased the acetylation of PERK. Therefore, CPNE1 is an important modifier that drives mitochondrial homeostasis to regulate myogenic cell proliferation and differentiation via the PERK-eIF2α pathway, which could be a valuable target for age-related sarcopenia.

Promotion of osteogenesis in BMSC under hypoxia by ATF4 via the PERK-eIF2α signaling pathway.

Mandibular distraction osteogenesis (MDO) is an endogenous tissue engineering technology in which bone marrow mesenchymal stem cells (BMSC) play a key role in MDO-related osteogenesis. Activating transcription factor 4 (ATF4) is involved in osteogenesis through activation of PERK (Protein kinase R-like endoplasmic reticulum kinase) in endoplasmic reticulum stress (ERS) condition under hypoxia. However, the specific role of ATF4 in MDO with BMSC remains unknown. The aim of this study was to explore the effects of ATF4 in MDO with BMSC under hypoxia. Briefly, canine BMSCs were cultured in a hypoxic chamber, and effects of hypoxia were evaluated using cell migration assay and Alizarin Red S staining. Expression levels of protein kinase R-like endoplasmic reticulum kinase, eukaryotic translation initiation factor 2α, ATF4, osteocalcin, and bone sialoprotein were evaluated using quantitative polymerase chain reaction and western blotting. BMSCs were transduced with the ATF4-small interfering RNA lentivirus. The effects were evaluated using all the aforementioned experiments. The results showed that hypoxia promoted migration, osteoblast differentiation, and ATF4 expression in BMSC. ATF4 knockdown in BMSC significantly inhibited migration and osteoblast differentiation abilities, while hypoxia reversed these effects to some extent. In addition, the molecular mechanism partly depended on the ERS signaling pathway, with ATF4 as the key factor. In summary, we presented a novel mechanism of ATF4-mediated regulation of BMSC under hypoxia.

[Hydrogen Sulfide Ameliorates Myocardial Injury Caused by Sepsis Through Suppressing ROS-Mediated Endoplasmic Reticulum Stress].

Objective: To investigate the effect of hydrogen sulfide (H 2S) on reactive oxygen species (ROS)-mediated endoplasmic reticulum stress in myocardial injury caused by sepsis. Methods: A sepsis model was induced in Sprague-Dawley (SD) rats by cecal ligation and puncture (CLP). The rats were randomly divided into sham operation (sham) group, sepsis (CLP) group, and sepsis+sodium hydrosulfide (NaHS) (CLP+NaHS) group. The left ventricular function of the rats was observed with echocardiography and their plasma H 2S levels were measured. Lactate dehydrogenase (LDH), malondialdehyde (MDA), glutathione (GSH) levels were measured and HE staining was done to evaluate the level of myocardial oxidative stress in rats. HE staining was done to observe the morphological changes of rat myocardium, and transmission electron microscope was used to observe the ultrastructure of myocardial mitochondria. Western blot was done to examine changes in the expression of two endogenous hydrogen sulfide synthases, cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfur transferase (3-MST), and changes in the expression of endoplasmic reticulum stress (ERS) marker proteins, including phosphorylated (p) protein kinase R-like endoplasmic reticulum kinase (p-PERK), p-eukaryotic translation initiation factor 2α (p-eIF2α), p-inositol requires enzyme 1α (IRE1α), recombinant activating transcription factor 4 (ATF4), and C/EBP homologous protein (CHOP). TUNEL staining was performed to observe the changes of cardiomyocyte apoptosis in rats. Results: Left ventricular ejection fraction (LVEF), left ventricular shortening fraction (LVFS) and plasma H 2S decreased in septic rats ( P<0.05). Plasma H 2S exhibited linear correlation with LVEF and LVFS ( r 2=0.62 and r 2=0.64, all P<0.05). The ROS levels were significantly elevated in rats of the CLP group. In addition, these rats showed increased level of LDH ( P<0.05), increased expression of MDA ( P<0.05), and decreased expression of GSH ( P<0.05). Inflammatory cell infiltration and cardiomyocyte edema were observed in HE staining. Transmission electron microscopic observation revealed significant mitochondrial damage, observable mitochondrial edema, and cristae structure dissolution. The Western blot results showed that the expression levels of CSE and 3-MST decreased ( P<0.05), while the ERS marker proteins, including p-PERK, p-eIF2, IRE1α, ATF4, and CHOP, were expressed at increased levels ( P<0.05). TUNEL staining showed significant increase of apoptosis in cardiomyocytes ( P<0.05). After NaHS treatment, LVEF and LVFS increased ( P<0.05) and plasma H 2S increased in septic rats ( P<0.05). Myocardial oxidative stress levels decreased. HE staining and transmission electron microscopy showed improved myocardial morphology. Mitochondrial damage was reduced and CSE and 3-MST levels were significantly increased ( P<0.05). The expression of p-PERK, p-eIF2α, p-IRE1α, and CHOP proteins decreased ( P<0.05). A decrease in cardiomyocyte apoptosis levels was observed by TUNEL staining ( P<0.05). Conclusion: H 2S reduces septic cardiomyocyte apoptosis by inhibiting ROS-mediated ERS, thereby improving myocardial dysfunction in sepsis.

Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA)-mediated ER stress crosstalk with autophagy is involved in tris(2-chloroethyl) phosphate stress-induced cardiac fibrosis.

Excessive organophosphate flame retardant (OPFR) use in consumer products has been reported to increase human disease susceptibility. However, the adverse effects of tris(2-chloroethyl) phosphate (TCEP) (a chlorinated alkyl OPFR) on the heart remain unknown. In this study, we tested whether cardiac fibrosis occurred in animal models of TCEP (10 mg/kg b.w./day) administered continuously by gavage for 30 days and evaluated the specific role of sarco/endoplasmic reticulum Ca2+ ATPase (SERCA). First, we confirmed that TCEP could trigger cardiac fibrosis by histopathological observation and cardiac fibrosis markers. We further verified that cardiac fibrosis occurred in animal models of TCEP exposure accompanied by SERCA2a, SERCA2b and SERCA2c downregulation. Notably, inductively coupled plasma-mass spectrometry (ICP-MS) analysis revealed that the cardiac concentrations of Ca2+ increased by 45.3% after TCEP exposure. Using 4-Isopropoxy-N-(2-methylquinolin-8-yl)benzamide (CDN1163, a small molecule SERCA activator), we observed that Ca2+ overload and subsequent cardiac fibrosis caused by TCEP were both alleviated. Simultaneously, the protein levels of endoplasmic reticulum (ER) markers (protein kinase R-like endoplasmic reticulum kinase (PERK), inositol requiring protein 1α (IRE1α), eukaryotic initiation factor 2 α (eIF2α)) were upregulated by TCEP, which could be abrogated by CDN1163 pretreatment. Furthermore, we observed that CDN1163 supplementation prevented overactive autophagy induced by TCEP in the heart. Mechanistically, TCEP could lead to Ca2+ overload by inhibiting the expression of SERCA, thereby triggering ER stress and overactive autophagy, eventually resulting in cardiac fibrosis. Together, our results suggest that the Ca2+ overload/ER stress/autophagy axis can act as a driver of cardiotoxicity induced by TCEP.

Insulin-like growth factor-1 reduces hyperoxia-induced lung inflammation and oxidative stress and inhibits cell apoptosis through PERK/eIF2α/ATF4/CHOP signaling.

Background: Insulin-like growth factor-1 (IGF-1), a member of the insulin family, has a high degree of homology with insulin and exhibits anti-inflammatory and anti-oxidative stress properties. However, the potential protective effect of IGF-1 on hyperoxia-induced lung injury remains unknown. In this study, we aimed to explore the effects and mechanism of action of IGF-1 in hyperoxia-induced lung injury in neonatal rats. Materials and Methods: Hematoxylin-eosin staining was used to observe pathological changes in lung tissue; transmission electron microscopy was used to examine the ultrastructure, and ELISA was used to detect the level of pro-inflammatory cytokines in bronchoalveolar lavage fluid. Further, malondialdehyde, glutathione, and superoxide dismutase activities in lung tissue were evaluated. TUNEL staining was used to detect cell apoptosis, and western blot analysis was used to detect the expression of Bax, Bcl-2, Caspase-3, p-PERK, p-eIF2α, ATF4, and CHOP in the lung tissue. Moreover, the wet/dry weight ratio of lung tissue was determined. Results: Intraperitoneal injection of IGF-1 effectively reduced lung tissue damage induced by hyperoxia; production of inflammatory cells and release of pro-inflammatory cytokines, oxidative stress, and cell apoptosis. Further, IGF-1 down-regulated the expression of ATF4, CHOP, and Bax/Bcl-2, and inhibited the phosphorylation of PERK and eIF2α. Conclusion: The results suggest that IGF-1 reduces hyperoxia-induced lung inflammation and oxidative stress in neonatal rats through the PERK/eIF2α/ATF4/CHOP signaling pathway and inhibits cell apoptosis.

Knockdown of ENTPD5 inhibits tumor metastasis and growth via regulating the GRP78/p-eIF-2α/CHOP pathway in serous ovarian cancer.

BACKGROUND: Dysregulation of Ectonucleoside Triphospahate Diphosphohydrolase 5 (ENTPD5) in tumors might be associated with tumor progression, while the role of ENTPD5 in the growth and metastasis of serous ovarian cancer (SOC) is still unclear. METHODS: ENTPD5 expression patterns in ovarian cancer tissues were analyzed by qRT-PCR and immunohistochemistry assay (IHC). Two SOC cell lines, SKOV3 and OVCAR8, were stably transfected with lentivirus to build knockdown and overexpression cell lines. Clone formation assay, collagen gel droplet culture technology, wound healing assay and flow cytometry were used to assess the migration and growth traits of SOC cells. Expression levels of ENTPD5, glucose regulated protein 78 (GRP78), eukaryotic translation initiation factor 2 alpha (eIF-2α), phosphorylated -eIF-2α and, C/EBP homologous protein (CHOP) in SOC cells were detected by Western blot. RESULTS: Compared to fallopian tube tissues, the expression of ENTPD5 was significantly higher in tumor tissues obtained from SOC patients, and positively correlated with clinical stage and metastasis. ENTPD5 knockdown robustly inhibited cell proliferation, migration, whereas ENTPD5 overexpression elicited the opposite effect on SOC cells. ENTPD5 knockdown arrested cell cycle in G0/G1 phase and increased apoptosis. Importantly, ENTPD5 knockdown was associated with significantly decreased protein levels for GRP78, CHOP, and p-eIF-2α, suggesting possible involvement of ENTPD5 in endoplasmic reticulum stress (ERS). CONCLUSIONS: Our study demonstrates that ENTPD5 knockdown inhibited SOC cell proliferation, migration and restrained the activation of the GRP78/p-eIF-2α/CHOP pathway, which provides a potentially effective therapeutic target for the treatment of SOC.

Flurochloridone Induced Cell Apoptosis via ER Stress and eIF2α-ATF4/ATF6-CHOP-Bim/Bax Signaling Pathways in Mouse TM4 Sertoli Cells.

Flurochloridone (FLC), as a novel herbicide, has been widely used in many countries since 1980s. Current studies have shown that FLC has toxic effects on male reproduction and its target organ is testis, while the underlying mechanism is still unknown. Mouse testis Sertoli cell line TM4 cells were used as an in vitro model and treated with FLC at different doses (40, 80, 160 µM) for different times (6, 12, 24 h). Cell viability, cytotoxicity and apoptotic cells were detected by CCK-8 assay, LDH leakage assay and flow cytometry. The protein levels of GRP78, phosphorylated-eIF2α, ATF4, ATF6, CHOP, Bim and Bax were observed by Western Blot and Immunofluorescence staining. FLC inhibited cell viability and induced cytotoxicity in dose-dependent way in TM4 cells. The percentage of apoptotic cells were 6.2% ± 0.6%, 7.3% ± 0.3%, 9.8% ± 0.4%, 13.2% ± 0.2%, respectively. The expression levels of ER stress and UPR related proteins were activated over dose. Meanwhile, the pro-apoptotic proteins (Bim and Bax) were also up-regulated in dose-dependent. After pretreated with ISRIB, the inhibitor of eIF2α phosphorylation, the elevated expression of GRP78, phosphorylated-eIF2α, ATF4, ATF6, CHOP and Bim was down to normal level accordingly. In conclusion, FLC induced apoptosis in TM4 cells mediated by UPR signaling pathways.

Phillygenin activates PKR/eIF2α pathway and induces stress granule to exert anti-avian infectious bronchitis virus.

The prevalence of avian infectious bronchitis virus (IBV) is still one of causes inducing severe losses of production in the poultry industry worldwide. Vaccination does not completely prevent IBV infection and spread due to immune failure and viral mutations. ForsythiaeFructus and its compounds have been widely used in a lot of prescriptions of the traditional Chinese medicine for a long history, and it is well-known as safety and efficiency in heat-clearing and detoxifying. This study aims to investigate the anti-IBV activity and mechanism of phillygenin. The results showed that phillygenin inhibited IBV replication by disturbing multiple stages of the virus life cycle, including viral adsorption, invasion, internalization, and release in Vero cells. After being treated with 100, 125 and 150 µg/mL phillygenin, the expression of G3BP1 was significantly increased and the phosphorylation of PKR/eIF2α was activated, which increased stress granule, thereby triggering the antiviral response in Vero cells. The anti-virus activity of PHI was decreased when G3BP1 was interfered by si-RNA, and G3BP1 was down-regulated when PKR/eIF2α was interfered by si-RNA. In conclusion, our findings indicate that phillygenin activates PKR/eIF2α pathway and induces stress granule formation to exert anti-IBV, which holds promise to develop into a novel anti-IBV drug. Further study in vivo is needed to explore phillygenin as a potential and effective drug to prevent IB in poultry.

The effect and apoptosis mechanism of 6-methoxyflavone in HeLa cells.

INTRODUCTION: Tumour cell apoptosis is a crucial indicator for judging the antiproliferative effects of anti-cancer drugs. The detection of optical and macromolecular biomarkers is the most common method for assessing the level of apoptosis. We aimed to explore the anti-tumour mechanisms of 6-methoxyflavone. MATERIALS AND METHODS: Three optical methods, including the percentage of apoptotic cells, cell morphology, and subcellular ultrastructure changes, were obtained using flow cytometry, inverted fluorescence microscopy, and transmission electron microscope imaging. The mRNA or protein expression of macromolecular biomarkers related to common apoptotic pathways was determined via polymerase chain reactions or western blot assays. The functional role of the core gene biomarker was investigated through overexpression, knockdown, and phosphorylation inhibitor (GSK2656157). RESULTS: Transcriptome sequencing and the optical biomarkers assays demonstrated that 6-methoxyflavone could induce apoptosis in HeLa cells. The expression of macromolecular biomarkers indicated that 6-methoxyflavone induced apoptosis through the PERK/EIF2α/ATF4/CHOP pathway. Phosphorylated PERK was identified as the core biomarker of this pathway. Both overexpression and GSK2656157 significantly altered the expression level of phosphorylated PERK in 6-methoxyflavone-treated HeLa cells. DISCUSSION AND CONCLUSION: Macromolecular biomarkers, such as phosphorylated PERK and phosphorylated EIF2α are of great significance for assessing the therapeutic effects of 6-methoxyflavone.

Osthole Exerts Inhibitory Effects on Hypoxic Colon Cancer Cells via EIF2[Formula: see text] Phosphorylation-mediated Apoptosis and Regulation of HIF-1[Formula: see text].

Hypoxic microenvironment and dysregulated endoplasmic reticulum stress/unfolded protein response (UPR) system are considered important factors that promote cancer progression. Although osthole extracted from Cnidium monnieri(Fructus Cnidii) has been confirmed to exhibit an anticancer activity in various cancers, the effects of osthole in hypoxic colon cancer cells have not been explored. Therefore, the aim of this study was to examine whether osthole has an inhibitory effect on hypoxic colon cancer HCT116 cells and further investigate the underlying molecular mechanisms. Treatment with osthole significantly attenuated the cell viability, proliferation, and migration in hypoxic HCT116 cells. Osthole also activated UPR signaling such as phospho-eukaryotic initiation factor 2 alpha (EIF2[Formula: see text]/ATF4/CHOP/DR5 cascade accompanied by upregulation of pro-apoptotic proteins. Moreover, the tubule-like formation of human umbilical vein endothelial cells, the secretion of vascular endothelial growth factor A, and the expression and activity of hypoxia-inducible factor-1[Formula: see text] (HIF-1[Formula: see text] in hypoxic HCT116 cells were markedly suppressed by osthole. However, suppressing EIF2[Formula: see text] phosphorylation with salubrinal or ISRIB markedly reversed the effects of osthole on the expressions of pro-apoptotic proteins and HIF-1[Formula: see text]. Co-treatment of hypoxic HCT116 cells with osthole greatly increased the sensitivity to cisplatin and the expressions of phospho-EIF2[Formula: see text] and cleaved caspase 3. Collectively, the inhibitory effect of osthole in hypoxic HCT116 cells may be associated with EIF2[Formula: see text] phosphorylation-mediated apoptosis and translational repression of HIF-1[Formula: see text]. Taken together, osthole may be a potential agent in the treatment of colon cancer.

Attenuation of Activated eIF2α Signaling by ISRIB Treatment After Spinal Cord Injury Improves Locomotor Function.

Following spinal cord injury (SCI), multiple signaling cascades are activated instantaneously in the injured segments of the spinal cord to create a complex and pathogenic microenvironment, making it difficult to treat SCI. Nevertheless, the significance of the integrated stress response (ISR) to the series of physiological and pathological changes that occur after SCI remains unclear. Through western blotting (WB), we determined that the autophosphorylation of stress receptors (GCN2, PERK, PKR, and HRI) was enhanced after SCI, leading to increased phosphorylation of eIF2α at Ser51. Strikingly, we found that eIF2α was highly phosphorylated at 1 day post injury (dpi) and that this hypophosphorylation was maintained thereafter in the spinal cord, especially in neurons, which suggests that intervening with eIF2α phosphorylation may be a treatment strategy for SCI. Therefore, we employed the small molecule ISRIB, which inhibits eIF2α phosphorylation when the ISR is activated at moderate or low levels but not when the ISR is highly activated. Daily intraperitoneal injection of ISRIB significantly inhibited ISR signaling after SCI, reduced the cytosolic localization of RNA-binding proteins, and decreased neuronal apoptosis. Histological and functional experiments further demonstrated that treatment with ISRIB after SCI effectively curbed morphological deterioration and promoted the recovery of locomotor function. In summary, the ISR plays an important role in SCI, and ISRIB is a promising drug for the treatment of SCI.

Glucagon and Insulin Cooperatively Stimulate Fibroblast Growth Factor 21 Gene Transcription by Increasing the Expression of Activating Transcription Factor 4.

Previous studies have shown that glucagon cooperatively interacts with insulin to stimulate hepatic FGF21 gene expression. Here we investigated the mechanism by which glucagon and insulin increased FGF21 gene transcription in primary hepatocyte cultures. Transfection analyses demonstrated that glucagon plus insulin induction of FGF21 transcription was conferred by two activating transcription factor 4 (ATF4) binding sites in the FGF21 gene. Glucagon plus insulin stimulated a 5-fold increase in ATF4 protein abundance, and knockdown of ATF4 expression suppressed the ability of glucagon plus insulin to increase FGF21 expression. In hepatocytes incubated in the presence of insulin, treatment with a PKA-selective agonist mimicked the ability of glucagon to stimulate ATF4 and FGF21 expression. Inhibition of PKA, PI3K, Akt, and mammalian target of rapamycin complex 1 (mTORC1) suppressed the ability of glucagon plus insulin to stimulate ATF4 and FGF21 expression. Additional analyses demonstrated that chenodeoxycholic acid (CDCA) induced a 6-fold increase in ATF4 expression and that knockdown of ATF4 expression suppressed the ability of CDCA to increase FGF21 gene expression. CDCA increased the phosphorylation of eIF2α, and inhibition of eIF2α signaling activity suppressed CDCA regulation of ATF4 and FGF21 expression. These results demonstrate that glucagon plus insulin increases FGF21 transcription by stimulating ATF4 expression and that activation of cAMP/PKA and PI3K/Akt/mTORC1 mediates the effect of glucagon plus insulin on ATF4 expression. These results also demonstrate that CDCA regulation of FGF21 transcription is mediated at least partially by an eIF2α-dependent increase in ATF4 expression.