RESUMO
Environmental changes can trigger endoplasmic reticulum (ER) stress and misfolded protein accumulation, potentially leading to pre-eclampsia (PE). Amyloid-ß (Aß) is a crucial misfolded protein that can overactivate autophagy. Our study assessed the expression of Aß1-42 and autophagic activity in PE placental tissues and trophoblasts under ER stress. Placental tissues were surgically collected from normal pregnant women (NP) and pregnant women with late-onset PE (LOPE) delivering through cesarean section. The expression levels of Aß1-42 were detected in both PE and NP placental tissues, as well as in tunicamycin (TM)-induced HTR-8/SVneo cells. Autophagy-related proteins, such as Beclin-1, the ratio of LC3-II to LC3-I, ATG5, and SQSTM1/p62 in the placental tissues and HTR-8/SVneo cells were measured by Western blot. The number and morphology of autophagosomes were observed using transmission electron microscopy (TEM). Potential targets associated with the unfolded protein response (UPR) in the placental tissues of NP and PE cases were screened using PCR Arrays. The misfolded protein was significantly upregulated in the PE group. In both PE placental tissues and TM-induced HTR-8/SVneo cells, not only was Aß1-42 upregulated, but also Beclin-1, ATG5, and LC3BII/I were significantly increased, accompanied by an increase in autophagosome count, while SQSTM1/P62 was downregulated. A total of 17 differentially expressed genes (DEGs) associated with the UPR were identified, among which elevated calnexin (CANX) was validated in the placenta from both PE and TM-induced HTR-8/SVneo cells. Autophagy is significantly upregulated in PE cases due to ER stress-induced Aß1-42 accumulation, likely mediated by autophagy-related proteins involved in the UPR.
Assuntos
Peptídeos beta-Amiloides , Autofagia , Estresse do Retículo Endoplasmático , Fragmentos de Peptídeos , Pré-Eclâmpsia , Trofoblastos , Tunicamicina , Humanos , Pré-Eclâmpsia/metabolismo , Pré-Eclâmpsia/genética , Pré-Eclâmpsia/patologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Feminino , Gravidez , Peptídeos beta-Amiloides/metabolismo , Linhagem Celular , Tunicamicina/farmacologia , Tunicamicina/efeitos adversos , Trofoblastos/metabolismo , Trofoblastos/efeitos dos fármacos , Trofoblastos/patologia , Adulto , Placenta/metabolismo , Placenta/efeitos dos fármacos , Placenta/patologia , Resposta a Proteínas não Dobradas/efeitos dos fármacosRESUMO
Renal injury is a fatal complication in critically ill patients with sepsis. As an ultrashort-acting synthetic opioid derivative, remifentanil has been reported to mitigate renal injury and sepsis. Nevertheless, whether remifentanil also suppresses sepsis-triggered renal injury is uncertain. The aim of this study was to investigate the effect of remifentanil on endoplasmic reticulum stress (ERS) and inflammatory response in an in vitro lipopolysaccharide (LPS)-stimulated renal tubular epithelial cell (HK-2) model and its mechanism. The viability of HK-2 cells with the absence or presence of LPS treatment was surveyed by cell counting kit-8 assay. Under the condition of LPS treatment, apoptosis was appraised by TUNEL assay and western blot. Levels of inflammatory factors were estimated though corresponding kits. Western blot tested the expression of toll-like receptor 4 (TLR4)/nuclear factor-kappaB (NF-κB) signaling-associated proteins. Also, the expression of ERS-related proteins was detected by western blot. Further, ERS inducer tunicamycin (TM) was added and the aforementioned experiments were conducted again. The results underlined the protective effects of remifentanil on LPS-evoked viability injury, inflammation, activation of TLR4/NF-κB signaling and ERS in HK-2 cells. Moreover, the impacts of remifentanil on the biological events of LPS-insulted HK-2 cells were all reversed by TM administration. To conclude, remifentanil might have a remarkable ameliorative effect on sepsis-induced renal injury, which implied the potential of remifentanil-based drug therapy in sepsis-induced renal injury.
Assuntos
Injúria Renal Aguda , Sepse , Humanos , Lipopolissacarídeos , Receptor 4 Toll-Like/metabolismo , NF-kappa B/metabolismo , Remifentanil/efeitos adversos , Estresse do Retículo Endoplasmático , Tunicamicina/efeitos adversos , Analgésicos Opioides/farmacologia , Linhagem Celular , Sepse/complicações , Sepse/tratamento farmacológico , Injúria Renal Aguda/tratamento farmacológico , Injúria Renal Aguda/induzido quimicamente , ApoptoseRESUMO
Recent evidence suggests that endoplasmic reticulum (ER) stress plays a vital role in inflammatory bowel disease (IBD). Therefore, the aim of this study was to investigate the mechanism by which ER stress promotes inflammatory response in IBD. The expression of Gro-α, IL-8 and ER stress indicator Grp78 in colon tissues from patients with Crohn's disease (CD) and colonic carcinoma was analyzed by immunohistochemistry staining. Colitis mouse model was established by the induction of trinitrobenzene sulphonic acid (TNBS), and the mice were treated with ER stress inhibitor tauroursodeoxycholic acid (TUDCA). Then the body weight, colon length and colon inflammation were evaluated, and Grp78 and Gro-α in colon tissues were detected by immunohistochemistry. Epithelial cells of colon cancer HCT116 cells were treated with tunicamycin to induce ER stress. Grp78 was detected by Western blot, and chemokines were measured by PCR and ELISA. The expression levels of Grp78, Gro-α and IL-8 were significantly upregulated in intestinal tissues of CD patients. Mice with TNBS induced colitis had increased expression of Grp78 and Gro-α in colonic epithelia. TUDCA reduced the severity of TNBS-induced colitis. In HCT116 cells, tunicamycin increased the expression of Grp78, Gro-α and IL-8 in a concentration-dependent manner. Furthermore, p38 MAPK inhibitor significantly inhibited the upregulation of Gro-α and IL-8 induced by tunicamycin. In conclusion, ER stress promotes inflammatory response in IBD, and the effects may be mediated by the activation of p38 MAPK signaling pathway.
Assuntos
Colite , Doenças Inflamatórias Intestinais , Animais , Colite/induzido quimicamente , Colite/patologia , Estresse do Retículo Endoplasmático/fisiologia , Humanos , Doenças Inflamatórias Intestinais/metabolismo , Interleucina-8/efeitos adversos , Camundongos , Ácido Trinitrobenzenossulfônico/toxicidade , Tunicamicina/efeitos adversos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/farmacologiaRESUMO
Oxidative stress, defined as the excess production of reactive oxygen species (ROS) relative to antioxidant defense, plays a significant role in the development of cardiovascular diseases. Endoplasmic reticulum (ER) stress has emerged as an important source of ROS and its modulation could be cardioprotective. Previously, we demonstrated that miR-16-5p is enriched in the plasma of ischemic dilated cardiomyopathy (ICM) patients and promotes ER stress-induced apoptosis in cardiomyocytes in vitro. Here, we hypothesize that miR-16-5p might contribute to oxidative stress through ER stress induction and that targeting miR-16-5p may exert a cardioprotective role in ER stress-mediated cardiac injury. Analysis of oxidative markers in the plasma of ICM patients demonstrates that oxidative stress is associated with ICM. Moreover, we confirm that miR-16-5p overexpression promotes oxidative stress in AC16 cardiomyoblasts. We also find that, in response to tunicamycin-induced ER stress, miR-16-5p suppression decreases apoptosis, inflammation and cardiac damage via activating the ATF6-mediated cytoprotective pathway. Finally, ATF6 is identified as a direct target gene of miR-16-5p by dual-luciferase reporter assays. Our results indicate that miR-16-5p promotes ER stress and oxidative stress in cardiac cells through regulating ATF6, suggesting that the inhibition of miR-16-5p has potential as a therapeutic approach to protect the heart against ER and oxidative stress-induced injury.
Assuntos
Biomarcadores/sangue , Cardiomiopatia Dilatada/genética , MicroRNAs/genética , Miócitos Cardíacos/citologia , Tunicamicina/efeitos adversos , Adulto , Idoso , Cardiomiopatia Dilatada/sangue , Cardiomiopatia Dilatada/etiologia , Estudos de Casos e Controles , Linhagem Celular , Estresse do Retículo Endoplasmático , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Miócitos Cardíacos/química , Miócitos Cardíacos/efeitos dos fármacos , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
The unfolded protein response in the endoplasmic reticulum (UPRER) is involved in a number of metabolic diseases. Here, we characterize UPRER-induced metabolic changes in mouse livers in vivo through metabolic labeling and mass spectrometric analysis of lipid and proteome-wide fluxes. We induced UPRER by tunicamycin administration and measured synthesis rates of proteins, fatty acids and cholesterol, as well as RNA-seq. Contrary to reports in isolated cells, hepatic de novo lipogenesis and cholesterogenesis were markedly reduced, as were mRNA levels and synthesis rates of lipogenic proteins. H&E staining showed enrichment with lipid droplets while electron microscopy revealed ER morphological changes. Interestingly, the pre-labeling of adipose tissue prior to UPRER induction resulted in the redistribution of labeled fatty acids from adipose tissue to the liver, with replacement by unlabeled glycerol in the liver acylglycerides, indicating that the liver uptake was of free fatty acids, not whole glycerolipids. The redistribution of adipose fatty acids to the liver was not explicable by altered plasma insulin, increased fatty acid levels (lipolysis) or by reduced food intake. Synthesis of most liver proteins was suppressed under UPRER conditions, with the exception of BiP, other chaperones, protein disulfide isomerases, and proteins of ribosomal biogenesis. Protein synthesis rates generally, but not always, paralleled changes in mRNA. In summary, this combined approach, linking static changes with fluxes, revealed an integrated reduction of lipid and cholesterol synthesis pathways, from gene expression to translation and metabolic flux rates, under UPRER conditions. The reduced lipogenesis does not parallel human fatty liver disease. This approach provides powerful tools to characterize metabolic processes underlying hepatic UPRER in vivo.
Assuntos
Colesterol/metabolismo , Ácidos Graxos/sangue , Perfilação da Expressão Gênica/métodos , Redes Reguladoras de Genes/efeitos dos fármacos , Fígado/metabolismo , Tunicamicina/efeitos adversos , Tecido Adiposo/metabolismo , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Insulina/sangue , Lipogênese/efeitos dos fármacos , Masculino , Espectrometria de Massas , Camundongos , Modelos Animais , RNA-Seq , Resposta a Proteínas não DobradasRESUMO
Endoplasmic reticulum (ER) stress is associated with acute kidney injury (AKI) caused by various mechanisms, including antibiotics, non-steroidal anti-inflammatory drugs, cisplatin, and radiocontrast. Tunicamycin (TM) is a nucleoside antibiotic that induces ER stress and is a commonly used model of AKI. 4-phenylbutyrate (4-PBA) is a chemical chaperone and histone deacetylase (HDAC) inhibitor and has been shown to protect the kidney from ER stress, apoptosis, and structural damage in a tunicamycin model of AKI. The renal protection provided by 4-PBA is attributed to its ability to prevent misfolded protein aggregation and inhibit ER stress; however, the HDAC inhibitor effects of 4-PBA have not been examined in the TM-induced model of AKI. As such, the main objective of this study was to determine if histone hyperacetylation provides any protective effects against TM-mediated AKI. The FDA-approved HDAC inhibitor vorinostat was used, as it has no ER stress inhibitory effects and therefore the histone hyperacetylation properties alone could be investigated. In vitro work demonstrated that vorinostat inhibited histone deacetylation in cultured proximal tubular cells but did not prevent ER stress or protein aggregation induced by TM. Vorinostat induced a significant increase in cell death, and exacerbated TM-mediated total cell death and apoptotic cell death. Wild type male mice were treated with TM (0.5 mg/kg, intraperitoneal injection), with or without vorinostat (50 mg/kg/day) or 4-PBA (1 g/kg/day). Mice treated with 4-PBA or vorinostat exhibited similar levels of histone hyperacetylation. Expression of the pro-apoptotic protein CHOP was induced with TM, and not inhibited by vorinostat. Further, vorinostat did not prevent any renal damage or decline in renal function caused by tunicamycin. These data suggest that the protective mechanisms found by 4-PBA are primarily due to its molecular chaperone properties, and the HDAC inhibitors used did not provide any protection against renal injury caused by ER stress.
Assuntos
Injúria Renal Aguda , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Tunicamicina/efeitos adversos , Vorinostat/farmacologia , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/prevenção & controle , Animais , Linhagem Celular , Modelos Animais de Doenças , Masculino , Camundongos , Agregação Patológica de Proteínas/induzido quimicamente , Agregação Patológica de Proteínas/metabolismo , Agregação Patológica de Proteínas/patologia , Agregação Patológica de Proteínas/prevenção & controle , Tunicamicina/farmacologiaRESUMO
Both endoplasmic reticulum (ER) stress and autophagy have been implicated in chronic kidney injury and renal fibrosis. However, the relationship and regulatory mechanisms between ER stress and autophagy under this condition remain largely unknown. In this study, we first established a mouse model of ER stress-induced chronic kidney injury by 2 weekly injections of a low dose of tunicamycin (TM), a classical ER stress inducer. This model showed the induction of ER stress, autophagy, fibrosis and apoptosis in kidney tissues. In vitro, TM also induced ER stress, autophagy, fibrosis and apoptosis in HK-2 human kidney proximal tubular cells and BUMPT-306 mouse kidney proximal tubular cells. In these cells, autophagy inhibitor suppressed TM-induced fibrotic changes and apoptosis, suggesting an involvement of autophagy in ER stress-associated chronic kidney injury. PERK inhibitor ameliorated autophagy, fibrotic protein expression and apoptosis in TM-treated cells, indicating a role of the PERK/eIF2α pathway in autophagy activation during ER stress. Similar results were shown in TGF-ß1-treated HK-2 cells. Interestingly, in both TM- or TGF-ß1-treated kidney proximal tubular cells, inhibition of autophagy exaggerated ER stress, suggesting that autophagy induced by ER stress provides a negative feedback mechanism to reduce the stress. Together, these results unveil a reciprocal regulation between ER stress and autophagy in chronic kidney injury and fibrosis.
Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Túbulos Renais Proximais/patologia , Insuficiência Renal Crônica/induzido quimicamente , Tunicamicina/efeitos adversos , Animais , Linhagem Celular , Modelos Animais de Doenças , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Fibrose , Humanos , Túbulos Renais Proximais/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/efeitos dos fármacos , Transfecção , Fator de Crescimento Transformador beta1/farmacologia , eIF-2 Quinase/metabolismoRESUMO
BACKGROUND: We aimed to determine sphingolipid levels and examine apoptotic pathways in human retinal pigment epithelial cells (ARPE-19) undergoing endoplasmic reticulum (ER) stress. METHODS: Cells were treated with tunicamycin (TM) to induce ER stress and tauroursodeoxycholic acid (TUDCA), an ER stress inhibitor, was administered to decrease cytotoxicity. Cell viability was measured by MTT assay. Levels of C16-C24 sphingomyelins (SM) and C16-C24 ceramides (CERs) were determined by LC-MS/MS. Glucose-regulated protein 78-kd (GRP78) and nuclear factor kappa-b subunit 1 (NFκB1) gene expressions were evaluated by quantitative PCR analysis, while GRP 78, NF-κB p65, cleaved caspase-3 and caspase-12 protein levels were assesed by immunofluorescence. Ceramide-1-phosphate (C1P) levels were determined by immunoassay, while caspase -3 and -12 activity in cell lysates were measured via a fluorometric method. RESULTS: Induction of ER stress in TM treated groups were confirmed by significantly increased mRNA and protein levels of GRP78. TM significantly decreased cell viability compared to controls. Treatment with TUDCA along with TM significantly increased cell viability compared to the TM group. A significant increase was observed in C22-C24 CERs, C1P, caspase-3, caspase-12, NFκB1 mRNA and NF-κB p65 protein levels in cells treated with TM compared to controls. Administration of TUDCA lead to a partial decrease in GRP78 expression, NFκB1 mRNA, NF-κB p65 protein, C22-C24 CERs and C1P levels along with a decrease in caspase-3 and -12 activity. CONCLUSIONS: The results of this study reveal the presence of increased long chain CERs, C1P and apoptotic markers in retinal cells undergoing ER stress.
Assuntos
Epitélio Pigmentado da Retina/citologia , Transdução de Sinais/efeitos dos fármacos , Esfingolipídeos/análise , Ácido Tauroquenodesoxicólico/farmacologia , Tunicamicina/efeitos adversos , Apoptose/efeitos dos fármacos , Linhagem Celular , Ceramidas/análise , Cromatografia Líquida , Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Subunidade p50 de NF-kappa B/genética , Epitélio Pigmentado da Retina/metabolismo , Esfingomielinas/análise , Espectrometria de Massas em TandemRESUMO
Melatonin 2-hydroxylase (M2H) catalyzes the conversion of melatonin into 2hydroxymelatonin (2OHM), which is present in plants at a higher concentration than melatonin. Although M2H has been cloned, the in vivo function of its product is unknown. Here, we generated stable T2 homozygous transgenic rice plants in which expression of endogenous M2H was suppressed (RNAi lines). However, we failed to generate M2H overexpression transgenic rice due to failure of somatic embryogenesis. The M2H transcript level showed a diurnal rhythm with a peak at night concomitantly with the peak concentration of 2OHM. RNAi rice showed a reduced M2H mRNA level and 2OHM and melatonin concentrations. The unexpected decrease in the melatonin concentration was caused by redirection of melatonin into cyclic 3hydroxymelatonin via a detour catabolic pathway. Thus, the decrease in the melatonin concentration in M2H RNAi rice led to slowed seedling growth and delayed germination. By contrast, the transient increase in the melatonin concentration was of greater magnitude in the M2H RNAi than the wild-type rice upon cadmium treatment due to possible suppression of melatonin degradation. Due to its higher concentration of melatonin, the M2H RNAi rice displayed tolerance to senescence, salt, and tunicamycin stresses. Therefore, the increase in the melatonin concentration caused by suppression of melatonin degradation or by overexpression of melatonin biosynthetic genes enhances stress tolerance in rice.
Assuntos
Melatonina/metabolismo , Oxigenases de Função Mista/genética , Oryza/crescimento & desenvolvimento , Estresse Fisiológico , Cádmio/efeitos adversos , Ritmo Circadiano , Regulação da Expressão Gênica de Plantas , Melatonina/análogos & derivados , Oxigenases de Função Mista/metabolismo , Oryza/genética , Oryza/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/metabolismo , Interferência de RNA , Estresse Salino , Tunicamicina/efeitos adversosRESUMO
Chemical proteasome inhibition has been a valuable animal model of neurodegeneration to uncover roles for the ubiquitin-proteasome system in the central nervous system. However, little is known about the effects of chemical proteasome inhibitors on retinal integrity. Therefore, we characterized the effects of structurally different chemical proteasome inhibitors on the retinal morphology and the mechanisms of their action in the normal adult rat eyes. Intravitreal injection of MG-262 and other proteasome inhibitors led to inner retinal degeneration. MG-262-induced inner retinal degeneration was accompanied by reduced proteasome activity, increased poly-ubiquitinated protein levels, and increased positive immunostaining of ubiquitin, 20S proteasome subunit and GADD153/CHOP in the retina. Its retinal degenerative effect was also associated with reduced retinal neurofilament light chain gene expression, reflecting retinal ganglion cell death. MG-262-induced neurofilament light chain downregulation was largely resistant to pharmacological modulation including endoplasmic reticulum stress, apoptosis or MAP kinase inhibitors. Thus, this study provides further evidence of roles for the ubiquitin-proteasome system in the maintenance of the retinal structural integrity. Chemical proteasome inhibition may be used as a novel animal model of inner retinal degeneration, including retinal ganglion cell loss, which warrants further analysis of the molecular mechanisms underlying its retinal degenerative effect.
Assuntos
Complexo de Endopeptidases do Proteassoma/genética , Inibidores de Proteassoma/farmacologia , Retina/patologia , Degeneração Retiniana/patologia , Animais , Apoptose/efeitos dos fármacos , Ácidos Borônicos/efeitos adversos , Ácidos Borônicos/farmacologia , Modelos Animais de Doenças , Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Humanos , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Ratos , Retina/efeitos dos fármacos , Degeneração Retiniana/induzido quimicamente , Degeneração Retiniana/metabolismo , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/patologia , Tapsigargina/efeitos adversos , Tapsigargina/farmacologia , Tunicamicina/efeitos adversos , Tunicamicina/farmacologiaRESUMO
AIMS: Signal transducer and activator of transcription (STAT)-3 lies at the convergence point of key pathways involved in many malignancies including oral squamous cell carcinoma (OSCC). Endoplasmic reticulum stress (ERS) and the unfolded protein response have been shown to be involved in the pathogenesis and progression of different cancers by influencing key cellular processes such as apoptosis. We investigated the differential expression of STAT3 pathway-related genes and proteins under ERS in OSCC. METHODS: Three normal oral keratinocyte (NOK) and three OSCC cell lines were subjected to tunicamycin to induce ERS for 24â¯h or to the vehicle medium as control. A pathway-focussed array was used to analyse the modulation of STAT3 pathway gene expression under ERS using qPCR. The expression of key regulated proteins was investigated in the cell lines using immunocytochemistry and in 76 OSCC and 9 normal oral mucosa (NOM) tissue samples using tissue microarray technology and immunohistochemistry. RESULTS: ERS resulted in up-regulation of interleukin-6 receptor (IL6R) gene in NOK cell lines (pâ¯=â¯0.001) and IL5 (pâ¯=â¯0.005) and IL22 (pâ¯=â¯0.024) in OSCC cell lines. Greater STAT3 (pâ¯=â¯0.019) and leukaemia inhibitory factor receptor (pâ¯=â¯0.042) protein expression was observed in treated than untreated NOK cell lines. CONCLUSIONS: The gene and protein regulation patterns show that ERS plays a role in modifying the tumour microenvironment in OSCC by up-regulating tumour-promoting cytokines.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Citocinas/metabolismo , Estresse do Retículo Endoplasmático , Neoplasias Bucais/metabolismo , Neoplasias Bucais/patologia , Tunicamicina/efeitos adversos , Regulação para Cima , Carcinoma de Células Escamosas/genética , Linhagem Celular Tumoral , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Bucais/genética , Fator de Transcrição STAT3/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Elevated soluble (s) CD163 and free hemoglobin (Hb) levels predict fatty liver progression; however, the molecular mechanisms underlying Hb metabolism and liver injury remain undefined. We investigated the effects of endoplasmic reticular (ER) stress on red blood cell (RBC) rheology and free Hb recycling pathways. ER stress was induced in Sprague-Dawley rats by an intraperitoneal injection of tunicamycin (TM) (50, 100, and 200 µg/100 g body weight (BW)) or an intravenous injection of Hb (5 mg/100 g BW). A TM injection increased sCD163 levels, attenuated free Hb uptake, and maintained RBC aggregability. An Hb injection increased serum LVV-hemorphin-7 and total bilirubin levels, but this effect was suppressed by TM. A Western blot analysis showed that ER stress suppressed Hb degradation in the liver through downregulation of globin degradation proteins cathepsin D and glyoxalase-1, as well as heme degradation protein heme oxyganase-1 and keap-1 expression. An ER stress activator also increased the translocation of nuclear factor (NF)-κB (p65) and nuclear factor-erythroid 2-related factor 2 (Nrf2) to nuclei. In conclusion, ER stress triggers ineffective Hb metabolism via altering globin and heme iron degradation pathways. Inability to recycle and metabolize free Hb may underlie the association between iron dysfunction and liver injury.
Assuntos
Estresse do Retículo Endoplasmático/fisiologia , Hemoglobinas/metabolismo , Fígado/patologia , Tunicamicina/efeitos adversos , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Bilirrubina/sangue , Catepsina D/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Eritrócitos/metabolismo , Heme/metabolismo , Hemoglobinas/administração & dosagem , Injeções Intraperitoneais , Injeções Intravenosas , Ferro/sangue , Lactoilglutationa Liase/metabolismo , Fígado/efeitos dos fármacos , Masculino , Fragmentos de Peptídeos/sangue , Ratos , Ratos Sprague-Dawley , Receptores de Superfície Celular/metabolismo , Tunicamicina/administração & dosagemRESUMO
BACKGROUND: The prevalence of Non-alcoholic fatty liver disease (NAFLD) including non-alcoholic steatohepatitis (NASH) has increased by 15-39% worldwide, but no pharmaceutical therapeutics exists. HYPOTHESIS/PURPOSE: This study investigated anti-hepatosteatotic effect of CGplus (a standardized herbal composition of Artemisia iwayomogi, Amomum xanthioides, and Salvia miltiorrhiza) and its underlying mechanisms in a tunicamycin-induced NASH model. METHODS: C57/BL6J male mice were orally administrated CGplus (50, 100, or 200â¯mg/kg), dimethyl dimethoxy biphenyl dicarboxylate (DDB, 50â¯mg/kg) or distilled water daily for 5 days. 18â¯h after a single injection of tunicamycin (ip, 2â¯mg/kg), the parameters for hepatic steatosis and inflammation were measured. RESULTS: Pretreatment with CGplus significantly attenuated the accumulation of triglycerides and total cholesterol as well as lipid peroxidation, evidenced by quantitative and histopathological analyses in liver tissues. The elevations of serum aspartate transaminase, alanine transaminase and lactate dehydrogenase were significantly ameliorated by CGplus. Also, it normalized the altered activities of pro- (TNF-α, IL-1ß and IL-6), anti-inflammatory (IL-10) cytokines and lipid metabolism-related molecules in protein and gene expression analyses. CONCLUSION: Our data present experimental evidence for the potential of CGplus as an herbal therapeutic against NAFLD and NASH. Its underlying mechanisms may involve the modulations of pro- and anti-inflammatory cytokines, but further study is required especially for the actions of CGplus on lipid metabolisms.
Assuntos
Medicamentos de Ervas Chinesas/química , Medicamentos de Ervas Chinesas/farmacologia , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Substâncias Protetoras/farmacologia , Tunicamicina/efeitos adversos , Alanina Transaminase/sangue , Animais , Aspartato Aminotransferases/sangue , Citocinas/metabolismo , Modelos Animais de Doenças , Metabolismo dos Lipídeos/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/induzido quimicamente , Triglicerídeos/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The involvement of endoplasmic reticulum (ER) stress in endothelial dysfunction and diabetes-associated complications has been well documented. Inhibition of ER stress represents a promising therapeutic strategy to attenuate endothelial dysfunction in diabetes. Recent attention has focused on the development of small molecule inhibitors of ER stress to maintain endothelial homeostasis in diabetes. Here we have developed a reliable, robust co-culture system that allows a study on the endothelial cells and pancreatic ß-cells crosstalk under ER stress and validated using a known ER stress modulator, quercetin. Furthermore, sensitizing of endothelial cells by quercetin (25 µM) confers protection of pancreatic ß-cells against ER stress through nitric oxide (NOâ) signaling. In addition, increased intracellular insulin and NOâ-mediated cyclic 3',5'-guanosine monophosphate (cGMP) levels in pancreatic ß-cells further confirmed the mechanism of protection under co-culture system. In addition, the potential protein targets of quercetin against ER stress in the endothelial cells were investigated through proteomic profiling and its phosphoprotein targets through Bioplex analysis. On the whole, the developed in vitro co-culture set up can serve as a platform to study the signaling network between the endothelial and pancreatic ß-cells as well as provides a mechanistic insight for the validation of novel ER stress modulators.
Assuntos
GMP Cíclico/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Óxido Nítrico/metabolismo , Quercetina/farmacologia , Animais , Técnicas de Cocultura , Diabetes Mellitus Experimental/tratamento farmacológico , Estresse do Retículo Endoplasmático/fisiologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Insulina/sangue , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Óxido Nítrico Sintase Tipo III/metabolismo , Fosfoproteínas/metabolismo , Quercetina/análise , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Tunicamicina/efeitos adversosRESUMO
RNA repair enzymes catalyze rejoining of an RNA molecule after cleavage of phosphodiester linkages. RNA repair in budding yeast is catalyzed by two separate enzymes that process tRNA exons during their splicing and HAC1 mRNA exons during activation of the unfolded protein response (UPR). The RNA ligase Trl1 joins 2',3'-cyclic phosphate and 5'-hydroxyl RNA fragments, creating a phosphodiester linkage with a 2'-phosphate at the junction. The 2'-phosphate is removed by the 2'-phosphotransferase Tpt1. We bypassed the essential functions of TRL1 and TPT1 in budding yeast by expressing "prespliced," intronless versions of the 10 normally intron-containing tRNAs, indicating this repair pathway does not have additional essential functions. Consistent with previous studies, expression of intronless tRNAs failed to rescue the growth of cells with deletions in components of the SEN complex, implying an additional essential role for the splicing endonuclease. The trl1Δ and tpt1Δ mutants accumulate tRNA and HAC1 splicing intermediates indicative of RNA repair defects and are hypersensitive to drugs that inhibit translation. Failure to induce the unfolded protein response in trl1Δ cells grown with tunicamycin is lethal owing to their inability to ligate HAC1 after its cleavage by Ire1. In contrast, tpt1Δ mutants grow in the presence of tunicamycin despite reduced accumulation of spliced HAC1 mRNA. We optimized a PCR-based method to detect RNA 2'-phosphate modifications and show they are present on ligated HAC1 mRNA. These RNA repair mutants enable new studies of the role of RNA repair in cellular physiology.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , RNA Ligase (ATP)/genética , Splicing de RNA , RNA/genética , Proteínas Repressoras/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Éxons/genética , Íntrons/genética , Mutação , RNA Mensageiro/genética , RNA de Transferência/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/fisiologia , Tunicamicina/efeitos adversos , Resposta a Proteínas não Dobradas/efeitos dos fármacosRESUMO
Nonsense-mediated mRNA decay (NMD) degrades mRNAs carrying a premature termination codon (PTC) in eukaryotes. Cellular stresses, including endoplasmic reticulum (ER) stress, inhibit NMD, and up-regulate PTC-containing mRNA (PTC-mRNA) levels in several cell lines. However, whether similar effects exist under in vivo conditions that involve systemic nutritional status is unclear. Here, we compared the effects of pharmacological induction of ER stress with those of nutritional interventions on hepatic PTC-mRNA levels in mice. In mouse livers, the ER stress inducer tunicamycin increased PTC-mRNA levels of endogenous marker genes. Tunicamycin decreased body weight and perturbed nutrient metabolism in mice. Food restriction or deprivation mimicked the effect of tunicamycin on weight loss and metabolism, but did not increase PTC-mRNA levels. Hyperphagia-induced obesity also had little effect on hepatic PTC-mRNA levels. Meanwhile, in mouse liver phosphorylation of eIF2α, a factor that regulates NMD, was increased by both tunicamycin and nutritional interventions. Hepatic expression of GRP78, a central chaperone in ER stress responses, was increased by tunicamycin but not by the nutritional interventions. In cultured liver cells (Hepa), exogenous overexpression of a phosphomimetic eIF2α failed to increase PTC-mRNA levels. However, GRP78 overexpression in Hepa cells increased PTC-mRNA and PTC-mRNA-derived protein levels. ER stress promoted localization of GRP78 to mitochondria, and exogenous expression of a GRP78 fusion protein targeted to mitochondria mimicked the effect of wild type GRP78. These results indicate that GRP78, but not nutritional status, is a potent up-regulator of hepatic PTC-mRNA levels during induction of ER stress in vivo. J. Cell. Biochem. 118: 3810-3824, 2017. © 2017 Wiley Periodicals, Inc.
Assuntos
Códon de Terminação , Estresse do Retículo Endoplasmático , Proteínas de Choque Térmico/biossíntese , Fígado/metabolismo , Degradação do RNAm Mediada por Códon sem Sentido , Obesidade/metabolismo , Animais , Chaperona BiP do Retículo Endoplasmático , Células HEK293 , Proteínas de Choque Térmico/genética , Humanos , Hiperfagia/induzido quimicamente , Hiperfagia/genética , Hiperfagia/metabolismo , Hiperfagia/patologia , Fígado/patologia , Masculino , Camundongos , Camundongos Obesos , Células NIH 3T3 , Obesidade/induzido quimicamente , Obesidade/genética , Obesidade/patologia , Tunicamicina/efeitos adversos , Tunicamicina/farmacologiaRESUMO
Autophagy and the unfolded protein response (UPR) both promote activation of hepatic stellate cells (HSC), however the link between the two stimuli remains unclear. Here we have explored the role of X-box binding protein 1 (XBP1), one of three UPR effector pathways and sought to establish the interdependence between autophagy and the UPR during HSC activation. XBP1 induction accompanied both culture-based HSC activation and ER stress induced by tunicamycin. Ectopic overexpression of XBP1 induced collagen 1-alpha expression in HSCs, which was inhibited by knockdown of ATG7, a critical autophagy mediator. Genome-wide transcriptomic profiling indicated an upregulation of collagen synthesis pathways, but not of the transforming growth factor (TGF)-b pathway, a canonical fibrogenic driver, suggesting that XBP1 activates a specific subset of fibrogenesis pathways independent of TGF-ß1. XBP1 target gene signatures were significantly induced in rodent liver fibrosis models (n = 3-5) and in human samples of non-alcoholic fatty liver disease (NAFLD) (n = 72-135). Thus, XBP1-mediated UPR contributes to fibrogenic HSC activation and is functionally linked to cellular autophagy.
Assuntos
Autofagia/fisiologia , Células Estreladas do Fígado/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Resposta a Proteínas não Dobradas/fisiologia , Proteína 1 de Ligação a X-Box/metabolismo , Animais , Proteína 7 Relacionada à Autofagia/genética , Linhagem Celular , Colágeno Tipo I/biossíntese , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Células HEK293 , Humanos , Cirrose Hepática/patologia , Camundongos , Camundongos Endogâmicos C57BL , Interferência de RNA , RNA Interferente Pequeno , Tunicamicina/efeitos adversos , Proteína 1 de Ligação a X-Box/genéticaRESUMO
Endoplasmic reticulum (ER) stress is a critical factor involved in the pathogenesis of Alzheimer's disease (AD). Vitamin D and resveratrol are two nutritional factors that have reported neuroprotective effects, and findings from cellular models suggest that resveratrol could potentiate vitamin D's effects. We aimed to determine the effects of vitamin D & resveratrol on ER stress mediated neurodegeneration and whether synergistic effects existed. Tunicamycin and Aß25-35 was utilized to induce ER stress in SH-SY5Y cells, cells were then incubated with vitamin D and resveratrol. The combination of vitamin D & resveratrol completely reversed tunicamycin and Aß25-35 induced cytotoxicity in SH-SY5Y cells, as well as elevation in ER stress markers (i.e.GRP78, p-eIF2α and CHOP), insulin signaling disruption (i.e. elevation in p-IRS-1serine307 and reduction in p-Akt serine473) and tau phosphorylation (i.e. reduction in p-GSK3ß serine9, and elevation in p-Tau serine396 &404). Further studies are required to clarify whether the observed synergistic effects in the present study would also existed in vivo, this will lay scientific foundation whether the combination of vitamin D with resveratrol might be an effective maneuver in the treatment of AD in human subjects.
Assuntos
Calcitriol/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Insulina/metabolismo , Neuroblastoma/prevenção & controle , Fármacos Neuroprotetores/farmacologia , Estilbenos/farmacologia , Proteínas tau/metabolismo , Peptídeos beta-Amiloides/metabolismo , Antibacterianos/farmacologia , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Western Blotting , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Chaperona BiP do Retículo Endoplasmático , Humanos , Neuroblastoma/induzido quimicamente , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Fragmentos de Peptídeos/metabolismo , Fosforilação/efeitos dos fármacos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Resveratrol , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Células Tumorais Cultivadas , Tunicamicina/efeitos adversos , Vitaminas/farmacologiaRESUMO
Endoplasmic reticulum (ER) stress is associated with the pathogenesis of hepatic steatosis. Alisma orientale Juzepzuk is a traditional medicinal herb for diuretics, diabetes, hepatitis, and inflammation. In this study, we investigated the protective effects of methanol extract of the tuber of Alisma orientale (MEAO) against ER stress-induced hepatic steatosis in vitro and in vivo. MEAO inhibited the tunicamycin-induced increase in luciferase activity of ER stress-reporter constructs containing ER stress response element and ATF6 response element. MEAO significantly inhibited tunicamycin-induced ER stress marker expression including GRP78, CHOP, and XBP-1 in tunicamycin-treated Human hepatocellular carcinoma (HepG2) cells and the livers of tunicamycin-injected mice. It also inhibited tunicamycin-induced accumulation of cellular triglyceride. Similar observations were made under physiological ER stress conditions such as in palmitate (PA)-treated HepG2 cells and the livers of high-fat diet (HFD)-induced obese mice. MEAO repressed hepatic lipogenic gene expression in PA-treated HepG2 cells and the livers of HFD obese mice. Furthermore, MEAO repressed very low-density lipoprotein receptor (VLDLR) expression and improved ApoB secretion in the livers of tunicamycin-injected mice or HFD obese mice as well as in tunicamycin or PA-treated HepG2 cells. Alismol, a guaiane-type sesquiterpenes in Alisma orientale, inhibited GRP78 expression in tunicamycin-treated HepG2 cells. In conclusion, MEAO attenuates ER stress and prevents hepatic steatosis pathogenesis via inhibition of expression of the hepatic lipogenic genes and VLDLR, and enhancement of ApoB secretion.
Assuntos
Alisma/química , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Fígado Gorduroso/metabolismo , Extratos Vegetais/farmacologia , Animais , Apolipoproteínas B/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Dieta Hiperlipídica , Modelos Animais de Doenças , Chaperona BiP do Retículo Endoplasmático , Fígado Gorduroso/tratamento farmacológico , Fígado Gorduroso/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Células Hep G2 , Humanos , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Camundongos , Camundongos Obesos , Substâncias Protetoras/farmacologia , Receptores de LDL/genética , Receptores de LDL/metabolismo , Triglicerídeos/metabolismo , Tunicamicina/efeitos adversosRESUMO
Epithelial-to-mesenchymal transition (EMT) and apoptosis of peritoneal mesothelial cells are known to be the earliest mechanisms of peritoneal fibrosis in peritoneal dialysis (PD). Endoplasmic reticulum (ER) stress with an unfolded protein response is regarded to have a role in the development of organ fibrosis. To investigate the potential role of ER stress as a target to prevent and/or delay the development of peritoneal fibrosis, we examined the effect of ER stress on EMT or apoptosis of human peritoneal mesothelial cells (HPMCs) and elucidated the mechanisms underlying the protective effect of ER stress preconditioning on TGF-ß1-induced EMT. ER stress inducers, tunicamycin (TM) and thapsigargin (TG), induced EMT with Smad2/3 phosphorylation, an increased nuclear translocation of ß-catenin and Snail expression. Low concentrations of TM and TG did not induce apoptosis within 48 h; however, high concentrations of TM- (>1 ng/ml) and TG- (>1 nM) induced apoptosis at 12 h with a persistent increase in C/EBP homologous protein. TGF-ß1 induced EMT and apoptosis in HPMCs, which was ameliorated by taurine-conjugated ursodeoxycholic acid, an ER stress blocker. Interestingly, pre-treatment with TM or TG for 4 h also protected the cells from TGF-ß1-induced EMT and apoptosis, demonstrating the role of ER stress as an adaptive response to protect HPMCs from EMT and apoptosis. Peritoneal mesothelial cells isolated from PD patients displayed an increase in GRP78/94, which was correlated with the degree of EMT. These findings suggest that the modulation of ER stress in HPMCs could serve as a novel approach to ameliorate peritoneal damage in PD patients.