RESUMO
BACKGROUND: Osteonecrosis of the femoral head (ONFH) seriously affects the quality of life and labor ability of patients. It is urgent and vital to find the methods for necrosis clinical treatment. OBJECTIVE: This study aims to study the potential protective effects of Alendronate in the early stage of femur head necrosis. METHODS: Ten clinal ONFH tissue samples were employed. H&E staining was employed for the observation of the pathological characteristics of ONFH. The rat model (n=12) was established by the treatment of liquid nitrogen and then treated with Alendronate. The protein expression of BMP2, EIF2AK3, EIF2A and ATF4 were detected via Western blotting and IHC. RESULTS: Fibrin and necrotizing granulation tissue were observed in ONFH tissues with lymphocytes and plasma cells infiltrating in the necrotic area, exhibiting the inflammatory muscle with abnormal shape and color. In the Model group, the BMP2 and ATF4 were mainly distributed in the cell boundaries. The relative protein expression of BMP2, EIF2AK3, EIF2A, ATF4 was decreased in the Model group, compared to the NC group, which was partially recovered by the Alendronate application. CONCLUSION: Alendronate application partially reversed the suppression of expression of BMP2, EIF2AK3, EIF2A, ATF4 caused by liquid nitrogen. Alendronate could be a promising strategy of curing ONFH via targeting BMP2/EIF2AK3/EIF2A/ATF4 pathway.
Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Alendronato/farmacologia , Proteína Morfogenética Óssea 2/metabolismo , Necrose da Cabeça do Fêmur/tratamento farmacológico , Regulação para Cima/efeitos dos fármacos , eIF-2 Quinase/metabolismo , Fator 4 Ativador da Transcrição/antagonistas & inibidores , Fator 4 Ativador da Transcrição/genética , Animais , Proteína Morfogenética Óssea 2/antagonistas & inibidores , Proteína Morfogenética Óssea 2/genética , Feminino , Necrose da Cabeça do Fêmur/metabolismo , Necrose da Cabeça do Fêmur/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Nitrogênio/farmacologia , Osteonecrose/tratamento farmacológico , Osteonecrose/metabolismo , Osteonecrose/patologia , Ratos , Ratos Sprague-Dawley , eIF-2 Quinase/antagonistas & inibidores , eIF-2 Quinase/genéticaRESUMO
Traumatic spinal cord injury (SCI) is a devastating neurological condition that involves both primary and secondary tissue loss. Various cytotoxic events including hypoxia, hemorrhage and blood lysis, bioenergetic failure, oxidative stress, endoplasmic reticulum (ER) stress, and neuroinflammation contribute to secondary injury. The HIF prolyl hydroxylase domain (PHD/EGLN) family of proteins are iron-dependent, oxygen-sensing enzymes that regulate the stability of hypoxia inducible factor-1α (HIF-1α) and also mediate oxidative stress caused by free iron liberated from the lysis of blood. PHD inhibition improves outcome after experimental intracerebral hemorrhage (ICH) by reducing activating transcription factor 4 (ATF4)-driven neuronal death. As the ATF4-CHOP (CCAAT-enhancer-binding protein homologous protein) pathway plays a role in the pathogenesis of contusive SCI, we examined the effects of PHD inhibition in a mouse model of moderate T9 contusive SCI in which white matter damage is the primary driver of locomotor dysfunction. Pharmacological inhibition of PHDs using adaptaquin (AQ) moderately lowers acute induction of Atf4 and Chop mRNAs and prevents the acute decline of oligodendrocyte (OL) lineage mRNAs, but does not improve long-term recovery of hindlimb locomotion or increase chronic white matter sparing. Conditional genetic ablation of all three PHD isoenzymes in OLs did not affect Atf4, Chop or OL mRNAs expression levels, locomotor recovery, and white matter sparing after SCI. Hence, PHDs may not be suitable targets to improve outcomes in traumatic CNS pathologies that involve acute white matter injury.
Assuntos
Fator 4 Ativador da Transcrição/antagonistas & inibidores , Estresse do Retículo Endoplasmático , Locomoção , Pró-Colágeno-Prolina Dioxigenase/antagonistas & inibidores , Recuperação de Função Fisiológica , Traumatismos da Medula Espinal/fisiopatologia , Fator de Transcrição CHOP/antagonistas & inibidores , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Traumatismos da Medula Espinal/metabolismo , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismoRESUMO
Intracerebral hemorrhage (ICH) is the second largest type of stroke, with high mortality and morbidity, and most patients have severe sequelae. Brain injury induced by ICH includes primary damage and secondary damage, and the secondary brain injury is the main reason of neurological impairment. The hallmark of secondary brain injury is cell death. Necroptosis is a type of the cell death and plays vital roles in various neurological diseases, but the roles of necroptosis in ICH are still not fully known. Microglia cell is the type of immune cell, plays protective roles in nerve damage and modulates the activity of neurons through secreting exosomes. Exosome-contained miRNAs are also involved in the regulating neuronal activity. However, the roles and the mechanisms of microglia-secreted exosomes miRNAs in ICH neurons necroptosis need to further explore. In this study, ICH model was construct in rats and cells. Injury of cells in brain was detected by PI staining. Necroptosis in rats and cells was detected by western blot and flow cytometry. The expression of miR-383-3p was detected by RT-qPCR. The roles of activated microglia-secreted exosomes and exosome-contained miR-383-3p were detected through co-culturing medium or exosomes with neurons. The target gene of miR-383-3p was determined by luciferase assay and the expression of target gene was detected by western blot. Rescue experiments were used to confirm the mechanism of miR-383-3p in neurons necroptosis. The miR-383-3p role was verified in vivo through injecting miR-383-3p mimic into ICH rats. Here, we found that the necroptosis of neurons was increased in ICH rats through detecting the expression of RIP1 and RIP3 and PI staining. Microglia that activated by ICH promote neurons necroptosis through secreting exosomes and transferring miR-383-3p into neurons. In mechanism, miR-383-3p negatively regulated the expression of ATF4 and then promoted the necroptosis of neurons. Overall, our results provide a novel molecular basis to neurons necroptosis in ICH and may provide a new strategy to retard the secondary brain injury of ICH.
Assuntos
Fator 4 Ativador da Transcrição/antagonistas & inibidores , Hemorragia Cerebral/fisiopatologia , Exossomos/metabolismo , MicroRNAs/metabolismo , Microglia/metabolismo , Necroptose/fisiologia , Animais , Células Cultivadas , Técnicas de Cocultura , Masculino , Neurônios/metabolismo , Ratos Sprague-DawleyRESUMO
Endoplasmic reticulum (ER) stress plays a critical role in pancreatic ß cell destruction which leads to the pathogenesis of type 1 diabetes mellitus (T1DM). Vitamin D (VD) has been reported to reduce the risk of T1DM; however, it remains unknown whether VD affects ER stress in pancreatic ß cells. In this study, we investigated the role of the active form of VD, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], in ER stress-induced ß cell apoptosis and explored its potential mechanism in mouse insulinoma cell line mouse insulinoma 6 (MIN6). The results of cell counting kit-8 (CCK8) and flow cytometric analyses showed that 1,25-(OH)2D3 caused a significant increase in the viability of MIN6 cells injured by H2O2. The protein kinase like ER kinase (PERK) signal pathway, one of the most conserved branches of ER stress, was found to be involved in this process. H2O2 activated the phosphorylation of PERK, upregulated the activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP) expression, and subsequently initiated cell apoptosis, which were significantly reversed by 1,25-(OH)2D3 pretreatment. In addition, GSK2606414, a specific inhibitor of PERK, suppressed PERK phosphorylation and reduced the expressions of ATF4 and CHOP, leading to a significant decrease in ß cell apoptosis induced by H2O2. Taken together, the present findings firstly demonstrated that 1,25-(OH)2D3 could prevent MIN6 cells against ER stress-associated apoptosis by inhibiting the PERK-ATF4-CHOP pathway. Therefore, our results suggested that 1,25-(OH)2D3 might serve as a potential therapeutic target for preventing pancreatic ß cell destruction in T1DM.
Assuntos
Fator 4 Ativador da Transcrição/antagonistas & inibidores , Calcitriol/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição CHOP/antagonistas & inibidores , eIF-2 Quinase/antagonistas & inibidores , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Diabetes Mellitus Tipo 1/tratamento farmacológico , Diabetes Mellitus Tipo 1/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Peróxido de Hidrogênio/toxicidade , Indóis/farmacologia , Células Secretoras de Insulina/citologia , CamundongosRESUMO
Arginine deprivation therapy (ADT) is a new metabolic targeting approach with high therapeutic potential for various solid cancers. Combination of ADT with low doses of the natural arginine analog canavanine effectively sensitizes malignant cells to irradiation. However, the molecular mechanisms determining the sensitivity of intrinsically non-auxotrophic cancers to arginine deficiency are still poorly understood. We here show for the first time that arginine deficiency is accompanied by global metabolic changes and protein/membrane breakdown, and results in the induction of specific, more or less pronounced (severe vs. mild) ER stress responses in head and neck squamous cell carcinoma (HNSCC) cells that differ in their intrinsic ADT sensitivity. Combination of ADT with canavanine triggered catastrophic ER stress via the eIF2α-ATF4(GADD34)-CHOP pathway, thereby inducing apoptosis; the same signaling arm was irrelevant in ADT-related radiosensitization. The particular strong supra-additive effect of ADT, canavanine and irradiation in both intrinsically more and less sensitive cancer cells supports the rational of ER stress pathways as novel target for improving multi-modal metabolic anti-cancer therapy.
Assuntos
Canavanina/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Tolerância a Radiação/efeitos dos fármacos , Raios X , Fator 4 Ativador da Transcrição/antagonistas & inibidores , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Apoptose/efeitos dos fármacos , Arginina/deficiência , Arginina/metabolismo , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Meios de Cultura/química , Endorribonucleases/antagonistas & inibidores , Endorribonucleases/genética , Endorribonucleases/metabolismo , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacos , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Fator de Transcrição CHOP/antagonistas & inibidores , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismoRESUMO
The underlying pathological mechanisms of glaucomatous trabecular meshwork (TM) damage and elevation of intraocular pressure (IOP) are poorly understood. Here, we report that the chronic endoplasmic reticulum (ER) stress-induced ATF4-CHOP-GADD34 pathway is activated in TM of human and mouse glaucoma. Expression of ATF4 in TM promotes aberrant protein synthesis and ER client protein load, leading to TM dysfunction and cell death. These events lead to IOP elevation and glaucomatous neurodegeneration. ATF4 interacts with CHOP and this interaction is essential for IOP elevation. Notably, genetic depletion or pharmacological inhibition of ATF4-CHOP-GADD34 pathway prevents TM cell death and rescues mouse models of glaucoma by reducing protein synthesis and ER client protein load in TM cells. Importantly, glaucomatous TM cells exhibit significantly increased protein synthesis along with induction of ATF4-CHOP-GADD34 pathway. These studies indicate a pathological role of ATF4-CHOP-GADD34 pathway in glaucoma and provide a possible treatment for glaucoma by targeting this pathway.
Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Estresse do Retículo Endoplasmático , Glaucoma de Ângulo Aberto/metabolismo , Biossíntese de Proteínas , Fator 4 Ativador da Transcrição/antagonistas & inibidores , Fator 4 Ativador da Transcrição/genética , Animais , Humor Aquoso/metabolismo , Morte Celular , Células Cultivadas , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/genética , Glaucoma de Ângulo Aberto/tratamento farmacológico , Glaucoma de Ângulo Aberto/patologia , Humanos , Camundongos , Hipertensão Ocular/tratamento farmacológico , Hipertensão Ocular/metabolismo , Hipertensão Ocular/patologia , Nervo Óptico/metabolismo , Nervo Óptico/patologia , Biossíntese de Proteínas/efeitos dos fármacos , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismo , Células Ganglionares da Retina/metabolismo , Células Ganglionares da Retina/patologia , Transdução de Sinais , Malha Trabecular/efeitos dos fármacos , Malha Trabecular/metabolismo , Malha Trabecular/patologia , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismoRESUMO
Norditerpenoids and dinorditerpenoids represent diterpenoids widely distributed in the genus Podocarpus with notable chemical structures and biological activities. We previously reported that nagilactone E (NLE), a dinorditerpenoid isolated from Podocarpus nagi, possessed anticancer effects against lung cancer cells in vitro. In this study we investigated the in vivo effect of NLE against lung cancer as well as the underlying mechanisms. We administered NLE (10 mg·kg-1·d-1, ip) to CB-17/SCID mice bearing human lung cancer cell line A549 xenograft for 3 weeks. We found that NLE administration significantly suppressed the tumor growth without obvious adverse effects. Thereafter, RNA sequencing (RNA-seq) analysis was performed to study the mechanisms of NLE. The effects of NLE on A549 cells have been illustrated by GO and pathway enrichment analyses. CMap dataset analysis supported NLE to be a potential protein synthesis inhibitor. The inhibitory effect of NLE on synthesis of total de novo protein was confirmed in Click-iT assay. Using the pcDNA3-RLUC-POLIRES-FLUC luciferase assay we further demonstrated that NLE inhibited both cap-dependent and cap-independent translation. Finally, molecular docking revealed the low-energy binding conformations of NLE and its potential target RIOK2. In conclusion, NLE is a protein synthesis inhibitor with anticancer activity.
Assuntos
Fator 4 Ativador da Transcrição/antagonistas & inibidores , Antineoplásicos/farmacologia , Inibidor de Quinase Dependente de Ciclina p21/antagonistas & inibidores , Diterpenos/farmacologia , Fator 2 Relacionado a NF-E2/antagonistas & inibidores , Inibidores da Síntese de Proteínas/farmacologia , Fator de Transcrição STAT3/antagonistas & inibidores , Células A549 , Fator 4 Ativador da Transcrição/biossíntese , Fator 4 Ativador da Transcrição/genética , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/isolamento & purificação , Proliferação de Células/efeitos dos fármacos , Biologia Computacional , Inibidor de Quinase Dependente de Ciclina p21/biossíntese , Inibidor de Quinase Dependente de Ciclina p21/genética , Diterpenos/administração & dosagem , Diterpenos/isolamento & purificação , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Injeções Intraperitoneais , Masculino , Camundongos , Camundongos SCID , Simulação de Acoplamento Molecular , Fator 2 Relacionado a NF-E2/biossíntese , Fator 2 Relacionado a NF-E2/genética , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Inibidores da Síntese de Proteínas/administração & dosagem , Inibidores da Síntese de Proteínas/isolamento & purificação , Fator de Transcrição STAT3/biossíntese , Fator de Transcrição STAT3/genética , Células Tumorais CultivadasRESUMO
Identifying disease-causing pathways and drugs that target them in Parkinson's disease (PD) has remained challenging. We uncovered a PD-relevant pathway in which the stress-regulated heterodimeric transcription complex CHOP/ATF4 induces the neuron prodeath protein Trib3 that in turn depletes the neuronal survival protein Parkin. Here we sought to determine whether the drug adaptaquin, which inhibits ATF4-dependent transcription, could suppress Trib3 induction and neuronal death in cellular and animal models of PD. Neuronal PC12 cells and ventral midbrain dopaminergic neurons were assessed in vitro for survival, transcription factor levels and Trib3 or Parkin expression after exposure to 6-hydroxydopamine or 1-methyl-4-phenylpyridinium with or without adaptaquin co-treatment. 6-hydroxydopamine injection into the medial forebrain bundle was used to examine the effects of systemic adaptaquin on signaling, substantia nigra dopaminergic neuron survival and striatal projections as well as motor behavior. In both culture and animal models, adaptaquin suppressed elevation of ATF4 and/or CHOP and induction of Trib3 in response to 1-methyl-4-phenylpyridinium and/or 6-hydroxydopamine. In culture, adaptaquin preserved Parkin levels, provided neuroprotection and preserved morphology. In the mouse model, adaptaquin treatment enhanced survival of dopaminergic neurons and substantially protected their striatal projections. It also significantly enhanced retention of nigrostriatal function. These findings define a novel pharmacological approach involving the drug adaptaquin, a selective modulator of hypoxic adaptation, for suppressing Parkin loss and neurodegeneration in toxin models of PD. As adaptaquin possesses an oxyquinoline backbone with known safety in humans, these findings provide a firm rationale for advancing it towards clinical evaluation in PD.
Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Proteínas de Ciclo Celular/biossíntese , Transtornos Parkinsonianos/metabolismo , Transtornos Parkinsonianos/prevenção & controle , Piridinas/farmacologia , Quinolinas/farmacologia , Fator de Transcrição CHOP/metabolismo , Fator 4 Ativador da Transcrição/antagonistas & inibidores , Animais , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Células Cultivadas , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Oxidopamina/toxicidade , Células PC12 , Transtornos Parkinsonianos/induzido quimicamente , Piridinas/uso terapêutico , Quinolinas/uso terapêutico , Ratos , Fator de Transcrição CHOP/antagonistas & inibidoresRESUMO
Activating transcription factor 4 (ATF4) promotes bone formation in human bone marrow mesenchymal stem cells. However, the underlying mechanisms of ATF4 in high glucose-induced injury of osteoblast still remain unclear. Small interfering RNA and plasmid targeting ATF4 were used to transfect MC3T3-E1 cells to knock down and overexpress ATF4 using Lipofectamin 3000. Cell viability, alkaline phosphatase (ALP) activity and levels were determined by MTT, ALP kit assay, quantitative real-time (qRT)-PCR and Western blot. Osteocalcin (OCN) expression was determined by ELISA, PCR and Western blot. The mRNA and protein levels of ATF4, glucose regulated protein 78 kDa (GRP78) and C/EBP homologous protein (CHOP) were detected by PCR and Western blot. In the current study, viabilities of MC3T3-E1 cells were inhibited by high glucose. Meanwhile, the mRNA and protein levels of ATF4 were effectively up-regulated in high glucose-incubated MC3T3-E1 cells. By conducting functional experiments, silencing ATF4 induced by small interfering RNA partially reversed the inhibitory effects of high glucose on viabilities of MC3T3-E1 cells. We also found that the expressions of ER stress-related proteins (ATF4, GRP78 and CHOP) were higher in high glucose-treated MC3T3-E1 cells but were inhibited by siATF4. However, overexpression of AFT4 had opposite results, and high glucose attenuated the protein levels of osteogenic marker genes ALP and OCN, which were further inhibited by ATF4 knockout gene. Thus, ATF4 was a necessary gene for high glucose to inhibit the proliferation and differentiation of MC3T3-E1 cells.
Assuntos
Fator 4 Ativador da Transcrição/genética , Diferenciação Celular/genética , Células-Tronco Mesenquimais/metabolismo , Osteogênese/genética , Fator 4 Ativador da Transcrição/antagonistas & inibidores , Fosfatase Alcalina/genética , Animais , Proliferação de Células/genética , Sobrevivência Celular/efeitos dos fármacos , Chaperona BiP do Retículo Endoplasmático , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Técnicas de Inativação de Genes , Inativação Gênica , Glucose/toxicidade , Proteínas de Choque Térmico/genética , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Osteoblastos/efeitos dos fármacos , Osteocalcina/genética , RNA Mensageiro/genética , Fator de Transcrição CHOP/genéticaRESUMO
Understanding the intrinsic mediators that render CD8+ T cells dysfunctional in the tumor microenvironment is a requirement to develop more effective cancer immunotherapies. Here, we report that C/EBP homologous protein (Chop), a downstream sensor of severe endoplasmic reticulum (ER) stress, is a major negative regulator of the effector function of tumor-reactive CD8+ T cells. Chop expression is increased in tumor-infiltrating CD8+ T cells, which correlates with poor clinical outcome in ovarian cancer patients. Deletion of Chop in T cells improves spontaneous antitumor CD8+ T cell immunity and boosts the efficacy of T cell-based immunotherapy. Mechanistically, Chop in CD8+ T cells is elevated primarily through the ER stress-associated kinase Perk and a subsequent induction of Atf4; and directly represses the expression of T-bet, a master regulator of effector T cell function. These findings demonstrate the primary role of Chop in tumor-induced CD8+ T cell dysfunction and the therapeutic potential of blocking Chop or ER stress to unleash T cell-mediated antitumor immunity.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Carcinoma Epitelial do Ovário/genética , Estresse do Retículo Endoplasmático/genética , Regulação Neoplásica da Expressão Gênica/imunologia , Neoplasias Ovarianas/genética , Proteínas com Domínio T/genética , Fator de Transcrição CHOP/genética , Fator 4 Ativador da Transcrição/antagonistas & inibidores , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/imunologia , Transferência Adotiva , Animais , Linfócitos T CD8-Positivos/patologia , Carcinoma Epitelial do Ovário/imunologia , Carcinoma Epitelial do Ovário/mortalidade , Carcinoma Epitelial do Ovário/terapia , Linhagem Celular Tumoral , Feminino , Humanos , Imunidade Celular , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/patologia , Camundongos , Camundongos Knockout , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/mortalidade , Neoplasias Ovarianas/terapia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Análise de Sobrevida , Proteínas com Domínio T/imunologia , Fator de Transcrição CHOP/antagonistas & inibidores , Fator de Transcrição CHOP/imunologia , Microambiente Tumoral/genética , Microambiente Tumoral/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto , eIF-2 Quinase/antagonistas & inibidores , eIF-2 Quinase/genética , eIF-2 Quinase/imunologiaRESUMO
Hyperglycemia associated ER stress has been found as a critical contributor in the pathogenesis of type 2 diabetes mellitus. However, reports regarding molecular mechanisms involved are limited. This study was aimed to identify the role of ER stress in regulating hepatic glucose metabolism and its link with oxidative stress. Further, this study explores the novel role of Morin, a flavonol, in modulating ER stress in STZ/nicotinamide induced type 2 diabetic male Wistar rats. Results demonstrate that hyperglycemia induced ER stress in rats and significantly lowered the expression of glucose transporter proteins resulting in impaired glucose metabolism during diabetes. Morin was found to downregulate PERK-eIF2α-ATF4 pathway by interacting with PERK protein as confirmed through pull-down assay. Additionally, Morin maintained the reducing environment in ER and enhanced PDI activity compared to diabetic rats. Morin prevented cell death by suppressing the expression of PERK dependent pro-apoptotic proteins including ATF4 and CHOP. Findings from this study affirm the role of ER stress in hyperglycemia induced gluco-metabolic aberrations and liver injury as confirmed by ISRIB, a standard chemical ER stress inhibitor. Notably, Morin promoted deactivation of UPR sensors and upregulated PDI activity endorsing its anti-ER stress potential which may allow the development of new therapeutic avenues to target hyperglycemic hepatotoxicity.
Assuntos
Fator 4 Ativador da Transcrição/antagonistas & inibidores , Antioxidantes/farmacologia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Flavonoides/farmacologia , Hepatopatias/tratamento farmacológico , eIF-2 Quinase/antagonistas & inibidores , Fator 4 Ativador da Transcrição/metabolismo , Animais , Antioxidantes/uso terapêutico , Apoptose/efeitos dos fármacos , Doença Crônica , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Tipo 2/complicações , Regulação para Baixo , Flavonoides/uso terapêutico , Hiperglicemia/complicações , Hepatopatias/metabolismo , Masculino , Ratos , Ratos Wistar , Estreptozocina , eIF-2 Quinase/metabolismoRESUMO
BACKGROUND: Multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), are inflammatory demyelinating and neurodegenerative diseases of the CNS. Although recent studies suggest the neuroprotective effects of oligodendrocytes in neurodegenerative diseases, it remains unknown whether oligodendrocyte death induced by inflammatory attacks contributes to neurodegeneration in MS and EAE. Upon endoplasmic reticulum (ER) stress, activation of pancreatic ER kinase (PERK) promotes cell survival through induction of activating transcription factor 4 (ATF4) by phosphorylating eukaryotic translation initiation factor 2α (eIF2α). We have generated a mouse model that allows for temporally controlled activation of PERK specifically in oligodendrocytes. Our previous study has demonstrated that PERK activation specifically in oligodendrocytes attenuates EAE disease severity and ameliorates EAE-induced oligodendrocyte apoptosis, demyelination, and axon degeneration, without altering inflammation. METHODS: We determined whether oligodendrocyte-specific PERK activation reduced neuron loss in the CNS of EAE mice using the mouse model that allows for temporally controlled activation of PERK specifically in oligodendrocytes. We further generated a mouse model that allows for inactivation of ATF4 specifically in oligodendrocytes, and determined the effects of ATF4 inactivation in oligodendrocytes on mice undergoing EAE. RESULTS: We showed that protection of oligodendrocytes resulting from PERK activation led to attenuation of neuron loss in the CNS gray matter of EAE mice. Surprisingly, we found that ATF4 inactivation specifically in oligodendrocytes did not alter EAE disease severity and had no effect on oligodendrocyte loss, demyelination, axon degeneration, neuron loss, and inflammation in EAE mice. CONCLUSIONS: These findings suggest the neuroprotective effects of PERK activation in oligodendrocytes in EAE, and rule out the involvement of ATF4 in oligodendrocytes in the development of EAE. These results imply that the protective effects of PERK activation in oligodendrocytes in MS and EAE are not mediated by ATF4.
Assuntos
Fator 4 Ativador da Transcrição/antagonistas & inibidores , Encefalomielite Autoimune Experimental/patologia , Oligodendroglia/efeitos dos fármacos , Animais , Axônios/patologia , Proliferação de Células , Doenças Desmielinizantes/patologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Degeneração Neural/patologia , Neurônios/patologia , Linfócitos T/efeitos dos fármacos , eIF-2 Quinase/antagonistas & inibidores , eIF-2 Quinase/genéticaRESUMO
Endoplasmic reticulum (ER) stress results from imbalances in unfolded/misfolded proteins, contributing to a wide variety of human diseases. To better understand the mechanisms involved in the cellular response to ER stress in cardiomyocytes, we previously conducted a genome-wide screening in an in vitro ER stress model of rat cardiomyocytes, which highlighted amino acid transporter heavy chain, member 2 (SLC3A2) as an important factor in ER stress. In the present study, we characterized the role of SLC3A2 during the unfolded protein response (UPR), as one of the primary pathways activated during ER stress. First, we confirmed the induction of Slc3a2 mRNA expression following treatment with various ER stress inducers in rat cardiomyocytes (H9C2) and neural cells (PC12). Knockdown of Slc3a2 expression with small interfering RNA (siRNA) revealed that the encoded protein functions upstream of three important UPR proteins: ATF4, ATF6, and XBP1. siRNA-mediated knockdown of both SLC3A2 and mammalian target of rapamycin 1 (mTOR1) revealed that mTOR1 acts as a mediator between SLC3A2 and the UPR. RNA sequencing was then performed to gain a more thorough understanding of the function of SLC3A2, which identified 23 highly differentially regulated genes between the control and knockdown cell lines, which were related to the UPR and amino acid transport. Notably, flow cytometry further showed that SLC3A2 inhibition also enhanced the apoptosis of rat cardiomyocytes. Taken together, these results highlight SLC3A2 as a complex, multifunctional signaling protein that acts upstream of well-known UPR proteins with anti-apoptotic properties, suggesting its potential as a therapeutic target for ER stress-related diseases.
Assuntos
Apoptose , Estresse do Retículo Endoplasmático , Cadeia Pesada da Proteína-1 Reguladora de Fusão/metabolismo , Resposta a Proteínas não Dobradas , Fator 4 Ativador da Transcrição/antagonistas & inibidores , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Regulação para Baixo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Cadeia Pesada da Proteína-1 Reguladora de Fusão/antagonistas & inibidores , Cadeia Pesada da Proteína-1 Reguladora de Fusão/genética , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Células PC12 , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ratos , Serina-Treonina Quinases TOR/metabolismo , Tapsigargina/farmacologia , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Proteína 1 de Ligação a X-Box/antagonistas & inibidores , Proteína 1 de Ligação a X-Box/genética , Proteína 1 de Ligação a X-Box/metabolismoRESUMO
Angiogenesis, the formation of new blood vessels by endothelial cells (ECs), is an adaptive response to oxygen/nutrient deprivation orchestrated by vascular endothelial growth factor (VEGF) upon ischemia or exercise. Hypoxia is the best-understood trigger of VEGF expression via the transcription factor HIF1α. Nutrient deprivation is inseparable from hypoxia during ischemia, yet its role in angiogenesis is poorly characterized. Here, we identified sulfur amino acid restriction as a proangiogenic trigger, promoting increased VEGF expression, migration and sprouting in ECs in vitro, and increased capillary density in mouse skeletal muscle in vivo via the GCN2/ATF4 amino acid starvation response pathway independent of hypoxia or HIF1α. We also identified a requirement for cystathionine-γ-lyase in VEGF-dependent angiogenesis via increased hydrogen sulfide (H2S) production. H2S mediated its proangiogenic effects in part by inhibiting mitochondrial electron transport and oxidative phosphorylation, resulting in increased glucose uptake and glycolytic ATP production.
Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Aminoácidos Sulfúricos/deficiência , Sulfeto de Hidrogênio/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator 4 Ativador da Transcrição/antagonistas & inibidores , Fator 4 Ativador da Transcrição/genética , Aminoácidos Sulfúricos/metabolismo , Animais , Cistationina gama-Liase/metabolismo , Modelos Animais de Doenças , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Isquemia/metabolismo , Isquemia/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Fisiológica , Condicionamento Físico Animal , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
The stress-induced unfolded protein response (UPR) in the endoplasmic reticulum (ER) involves various signaling cross-talks and controls cell fate. B-cell receptor (BCR) signaling, which can trigger UPR, induces gammaherpesvirus lytic replication and serves as a physiological mechanism for gammaherpesvirus reactivation in vivo However, how the UPR regulates BCR-mediated gammaherpesvirus infection is unknown. Here, we demonstrate that the ER stressors tunicamycin and thapsigargin inhibit BCR-mediated murine gammaherpesvirus 68 (MHV68) lytic replication by inducing expression of the UPR mediator Bip and blocking activation of Akt, ERK, and JNK. Both Bip and the downstream transcription factor ATF4 inhibited BCR-mediated MHV68 lytic gene expression, whereas UPR-induced C/EBP homologous protein (CHOP) was required for and promoted BCR-mediated MHV68 lytic replication by suppressing upstream Bip and ATF4 expression. Bip knockout was sufficient to rescue BCR-mediated MHV68 lytic gene expression in CHOP knockout cells, and this rescue was blocked by ectopic ATF4 expression. Furthermore, ATF4 directly inhibited promoter activity of the MHV68 lytic switch transactivator RTA. Altogether, we show that ER stress-induced CHOP inhibits Bip and ATF4 expression and that ATF4, in turn, plays a critical role in CHOP-mediated regulation of BCR-controlled MHV68 lytic replication. We conclude that ER stress-mediated UPR and BCR signaling pathways are interconnected and form a complex network to regulate the gammaherpesvirus infection cycle.
Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Linfócitos B/virologia , Estresse do Retículo Endoplasmático , Gammaherpesvirinae/fisiologia , Proteínas de Choque Térmico/metabolismo , Receptores de Antígenos de Linfócitos B/agonistas , Fator de Transcrição CHOP/metabolismo , Fator 4 Ativador da Transcrição/antagonistas & inibidores , Fator 4 Ativador da Transcrição/genética , Animais , Antivirais/farmacologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linhagem Celular Transformada , Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Gammaherpesvirinae/efeitos dos fármacos , Gammaherpesvirinae/crescimento & desenvolvimento , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Inativação de Genes , Proteínas de Choque Térmico/antagonistas & inibidores , Proteínas de Choque Térmico/genética , Lisogenia/efeitos dos fármacos , Camundongos , Chaperonas Moleculares/antagonistas & inibidores , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tapsigargina/farmacologia , Fator de Transcrição CHOP/antagonistas & inibidores , Fator de Transcrição CHOP/genética , Tunicamicina/farmacologia , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/genética , Proteínas Virais/metabolismo , Ativação Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
CONTEXT: Molecular pathogenesis of chronic alcoholism is linked to increased endoplasmic reticulum stress. Ethanol is a competitive inhibitor of vitamin A metabolism and vitamin A supplementation aggravates existing liver problems. Hence, we probed into the impact of supplementation of all trans retinoic acid (ATRA), the active metabolite of vitamin A on ethanol-induced endoplasmic reticulcum stress. METHODS: Male Sprague-Dawley rats were divided into four groups - I: Control; II: Ethanol; III: ATRA; IV: ATRA + Ethanol. After 90 days the animals were sacrificed to study markers of lipid peroxidation in hepatic microsomal fraction and expression of ER stress proteins and apoptosis in liver. RESULTS AND CONCLUSION: Ethanol caused hepatic hyperlipidemia, enhanced microsomal lipid peroxidation, upregulated expression of unfolded protein response associated proteins and that of apoptosis. Ethanol also led to downregulation of retinoid receptors. ATRA supplementation reversed all these alterations indicating the decrease in ethanol-induced endoplasmic reticulum stress.
Assuntos
Suplementos Nutricionais , Estresse do Retículo Endoplasmático , Fígado Gorduroso Alcoólico/prevenção & controle , Fígado/metabolismo , Substâncias Protetoras/uso terapêutico , Receptores do Ácido Retinoico/agonistas , Tretinoína/uso terapêutico , Fator 4 Ativador da Transcrição/agonistas , Fator 4 Ativador da Transcrição/antagonistas & inibidores , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Animais , Apoptose/efeitos dos fármacos , Biomarcadores/metabolismo , Citocromo P-450 CYP2E1/química , Citocromo P-450 CYP2E1/genética , Citocromo P-450 CYP2E1/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Etanol/toxicidade , Fígado Gorduroso Alcoólico/enzimologia , Fígado Gorduroso Alcoólico/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/enzimologia , Masculino , Ratos Sprague-Dawley , Receptores do Ácido Retinoico/antagonistas & inibidores , Receptores do Ácido Retinoico/genética , Receptores do Ácido Retinoico/metabolismo , Receptores X de Retinoides/agonistas , Receptores X de Retinoides/antagonistas & inibidores , Receptores X de Retinoides/genética , Receptores X de Retinoides/metabolismo , Fator de Transcrição CHOP/agonistas , Fator de Transcrição CHOP/antagonistas & inibidores , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo , Tretinoína/antagonistas & inibidores , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Proteína 1 de Ligação a X-Box/agonistas , Proteína 1 de Ligação a X-Box/antagonistas & inibidores , Proteína 1 de Ligação a X-Box/genética , Proteína 1 de Ligação a X-Box/metabolismoRESUMO
Anoikis resistance is a crucial step in the process of tumor metastasis. This step determines whether the tumor cells will survive when they become detached from the extracellular matrix. However, the specific mechanism of tumor cells to bypass anoikis and become resistant remains to be elucidated. The present study aimed to determine the internal mechanism of bypassing anoikis through comparison of human osteosarcoma cell lines with human normal cell lines. High activating transcription factor 4 (ATF4) and myelocytomatosis oncogene (MYC) expression levels were observed in MG63 and U2 OS human osteosarcoma cell lines. It is possible that ATF4 and MYC contribute to tumor progression. Subsequently, the expression levels of ATF4 and MYC in HUVEC and CHON001 human normal cell lines were upregulated and their adhesion abilities were reduced; whereas their ability to bypass anoikis increased significantly. Simultaneously, after we Following a knockdown ofATF4 and MYC expression levels in MG63 and U2 OS human osteosarcoma cell lines, their adhesion ability increased and their ability to bypassing anoikis was significantly reduced. Upregulation of MYC resulted in an upregulation of ATF4, and chromatin immunoprecipitation and luciferase reporter gene technology demonstrated that MYC binds to the promoter of ATF4. These findings suggest that ATF4 regulated by MYC might contribute to resistance to anoikis in human osteosarcoma cells.
Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Anoikis , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fator 4 Ativador da Transcrição/antagonistas & inibidores , Fator 4 Ativador da Transcrição/genética , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Adesão Celular , Linhagem Celular Tumoral , Células Endoteliais da Veia Umbilical Humana , Humanos , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Regiões Promotoras Genéticas , Ligação Proteica , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Regulação para CimaRESUMO
Leishmania parasites utilize adaptive evasion mechanisms in infected macrophages to overcome host defenses and proliferate. We report here that the PERK/eIF2α/ATF4 signaling branch of the integrated endoplasmic reticulum stress response (IERSR) is activated by Leishmania and this pathway is important for Leishmania amazonensis infection. Knocking down PERK or ATF4 expression or inhibiting PERK kinase activity diminished L. amazonensis infection. Knocking down ATF4 decreased NRF2 expression and its nuclear translocation, reduced HO-1 expression and increased nitric oxide production. Meanwhile, the increased expression of ATF4 and HO-1 mRNAs were observed in lesions derived from patients infected with the prevalent related species L.(V.) braziliensis. Our data demonstrates that Leishmania parasites activate the PERK/eIF2α/ATF-4 pathway in cultured macrophages and infected human tissue and that this pathway is important for parasite survival and progression of the infection.
Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Leishmaniose Cutânea/patologia , Fator 4 Ativador da Transcrição/antagonistas & inibidores , Fator 4 Ativador da Transcrição/genética , Animais , Estresse do Retículo Endoplasmático , Células HEK293 , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Humanos , Leishmania/patogenicidade , Leishmaniose Cutânea/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos C57BL , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Óxido Nítrico/metabolismo , Fosforilação , Células RAW 264.7 , Interferência de RNA , RNA Interferente Pequeno/metabolismoRESUMO
The Helicobacter pylori vacuolating cytotoxin (VacA) can promote progressive vacuolation and gastric injury and may be associated with human gastric cancer. Increasing evidence indicates that autophagy is involved in the cell death induced by VacA, but the specific mechanisms need to be further elucidated. We show here that VacA could induce autophagy and increase cell death in human gastric cancer cell lines. Further investigations revealed that inhibition of autophagy could decrease the VacA-induced cell death in AGS cells. Furthermore, numerous dilated endoplasmic reticula (ER) were observed, and the phosphorylation of a subunit of eukaryotic translation initiation factor 2 subunit 1 also increased in the VacA-treated AGS cells, while repression of ER stress could reduce autophagy and cell death through knockdown of activating transcription factor 4 and DNA-damage-inducible transcript 3. In addition, the expression of pseudokinase tribbles homolog 3 (TRIB3) upon ER stress was triggered by VacA, and knockdown of TRIB3 could also decrease VacA-induced cell death. Finally, inhibition of autophagy could decrease VacA s1m1 -induced cell death and apoptosis, and apoptosis inhibitor Z-VAD had no significant effect on autophagy induced by VacA s1m1 . Thus, these results suggested that VacA causes autophagic cell death via ER stress in gastric epithelial cells.
Assuntos
Fator 4 Ativador da Transcrição/genética , Autofagia/efeitos dos fármacos , Proteínas de Bactérias/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Helicobacter pylori/química , Fator de Transcrição CHOP/genética , Fator 4 Ativador da Transcrição/antagonistas & inibidores , Fator 4 Ativador da Transcrição/metabolismo , Adenina/análogos & derivados , Adenina/farmacologia , Clorometilcetonas de Aminoácidos/farmacologia , Animais , Autofagia/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/patologia , Retículo Endoplasmático/ultraestrutura , Células Epiteliais , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Helicobacter pylori/metabolismo , Helicobacter pylori/patogenicidade , Humanos , Macrolídeos/farmacologia , Camundongos , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais , Tapsigargina/farmacologia , Fator de Transcrição CHOP/antagonistas & inibidores , Fator de Transcrição CHOP/metabolismo , Vacúolos/efeitos dos fármacos , Vacúolos/patologia , Vacúolos/ultraestruturaRESUMO
OBJECTIVE: Fragility fracture is one of the common complications of osteoporosis. Elevated oxidative stress-induced apoptosis is thought to be one of the unfavorable factors to osteoblastic dysfunction, which increased the risk of bone fracture. However, the molecular mechanisms for oxidative stress-induced osteoblast cells apoptosis still need to be elucidated. This study aims to investigate the protective function of miR-214 in H2O2-induced apoptosis of MC3T3-E1 osteoblasts. MATERIALS AND METHODS: MC3T3-E1 cells were treated with 400 µM H2O2. Flow cytometry was adopted to detect the apoptosis rate; malondialdehyde (MDA) and glutathione peroxidase (Gpx) levels were used to determine the reactive oxygen species (ROS) level. Reverse transcription-polymerase chain reaction (RT-PCR) was employed to test the expression level of miR-214 and ATF4. After transfected MC3T3-E1 cells with miR-214 mimics and inhibitor, RT-PCR was used to detect activating transcription factor 4 (ATF4) expression level. RESULTS: H2O2 treatment increased ROS induced intracellular oxidative injury. Flow cytometry showed that 400 µM H2O2 induced the apoptosis of MC3T3-E1 cells. Moreover, RT-PCR showed decreased expression level of MiR-214. Furthermore, the apoptosis induced by high ROS level was reversed by increased miR-214 expression level. The regulatory ability of MiR-214 to apoptosis is by regulating ATF4 expression. CONCLUSIONS: miR-214 plays a protective role in H2O2 induced MC3T3 osteoblasts apoptosis and its protective effect is proceeded by regulating ROS level and ATF4 expression level.