RESUMEN
BACKGROUND: Ischemia- reperfusion (I/R) injury-induced oxidative stress is a key factor in the pathogenesis of pressure ulcer formation. Ferroptosis is an iron-dependent programmed cell death that connects oxidative stress and inflammation in various diseases. Recent studies revealed the protective effect of inhibition of ferroptosis in I/R injury. However, the role of ferroptosis in cutaneous I/R injury remains elusive. OBJECTIVE: To assess the role of ferroptosis in the progression of cutaneous I/R injury. METHODS: Cutaneous I/R injury experiments and histopathological studies were performed in wild-type mice with or without exposure to volatile ferroptosis inhibitor, TEMPO (2,2,6,6-Tetramethylpiperidine-1-oxyl). The suppressive effects of TEMPO on ferroptosis inducing cell death and oxidative stress were examined in vitro. RESULTS: Inhibition of ferroptosis with TEMPO significantly reduced ulcer formation after cutaneous I/R injury. Fluctuated ferroptosis markers, such as GPX4, ACSL4, and 4-HNE expression in the I/R skin site, were reversed by TEMPO treatment. Inhibition of ferroptosis reduced apoptosis, CD3+ infiltrating lymphocytes, and improved vascularity in the I/R skin site. Inhibition of ferroptosis also suppressed the enhancement of Nrf2 activation. In vitro, ferroptosis and the activation of ferroptosis-related gene expression by RSL3 stimulation were markedly ameliorated by TEMPO treatment in mouse fibroblasts. Inhibiting ferroptosis also suppressed the elevation of the mRNA levels of NOX2 and HO-1 caused by ferroptosis. CONCLUSION: Cutaneous I/R injury-induced ferroptosis likely promotes cell death, vascular loss, infiltration of inflammatory cells, and oxidative stress. The inhibition of ferroptosis with TEMPO might have potential clinical application as novel therapeutic agent for cutaneous I/R injury.
RESUMEN
Mammalian cells have three types of endoplasmic reticulum (ER) stress-sensing molecules: ATF6, IRE1, and PERK. Among these, ATF6 is unique in that it is processed in an ER-stress-specific manner and functions as a transcription factor for the activation of anti-ER stress genes (such as BiP). ATF6 is known to have two homologues, ATF6α and ATF6ß, and a greater understanding of their functions has been achieved through analyses using cultured cells. Physiological functions are also gradually being investigated in mice lacking ATF6α or ATF6ß. However, little is known about the effects on mouse organisms of the deletion of both the ATF6α and ATF6ß genes, since such double-knockout (DKO) mice suffer embryonic lethality at an early developmental stage. In this study, we generated and analyzed ATF6 DKO mice in which embryonic lethality was evaded by using Cre/loxP technology. Pancreatic ß cell-specific ATF6 DKO mice were born normally and lived without dysregulation of blood-glucose levels but had a reduced tolerance to glucose. Islets isolated from ATF6 DKO mice also showed low production and secretion of insulin and mild enhancement of IRE1 and PERK activity. We further examined the developmental abnormalities of systemic ATF6 DKO mice. The phenotypes of ATF6α-/-; ATF6ß-/- mice were similar to those previously reported, but ATF6α+/-; ATF6ß-/- and ATF6α-/-; ATF6ß+/- mice showed embryonic lethality at middle developmental stages, unlike those reported. Analysis of embryonic fibroblasts derived from these mice revealed that ATF6α and ATF6ß have a gene-dose-dependent functional redundancy and display distinct differences in their ability to induce BiP expression. (250 words).
Asunto(s)
Retículo Endoplásmico , Factores de Transcripción , Ratones , Animales , Retículo Endoplásmico/metabolismo , Factores de Transcripción/metabolismo , Respuesta de Proteína Desplegada , Estrés del Retículo Endoplásmico , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Glucosa/metabolismo , Factor de Transcripción Activador 6/genética , Factor de Transcripción Activador 6/metabolismo , MamíferosRESUMEN
Ischemia-reperfusion (I/R) injury is a key player in the pathogeneses of pressure ulcer formation. Our previous work demonstrated that inducing the transcription factor SOX2 promotes cutaneous wound healing through EGFR signaling pathway enhancement. However, its protective effect on cutaneous I/R injury was not well-characterized. We aimed to assess the role of SOX2 in cutaneous I/R injury and the tissue-protective effect of SOX2 induction in keratinocytes (KCs) in cutaneous I/R injury. SOX2 was transiently expressed in KCs after cutaneous I/R injury. Ulcer formation was significantly suppressed in KC-specific SOX2-overexpressing mice. SOX2 in skin KCs significantly suppressed the infiltrating inflammatory cells, apoptotic cells, vascular damage, and hypoxic areas in cutaneous I/R injury. Oxidative stress-induced mRNA levels of inflammatory cytokine expression were suppressed, and antioxidant stress factors and amphiregulin were elevated by SOX2 induction in skin KCs. Recombinant amphiregulin administration suppressed pressure ulcer development after cutaneous I/R injury in mice and suppressed oxidative stress-induced ROS production and apoptosis in vitro. These findings support that SOX2 in KCs might regulate cutaneous I/R injury through amphiregulin production, resulting in oxidative stress suppression. Recombinant amphiregulin can be a potential therapeutic agent for cutaneous I/R injury.
Asunto(s)
Úlcera por Presión , Daño por Reperfusión , Animales , Ratones , Anfirregulina/genética , Anfirregulina/metabolismo , Apoptosis , Queratinocitos/metabolismo , Daño por Reperfusión/complicaciones , Daño por Reperfusión/genética , Daño por Reperfusión/metabolismo , Piel/metabolismoRESUMEN
Oxidative stress is a major risk factor for calcium oxalate nephrolithiasis. Reports suggest that oxidative stress response is induced in animals and humans with kidney stones. Keap1, Nrf2, and HO-1 are known as oxidative stress mediators. However, the association between oxidative stress response and stone formation is unclear. In this study, we analyzed oxidative stress response from the acute to the crystal formation phase when crystal formation was applied to renal crystal mice model and bioimaging mice and investigated the effect on crystal formation. In renal tissues, after glyoxylate administration, HO-1 increased for up to 6 h and returned to baseline at 24 h. This was observed following each daily dose until five days after the crystallization phase; however, the range of increase was attenuated. The possibility that Nrf2 activity influenced the number of crystals was considered in the experiment. Crystal formation increased in Nrf2-deficient mice and could be reduced by Nrf2 activators. In conclusion, the oxidative stress response via the Keap1-Nrf2 pathway may contribute to crystal formation. Particularly, this pathway may be a prospective target for drug development to prevent and cure nephrolithiasis.
Asunto(s)
Proteína 1 Asociada A ECH Tipo Kelch , Factor 2 Relacionado con NF-E2 , Nefrolitiasis , Estrés Oxidativo , Animales , Ratones , Proteína 1 Asociada A ECH Tipo Kelch/genética , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Riñón/metabolismo , Nefrolitiasis/genética , Nefrolitiasis/metabolismo , Nefrolitiasis/prevención & control , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Estrés Oxidativo/genéticaRESUMEN
Oxidative stress has been reported to play an important role in the pathogenesis of skin fibrosis in systemic sclerosis (SSc). We previously identified that botulinum toxin (BTX) injection suppresses pressure ulcer formation in a cutaneous ischemia-reperfusion injury mouse model by regulation of oxidative stress. However, the therapeutic possibility of BTX administration for preventing skin fibrosis in SSc is unclear. The objective of this study was to investigate the effect of BTX-B on skin fibrosis in a murine model of SSc and determine the underlying mechanism. We found that BTX-B injection significantly reduced dermal thickness and inflammatory cell infiltration in bleomycin-induced skin fibrosis lesion in mice. We also identified that the oxidative stress signal detected through bioluminescence in OKD48 mice after bleomycin injection in the skin was significantly decreased by BTX-B. Additionally, mRNA levels of oxidative stress associated factors (NOX2, HO-1, Trx2) were significantly decreased by BTX-B. Apoptotic cells in the lesional skin of bleomycin-treated mice were significantly reduced by BTX-B. Oxidant-induced intracellular accumulation of reactive oxygen species in SSc fibroblasts was also inhibited by BTX-B. In conclusion, BTX-B might improve bleomycin-induced skin fibrosis via the suppression of oxidative stress and inflammatory cells in the skin. BTX-B injection may have a therapeutic effect on skin fibrosis in SSc.
Asunto(s)
Esclerodermia Sistémica , Enfermedades de la Piel , Animales , Bleomicina , Modelos Animales de Enfermedad , Fibroblastos/patología , Fibrosis , Ratones , Estrés Oxidativo , Esclerodermia Sistémica/patología , Piel/patología , Enfermedades de la Piel/patologíaRESUMEN
Methylmercury (MeHg), an environmental toxicant, induces neuronal cell death and injures a specific area of the brain. MeHg-mediated neurotoxicity is believed to be caused by oxidative stress and endoplasmic reticulum (ER) stress but the mechanism by which those stresses lead to neuronal loss is unclear. Here, by utilizing the ER stress-activated indicator (ERAI) system, we investigated the signaling alterations in the unfolded protein response (UPR) prior to neuronal apoptosis in the mouse brain. In ERAI transgenic mice exposed to MeHg (25 mg/kg, S.C.), the ERAI signal, which indicates activation of the cytoprotective pathway of the UPR, was detected in the brain. Interestingly, detailed ex vivo analysis showed that the ERAI signal was localized predominantly in neurons. Time course analysis of MeHg exposure (30 ppm in drinking water) showed that whereas the ERAI signal was gradually attenuated at the late phase after increasing at the early phase, activation of the apoptotic pathway of the UPR was enhanced in proportion to the exposure time. These results suggest that MeHg induces not only ER stress but also neuronal cell death via a UPR shift. UPR modulation could be a therapeutic target for treating neuropathy caused by electrophiles similar to MeHg.
Asunto(s)
Encéfalo/efectos de los fármacos , Estrés del Retículo Endoplásmico/efectos de los fármacos , Compuestos de Metilmercurio/toxicidad , Respuesta de Proteína Desplegada/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Encéfalo/patología , Muerte Celular/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas/efectos de los fármacos , Neuronas/patología , Estrés Oxidativo/efectos de los fármacos , Análisis Espacio-Temporal , Factores de TiempoRESUMEN
Ischemia-reperfusion (I/R) is associated with various pathogenic conditions, and there has been increasing evidence that cutaneous I/R injury is associated with the pathogenesis of pressure ulcers (PUs), especially at the early stage presenting as non-blanchable erythema. Several studies demonstrated that oxidative stress is a key player in I/R injury, and the inhibition of oxidative stress may be capable of protecting tissue damage after I/R injury in various organs including skin. Dimethyl fumarate (DMF) approved by the Food and Drug Administration is Nrf2 activator, and recent studies revealed the antioxidative and anti-inflammatory effects of DMF on I/R injury in animal models. Our objective was to assess the effects of oral administration of DMF on the development of PUs after cutaneous I/R injury in mice. We found that DMF administration significantly decreased the size of PUs after cutaneous I/R. Cutaneous I/R-induced oxidative stress was also significantly inhibited by DMF in OKD48 mice, in which oxidative stress can be visually assessed. In addition, DMF treatment decreased hypoxic area, the numbers of apoptotic cells, and vascular loss in I/R area. DMF treatment suppressed the infiltration of MPO+ neutrophils and the production of proinflammatory cytokines in I/R site after cutaneous I/R injury. in vitro experiments, DMF treatment suppressed the production of reactive oxygen species in pericyte-like cells. These results suggest that DMF treatment might prevent the formation of PUs induced by cutaneous I/R injury via suppressing oxidative stress and subsequent inflammation. DMF treatment during the early phase of decubitus ulcers might protect against further progression.
Asunto(s)
Dimetilfumarato/farmacología , Úlcera por Presión/etiología , Úlcera por Presión/prevención & control , Daño por Reperfusión/complicaciones , Administración Oral , Animales , Dimetilfumarato/administración & dosificación , Modelos Animales de Enfermedad , Ratones , Ratones Endogámicos C57BL , Estrés OxidativoRESUMEN
The Cre/loxP system is an indispensable tool for temporal and spatial control of gene function in mice. Many mice that express Cre and carry loxP sites in their genomes have been bred for functional analysis of various genes in vivo. Also, several reporter mice have been generated for monitoring of recombination by the Cre/loxP system. We have developed a Cre reporter gene with DsRed1 and Venus that exhibits a strong red fluorescence before and a strong green fluorescence after Cre/loxP-mediated recombination in experiments using NIH3T3 cells. However, a transgenic mouse introduced with the same reporter gene exhibits a weak red fluorescence before and a strong green fluorescence after Cre/loxP-mediated recombination. This property manifested ubiquitously in this mouse model and was maintained stably in mouse-derived fibroblasts. Use of the mouse model exhibiting the stronger red fluorescence might result in confusion of the Cre-dependent signal with false signals, because the Venus signal includes some fluorescence in the red region of the spectrum and the DsRed1 signal includes some fluorescence in the green region. However, we fortuitously obtained reporter mice that exhibit a weaker red fluorescence before Cre/loxP-mediated recombination. The use of this mouse model would decrease concern regarding errors in the identification of signals and should increase certainty in the detection of Cre activity in vivo.
Asunto(s)
Fluorescencia , Proteínas Fluorescentes Verdes , Integrasas , Modelos Genéticos , Recombinación Genética , Animales , Fibroblastos , Genes Reporteros/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Células 3T3 NIHRESUMEN
Several studies have demonstrated potential roles for apelin/APJ signaling in the regulation of oxidative stress associated with ischemia-reperfusion (I/R) injury in several organs. Objective was to assess the role of apelin/APJ signaling in the development of pressure ulcers (PUs) formation after cutaneous I/R injury in mice. We identified that cutaneous I/R injury increased the expression of apelin in the skin at I/R site. Administration of apelin significantly inhibited the formation of PUs. The reductions of blood vessels, hypoxic area and apoptosis in I/R site were inhibited by apelin injection. Oxidative stress signals in OKD48 mice and the expressions of oxidative stress related genes in the skin were suppressed by apelin injection. H2O2-induced intracellular ROS and apoptosis in endothelial cells and fibroblasts were suppressed by apelin in vitro. Furthermore, MM07, biased agonist of APJ, also significantly suppressed the development of PUs after cutaneous I/R, and the inhibitory effect of MM07 on PUs formation was higher than that in apelin. We conclude that apelin/APJ signaling may inhibit cutaneous I/R injury-induced PUs formation by protecting the reduction of vascularity and tissue damage via suppression of oxidative stress. Exogenous application of apelin or MM07 might have therapeutic potentials against the development of PUs.
Asunto(s)
Receptores de Apelina/metabolismo , Apelina/metabolismo , Daño por Reperfusión/metabolismo , Transducción de Señal , Piel/irrigación sanguínea , Piel/metabolismo , Úlcera/etiología , Úlcera/metabolismo , Animales , Apelina/genética , Receptores de Apelina/genética , Apoptosis/genética , Citocinas/genética , Citocinas/metabolismo , Modelos Animales de Enfermedad , Técnica del Anticuerpo Fluorescente , Expresión Génica , Ratones , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Daño por Reperfusión/etiología , Daño por Reperfusión/patología , Úlcera/patologíaRESUMEN
BACKGROUND: There is growing evidence that vasculopathy-induced hypoxia and oxidative stress enhance the process of fibrosis in systemic sclerosis (SSc). Kaempferol is a natural flavonoid widely found in various vegetables and fruits, and has been reported to have excellent antioxidant activity. OBJECTIVE: Objective was to elucidate the effect of kaempferol on skin fibrosis and the mechanism of the inhibitory regulation of fibrosis by kaempferol. METHODS: We assessed the effect of intraperitoneally administered kaempferol on bleomycin-induced dermal fibrosis in mice. The effect of kaempferol on oxidative stress in bleomycin-treated mice and SSc fibroblasts was assessed in vivo and in vitro. RESULTS: We identified that kaempferol injection significantly inhibited bleomycin-induced dermal fibrosis in mice. The number of αSMA+ myofibroblasts, CD3+ T-cells, and CD68+ macrophages in lesional skin was significantly decreased by kaempferol injections. Kaempferol administration also significantly suppressed the bleomycin-induced oxidative stress signal in OKD48 mice. Additionally, mRNA levels of oxidative stress-associated factors, such as HO-1 and NOX2, as well as inflammatory and pro-fibrotic cytokines, including IL-6, TGF-ß and TNFα in sclerotic skin were significantly decreased by kaempferol. Kaempferol also reduced bleomycin-induced TUNEL+ apoptotic cells in the lesional skin of bleomycin-treated mice. Furthermore, the oxidant-induced intracellular accumulation of reactive oxygen species (ROS) in SSc fibroblasts was inhibited by kaempferol treatment. In addition, the oxidant-induced apoptosis of SSc fibroblasts was decreased by kaempferol in vitro. CONCLUSION: Kaempferol might improve bleomycin-induced fibrosis by reducing oxidative stress, inflammation, and oxidative cellular damage. Administration of kaempferol might be an alternative treatment for skin fibrosis in SSc.
Asunto(s)
Antioxidantes/administración & dosificación , Fibroblastos/efectos de los fármacos , Quempferoles/administración & dosificación , Esclerodermia Sistémica/tratamiento farmacológico , Piel/patología , Animales , Apoptosis/efectos de los fármacos , Biopsia , Bleomicina/toxicidad , Modelos Animales de Enfermedad , Femenino , Fibroblastos/patología , Fibrosis , Humanos , Inyecciones Intraperitoneales , Ratones , Estrés Oxidativo/efectos de los fármacos , Cultivo Primario de Células , Especies Reactivas de Oxígeno/metabolismo , Esclerodermia Sistémica/patología , Piel/efectos de los fármacosRESUMEN
BACKGROUND: Zinc deficiency is believed to be a predisposing factor for the development and intractable healing of pressure ulcers (PUs); however, the mechanisms of this association have not been elucidated. OBJECTIVE: Objective was to elucidate the mechanisms of the formation of severe and prolonged PUs under the zinc deficiency condition. METHODS: We assessed PUs formation after cutaneous ischemia-reperfusion (I/R) injury in mice fed with a zinc-adequate (ZA) or a zinc-deficient (ZD) diet from 2 weeks before I/R injury. Wound size, vascular damage, apoptotic cells, adenosine triphosphate (ATP) amount, and the number of Langerhans cells (LCs) in I/R area were analyzed. We evaluated the extent of oxidative stress in I/R area in OKD48 mice through bioluminescence detection. RESULTS: We found that dietary zinc deficiency caused the formation of severe and prolonged PUs in mice. Zinc deficiency increased the vascular disorder, oxidative stress, and apoptosis induced by cutaneous I/R injury. I/R injury-induced oxidative stress signals were significantly higher in ZD OKD48 mice than in ZA OKD48 mice. Additionally, zinc deficiency reduced the number of LCs and increased the amount of ATP in cutaneous I/R-injured skin. Oral supplementation of zinc improved zinc deficiency-associated PUs. CONCLUSION: Zinc deficiency might increase cutaneous I/R injury-induced vascular damages, oxidative stress, and apoptosis, as well as ATP amount in I/R area due to the loss of LCs. These mechanisms might partly account for zinc deficiency-induced formation of severe and prolonged PUs. Oral supplementation of zinc might be a reasonable therapeutic choice for patients with PUs and zinc deficiency.
Asunto(s)
Adenosina Trifosfato/metabolismo , Úlcera por Presión/patología , Piel/patología , Zinc/deficiencia , Animales , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Humanos , Ratones , Estrés Oxidativo/efectos de los fármacos , Úlcera por Presión/etiología , Daño por Reperfusión/complicaciones , Zinc/administración & dosificaciónRESUMEN
In the version of this Article originally published, the competing interests statement was missing. The authors declare no competing interests; this statement has now been added in all online versions of the Article.
RESUMEN
Maintenance of endoplasmic reticulum (ER) proteostasis is controlled by a signalling network known as the unfolded protein response (UPR). Here, we identified filamin A as a major binding partner of the ER stress transducer IRE1α. Filamin A is an actin crosslinking factor involved in cytoskeleton remodelling. We show that IRE1α controls actin cytoskeleton dynamics and affects cell migration upstream of filamin A. The regulation of cytoskeleton dynamics by IRE1α is independent of its canonical role as a UPR mediator, serving instead as a scaffold that recruits and regulates filamin A. Targeting IRE1α expression in mice affected normal brain development, generating a phenotype resembling periventricular heterotopia, a disease linked to the loss of function of filamin A. IRE1α also modulated cell movement and cytoskeleton dynamics in fly and zebrafish models. This study unveils an unanticipated biological function of IRE1α in cell migration, whereby filamin A operates as an interphase between the UPR and the actin cytoskeleton.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Movimiento Celular , Endorribonucleasas/metabolismo , Fibroblastos/metabolismo , Filaminas/metabolismo , Neuronas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Endorribonucleasas/deficiencia , Endorribonucleasas/genética , Evolución Molecular , Femenino , Filaminas/genética , Células HEK293 , Humanos , Cinética , Masculino , Ratones , Ratones Noqueados , Neuronas/patología , Heterotopia Nodular Periventricular/genética , Heterotopia Nodular Periventricular/metabolismo , Heterotopia Nodular Periventricular/patología , Fosforilación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/genética , Transducción de Señal , Respuesta de Proteína Desplegada , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
BACKGROUND: We previously identified that botulinum toxin A (BTX-A) suppressed pressure ulcer (PU) formation after cutaneous ischemia-reperfusion (I/R) injury; however, regulation of cutaneous I/R-induced oxidative and endoplasmic reticulum (ER) stress by BTX-B was not investigated. Additionally, the efficacy of BTX-B injection has never been examined. OBJECTIVE: Objective was to assess the effects of BTX-B on the formation of PU by cutaneous I/R injury, and the regulation of oxidative and ER stress in I/R injury by BTX-B. METHODS: BTX-B was subcutaneously injected into I/R area, and wound size, vascular damage, hypoxic area, and apoptotic cells in I/R area were analyzed. We evaluated the extent of oxidative and ER stress in I/R area by using OKD48 mice and ERAI mice, respectively, which enabled evaluating oxidative and ER stress through bioluminescence detection. RESULTS: BTX-B injection significantly suppressed the formation of PU by cutaneous I/R injury. Cutaneous I/R-induced vascular damage, hypoxic area, and number of oxidative-damaged cells and apoptotic cells were suppressed by BTX-B injection. BTX-B administration significantly inhibited I/R-induced oxidative stress signal in OKD48 mice. BTX-B reduced the I/R-induced oxidative stress-associated factors. BTX-B significantly inhibited the oxidant-induced reactive oxygen species and apoptosis of endothelial cells and fibroblasts. BTX-B significantly inhibited I/R-induced ER stress signal in ERAI mice. Cutaneous I/R injury-induced ER stress-response factors and GRP78/BiP and CHOP-positive cells in I/R area were significantly decreased by BTX-B injection. CONCLUSION: BTX-B injection might have protective effects against PU formation after cutaneous I/R injury by reducing vascular damage, hypoxia-induced oxidative and ER stress, and apoptosis.
Asunto(s)
Toxinas Botulínicas Tipo A/uso terapéutico , Estrés del Retículo Endoplásmico/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Úlcera por Presión/tratamiento farmacológico , Daño por Reperfusión/complicaciones , Animales , Apoptosis/efectos de los fármacos , Toxinas Botulínicas Tipo A/farmacología , Modelos Animales de Enfermedad , Chaperón BiP del Retículo Endoplásmico , Fibroblastos , Células Endoteliales de la Vena Umbilical Humana , Humanos , Inyecciones Subcutáneas , Ratones , Ratones Endogámicos C57BL , Células 3T3 NIH , Oxidantes/farmacología , Úlcera por Presión/etiología , Úlcera por Presión/patología , Especies Reactivas de Oxígeno/metabolismo , Daño por Reperfusión/patología , Piel/irrigación sanguínea , Piel/efectos de los fármacos , Piel/patología , Resultado del TratamientoRESUMEN
Cutaneous ischemia-reperfusion (I/R) injury is associated with the early pathogenesis of cutaneous pressure ulcers (PUs). The objective of this study was to investigate the effect of mesenchymal stem cells (MSCs) injection on the formation of PUs after I/R injury and determine the underlying mechanisms. We found that the subcutaneous injection of MSCs into areas of I/R injured skin significantly suppressed the formation of PUs. I/R-induced vascular damage, hypoxia, oxidative DNA damage, and apoptosis were decreased by MSCs injection. Oxidative stress signals detected after I/R in OKD48 (Keap1-dependent oxidative stress detector, No-48-luciferase) mice were decreased by the injection of MSCs. In cultured fibroblasts, MSCs-conditioned medium significantly inhibited oxidant-induced reactive oxygen species (ROS) generation and apoptosis. Furthermore, endoplasmic reticulum (ER) stress signals detected after I/R in ERAI (ER stress-activated indicator) mice were also decreased by the injection of MSCs. These results suggest that the injection of MSCs might protect against the development of PUs after cutaneous I/R injury by reducing vascular damage, oxidative cellular damage, oxidative stress, ER stress, and apoptosis.
Asunto(s)
Estrés del Retículo Endoplásmico , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/citología , Estrés Oxidativo , Úlcera por Presión/prevención & control , Sustancias Protectoras , Daño por Reperfusión/complicaciones , Animales , Apoptosis , Masculino , Ratones , Ratones Endogámicos C57BL , Oxidación-Reducción , Úlcera por Presión/etiología , Úlcera por Presión/metabolismo , Úlcera por Presión/patología , Especies Reactivas de Oxígeno , Enfermedades de la Piel/complicacionesRESUMEN
Activating transcription factor 4 (ATF4) is a translationally activated protein that plays a role in cellular adaptation to several stresses. Because these stresses are associated with various diseases, the translational control of ATF4 needs to be evaluated from the physiological and pathological points of view. We have developed a transgenic mouse model to monitor the translational activation of ATF4 in response to cellular stress. By using this mouse model, we were able to detect nutrient starvation response, antivirus response, endoplasmic reticulum (ER) stress response, and oxidative stress in vitro and ex vivo, as well as in vivo. The reporter system introduced into our mouse model was also shown to work in a stress intensity-dependent manner and a stress duration-dependent manner. The mouse model is therefore a useful tool for imaging ATF4 translational activation at various levels, from cell cultures to whole bodies, and it has a range of useful applications in investigations on the physiological and pathological roles of ATF4-related stress and in the development of clinical drugs for treating ATF4-associated diseases.
Asunto(s)
Imagen Molecular , Biosíntesis de Proteínas , Estrés Fisiológico , Factor de Transcripción Activador 4/metabolismo , Animales , Fibroblastos , Expresión Génica , Genes Reporteros , Humanos , Ratones , Ratones TransgénicosRESUMEN
Inflammation is a biological response associated with symptoms of various diseases, and its study is important in gaining an understanding of the pathological conditions of such diseases and in making strategic plans for promoting healing. It is therefore essential to develop technologies for the detection of inflammatory conditions. Interleukin-1ß (IL-1ß) is a proinflammatory cytokine produced and secreted mainly by monocytes and macrophages in response to inflammatory stimulation. The activation of IL-1ß is regulated through transcriptional induction by the promoter and post-translational processing by the inflammasome. Here we have developed a reporter gene to monitor the activation status of IL-1ß by using a dual regulation system and, by using the reporter gene, we have established a mouse model that permits low-invasive visualization of the inflammatory status. Previous reporter systems dependent on the transcription or processing of IL-1ß show problems in terms of background noise or signal specificity. Our reporter system overcomes these weaknesses by combining advantages from regulation by a promoter and processing of IL-1ß. Our mouse model detected specific physiological inflammation in the liver and pancreas caused by hepatitis or pancreatitis models, respectively. Our reporter gene and mouse model are therefore expected to become useful bioresources for future medical science.
Asunto(s)
Hepatitis/inmunología , Interleucina-1beta/fisiología , Pancreatitis/inmunología , Animales , Modelos Animales de Enfermedad , Genes Reporteros , Hepatitis/patología , Inflamasomas/metabolismo , Lipopolisacáridos/farmacología , Hígado/inmunología , Hígado/metabolismo , Hígado/patología , Luciferasas/biosíntesis , Luciferasas/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Páncreas/inmunología , Páncreas/metabolismo , Páncreas/patología , Pancreatitis/patología , Células RAW 264.7RESUMEN
Insulin biosynthesis has been well characterized with respect to transcriptional and post-translational regulation. However, the relationship between translational regulation of insulin and protein quality control in the endoplasmic reticulum (ER) remains to be clarified. Here we carried out forced expression of insulin in non-insulin-producing cells and compared activation level of ER stress-responsive molecules between insulin-producing cells and non-insulin-producing cells under normal culture condition or ER stress condition. Forced expression of insulin in non-insulin-producing cells caused severe ER stress with striking translational attenuation through phosphorylation of eIF2α by activation of protein kinase RNA-like endoplasmic reticulum kinase (PERK), resulting in inhibition of insulin production at the protein level. We also found that GADD34 and CReP are highly expressed in the cells that endogenously produce insulin and that eIF2α shows constitutively low phosphorylation level in these cells although PERK is constitutively activated under both normal culture conditions and physiological conditions in the same cells. Inhibition of eIF2α phosphatase further decreased insulin level in pancreatic ß cells. These findings suggest that eIF2α phosphorylation level is kept low by GADD34- and/or CReP-regulated phosphatases in pancreatic ß cells and that cancellation of phospho-eIF2α-dependent translational inhibition by the molecular mechanism contributes to mass production of insulin in pancreatic ß cells.
Asunto(s)
Factor 2 Eucariótico de Iniciación/metabolismo , Células Secretoras de Insulina/fisiología , Insulina/biosíntesis , Proteína Fosfatasa 1/metabolismo , Animales , Técnicas de Cultivo de Célula , Retículo Endoplásmico/metabolismo , Factor 2 Eucariótico de Iniciación/genética , Células HEK293 , Células HeLa , Humanos , Insulina/genética , Células Secretoras de Insulina/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Células 3T3 NIH , Fosforilación , Biosíntesis de Proteínas , Proteína Fosfatasa 1/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal , eIF-2 Quinasa/genética , eIF-2 Quinasa/metabolismoRESUMEN
Cerebral malaria is a fatal complication of malaria. Conventional methods for evaluating experimental cerebral malaria have several drawbacks. Therefore, we aimed to develop an easy-to-use method for evaluating experimental cerebral malaria using OKD48 (Keap1-dependent Oxidative stress Detector, No-48-luciferase) mice to evaluate oxidative stress. OKD48 mice infected with Plasmodium berghei ANKA strain (PbA) suffered from experimental cerebral malaria and oxidative stress was successfully detected in the brains of living OKD48 mice developing experimental cerebral malaria. Oxidative stress in the brain was dependent on the development of experimental cerebral malaria, as prevention of experimental cerebral malaria did not elicit oxidative stress. We provide a novel evaluation method for experimental cerebral malaria using oxidative stress indicator OKD48 mice.